Adrenal Zona Glomerulosa Targeting in Transgenic Mice
By
Jeniel Parmar
Submitted to the Faculty of the School of Graduate Studies
of the Medical College of Georgia
in partial fulfillment of the Requirements of the
Degree of Doctor Philosophy
December, 2009
V Adrenal Zona Glomerulosa Targeting in Transgenic Mice
This dissertation is submitted by Jeniel Parmar and has been examined and approved by an appointed committee of faculty of the School of Graduate Studies of the Medical College of Georgia.
The signatures which appear below verify the fact that all the required changes have been incorporated and that the thesis/dissertation has received final approval with reference to content, form, and accuracy of presentation.
This dissertation is therefore in partial fulfillment of the requirement for the degree of Doctor of Philosophy.
Date Major Advisor - William E. Rainey, 8
Dept. Chair - R Clinton Webb, PhD
Dean, School of Gr te Studies - Gretchen Caughman, PhD
VI JENIEL PARMAR Adrenal Zona Glomerulosa Targeting in Transgenic Mice (Under the guidance of WILLIAM E. RAINEY II~ Ph.D.)
The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11 B2)-. CYP11 B2 is primarily expressed in the zona glomerulosa (ZG). of the adrenal cortex. Adrenocortical, expression of CYP11 B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Con$iderable in vitro analyses have been performed towards defining the mechanisms that control CYP11 B2 expression. Previous studies from our laboratory and others have identified ·several cis-regulatory elements on the 5' · flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11 B2 gene transcription. Moreover, key· transcription factors that bind these cis-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified.
Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP 11 B2 gene.
However, thus far, all evidence is based on in. vitro analyses of transcriptional regulation, which does not always depict in vivo_occurrences.
I To initiate our in vivo assessment of CYP1182 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP1182 genes, which interestingly revealed high sequence similarity in the ·5' flanking promoter region of the CYP1182 gene. This result suggested that the cis regulatory regions identified by in vitro analyses likely plays an important role in
CYP1182 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11 b2 promoter driving expression of a Lacz reporter gene.· Importantly, 4 founder Tg mouse lines revealed Lacz expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed Lacz mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein
(the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of Lacz exclusively in the adrenal ZG of Tg mice.
Moreover, with the expression of Lacz properly restricted to adrenal ZG, we concluded that regions required for Cyp11 b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment. will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation.
II While developing Tg mice, we also focused on characterization and development
of novel adrenocortical cell lines. As aforementioned, in vitro culture models have
allowed a multitude of studies that have broadened our understanding of normal
adrenocortical endocrine function. Primary cultures of adrenocortical cells have
been an excellent source for in vitro studies. However, the eventual onset of
senescence in primary cultures of cells creates a recurring need for the costly
· and difficult isolations of fresh adrenocortical cells. Hence, the use of primary
cultures has been increasingly supplemented by immortalized cell lines. We
utilized an adrenocortical carcinoma to develop a human adrenocortical cell line.
We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15).
HAC15 represents only the second human adrenocortical cell line available that
exhibits physiological hormonal responses, steroid.ogenesis, and expression of
steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and
ACTH makes it the first adrenal cell line capable of responding to the three main
physiologic regulators of the adrenal cortex.
III INDEX WORDS: Adrenal cortex, aldosterone, aldosterone synthase, transgenic mice, Lacz, cell lines, in vitro model systems.
NOMENCLATURE GUIDLINES: Throughout this dissertation, names of all human genes and proteins will be fully capitalized (e.g., CYP11 B2, CYP11 B1, and CYP17)-. The names of all rodent genes and proteins will be depicted with only the first letter capitalized (e.g., Cyp11 b2, Cyp11 b1, and Cyp17).
IV TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ...... IX
LIST OF FIGURES ...... X
LIST OF TABLES ...... XI I
LIST OF ABBREVIATIONS ...... ■- •• ___ •••••••••••••• XI U
I. INTRODUCTION
A. Statement of the Problem: CYP1182 Gene Regulation ...... _.. 1
1) Specific Aim I ...... 3
2) Review of Literature and Rationale ...... 4 ·
B. Statement of the Problem: A Paucity of Adrenal Cell Lines .... 20
1) Specific Aim 11 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 22
2) Review of Literature and Rationale ...... 23
II. MANUSCRIPTS·
A. Peer Reviewed Articles ...... 30
1) "Gene targeting to the mouse adrenal zona glomerulosa"
VII 2) "Development of an ACTH Responsive Human Adrenocortical Carcinoma Cell Line"
8. Invited Review Article ...... 76
"Comparisons of Adrenocortical Cell Lines as in vitro Test
Systems"
C. Book Chapter ...... 107
"Adrenocortical Cell Lines"
Ill. DISCUSSION
A. Specific Aim I: CYP1182 Gene Regulation ...... 146
B. Specific Aim II: A Paucity of Adrenal Cell Lines ...... 155
IV. BIBLIOGRAPHY ...... ~ ...... 162
VIII ACKNOWLEDGEMENTS
I wish to express my sincerest gratitude to Dr. William E. Rainey for taking me under his wing and guiding me throughout my graduate career. Seldom does one have the opportunity to learn from a true advisor. I deeply value the wisdom and scientific expertise that he bestowed upon me. I will miss his enthusiasm and ceaseless support. The experience will surely never be forgotten.
I am indebted to Dr. Tsugio Seki for his kind technical as well as advisory support.
I would also like to thank my committee members Dr. Edward Inscho for never letting me forget about the kidneys, Dr. Wendy Bollag for regularly reminding me about the importance of primary cell cultures, and Dr. Lawrence Layman for teaching me the value of statistical analyses. My appreciation is extended to the readers of my thesis Dr. Michael Edwards and Dr. Celso Gomez-Sanchez for willingly provide selfless support.
I would like to thank the faculty of the department of Physiology at the Medical College of Georgia for providing me with an exceptional education. I would also like to thank the past and present Rainey lab members and our department management including Anthony, Jennifer, Daniel, Claudia, Rebecca, Nick, Bobbie, Kathy, Faye, Valerie, and Brenda, who have all been a distinguished part of my life in the past several years.
Finally, a special word of appreciation must be extended to my family. I would like to thank my parents for their seemingly inexhaustible faith and understanding· during my education. I would like to thank my best friend and brother, Hiren, and his wife, Jigna, for their eternal support and their cheerful and enthusiastic endorsements. I would also like to thank my friend, companion and wife, Ami, for her love and compassion.
IX LIST OF FIGURES
Figure Page
1. Phylogenetic an.cestral lineages ...... 5
2. H&E stained mouse adrenocortical tissue ...... 10
3. Major adrenocortical steroidogenic pathways ...... 12
4. Glucocorticoid suppressible hyperaldosteronism model ...... - ...... 16
5. Cyp11 b2 evolutionary conserved regions (ECR) ...... 18
6. Cyp11 b2 ECR analysis and Tg construct (manuscript A 1) ...... 53
7. Cyp11 b2 transcript quantity (manuscript A1 } ...... · ...... •...... 54
8. Cyp11 b2 localization in adrenal cortex (manuscript A 1) ...... 55
9. X-gal stained Tg adrenals under LS diet (manuscriptA1) ...... ' ...... 56
10.Co-localization of Cyp11b2 and X-gal in Tg mice (manuscript A1} ...... 57
11.Amplification curve of Cyp11b2 (manuscriptA) ...... : ...... 58
12.HAC15 and H295R cell comparison (manuscript A2) ...... ~ ...... - .... 74
13. HAC15 cell line enzymatic expression assessment (manuscript A2) ...... ~ .. 75
14. Human a_drenal steroid biosynthetic pathways (manuscrip~ _,B) ...... 104
15. Human adrenal cell signaling pathways (manuscript B) ...... · ...... 105
16. Rodent adrenal steroid biosynthetic (manuscript 8) ...... ~ ...... 106
17. Intracellular adrenocortical cell signaling (manuscript C) ...... 140
18. Phase contrast images of H295R and HAC cells (manuscript C) ...... 141
X 19. Human adrenocortical enzymatic pathway (manuscript C)-...... 142
20. Development and isolation of HAC clones (manuscript C) ...... 143
21. Cytogenetic profiles depicted through karyotypes (m~nuscript C}...... 144
22. Murine adrenocortical enzymatic pathway (manuscript C)...... 145
XI ·LIST OF TABLES
Table Page
I. Plasma aldosterone concentrations in WT mice (manuscript A 1) ...... 49
II. Plasma aldosterone concentrations in Tg mice (manuscript A 1) ...... 49
m. Relative transgene mRNA expression (manuscript A 1) ...... 49
N. Rodent and bovine adr~na~ cell lines (manuscript B)...... 101
V. Human adrenal cell lines (manuscript B)...... 102
VI. All adrenocortical cell lines (manuscript C)...... 136
VII. Cytogenetic analyses of HAC15 and H295R cells (manuscript C)...... 138
XII LIST OF ABBREVIATIONS
• ACTH: adrenocorticotropin • NSAID: non-Steroidal anti-inflammatory
• ANOVA: analysis of variance drugs
• Ang 11: angiotensin 11 • PAGE: polyacrylamide gel
• ATCC: american Type Culture electrophoresis
Collection • PCR: polymerase chain reaction
• cAMP: cyclic AMP • RAS: rennin-angiontensin-system
• cDNA: complementary DNA • RIA: radioimmunoas_say
• CRH: corticotropin~releasing hormone • RT-PGR : reverse transcription-PCR
• CYP 11 B 1 : 11 ~ -hyd roxylase • mRNA: messenger RNA
• CYP1182: aldosterone synthase • rRNA: ribosomal RNA
• DNAase: deoxyribonuclease . • RNase: ribonuclease
• DMSO: dimethylsulfoxide • SE: standard error
• EDTA: ethylenediaminetetraacetic acid • T g: transgenic
• ELISA: enzyme-linked immunosorbent · • qPCR: quantitative PCR
assay • ZF: zona fasciculata
• EMSA: elecrophoretic mobility shift • ZG: zona glomerulosa
assay · • ZR: zona reticularis
• HAC: human adrenocortical carcinoma
• K+: potassium
XIIl I. INTRODUCTION
A. Statement of the problem: CVP11 B2 gene regulation
In humans, the biosynthesis of cortisol and aldosterone is dependent on two key en:zymes: 11 ~-hydroxylase (CYP11 B1) and aldosterone synthase (CYP11 B2}. In humans the coding sequences of these two genes are 95 % identical, their non coding intronic sequences are 90 % identical, and their encoded amino acid
5 8 sequences are 93 % identical - • Due to the high sequence homology shared by the two isozymes, only mouse and rat aldosterone synthase antibodies have
9 been developed, to date, which can exclusively detect the respective protein •
Interestingly, despite this similarity, CYP11 B1 is exclusively expressed in the zona fasciculata (ZF), while CYP11 B2 is exclusively expressed in the zona glomerulosa (ZG). The molecular mechanisms that cause tissue-specific expression and repression of CYP11 B2 are unknown. However, it is clear that the sequence similarity between the 5' flanking promoter regions of CYP11 B2
10 11 and CYP11 B1 genes is significantly reduced • • Perhaps the promoter regions of the two genes harbor discrete transcription factor binding sites that allow for the differential regulation of transcription. Therefore, we hypothesized that this dissimilarity accounts for the differential zone-specific distribution of these two
12 14 genes within the adrenal cortex - • Defining the mechanisms that allow zone specific expression and repression of CYP 11 B2 should provide important
1 information with regard to its aberrant expression in human adrenal diseases.
Dys regulation of CYP 11 B2 expression plays a key role in aldosterone-producing·
15 16 _adenomas (APA} and glucocorticoid suppressible hyperaldosteronism (GSH} , both of which lead to primary aJdosteronism (PA), the most common cause of endocrine hypertension.
A DNA sequence comparison between mouse and rat CYP11 B2 genes revealed high sequence similarity in the 5' flanking promoter region of the
CYP11 B2 gene. Aforementioned, the greatest dissimilarity between the highly conserved CYP11 B1 and CYP11 B2 genes lies within the 5' flanking promoter region; concurrenUy, the same promoter region of CYP11 B2 in a number _of species is also highly similar. Collectively, this suggests that as CYP11 B1 and
CYP11 B2 evolutionarily diverged into two distinct enzymes, the zone-specific regulation of the two isozymes must have been established and maintained in closely related species. One aim has been proposed to determine the CYP11 B2 promoter regions that are required for its tissue-specific expression, as well as repression, within the adrenocortical zones. These studies should broaden our knowledge of adrenocortical zonation under physiological conditions and provide insights into human adrenal diseases.
-2 1) Specific aim I
As in humans, Cyp11 b2 in mice is solely expressed in the ZG and its expression
is repressed in the adjacent ZF and zona reticularis (ZR), of the adrenal cortex.
· Herein, an in vivo model to define the regions responsible for the ZG-specific
expression of Cyp11 b2 gene was sought.
Hypothesis: The regulatory regions within the 5' flanking promoter of Gyp 11 b2
are sufficient for restricted adrenal ZG-specific expression.
a. We demonstrated that the 985 bp Cyp11 b2 promoter region was sufficient
for ZG-specific targeting. The 985 bp Cyp11 b2 promoter-driven Lacz
reporter gene was detected solely in_ the ZG of the adrenal cortex. J3-
galactosidase (x-gal) staining analysis was confirmed by quantitative real
time PCR (qPCR}.
b. The 985 bp Cyp11 b2 promoter region was also sufficient for ZF- and ZR
specific repression. Lacz was -not expressed in ZF and ZR. Upon qPCR
examination of 16 key mouse tissues, no aberrant extra-adrenal Lacz
transcript was found.
3 2) Brief literature review and rationale: CVP11 B2 gene regulation
The adrenal gland, an integral part of the endocrine system, evolved to regulate essential physiological functions. According· to the earliest descriptions of adrenal function, Agnathans (lampreys) were among the first animals to have developed adrenal steroid hormone receptors-the ancestral
17 18 corticosteroid receptor (CR)-about 470 million years ago (mya} • • The ligand for this ancestral CR is yet to be elucidated; consequently, the function of CR remains to·- be established in lampreys. Subsequently, a whole genome wide duplication event is believed to have occurred in the CR lineage giving rise to
.. glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)- homologues in the Gnathostomatas (sharks and skates) 440 mya 19 (Fig._ 1A). Both GR and MR
3 have been cloned and characterized in the ancestral skate, Raja erinacea •
Moreover, a number of studies with sharks and skates indicate the formation of
20 21 corticosterone and 1a-OH corticosterone, capable of binding GR and MR • •
Hence corticosterone is believed to have emerged as a ligand for MR _and GR; importantly, these results suggest the apparent presence of a key enzyme capable of 11 (3-hyroxylase activity since 440 mya. This · ancestral enzyme capable of 11 (3-hydroxylation was designated to ~he cytochrome P450, family 11, and subfamily B (CYP11 B). A distinct role for the CYP11 B enzyme expands with
22 23 the appearance of cortisol in Osteichthyes (bony fish) 420 mya • • Presumably as evolution progressed, stress due to a vast array of extreme environmental and physiological changes necessitated the emergence of cortisol for assistance in .
4 A GR 300 F, Aldo CYPH 81 4¥" MR mya ·Doc CYP11B2 Amniotes · Reptiles; birds, and mammals
GR 350 F, Aldo CYP11 B Tetrapods Amphibians MR mya- DOC
GR 400 F, Aldo CYP11 B Sarcopterygii _Lung fish ,, ' MR mya DOC GR 420 F CYP11 B Osteichthyes Bony fish ,.. MR mya Doc
_,_. GR 440 1a-OH CYP B 11 MR mya B, B Gnathostomatas Cartilaginous fish-Sharks and Skates
41Q ( 470· CR ??? ??? . Agnathans · Jawless fish-Lampreys and Hagfish mya
B Frog CYP118 Bovine CYP11 B
CYP11B Human CYP11 B1
Human CYP11B2
Mouse CYP11 B1 Rat CYP11B1 Rat CYP11B3 Mouse CYP11 B2
Rat CYP11 B2 ·
50 40 30 20 10 0 Time (million years) Present day _
Fig. 1: Phylogenetic ancestral lineages. Panel A: evolutionarily · led major transitions in adrenal steroid· hormone receptors,. adrenal steroid hormones, and 1 3 adrenocortical enzymes · • Functional role of the adrenal gland increases with growth in complexity of vertebrate evolution. Abbreviations: · corticosteroid receptors (CR)-, glucocorticoid receptors (GR), mineralocorticoid receptors (MR), cortisol (F), aldosterone (Aldo), deoxycorticosterone (DOC), and corticosterone 4 (B). Panel B: mammalian phylogenetic tree of the CYP11 B subfamily •
. - 5 coping with perpetual stress. In terms of migration of aquatic vertebrates from
water to land roughly 400 mya, the need-- for mineral and water homeostasis at
the systemic and cellular levels must have risen, which necessitated- a salt- and
water-retaining hormone. In both African and Australian Sarcopterygii (lungfish),
aldosterone, cortisol, and DOC are detected in plasma by double-isotope
24 25 derivative assay (DIDA) and radio-immunoassay (RIA) • • At the outset, the
mineralocorticoid effect of aldosterone exerted at the levels of gills in these
lungfish, where it controls the chloride ion exchange and Na+/K+ ATPase activity
26 • Studies also indicate the ·control of aldosterone secretion by a functioning . ~
renin-angiotensin-system (RAS) as both angiotensin I (Ang I). and Ang 11
24 27 stimulate the release of aldosterone in lungfish • • Intriguingly, this suggests
that the RAS likely played an important role in the aquatic to terrestrial transition
) of vertebrate evolution. It would seem reasonable to- presume that aldosterone
provided a selective sodium- and water-sparing advantage to the first aquatic
invaders venturing into the terrestrial habitats, where both were fluctuating
resources. A comparative genomic approach supports the hypothesis that
phylogeny subsequent to lungfish paved the way 400 mya and gave rise to
28 29 Tetrapods (amphibians) and Amniotes (reptiles, birds, and mammals) • • These
tremendous biological changes enforced the distribution of MR and GR to
various anatomical locations in an array of species according to their habitation.
Aldosterone and corticosterone promote transepithelial sodium and water re
absorption from the skin and urinary bladders of Anurans (frogs and toads},
31 which do not drink water3°· • Reptiles have nasal salt secreting gland in which
6 corticosterone increases sodium secretion while aldosterone decreases it to
2 stimulate sodium- and water-reabsorption through kidney and urinary bladder3 •
Birds primarily utilize both aldosterone and corticosterone' to· induce sodium
33 secretion and excretion from the nasal glands as they lack urinary bladders •
Hence, corticosteroids may target various osmoregulatory organs and have variable actions in fish, amphibians, reptiles, and birds as compared to mammals.
Common features between mammalian and fish adrenocortical function are likely to predate our understanding of corticosteroid function back 400 mya, · when mammals and fish last shared a common ancestor. Nevertheless, most
(but not all) mammalian species, including humans, have resumed the adrenal synthesis of cortisol as the primary glucocorticoid and aldosterone as the primary mineralocorticoid, since earliest seen in lungfish. Synthesis of corticosteroids in lungfish is made possible by a single CYP11 B enzyme. Presumably, as the role of aldosterone as the salt- and water-sparing . hormone became critical for survival in the terrestrial mammals, in some species, the CYP11 B gene duplicated to originate the CYP11 B 1 and CYP1182 genes (Fig. 1B). The complexity of this system is increased even more in rat where a duplication has
34 led to a third gene, CYP1183 (Fig. 18) • The enzyme CYP1181, incapable of synthesizing aldosterone, was labeled 11 J3-hydroxylase as it 11 J3-hydroxylates
11-deoxycorticoids to yield cortisol and corticosterone. The enzyme CYP1182 was labeled aldosterone synthase as ~t performed 11 J3-hydroxylation, 18- hydroxlation and 18-oxidation to yield aldosterone. It is unclear why the ancestral
7 CYP11 B enzyme, capable of both aldosterone and cortisol synthesis, is still
expressed in some mammals such as cattle, sheep etc. Moreover, . some
mammalian species such as mice and rats have diverged about 100 mya,
utilizing corticosterone as their primary glucocorticoid instead of cortisol. Hence
the phylogenetic tree of the CYP 11 B gene subfamily remains elusive (Fig. 1B).
It is noteworthy that although terrestrial mammals rely on aldosterone for
sodium and water retention, marine mammals have an opposite osmoregulatory
role as they need to excrete the surplus of sodium. Immunoassay measurements
of Antarctic seals and whales show 20-100 times lower aldosterone secretion
35 36 than in terrestrial mammals • •
As the evolutionary tree expanded becoming more complex so did the
1 function, anatomical location, and zonation of the adrenal gland • Hardisty
et al. have shown in lampreys that putative adrenocortical cells are located as
37 loose pockets around the great dorsal vessels , representing the least
specialized system in terms of structural organization of the adrenal gland among
_all vertebrates. These adrenocortical cells have an ultra-structure of typical
steroid-secreting cells with abundant lipid droplets, extensive smooth
endoplasmic reticulum, and they are rich in tubular and transverse mitochondrial
18 cristae . Sharks and skates have islets of adrenocortical cells located between
38 the two kidneys, referred as the interrenal gland • Bony fish have patches of
dispersed adrenocortical cells located within their nephritic and hemopoatic
20 tissues • In African as well as Australian lungfish, adrenocortical cells appear in
8 gross· dissection as small, yellow-pigmented regions within the kidney, referred
39 as . the intrarenal glands • Histologic preparations demonstrate that these pigmented regions have irregularly organized clusters of lipid-contain1_ng. cells
40 41 and that these cells are immuno-reactive to adrenal-specific enzymes • • A positive enzymatic reaction during incubation of frozen sections is considered to be the strongest proof of potential for steroidogenesis.
In terms of location of adrenocortical cells, all possible anatomical locations discussed above are represented by different species of amphibians,
42 intrarenal and interrenal glands • Reptiles and birds represent the first vertebrates to have developed separate adrenal glands atop of the kidneys, however, their chromaffin cells are not encapsulated by the cortical cells, rather the . adrenal is a mixture of the two: In all mammals, the adrenal gland is subdivided into two different compartments-the outermost cortex and the innermost medulla. Consequently, only the mammals contain a well-defined adrenal cortex that surrounds the adrenal medulla, while all the lower vertebrates have mixed cortical and chromaffin cells.
The histological aspects of the adrenal cortex vary to some extent among different mammalian species. However, in general, it has been known since the end of the 19th century that the adrenal cortex is divided into three concentric
43 zones; ZG, ZF, and ZR (from_ outer- to inner-most} • Each zone has been histologically separated and characterized. The ZG appears to be irregularly structured with groups of rounded and columnar shaped cells (Fig. 2). The ZG . cells also appear to have round, dark nuclei and a much smaller cytoplasm as
9 compared to the other two zones. Cells in the ZF are polyhedral with a foamy appearance due to the abundance of lipid droplets (Fig. 2}. The ZF appears, to be radially arranged into cords of cells _separated by fenestrated sinusoidal
_capillaries. Cells of the ZR
appear to be smaller than
ZF and uneven~
arranged. The Nuclei of
cells in the ZR appear
well-rounded and due to
the small cell size there is
a larger nuclear to
cytoplasmic ratio (Fig. 2).
Including histological divisions, each zone clearly has biochemical differences that are responsible for their distinctive functions_~ Each zone is responsible for the production and biosynthesis of unique steroid hormones. In the ZG, cholesterol is used to synthesize mineralocorticoids (e.g., aldosterone) in response to Ang II and K+ stimulation. In the ZF,, cholesterol is metabolized into glucocorticoids (e.g., cortisol in humans and corticosterone in mice) _under the regulation of adrenocorticotropin hormone (ACTH). In humans, the ZR utilizes cholesterol to synthesize the adrenal androgens (e.g., DHEA and DHEA-sulfate), regulated by
ACTH and other unknown factors. While histological and biochemical distinctions have been evident for quite some time, the molecular mechanisms that are
10 responsible for these differences remain unclear. Thus, the question remains as
to how the zones are able to regulate the exclusive production of specific steroid
hormones without a physical barrier separating each zone.
Functional zonation relies, in part, on the zone-specific expression of the
unique adrenocortical enzymes. Zonation functionally relates to production of
unique steroids, which result from the differential expression of steroid
metabolizing enzymes. Common to all adrenocortical pathways is the rate
limiting step; the translocation of cholesterol from the outer mitochondrial
membrane into the mitochondrion for side-chain cleavage by the cholesterol
side-chain cleavage enzyme (CYP11A1) (expressed throughout the adrenal
44 cortex) • On the contrary, CYP1182 is solely expressed in the_ ZG of the adrenal
cortex, and it represents a key regulatory enzyme in the aldosterone biosynthetic
11 45 pattiway ' • As shown in Figure 3, the three main adrenocortical steroid
hormones share certain enzymes within the biosynthetic pathway. In other
words, many of the intermediate enzymes needed to produce a particular steroid
· hormone are common to all of the zones. Taking this fact into consideration, if
there were even small ectopic expression of CYP1182 in the much larger ZF
zone, it would result in an excess of circulating aldosterone. As with CYP1182, if
CYP17 were expressed in the ZG, cortisol would be produced in the ZG. In
humans, the isozyme of CYP1182, CYP1181, is modestly expressed in ZG
46 cells , where it catalyzes 18-hydroxylation poorly and is unable to perform 18-
oxidation; therefore, CYP1181 is not able to produce aldosterone. The ZG of
11 47 mice and rats are .devoid of CYP1181 enzyme activity • Due to its role in glucocorticoid production, CYP1181 expression· is significantly elevated in the
8 ZF • Therefore, the spatial segregation of biosynthesis pathways through zonation of the adrenal is one way to control· the relative production rates of mineralocorticoids and glucocorticoids. The molecular mechanisms that cause
Cholesterol
• 11-DO~ Aldosterone
17a-hydroxy 17a-hydroxy 11-deoxycortis.ortisol Pregnenolone Progesterone
Location of enzymes: CHEA Mitochondrial
Smooth endoplasmic reticulum
Fig. 3: Major human adrenocortical steroidogenic pathways. Nomenclature for the steroidogenic enzymes are as follows: CYP11 A 1, cholesterol side-chain cleavage; CYP17, 17a-hydroxylase- 17, 20-lyase; HSD382, 3(3-hydroxysteroid dehydrogenase type II; CYP21, 21-hydroxylase; CYP1181, 11 (3-hydroxylase; CYP1182, aldosterone synthase. the zone-specific expression pattern of the adrenocortical enzymes are yet to be_ resolved. It has been demonstrated in vitro that the regulatory regions within the
5' flanking regions control the relative expression of these genes required for
6 11 steroidogenesis in each of the zones • •
12 Regulatory regions within the human CYP1182 promoter have been
11 12 studied and several elements have been identified ' • The earliest_ work on defining the regulatory regions of the adrenocorticaJ steroidogenic. enzymes was performed utilizing bovine ~nd porcine CYP11 B genes. Unlike humans and rodents, who express CYP11 B1 and CYP11 B2 genes, cattle, ·sheep, and pigs
4 48 only have one CYP11 B gene that is expressed in both ZG and ZF • . The same
CYP11 B gene product is responsible for the production of mineralocorticoids in the ZG and glucocorticoids in the ZF through mechanisms that are not yet clear.
However, considerable progress has been made towards understanding the regulation of CYP11 B gene transcription in the· adrenal cortex. Omura and colleagues have assessed the 5' flanking· CYP11 B gene promoter and identified
49 52 six different cis-regulatory elements, termed adrenal (Ad) 1-6 - • Through several in vitro deletion and mutation analyses of the .5' flanking region of the
CYP11 B promoter, Ad1 and Ad4 elements have been identified to be the most important regulators of gene transcription.
Consequently, as with the CYP11 B gene promoter, the 5-' flanking region of the CYP11 B1 and CYP11 B2 gene promoters have ·also been analyzed in
47 53 54 55 56 detail for rodents • • and humans • • Clyne and colleagu·es have utilized
H295R-a human adrenocortical carcinoma cell line-to assess the role .of the promoter region of the CYP11 B2 gene. H295R cells, transiently transfected and stimulated with Ang II or· K+, increase the expression of luciferase reporter constructs prepared with variable lengths of the CYP11 B2 promoter; similar
13 2 57 58 increases are ·observed with cAMP · and, Ca + stimulation • • Reporter. constructs of the CYP11 B2 promoter with serial deletions and mutations have been tested through electrophoretic mobility shift assays (EMSA), and DNase I footprinting experiments. Mutation of the Ad1 element at position -71 bp leads to a decreased induction by cAMP, Ang II, and K+, suggesting the importance of this element for both Ca2+- and cAMP-induced CYP11 B2 transcription. However, deletion of the Ad1 element does not terminate basal activity, suggesting requirements of other elements. Deletion or mutation of the Ad5 element at -129 bp in the CYP11 B2 promoter completely abolishes the basal and agonist stimulated transcription. DNA-protein interaction analyses· show that the Ad1 site resembles a cAMP response element (CRE) and binds CRE binding proteins
(CREB, ATF1 and ATF2), while the Ad4 sites bind steroidogenic factor-1 (SF-1) and Ad5 sites bind chicken ovalbumin upstream promoter transcription factor
54 56 59 61 (COUP-TF), among other transcription factors - • - •. Other Ad elements have been identified that bind transcription factors on the CYP11 B2 promoter; however, in vitro studies thus far do not support the role of these elements in
CYP11 B2 transcription. However, subsequent studies using the human
CYP11 B2 promoter have identified an additional cis-regulatory element that
55 62 binds the nuclear hormone receptor NURR1 • • In sum, three distinct cis regulatory elements on the CYP11 B2 promoter,· Ad1 (at -71/64), Ad5 (at -
129/114), and NBRE (at -766 /-759} are necessary for basal transcription; Ad5 site is also required for cAMP- and Ca2+-dependent signaling pathway induction.
14 · Analogous elements are present on the CYP1181 promoter; the Ad1 and
Ad4 sites are required for basal, as well as cAMP-ihduced, transcription of the
56 CYP1181 gene . The Ad1 site of the CYP11 B1 and CYP1182 promoters is nearly identical, differing in only two bp·, while the Ad4 site is exactly same between the two isozymes. As with CYP1182, CYP1181 Ad4 site binds SF-1; however, SF-1 plays a rather contrasting role in regulation of the two genes, as it promotes the transcription of CYP1181 and inhibits the transcription of CYP1182
54 61 through mechanisms that are still unknown • Collectively, the 5' flanking
promoter region of the CYP1181 and CYP1182 genes has evolutionarily
diverged just enough to control their zone-specific expression. Both genes rely
on the Ad1 site for basal transcription. The Ad5 site plays a major role in_
CYP1182 and not in CYP1181 transcription, while the Ad4 site plays a significant role in CYP1181 and not CYP1182 transcription. The NBRE and
NURR1 regulate CYP1182 but not CYP1181. Such differences in the cis
regulatory elements on the 5' flanking regions of the two genes are likely to play
an important role in differential regulation of these isozymes within the adrenal cortex.
Consequently, the significance of the differences between the two genes
is affirmed by a genetic disease, glucocorticoid supressable hyperaldosteronism
(GSH), a rare but well understood cause of primary aldosteronism (PA).
Normally, the two CYP11 B genes (CYP11 B1 ahd CYP1182) are 10 kilo bp (kbp)
63 apart on chromosome 8 ; interestingly, in GSH, an unequal meiotic crossover occurs that produces chromosomes carrying three CYP11 B genes: a wildtype
15 CYP11 B2, a wildtype CYP11 B1, and a chimeric CYP11 B2 gene that is under-the
14 64 control of CYP1181 5' flanking promoter region • (Fig.- 4}. Although rare, such
unequa~ meiotic crossovers occur among other genes leading. to several human
diseases. For example, a crossover on human chromosome 6 between 21-
C_YP1182 40-kb CYP11B1 l Chromosome Unequal meiotic 8 locus q24.3 :>; crossover l Chimeric gene CYP1182 CYP1181
Fig. 4: Glucocorticoid suppressible hyperaldosteronism (GSH) model. Crossover between CYP11 B2 and CYP11 B1 occurs during meiosis, resulting in one gamete that has a a chimeric gene with 5' flanking promoter sequence of CYP11 B1 and 3' seauence of CYP11 B2. Presence of a sinale chimeric aene could lead to GSH.
hydroxylase (CYP21) and a human leukocyte antigen family gene causes
65 congenital adrenal hyperplasia due to 21-hydroxylase deficiency • The unequal
crossover between CYP11 B1 and CYP11 B2 that leads to GSH produces a
chimeric gene that encodes an enzyme with aldosterone synthase activity, but
the gene is expressed in the ZF under the control of ACTH and not its typical
agonist (Ang 11)-. ACTH activates expression of the c~imeric enzyme increasing
the production of aldosterone, which can be inhibited upon administration of
glucocorticoids to suppr~ss circulating- ACTH levels. Importantly, in 1992, White
and colleagues showed that- the DNA sequence of this chimeric gene contains 5'
16 flanking regions of the CYP1181 gene _promoter, while the majority of the coding
6 sequence belongs to.the CYP1182 gene 1 • Since the chimeric gene is expressed-
. in the ZF under the control of ACTH while encoding. an enzyme with aldosterone
synthase activity, this strongly suggests that DNA sequences within the 5'
flanking promoter region of CYP11 B1 can _mediate its zone-specific expression.
One of my goals was to determine if a similar scenario is true for CYP1182.
As with GSH, another alternative mechanism leading to ectopic zonal
expression of CYP1182 has been reported in TASK1 (KCNK3; acid-sensitive K+
9 66 channel} deficient mice • • In these mice, CYP1182 is absent from the ZG and
expressed in the ZF of the female transgenic (Tg): mice, but not in male mice. In
addition, similar to GSH, these mice have ZF expression of both CYP11 B1 and
CYP1182 under the control of ACTH. This demonstrates another mechanism
leading to ectopic CYP1182 expression.
Recent literature reports inexplicable aldosterone increases in patients
67 69 with hypertension - ; which may in part be due to ·cYP1182 gene
70 74 dysregulation - • The exact mechanisms that cause low-renin expression of
adrenal CYP1182 in these patients have not been defined. Thus far, through in
vitro analyses, a wealth of knowledge has been attained regarding the normal
physiologic regulation· of the CYP1182 gene. However, progress towards
understanding the role of CYP1182 in human diseases continues to be slowed
by the lack of optimal in vivo models. As discussed in the subsequent pages, the
development of a better model to study CYP1182 was the goal of Aim two of my
research program.
17 Tg mice are an appropriate in vivo model to study CYP1182 gene
regulation. CYP1182 gene is expressed exclusively in the ZG of both humans
and mice, and its enzymatic regulation and activity are also highly similar
Fig. 5: Mouse Cyp11 b2 ECR analysis. A. Schematic representation of mouse Cyp11 b2 genomic structure. Boxes indicate positions of exons. The first exon contains translation start site, which is marked by an arrow from the box. 8 . The mouse-human (top) and mouse-rat (bottom) ECR analyses of the Cyp11 b2 promoter region obtained from the ECR Browser, which are presented in percent identity plots (pip-plots). The height of the colored areas corresponds to the percent identity of alignment (50 - 100 %), whereas the length corresponds to the length of alignments. Color indicates the nature of the ECRs (see legend).
between the two species. Previously, it was shown in Tg mice that 6.4 kbp of the
5' flanking promoter region of the mouse Cyp21 gene is sufficient to direct
75 expression of a reporter construct exclusively in the adrenal cortex • This
18 suggests that since the CYP21 promoter contains adrenocortical-specific locus control regions, such a ,region may also lie inside of the CYP 1182 promoter,
directing its ZG-specific adrenocortica~ expression. Targeting ZG utilizing the
CYP1182 gene has been attempted previously a bacterial artificial chromosome
(BAC) containing the entire CYP1182 gene or a large segment (>5 kbp) of the
76 gene promoter • However, the previous studies were not successful, possibly
due to the large amount of DNA that was utilized. Our analysis of the
evolutionarily conserved regions (ECR) between mouse and human CYP1182
promoters revealed homology within the -1 to -362 bp region, while greater
homology between the two rodents continued from -1 to -891 bp before reaching
a 104 bp gap and repetitive sequences (Fig. 5}. Therefore, Tg mice were utilized
in the specific aim of my project directed toward a better understanding of
CYP1182 gene regulation and the development of targeting strategies for the
mouse ZG. Our in vivo study is, in part, based on the 5' flanking cis-regulatory
regions of the CYP1182 promoter that have been identified through decades of
in vitro analyses. We presume that these key elements on the promoter region
should be important to physiologic regulation and ZG expression in Tg mice. We
believe that the adrenal ZG-specific expression of CYP1182 is -controlled· by the
promoter, which leads to its tightly regulated adrenocortical expression. This
study is of potential clinical importance, as it may become the first in vivo model
that advances our understanding of CYP1182 gene regulation.
19 B. Statement of the problem: A paucity of adrenal cell lines
Explorations into the physiology of the adrenal cortex have been hampered, in part, by the lack of available adrenal tissue ·samples and cell cultures. Primary adrenocortical cultures and adrenal cell lines allow critical in vitro analyses that could lead to- in vivo studies. Primary cultures are useful, as they are sufficient for transient in vitro analyses; however, handling and management of primary cultures becomes progressively problematic. Concerns regarding ·cell growth, response to agonists, and maintenance of steroidogenic capacity in primary adrenocortical cultures become more apparent with time. The ability of adrenal primary cultures to produce steroids or to respond to physiologic agonists, such as Ang II, ACTH, and K+, reduces over time. Expression of the enzymes involved in steroid hormone biosynthesis is reduced for most primary cultures. As a result, the steroids synthesized and released by primary cultures no longer resemble in vivo occurrences. Importantly, due to the above, there is a constant requirement for freshly acquired adrenal tissue for laboratories relying on primary cultures.
Collectively, the limitations of primary cultures have forced many laboratories to develop adrenocortical cell lines or use currently available cell lines.
Cell lines continuously allow provision of large numbers of viable cells without the need for animal sacrifice or acquisition of human tissue. Considerable progress in defining the basic molecular mechanisms involved in adrenocortical function has been made through the use of cell lines. Adrenocortical cell lines investigated thus far were generated from different sources, including cell
20 77 79 0O 84 · suspensions from adrenal tumors, • established _cell lines from tumors · ,
isolated immortalized cells following oncogenesis and cells harvested from Tg
animals85-88. Consequently, the difficulties associated· with development and
characterization of viable cell lines should not be overlooked. As with primary
cultures, the ability of cell lines to produce steroids or to respond to- agonists can
change over time; therefore, steroid synthesis and cellular responses to agonists
must be monitered on a regular basis. The expression of the enzymes involved in
steroid hormone biosynthesis can also change under culture conditions. Due to
such stringent criterion, it is difficult to develop and characterize viable cell lines
to study the adrenal. Hence, the rarity in available human adrenocortical cell lines
to study the physiology and pathophysiology of the human adrenal cortex is re~I.
Herein, we attempted the development and characterization of a human
adrenocortical cell line. One aim has been proposed to determine the CYP11 B2
promoter regions that are required for its tissue-specific expression, as well as
repression within the adrenocortical zones. These studies should broaden our
knowledge of adrenocortical zonation under physiological conditions and provide
insights into human adrenal diseases.
21 1) Specific aim II
An appropriate, well characterized human adrenocortical cell model system· would be critical for studies focused towards defining the molecular mechanisms that lead to zonation of the adrenal ·cortex. Development and utilization of a human adrenocortical cell model is sought to further our understanding of ·the adrenocortical function.
Hypothesis: Adrenocortical carcinomas are an appropriate source of viable cell lines.
a. We developed several monoclonal populations of cells from an
adrenocortical carcinoma. Among the 47 different clones developed,
human adrenocortical carcinoma clone 15 (HAC15) expressed ·all of the
genes involved in the adrenocortical pathway, including StAR, CYP11 A 1,
CYP17, CYP21, HSD3B2, CYP11B1, CYP1182, and SU_LT2A1. Upon
stimulation with Ang· 11 and K+, an increase in aldosterone levels, as well
as the expression level.s for all of the enzymes involved in its production
was obsetved. ACTH caused a modest increase in cortisol and DHEA,
along with increased expression of all of the enzymes in their respective
biosynthetic pathways. HAC15 emerged as an appropriate in vitro model
to study the regulation of adrenocortical function.
22 2) Brief literature review and rationale: A paucity of adrenal cell lines
The historical methods of developing cell lines and maintaining cell cultures. Discovered in the early 20th century, the concept of utilizing
89 immortalized cell lines is quite complex • Most cell lines are grown and maintained under a tightly controlled environment, with optimal temperature and gas mixture. Culture conditions vary widely for each cell line, and slight variations in conditions could result in different phenotypes being expressed. One of the key factors in cell culture systems is growth media, as most cell lines require a
9 specific combination of components that make up growth media ~. Growth media vary in pH, mineral content, amount of growth factors, and presence of a variety of nutrients. Improper growth media could lead to suboptimal cell growth and reduction in efficiency of in vitro analyses. The most important constituent to consider before beginning in vitro analyses is the origin and development of a cell line. There are several innovative techniques of developing cell lines; proper procedures need to be followed in order to complete the characterization and development process.
Oncogenes such as Kirsten murine scarcoma virus (KMSV) have proven
91 useful in establishing virally infected immortalized cell lines • Following infection of a host cell, KMSV releases its single-stranded RNA genome into the host cell.
Once inside, such an oncogene is reverse-transcribed into double-stranded (pro viral) DNA, which then becomes covalently integrated into the host cellular DNA.
23 The proteins encoded by oncogenes are capable of transforming cells by stimulating- uncontrolled mitosis and hence providing a permanently growing population of cetls. Auersperg et al. developed rat adrenal cell lines by infection with KMSV. Primary cultures of rat adrenal cells in early passage were infected
92 94 with virus and allowed to overgrow the normal cell population - • Ragazzon et al. recently established mouse adrenocortical tumor cell lines using similar genetically targeted oncogenesis. Two cell lines, namely ATC1 and ATC7-L,
95 were developed and exhibit a ZF-specific phenotype •
Genetically targeted tumorigenesis in. Tg mice to establish adrenal cell
87 88 96 98 lines has been utilized by several investigators • • - • Mellon et al. used a human CYP11 A promoter to target expression of Simian Virus 40 T-antigen
(SV40) as a transgene in mice, which resulted in adrenal tumors. Moreover,
87 96 these generated adrenal tumors were used to establish cell lines • •
Correspondingly, mouse inhibin a-subunit promoter has ·also been utilized to drive SV4O expression in mice to generate adrenal tumors. Several adrenocortical cell lines were also generated from mice bearing a trarisgene for a
88 temperature-sensitive form of SV40 • At permissive temperatures, the protein encoded by the SV40 gene is activated, and the host cells are transformed. This process is reversible once the optimal temperature is shifted. However, cell lines developed from oncogenesis and -tumorigenesis have, thus .far, .yielded suboptimal cell lines with limited steroidogenic responses and modest enzymatic stimulation.
24 Consequently, naturally occurring adrenocortical tumors are the best source for the development of viable adrenal cell lines. Adrenocortical tumors provide a large number of viable steroidogenic populations of rapidly dividing. cells. A human adrenocortical ·cell line, NCI-H295, was established from a woman diagnosed with an adrenocorticat carcinoma84; the excised tumor was 14 x 13 x 11 cm and was used to establish this cell line. Tumor tissue was finely minced and the resulting population of cells has been maintained in various growth media.
Human adrenocortical cell lines have provided a wealth of knowledge towards understanding the mech_anisms that control . adrenal
_steroidogenesis. To date, of the several cell lines that have been developed, the human adrenocortical cell line, NCI-H295, and its strains have proven to be the most valuable. Initially, gas chromatography/mass spectrometry (GC/MS) and radioimmunoassays were utilized to identify steroids in the NCI-H295; 30 different steroids were discovered. Based on secreted steroids, these cells appear to contain all of the adrenocortical enzymes. After several analyses, NCI
H295 adrenocortical cells express all of _the enzymes participating in normal human adrenal steroidogenesis, including CYP11A, HSD3B2, CYP11 B1, CYP21,
99 1 1 ·cYP17, CYP11 B2, and HSD3B2 - ~ • The NCI-H295 cell line is presently available from the American Type Culture Collection as ATCC CRL-10296 and wiH grow in suspension.
25 A population of cells, namely H295R, was screened and developed from
102 the parental NCI-H295 • In comparison to the p·arent NCI-H295 cell line, H295R cells grow as an adherent monolayer, and population doubling time is reduced from five to two days. In vivo Ang II acts on the adrenal ZG cells to increase production of aidosterone through type 1 Ang II (AT1)- receptors. Studies utilizing
H295R with radiolabeld Ang 11 in the presence of antagonists to the AT1 and AT2
· receptors established that the H295R cells utilize AT1 receptors almost
103 105 exclusively in aldosterone biosynthesis - • Another major physiologic regulator of adrenal aldosterone production is extracellular K+. Experiments utilizing
2 H295R cells have shown that K+ causes an increase in intracellular Ca + level leading to aldosterone biosynthesis and secretion. The H295R cells are currently being used as a model to better define the mechanisms of K+ regulation of
106 111 adrenal steroid production - . ACTH is the primary hormonal regulator of adrenal cortisol production. The H295R cell line is only mildly responsive to
ACTH, and most other strains and subpopulations of NCI-H295 are completely
82 unresponsive . H295R cells treated with ACTH respond acutely with an increase in adrenocortical steroid biosynthesis though the cell line lacks long-term
83 responsiveness .
These cells are useful in defining the role of enzymes controlling adrenocortical steroidogenesis. mRNA transcripts encoding the StAR gene, as well as the five forms of cytoch~ome P450 known to be involved in normal adrenal steroidogenesis (CYP11A1, CYP17, CYP21, CYP1182, and CYP1181),
112 116 are detectable in the H295R cells • • Experiments using the 5' flanking DNA
26 regions from CYP1181, .CYP1182, CYP17, and HSD382 have been performed .. 1 126 using these cells and several key transcription factors have been identified n• •
Taking adrenocortical steroidogenesis into consideration, the H295R cell line produces ·an steroid hormones under basal conditions, as well ~s upon
105 127 stimulation by Ang II, K+, and ACTH • • These cells ·have the ability to produce steroids that originate from all three zones of the adrenal cortex, including aldosterone (ZG), cortisol (ZF), DHEA (ZR), and DHEA-S (ZR). Hence, the H295R cell line is a potential model for studying steroid hormone
61 128 135 biosynthesis in all adrenocortical zones • • •
Subsequently, another strain of cells has been developed from the original
NCI-H295, designated H295A. Unlike the NCI-H295 cell line that grows in
1 suspension, H295A cells have been described to grow as a· monolayer8 • The method of isolation of the H295A strain was similar to that of the H295R, relying on the removal of non-attached cells with successive media changes. H295A cells lack Ang II, K+, and ACTH-stimulated steroidogenic responses and produce significantly less amount of steroids than the H295R cells. The AT1 receptor is
136 also highly expressed _in the H295R strain compared to H295A • Comparing the steroidogenic response to agonist and basal steroidogenesis, the H295R strain seems to be superior to the H295A strain.
The original NCI-H295 and its strains are useful models in defining the mechanisms controlling adrenocortical steroidogenesis, and they will continue to provide valuable information regarding the regulation of genes encoding steroidogenic enzymes. The regulation of genes in the steroidogenic pathway
27 has been demonstrated by a number of transcription factors identified in NCI
H295 and H295R119-12a, 136-141 _
Another human adrenal carcinoma-derived cell line, SW13, has been developed. Although adrenal in origin, SW13 cells produce no steroids; perhaps this cell line was derived from non-steroidogenic cells of the original carcinoma 142. Due to the lack of steroidogenesis, its usefulness as an adrenocortical model system is none. These cells are also available from the
American Type Culture Collection (CCL-105).
Rodent and bovine adrenocortical cell lines have also contributed to our understanding of the adrenal gland. Its is presumed that following exposure to an atomic blast, an adult LAF1 (C57L x A/HeJ) mouse developed an adrenal tumor143. Sato and colleagues captured the transplantable tumor and allowed it to grow in vitro and developed the Y1 mouse adrenocortical cell line 144. Although the major steroid pathways present in Y1 cells are unlike the normal physiologic mous~ adrenocortical steroid pathways, a wealth of information has been gained through the utilization of these cells. Y1 cells have proved to be very useful in assessing the roles of cAMP, protein kinase A, adenylyl cyclases, ACTH, and
ACTH receptor in the adrenal cortex145-151 . Y1 cells do not respond to Ang II, although transgene expression of the Ang 11 receptor has been described in the
Y1 cells 152. Other mouse and rat adrenocortical cell lines have been developed through various aforementioned techn·iques; however, due to the lack of
28 hormonal responsiveness and - in_significant steroidogenesis, they remain unimportant.
Due to the relative ease in obtaining fresh bovine adrenals, the need for development - of bovine adrenal cell lines has been limited. Hornsby and
53 colleagues utilized SV40 to create a series of clonal bovine adrenal cell lines 1 •
The addition of SV40 to the cells greatly enhanced the cell culture lifespan of the cells; however, c·ellular expression of adrenocortical enzymes was found to
154 require specialized growth conditions • Consequently, this area of research remains largely unexplored.
The aforementioned human and rodent cell lines have allowed a multitude of studies that has broadened our understanding of normal adrenocortical endocrine function, as well as the properties of adrenocortical tumors. However, most of the cell lines have several limitations. The mechanisms involving hormonal regulation of adrenocortical cell growth, morphology, arid steroidogenesis remains a topic of active research; therefore, additional viable adrenocortical cell lines are sought.
29 n. MANUSCRIPTS
A. Peer Reviewed Articles
1) Gene targeting to the mouse adrenal zona glomerulosa
1 1 2 Jeniel Parmar , Anthony M. Cohen , Celso E. Gomez-Sanchez , William E. Rainey1 and Tsugio Seki1
1 Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912 2Division of Endocrinology, G. V. Montgomery VA Medical Centerand University of Mississippi Medical Center, Jackson, MS 3911 O
Abbreviated Title: Adrenal glomerulosa targeting
Key words: Cyp11 b2, adrenal cortex, zona glomerulosa, in vivo targeting
Name/Address for Correspondence: Jeniel Parmar Department of Physio.logy Medical College of Georgia Augusta, Georgia 30912
Telephone: 1-706-721-8363 Fax: 1-706-721-8360( •E-mail: [email protected]
30 Abstract
Adrenal zona glomerulosa (ZG) cells synthesize and secrete the majority of
circulating aldosterone, which is one of the key players of the· renin-angiotensin
aldosterone-system (RAAS). Aldosterone plays a vital role in maintaining sodium
homeostasis, and its secretion is regulated, in large part, at the transcription level
of the aldosterone synthase (Gyp11 b2) gene in ZG cells. Although several
adrenocortical cell lines are available to study the mechanism regulating adrenal
gene transcription, lack of in vivo targeting strategies has made it difficult to apply
the in vitro findings to animal models. In this study, we isolated a 985 bp
Cyp11 b2 promoter fragment and successfully targeted adrenal ZG cells in vivo
using a reporter transgenic mouse model. In this Tg (Cyp11 b2-lacZ) mouse, the
lacZ reporter gene was detected only in the ZG cells and RAAS activation by low
sodium diet significantly upregulated ZG expression of lacZ. Moreover, the
transgene expression was not detectable in any of the 15 non-adrenal organs
assessed using quantitative PCR, indicating that this fragment exclusively targets
. adrenal ZG cells. This regulatory fragment will be an invaluable tool to study in
vivo the functional role of genes believed to play a pivotal role in adrenal
physiology and diseases.
31 Introduction
Aldosterone · is ·the principal mineralocorticoid synthesized in the zona glomerulosa (ZG) of the adrenal cortex. In humans and mice, aldosterone functions primari_ly to preserve cardiovascular homeostasis by regulating fluid
155 156 and electrolyte balance • . The principal glucocorticoid-cortisol in humans and corticosterone in mice-is synthesized in the zona fasciculata (ZF) of the
8 47 adrenal cortex • • The Cyp11 b2 and Cyp11 b1 genes share a high level of sequence similarity and the enzymes they encode, aldosterone synthase and
1113-hydroxylase, respectively, also possess high level of amino acid sequence
54 157 160 similarity • - • Despite these similarities, Cyp11 b2 is solely responsible for the
10 final step conversion of 11-deoxycorticosterone (DOC) to aldosterone , while
Cyp11 b1 converts DOC and 11-deoxycortisol to corticosterone (mice) and
56 cortisol (humans) respectively .
Studies utilizing transgenic mice have demonstrated that regulatory elements within the gene or in proximity of the gene can control tissue- and cell-specific
161 171 expression of certain genes - • Several genetically alte'red mouse studies have demonstrated that adrenal cortex targeted transgene expression is possible
163 172 177 through steroidogenic or non-steroidogenic gene promoters • - • Although the adrenal cortex has been targeted previously, these transgenic studies do not permit further adrenal cortex specific applications as most of these transgenic constructs have extra-adrenal targets. The exception is 21-hydroxylase driven
32 adrenocortical targeting by Morley et al (1996), however, this transgenic construct exhibited scattered-inconsistent targeting throughout the adrenal cortex and hence no further analyses have been conducted using this targeting
75 strategy • Attempts to develop a ZG-specific targeting strategy using a 5 kb promoter of mouse Cyp-11_ b2 gene have also been conducted, however this
177 approach essentially failed •
Herein, we have developed a new transgenic mouse mo~el using a 985 bp_
Cyp11 b2 promoter that specifically targets ZG. This DNA region was selected based on evolutionarily conserved region (ECR) analysis of the mouse, rat and human CYP11 B2 promoter sequences, which allowed us to reduce the length of the promoter from the previous study that attempted Cyp11b2 driven ZG targeting. We also utilized a lacZ reporter construct with enhanced stability in order to maximize· sensitivity of the -reporter gene expression in the mouse genome. Our results demonstrated that 985 bp Cyp11 b2 promoter-lacZ
(Cyp11 b2-lacZ) construct caused reporter gene expression solely in a ZG specific manner in transgenic mice. Furthermore, as with the endogenous
Cyp11 b2 gene, the expression of reporter lacZ was under the control of renin angiotensin-aldosterone system (RAAS).
33 Materials and Methods
Generation of transgenic mice
All animal procedures carried out in this study were reviewed and approved by
the Institutional Animal Care and Use Committee (IACUC) in the Medical College
of Georgia (MCG). All animals were housed in specific pathogen free (SPF)
facility.
An alignment of human, mouse, and rat aldosterone synthase (CYP11 B2)
genomic DNA sequences was obtained from the ECR Browser
178 . (http://ecrbrowser.dcode.org) • Based on the alignment analysis, a 985 bp
mouse Cyp11 b2 regulatory fragment was amplified from the RP23-323C12
bacterial artificial chromosome (Children's Hospital Oakland Research Institute)
using PCR. In order to generate transgenic (Tg} construct, the amplified fragment
was ligated to the SIB reporter cassette, which contains the SV40 splicing
donor/acceptor signals (SD/SA}, internal ribosomal entry sequence (I RES),
179 bacterial lacZ coding sequence,_ and SV40 late poly A signal • The Tg construct
was injected into B6C3 fertilized eggs by the MCG Mouse Embryonic Stem Cell
and Transgenesis Core Laboratories. Screening of founder lines were performed
by following the published protocol and the Tg lines were maintained as
hemizygous for transgene using C57BU6 mice (Harlan Laboratories) as
179 bleeders • Out of the seven Tg(Cyp11 b2-lacZ) founders that transmitted
34 transgene to the F1 generation, lines 5 and 6 that be·came available first were expanded for detailed analyses presented in this report. Later, additional lines 7 and 16 were analyzed for their lacZ reporter expression and found it essentially identical to the lines 5 and 6. None of the lines showed any detectable ectopic lacZ expression.
Of note, following the convention of the adrenal research community, mouse aldosterone synthase gene is designated as Cyp 11 b2 with italicization and its protein product, .aldosterone synthase, is designated as Cyp11 b2 without italicization.
Diet regimen and tissue sample collection
Only male mice were used in this study. All mice were separated from mothers at weaning age (3 weeks) and fed either normal sodium (NS, 0.3 % NaCl) or low sodium (LS, 0.03 % NaCl)- diets (Harlan Laboratories) ad libitum with unrestricted access to drinking water for 7 days. At the end of diet treatment, mice were instantly decapitated using a guillotine and trunk blood was collected into EDTA coated tubes for plasma aldosterone assay, followed by collection of sixteen major organs for immunohistochemical and molecular biological assays. These organs include adrenal glands, thoracic aorta, brain, eye, fat (visceral fat adjacent to spleen), heart, large intestine, small intestine, kidney, liver, lung, pancreas, skeletal muscle, spleen, testis, and thymus.
35 Plasma aldosterone measurement
Plasma aldosterone levels in mice were determined using the Diagnostic
180 Systems Laboratories RIA kit as previously described • Plasmas from mice fed
NS diet (NS mice) were diluted 1 : 2 or 1 : 3 with charcoal stripped mouse plasma (Bioreclamation)- to obtain an appropriate volume for assay. Plasmas from LS mice were diluted 1 : 20 with the same stripped plasma so that aldosterone concentrations fall within the kit's detection range. Samples for the standard curve were prepared by serially diluting the supplied control aldosterone with the same plasma.
Total RNA and cDNA preparation
After the diet treatment, tissues samples were homogenized using the FastPrep homogenizer (Thermo Fisher Scientific}, and total RNAs were extracted using the
RNeasy Mini plus kit (QIAGEN). Two µg each of total RNAs was reverse
83 transcribed using the High Capacity cDNA RT kit (Applied Biosystems) ..
Absolute quantif-ication of Cyp11 b2 transcript in male C57BU6 organs
Due to significant sequence homology between Cyp11 b2 and closely related steroid 11 ~-hydroxylase (Cyp11 b-1) genes, determining transcript levels of these two genes independently was challenging. In fact, one of the Applied Biosystems
Pre-developed TaqMan Gene Expression Assays for Cyp11b2, Mm0·1200453- g1, showed cross reactivity to Cyp11 b1 cDNA (data not shown}. Therefore, a
36 new Taqman assay for Cyp11 b2 was established. This assay showed a linear detection range over 7 logs of Cyp11 b2 cDNA inputs with a minimal cross reactivity to Cyp11 b1 cDNA (Supplemental Fig. 11 ). Detailed information and protocols for the Cyp11 b2 Taqman assay, real-time quantitative PCR (qPCR), nor~alization to 18S rRNA quantity, and data exclusion criteria are described in the Supplemental Materials and Methods.
Whole mount X-gal staining
Targeting ability of the Cyp11 b2 promoter fragment was visualized by X-gal staining. On application of X-gal staining solution, the lacZ gene product, 13- galactosidase, hydrolyzes X-gal to produce an insoluble blue dye. Hence, blue staining in cells indicates active transcription from the promoter. Whole mount
162 organ X-gal staining was performed as previously described . Harvested organs were sectioned into 1-2 mm thickness and stained for at least 8 h at 37
°C with constant rocking. Organ samples from wild type littermates were used as a negative control. The stained organs were photographed and then fixed with formalin overnight, followed by paraffin embedding and sectioning.
Histology and Cyp11 b2 immunohistochemistry
In order to localize Cyp11 b2 protein in adrenal sections, a novel polyclonal anti
"rabbit Cyp11 b2 (mouse} antibody was developed by immunizing rabbit with human CYP1182 peptide. The VECTASTAIN Elite ABC Kit (Vector Laboratories)
37 was utilized to stain mouse tissue sections. First, antigen retrieval was performed by submerging the slides in sodium citrate buffer (2· mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0) and heating the container using a 1450 W microwave at 30 % power level for 12 minutes. The sections were then blocked with 2.5 % horse serum, followed by· application of the primary antibody, which was diluted 1 : 75 with PBS containing 0.5 % BSA. After the secondary antibody application, localization of Cyp11 b2 protein was visualized by application of 3.3' diaminobenzidine (DAB), and the sections were counterstained with nuclear fast red (NFR) or methyl green (Vector Laboratories). A negative control with no primary antibody treatment showed no detectable background staining in adrenal sections.
Relative quantification of transgene
In order to determine the transgene expression level in Tg_ mice, a transgene specific Taqman assay with a sense primer (5'-aaaaggctgtgaactgaagacaga), anti-sense primer (5'-tccgtacaggccagttaactttc), and probe (5'-ccccctcgaggaac) was developed using the Primer Express 3.0 program (Applied Biosystems). This. assay was designed to encompass the exon boundary in SD/SA sequence so that the Tg construct in genome will not be amplified. The following PCR program were used for qPCR with the ABI 7500 Fast Real-Time PCR System (Applied
Biosystems): the initial denature at 50 °C for 2 min and 95 ~C for 10 min, followed by 40 cycle amplification at 95 °C for 15 s, 50 °C for 1 min, and 72 °C for 1 min.
Using the relative qPCR method with standard curve, the results were -first
38 normalized to 18S rRNA, and then fold changes relative to average NS adrenal value were calculated.
Statistical analysis
Statistical comparisons were assessed between the NS and LS animals by
utilizing non-parametric unpaired Mann Whitney U-test. Significance was
accepted at the 0.05 level of probability. Data are expressed as mean± SE.
39,. Results
Since ECRs in promoter sequences are most likely to possess gene regulatory
178 functions , the essential regulatory region for the mouse Cyp11 b2 gene (Fig:.
6A) was predicted using mouse, rat, and human CYP11 B2 genomic sequences.
Counting from the translation start site, sequence homology between mouse and
human CYP11 B2 promoters was observed in -1 to -362 bp region, while higher
homology between the two rodents continued from -1 to -891 bp before reaching
a 104 bp gap and repetitive sequences (Fig .. 6B). Utilizing the endogenous Xbal
site at -989 bp position, we isolated a 985 _bp mouse Cyp11b2 regulatory
fragment covering- -989 to -5 bp sequence and then connected to the lacZ
reporter cassette (Fig. 6C}. The construct was injected to fertilized mouse eggs
and several Tg(Cyp11 b2-lacZ) mouse lines were established.
In advance characterizing this novel Tg mouse model, we first determined
· endogenous Cyp11 b2 gene expression patterns at RNA and protein levels using
male C57BU6 mice. Since LS diet significantly induced the Cyp11 b2 gene and
hence aldosterone production (Table I) via activation of renin-angiotensin
system (HAAS}, organs from the mice fed normal sodium (NS) and low sodium
(LS) diets were collected and their Cyp11 b2 transcript contents were quantified.
In NS mice, the highest expression was observed in adrenal glands (3.35 x 106 ±
1. 71 x 106 copies/µg 18S rRNA}, which showed 260 times higher level than the
4 3 average of the other 15 organs (1.29 x 10 ± 3.49 x 10 ), indicating that adrenal
40 glands are the major organ for Cyp11 b2 expression in mice (Fig. 7 A). In LS mice,
9 8 the highest expression Was also obseived in adrenals (2.50 x 10 ± 8,.04 x 10 ), which was 42,744 times higher than the average of the 15 organs (5.83 x 104 ±
4 2.00· x 10 ), again indicating that the Cyp11 b2 is almost exclusively expressed in the adrenals (Fig. 7B). Compared to NS diet, LS diet induced 745 fold higher
Cyp11 b2 transcripts in adrenal glands· (p < 0.01 ). In addition·, thoracic aorta (20- fold, 1.52 x 104 ± 7.91 x 103 in NS vs 3.11 x 105 ± 2.66 x 105 in LS}, thymus {3.9 fold, 1.53 x 104 ± 3. 73 x 103 vs 5.98 x 104 ± 1.35 x 104), and kidney (2.9 fold, 4.01
3 2 4 3 x 10 ± 6.09 x 10 vs 1.16 x 10 ± 2.23 x 10 ) showed statistically significant
Cyp11 b2 induction by LS diet (p < 0.05). It is noteworthy that, although the amount of Cyp11 b2 transcript in LS aorta is the second highest among the 16 LS organs, it is about 1O times less than that of NS adrenal glands. These results suggest that our 985 bp Cyp11 b2 promoter fragment should target primarily the adrenal glands~
Furthermore, immunohistochemical staining of C57BU6 adrenal glands showed zona glomerulosa (ZG) specific localization of Cyp11 b2 (Fig. 8). As it has been reported 181 one week of LS diet stimulated ZG layer prqliferation/expansion (Fig.
8A and 8D) and significant accumulation of Cyp11 b2 protein in the ZG layer (Fig.
88 and BE). These qPCR and_ immunohistochemical analyses led us to characterize our Tg(Cyp11 b2-lacZ) mice by first investigating transgene expression in adrenal glands under LS diet.
41 Intriguingly, whole mount X-gal staining of Tg(Cyp11 b2-lacZ) adrenals revealed that, in all Tg lines 5, 6, 7, and 16, th~ reporter lacZ gene was highly expressed· in the outer layer of adrenal glands under LS diet (Fig .. 9). Littermate wild type controls showed little background staining after 8 h staining-, indicating that this staining was specifically induced by the transgene. The Tg mice fed NS diet also showed _very faint X-gal staining, which was hardly detectable under dissection microscopy (data not shown). Tg mice under NS and LS diets produced plasma aldosterone at comparable levels to the wild type C57BU6 mice {Table II), indicating that these Tg adrenals are functionally normal.
The reporter lacZ localization in adrenal glands was further characterized by
Cyp11 b2-immunohistochemistry and X-gal double staining. In consistent with the endogenous Cyp11 b2, under NS diet the blue staining was found only in the ZG layer and a few of these cells were expressing detectable levels of Cyp11 b2 protein (Fig. 1OA-C). Furthermore, LS diet dramatically upregulated the X-gal staining intensity and area in parallel to the endogenous Cyp11 b2 (Fig. 1OD-F).
Intriguingly, the X-gal staining was darker in more outer layer of ZG while the immunohistochemistry staining was more prominent in the inner layer of ZG. No detectable X-gal staining was observed in the adrenal capsule cells. These results indicated that 985 bp Cyp11 b2 promoter fragment was sufficient to target
ZG layer in adrenal glands and held a capability to respond RAAS activation.
42 Lastly, the upregulation of transgene in LS mice and its adrenal specificity was confirmed at RNA level by qPCR using Tg-lines 5 and 6-. ·Although transgene expression in some of the NS adrenals was not detectable due to a somewhat
low sensitivity of this assay, transgene was significantly induced by LS diet
(Table Ill}. Relative quantification of transgene showed 4,908 times higher transgene transcript in LS adrenal than in NS adrenals (p<0.01 }, indicating that the Cyp11 b2 promoter fragment is highly inducible by RAAS activation. There was no significant difference between Tg lines 5 and 6 in transgene expression
levels. Furthermore, the same qPCR assay revealed that none of the 15 non
adrenal organs listed in the Fig. 7 showed detectable transgene expression. This
observation was confirmed by the X-gal staining, which demonstrated no specific staining in any of these major organs (data not shown). Therefore, we concluded that the 985 bp Cyp11 b2 promoter fragment contains essential regulatory elements for Cyp11 b2 gene transcription, it is sufficient to recapitulate the
endogenous promoter activity, and it drives transgene expression exclusively in
ZG cells in vivo.
41 . Discussion
Targeting adrenal glands in transgenic mice has been explored by utilizing
promoter fragments from various genes including the Cyp11 b2. Previously,
Morley et al have generate.d 7 independent Tg lines with a construct harboring
5.4 kb of mouse Cyp11 b2 5' flanking region and the lacZ reporter gene, which
177 resulted in no detectable adrenal lacZ expression in any of the lines • In
contrast, we used a much shorter (less than 1 kb) region from the same gene to
.successfully target the adrenal ZG layer. These results may suggest that the
additional 4.4 kb region contains further regulatory element(s) that have silenced
the lacZ reporter expression in .the 5.4 kb Tg mice. This additional 4.4 kb
sequence, however, does not show any homology to human corresponding
regions and in fact about three quarters of this sequence is filled with various
types of repetitive sequences. Alternatively, it is possible that the 5.4 kb Tg mice
may also have expressed the reporter gene if LS diet had been administered .
. The NS adrenals from the current Tg mice showed only very faint X-gal staining
and it could have been easily missed if we did not pay extra attention. We may
owe our success to our use of the LS diet that activated the RAAS .
. In addition, in the same report Morley et al have described that a 5.0 kb Cyp11 b1
regulatory fragment, which is not under the control of RAAS, failed to target
177 adrenal glands • In another report, however, the same group successfully
targeted adrenal cortex with a 6.4 kb regulatory fragment of the steroid 21-
44 hydroxylase gene (Cyp21a1}. In this Tg(Cyp21a1-lacZ) mouse model, the lacZ expression was observed exclusively in adrenal cortex, though it was in a
182 variegated pattern throughout adrenal cortex and was not layer specific • Other groups have also reported adrenal targeting by using regulatory regions from
173 174 various genes, •including aldo-keto reductase 187 (Akr1 b7) . • , steroidogenic
163 172 183 184 factor 1 (SF-1, Nr~a1) • • , and inhibin a (lnha) • These regulatory fragments, however, also lead to transgene expression in non-adrenal organs.
The current 985 bp Cyp11 b2 sequence is unique and distinct from these fragments since it induces transgene expression selectively and exclusively in the adrenal ZG layer.
Although both endogenous Cyp11 b2 protein and the lacZ reporter gene were induced in ZG layer in LS adrenals, there was a slight difference in their staining
185 186 patterns. Consistent with the previous reports • , both wild-type C57BU6 and
Tg mice showed ZG cells to proliferate from 3-4 layers to a 12-15 layered zone.
Within this thickened ZG layer, the X-gal staining was more prominent on the outer· (capsule} side, whereas the Cyp11 b2 immunostaining was more intense on the inner (medulla) side (Fig. 8 and 1O}. This difference may be explained by the current hypotheses that adrenocortical stem cells in capsule or sub-capsular layer proliferate, differentiate, and move inward to generate adrenal cortical
175 cells • Upon LS diet administration, the ZG cells start both inward proliferation and activation of Cyp11 b2 promoter. This will lead to Cyp11 b2 and Lacz protein production in the outer ZG layer in Tg adrenals, which will result in aldosterone
45 production and intense X-gal staining, respectively. Since the inward blood flow
in adrenai cortex transports aldosterone toward the inside, inner ZG celts will sense a high leveJ of aldosterone, which should results in down-regulation of
Cyp11 b2 promoter activity in these cells and hence weaker X-gal staining-. The
Cyp11 b2 protein, however, may have been stabilized under the LS diet condition
and thus have accumulated in the inner ZG layer.
In addition to adrenal glands, Cyp11 b2 expression has been described in non
adrenat organs, including heart, brain and vasculature, under physiological
187 195 and/or pathological conditions - • In our study, we have dete~ted Cyp11b2 transcript in all organs tested (Fig. 7), whereas the levels were much lower in
non-adrenal organs. In addition, LS diet induced Cyp11 b2 upregulation in thoracic aorta, thymus, and kidney, while their fold changes were small. Although
these non-adrenal Cyp11 b2 expressions may have little impact on plasma
aldosterone levels, it may activate a local aldosterone-mineralocorticoid receptor
system, and hence it should not be overlooked. Nonetheless, our Tg mice
showed no detectable reporter gene expression at RNA and X-gal staining levels
in any of the non-adrenal tissues tested, which may be due to the limitations in
our assay sensitivities. The NS adrenal in Tg mice showed minimal transgene
expression at both qPCR and X-gal staining levels (Fig. 1O and Table Ill),
indicating that the sensitivities of our assays were not sufficient to detect non
adrenal transgene expression, which should be much lower than that of NS
adrenals (Fig. 7). Therefore, it is still unclear whether the current Cyp11 b2
46 promoter fragment contains the essential regulator elements for non-adrenal transgene expression. Further analyses under various pathological conditions
may shed some light on this issue in• the future.
In this study, we identified the Cyp11 b2 regulator fragment that exclusively targets adrenal ZG cells and responds to RAAS activation. This regulatory
fragment can be used in a T g construct .to specifically activate or silence any
gene in ZG. cells in vivo. Nevertheless, this regulatory fragment will be an
invaluable tool for the advancement of research associated with adrenal
development, physiology, and pathology.
47 . Acknowledgement
We appreciate the work of the Medical' College of Georgia Mouse Embryonic
Stem CeJI and Transgenesis Core Laboratories and- the Electron Microscopy &
Histology Core Laboratory.
48 Table I: Plasma aldosterone concentrations in C57BU6,mice (average pg/ml± SE)-
NS (n=5} , LS (n=6} NS vs LS
587.6 ± 38.4 11,367.5-± 4,160.9
Table II: Plasma aldosterone concentrations in Tg mice (average pg/ml± SE)
NS (n=5} LS (n=5} NS vs LS
669.6 ± 167.5 5,688.4 ± 581.4 p<0.01
Table Ill: Relative Cyp11 b2-lacZ mRNA expression in adrenal glands (fold change against NS)
# of # of samples Tg mice in detection Fold change ± Fold change ± Diet line analyzed range SE Line 5 vs 6 SE NS vs LS
Line 5 6 4 (67%) 0.93 ± 0.39 NS p = 0.69 1.00 ± 0.24 Line 6 5 4 (80%) 1.07 ± 0.34 p < 0.001 3,103.26 ± Line 5 7 7 (100%} 736.20 4,908.27 LS p = 0.32 * 1,456.70 6,713.29 ± Line 6 7 7 (100%) 2,750.85 Figure legends:
Fig. 6: Mouse Cyp-11 b2 ECR analysis and Tg construct. A. Schematic representation of mouse Cyp11 b2 genomic structure. Boxes indicate positions of exons. The first exon contains translation start site, which is marked by an arrow from the box. B. The mouse-human (top) and mouse-rat (bottom) ECR analyses
.of the Cyp11 b2 promoter region obtained from the ECR Browser, which are presented in percent identity plots (pip-plots)'. The height of the colored areas corresponds to the percent identity of alignment (50 - 100 % }, whereas the length corresponds to the length of alignments. Color indicates the nature of the ECRs
(see legend). Note that the shaded area corresponds to the promoter fragment in panel C. C. Schematic representation of the Cyp11 b2-lacZ construct that is used to establish Tg(Cyp11 b2-lacZ) mouse lines.
Fig. 7: Cyp11 b2 transcript quantity in 16· major organs in male C57BU6 mice. A.
Cyp11 b2 transcript quantity in mice fed NS diet. Due to a significantly high copy number in adrenals, a graph without adrenal data is re-plotted in the inset. Sk. muscle: skeletal muscle; S. intestine: small intestine; L. intestine: large intestine.
B. Cyp11 b2 transcript quantity in mice fed NS (solid bar) and LS (open bar) diets.
Note that, to aid NS vs LS mouse comparison, the same NS data in A are shown in this graph but in logarithmic scale. Statistically significant difference between
NS and LS mice were seen in the 4 organs (**: p<0-.01 and *: p<0.05). Numbers
50 of mice ·tested for each organ is shown in parentheses. The inset shows fold changes of transcript quantity in LS mice compared to that of NS mi_ce.
Fig. 8: Cyp11 b2 localization in adrenal cortex. Adrenals from male C57BU6 mice fed NS (A-C) and LS (D-F) diets are immunohistochemically stained with a novel rabbit polyclonal antibody. Mouse adrenal sections show histologically distinct 3 cortical layers by hematoxylin and eosin (H&E) staining (A and D) and Cyp11 b2 immunohistochemistry (brown) revealed its localization in the ZG layer (Band E).
Note that LS adrenals show much thicker ZG layer .with significantly intense
Cyp11 b2 immunostaining. Negative control staining performed without the primary antibody show little background (C and F). lmmunohistochemical sections in panels B, C, E, and F are counter stained with methyl green. ZG: zona glomerulosa; ZF: zona fasciculata; ZR: zona reticularis. All panels are in the same magnification. Scale bar in F: 0.5mm.
Fig. 9: X-gal stained Tg adrenals under LS diet. A. Adrenals from Tg and wild type littermate control (wt) were sectioned in half and then X-gal stained. Tg adrenal shows dark X-gal staining at the outer cortex layer. B and C. Magnified views of panel A. Scale bar in A: 0.5mm.
Fig. 10: Colocalization of Cyp11 b2 and X-gal staining in Tg adrenal cortex.
Adrenals from male Tg mice fed NS (A-C) and LS (D..:F) diets were X-gal stained
(A and D), followed by immune-staining for Cyp11 b2 (8, C, E, and F). Sections are counterstained with nuclear fast red. Panels A, B, D, and E are in the same magnification. Panels C and F shows high power view of Band E, respectively.
51 Fig·. 11 (supplemental figure}: Amplification cutve. using· descending pattern of
Cyp11 b2 transcript number as depicted · (right). Cyp11 b2 and Cyp11 b1 primer/probe were tested against Cyp11 b2 transcripts to assess the cross reactivity against the two highly similar genes. Standard cutves were constructed using 9 samples containing varying known quantities of Cyp11b2 transcripts.
During the real-time PCR amplification, the cycle at which these samples attain a particular level of product-the crossing point-is noted and plotted against the starting concentration of standard. The crossing points of samples are then measured and the starting concentration of target mRNA is extrapolated from the standard cutve.
52 Fig. 6:
C ----ti .· so/SA~IR.E~la~ I Cyp11 b2~1acz constru¢f
53 Fig.
7:
54 Fig. 8:
N$
LS
55 Fig. 9:
56 Fig. 10:
57 Fig. 11:
Cyp11 b2 copy numbers and corresponding cycle of threshold (Ct) values
Cyp11b2 Average Ct transcript copy value number
108 13.03 • 107 17.17 • 106 20.21 • 105 23.90 • 104 27.64 I 2 3 • 5 8 T 8 910' 111213U1S1617 1819 2021 22231{25 262728 • 103 31 .24 • 102 35.29
30 1 36.92 :;; • 10 .Q 25 E Undetermined ::, • C 20 QI Cyp11b1 Average Ct 0 transcript copy >. 15 value I.) number ·10 108 35.68 5 +,:=:,~1::;:;:.,...,::,=:-,,=~~:,.-...-~~~ 107 Undetermined 0 ..p,...... _.,...:_.....,,__.....; __ ...... ,.....:..-.::...-"r-' ...... • 0 2 3 4 5 G 7 s 9 • 106 Undetermined Cypft b2 transcript copy number (log scale)
58 II. MANUSCRIPTS
A. Peer Reviewed Articles
2) Development of an ACTH Responsive Human Adrenocortical Carcinoma (HAC) Cell Line
Jeniel Parmar, Rebecca E. Key and William E. Rainey
Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912. -
Abbreviated Title: Development of a human adrenocortical cell line
Key terms: Adrenal, ACTH, angiotensin II, aldosterone, cortisol, model system
Name/Address for Correspondence: Jeniel Parmar Department of Physiology Medical College of Georgia Augusta, Georgia 30912
Telephone: 1-706-721-8363 Fax: 1-706-721-8360 E-mail: [email protected]
59 Abstract
Context: The molecular mechanisms regulating adrenal steroidogenesis continue to be defined. The only current human adrenocortical cell line is the
NCI-H295 and its substrains. One of the strains, the H295R, has retained the ability to respond to angiotensin 11 (Ang 11) however, it lacks ACTH responsiveness. An ACTH responsive human adrenocortical model would add significantly to studies directed at defining the molecular control of corticosteroid biosynthesis.
Objective: Develop a human adrenal cell line that retained both Ang II and
ACTH regulated corticosteroid production.·
Design:· Human adrenocortical carcinoma (HAC) cells were isolated from an adrenal tumor removed from a girl presenting with virilization and hypertension.
Clonal populations of cells were established and characterized. HAC cells were treated with ACTH, Ang II and forskolin, followed by examination of steroidogenic enzyme mRNA expression using quantitative real-time PCR and steroid production.
Results: HAC clone 15 (HAC15) cells responded to treatment with ACTH, Ang II, and forskolin, with increased cortisol and aldosterone production. ACTH, Ang II,
60 and forskolin also increased expression of mRNA, encoding all enzymes needed for cortisol and aldosterone biosynthesis; namely StAR, CYP11A, CYP17,
HSD3B2, CYP21, CYP11 B1, and CYP11 B2. In addition, the cells expressed mRNA for ACTH receptor (MC2R) and Ang II receptor (AT1 R). MC2R protein was also expressed in HAC 15 cells.
Conclusion: The current study describes the development and characterization of an ACTH and Ang II responsive human adrenal cell line. The HAC15 cell line should provide an important model system for · defining the molecular mechanisms regulating aldosterone and cortisol production.
61 Introduction
Defining the molecular mechanisms and cellular signaling pathways that regulate the production of adreriocortical steroids is often limited by the .availability of appropriate model systems. One method of modeling the adrenal is through the
196 use of cell lines that retain adrenocortical differentiated functions • The list of permanent cell lines established from human adrenal tumors. is remarkably short,
84 196 consisting of the NCI-H295 and its substrains • • The NGI-H295, H295R and
H295A retain several differentiated functions, which make them useful for
12 57 84 102 105 110 defining the cellular mechanisms regulating steroid production • • • , , ,
127 196 203 • - • However, the NCI-H295 cells have little or no ACTH .response. Thus, there is currently no human adrenal cell model that responds to a sustained
ACTH stimulation.
In this report, we describe the establishment of a permanent human adrenocortical carcinoma . (HAC) cell line. These cells continue· to secrete glucocorticoids -and mineralocorticoids. Most importantly, these cells have retained responsiveness to·both ACTH and angiotensin II (Ang II).
62 Materials and Methods
HAC Cell Culture Establishment
Primary cultures of HAC- cells were isolated following surgical removal of an
adrenocortical carcinoma from an 11 month old female with. hypertension and
hirsutism. The use of tumor tissue was approved by the Institutional Review
Board of the Medical College of Georgia (Augusta, GA). Primary carcinoma cells
were isolated after the tumor was minced into small pieces and incubated in
DME/F12 (lnvitrogen, Carlsbad, CA) containing 0·.1 % collagenase (Roche,
Indianapolis, IN}. Digestion and mechanical dispersion were carried out for 1.5 h
at 37°C, with a final digestion of the dispersed cells in medium containing 0.01 %
DNase I. Following isolation, cells were frozen in DME/F12 medium, 50 % Nu
Serum (BD Biosciences, Franklin · Lakes, NJ), and 10 % dimethyl sulfoxide
(DMSO) (Sigma, St. Louis, MO). The frozen aliquots were later suspended in
_growth media consisting of DME/F12 medium supplemented with 10 % Cosmic
Calf Serum (CCS) (HyClone, Logan, UT) antibiotics and 1 % ITS Premix (BD)
and plated at cloning density. After three weeks, clones were isolated for
characterization. The most ACTH responsive steroidogenic clone, HAC clone 15
(HAC15), was used for this study. H295R cells, grown under similar conditions
were used for comparison studies. For experiments, cells were plated onto 12
dishes (400,000 cells per well) in growth medium. Cells were treated in low
63 serum medium (0.1 % CCS) with either Ang II (1 O nM) (Sigma), forskolin (1 O µM)
(Sigma), or ACTH (1 O nM} (Organon, Bedford, OH}.
Analysis of Steroids
Experimental media from HAC15 and H295R cells were analyzed for aldosterone and cortisol levels using specific immunoassays (Siemens Diagnostics,
Tarrytown, NY and Alpco Diagnostics, Salem, NH, respectively). Steroid data· was normalized to mg total cell protein.
Real-Time Reverse-Transcriptase Polymerase Chai'1 Reaction {qPCR)
Total cellular RNA was extracted using RNeasy Mini Kits (Qiagen, Valencia, CA).
Two µg of totai RNA was reverse transcribed using the High C_apacity cDNA,
Archive Kit (Applied Biosystems, Foster City, CA). cDNA of the specific mRNA encoding enzymes and ·receptors were examined using probe sets obtained from
TaqMan Gene Expression Assays (Applied Biosystems). Quantitative normalization of cDNA for each gene was performed using the expression of 18s rRNA.
64 Protein lmmunoblot Analysis
Cells were lysed and 40· µg of lysate was applied to polyacrylamide gel electrophoresis using 12 % bis-tris NuPage gels (lnvitrogen). Membranes were incubated overnight at 4°C, with antibody against MC2R (ACTH receptor},
(Chemicon International, Temecula, CA), at a 1:1000 dilution. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies
(Santa Cruz, Santa Cruz, CA). lmmunoreactive bands were visualized using the
ECL plus Western Detection System (GE Healthcare,. Piscataway, NJ) and a
GBOX image analyzer system (Syngene, Frederick, MD}.
Statistical Analysis
All experiments were repeated a minimum of three times, and within each experiment, variables were performed in triplicate. Statistical comparisons were analyzed using one-way ANOVA. Significance was accepted at the 0.05 level of probability.
65 Results
The effects of Ang II, ACTH, and forskolin on HAC15 cell production of aldosterone and cortisol were examined following treatment for 24 h. Treatment with ACTH and Ang II significantly increased HAC15 cortisol production by over
2-fold (Fig. 12A). HAC15 cells also increased aldosterone production by 3-fold in response to ACTH and 8-fold in response ·to Ang 11 (Fig. 12A)-. Bypassing the
ACTH receptor by treating with forskolin (10 mM) significantly increased both aldosterone production (2.5-fold) and cortisol production (3-fold) in HAC15 (Fig.
12A}.
Due to the ACTH response observed for HAC cells, we examined expression· of
MC2R mRNA and protein. Protein immunoblot analysis was used to examine
MC2R in cell lysates of H295R and HAC15 (Fig. 12B). MC2R expression was evident in the HAC15 resulting in an unambiguous band of approximately 38
· kDa. In contrast, MC2R expression was undetectable in the H295R cells. MC2R and AT1 R (type 1 Ang ll} mRNA levels were also compared between the HAC15 and H295R using qPCR. HAC15 mRNA for MC2R was elevated 9.25-fold as compared to the H295R cell line. AT1 R mRNA levels were similar in both cell_ lines (Fig. 12B}. ·
To further assess adrenal differentiated function in HAC15 cells, the expression of mRNA encoding enzymes involved in the production of aldosterone and
66 cortisol were examined (Fig. 13). Treatment with ACTH increased mRNA expression of all the enzymes needed for aldosterone and cortisol production, name~y StAR, CYP11A1, CYP17, CYP21, HSD3B2, CYP11B1·, and CYP11B2 mRNA as compared to untreated cells (Fig. 13}. Treatment with Ang. II and forskolin also caused an increase in mRNA expression of all the enzymes needed for aldosterone and cortisol production.
67 Discussion
We previously developed a human adrenocortical cell model, H295R, which
96 retains Ang U and forskolin responses 1 • These cells have been widely used to study steroid hormone production; however, they are not a good model for ACTH regulated steroidogenesis. Herein, we isolated a clonal pop·ulation of adrenocortical carcinoma cells that retained Ang 11, ACTH and forskolin responsiveness. These cells will provide an alternative to the H295 substrains and should act as a better model for ACTH response.
Adrenal cell lines have proven useful for defining basic mechanisms involved in adrenal function. There have been a number of rodent adrenal cell lines developed -using either adrenal tumors or transgenic mice with adrenal-targeted
196 oncogenes • The Y-1 _mouse adrenal cell line is the most widely used for
196 204 studying acute regulation of steroidogenic· enzymes • • However, the Y-1 cells have _lost the expression of 21 hydroxylase and therefore cannot produce cortisosteroids. Two other mouse adrenocortical cell lines, ATC1 and ATC7-L,
95 were developed using genetically targeted oncogenesis • Both cell lines retain a zona fasciculata phenotype, which is manifested at multiple levels, including· corticosterone secretion, _ACTH responsiveness, and expression . profiles of steroidogenic enzymes .. While each of these cell lines are useful for many adrenal research projects, mouse adrenal cell lines can not replicate the ability of
human adrenal cells to produce cortisol or adrenal androgens. Therefore, human
68 adrenal cell lines are needed for many studies directed at understanding the
human adrenal.
The NCI-H295 and its substrains are currently the only human adrenocortical cell
12 57 84 102 105 110 127 196 203 models available • • • • • • • - • Two strains of., H295 cells,
196 _designated H295R and H295A, have been selected to grow as a monolayer •
These cells. have been used to study steroidogenic enzyme expression and
steroid hormone production. In addition, the cells have been used to examine the
molecular mechanisms regulating transcription of CYP11 A 1, CYP11 B1,
12 57 198 203 CYP1182, CYP17, CYP21, and HSD3B2 • • - • The H295R cells are also
responsive to Ang 11 and potassium through the production of aldosterone and
57 105 the expression of CYP11 B2 • • Thus, thes·e cells have been widely used to
bett.er understand the molecular mechanisms regulating human adrenal cell
steroidogenesis.
The successful development of the original NCI-H295 cells from an
adrenocortical carcinoma led us to use a similar approach to develop the HAC15
cells. Our findings indicate that the HAC15 cells have similar steroidogenic
profiles as compared to the H295R. These findings include the production of all
the corticosteroids. Cortisol was produced at significantly higher levels than
aldosterone. However, this may be the due to low expression of CYP11 B2
(al_dosterone synthase), which requires Ang II treatment to increase . its
expression. The HAC15 cells also consistently produce significant amounts of
the adrenal androgen, dehydroepiandrostenedione (DHEA) (data not shown).
69 Further studies will be needed to determine if chronic hormonal treatment (Ang II or ACTH) can influence the HAC15 phenotype to be either glomerulosa, fascicuJata or reticularis like.
Similar to the NCI-H295 and its substrains, the HAC15 cells expressed mRNA encoding all enzymes needed for production of aldosterone, cortisol and DHEA.
All of these transcripts were responsive to both Ang II and ACTH. HAC15 cells were also responsive to extracellular potassium through an increase in aldosterone production and CYP11 B2 expression (data not shown). The major difference between the HAC15 and H295R appears to b~ their ACTH response.
MC2R is the well documented receptor to which ACTH binds and regulates
205 different cellular responses • While the H295R cells have a number of limitations, one of the most obvious is the lack of a sustained ACTH response.
The lack of responsiveness in H295R cells is not clearly understood. The cells have mRNA expression for MC2R, which is regulated by Ang II and cAMP
206 signaling systems • In addition, the H295R cells respond acutely to ACTH with
207 an activation of the ERK 1/2 phosphorylation • However, our direct comparison of· H295R and HAC15 cells demonstrated significantly higher_ levels of MC2R m RNA and protein expression in the HAC 15 cells. These data suggest that the differences between the HAC15 and H295R cells are at least, in part, due to differences in MC2R expression.
70 In summary, we have developed an adrenal cell line, HAC1~ that exhibits hormonal responses, steroidogenesis, and expression of steroid-metabolizing. enzymes. This cell line represents only the second human adrenocortical cell line
available. The ability of HAC15 cells to respond to Ang II, potassium and ACTH
make them the first adrenal cell line capable of responding to the three main
adrenocortical physiologic regulators.
Acknowledgements
We thank the expert editorial assistance of Ors. J. Ian Mason, Raymond J.
Rogers, and Ora Pescovitz.
71 · Figure Legends ·
Fig. 12: Panel A. Steroidogenic responsiveness of HAC15 and H295R cells to
agonist treatment. Cell lines were treated for 24 h with Ang II (10 nM), ACTH (1 0
nM}, or forskolin (10 µM). Cortisol and aldosterone concentrations were
determined by EIA and RIA, respectively. Values represent the mean ± SE of six
independent experiments. *** P < 0.001 for agonist-stimulated steroidogenesis
vs. basal. Panel B. HAC 15 cell expression of MC2R and AT1 R receptors. Protein
immunoblot analysis was used to examine expression of MC2R in cell lysates of
H295R and HAC15. 40 µg of protein was loaded per lane. Comparison of MC2R
and AT1 R mRNA levels in HAC15 to H295R was done following qPCR with the
H295R cells used as a calibrator. Values represent the mean ± SE of six
independent RNA samples. *** P < 0.001.
Fig. 13: HAC15 cell expression and regulation of genes encoding enzymes
involved in steroid hormone biosynthesis. HAC15 cells were treated with or
without Ang II (10 nM}, ACTH (10 nM), or forskolin (10 µM) for 24 h. mRNA level
for each enzyme was determined using qPGR. Values represent the mean ± SE
of six independent experiments. Nomenclature for the steroidogenic enzymes are
as follows: StAR: steroidogenic acute regulatory protein; CYP11 A 1: cholesterol
side-chain cleavage; CYP17: 17a-hydroxylase-17,20-lyase; HSD382: 3(3-
hydroxysteroid dehydrogenase type II; CYP21: 21-hydroxylase; CYP1181: 11 (3-
72 hydroxylase; CYP11 B2: aldosterone synthase. *** P < 0.001, ** P < 0.0,1, * P <
0.05.
73 Fig .. 1-2:
A 90 18
80 *** • Aldosterone 16 -C: " O· 0 Basal Ang II ACTH Forskolin B 12 -a: (kDa) LO H295 HAC1 0) 220 *** -MC2R 11 ~_,_ C\J I 120 10 JJ :i... 60 - AT1R Q) 3 > 9 JJ 0 40 z Q) 8 )> 0) C: a, MC2R 7 --0 ..c: C. (.) 6 (') :::r "'O m 0 5 :::::, (Q -<( 4 CD 0 z < a: 3 CD E -,; 2 I a: I\) C\J c.o (.) 01 ~ JJ 0 -74 H295R HAC15 Fig. 13: Cholesterol - CYP11A1 liiiilStAH tt * ** Basal Ang 11 ACTH Forskolin Pregnenolone 17a-Hydroxypregnenolone 40 2.5 .,..... HSD382 CYP17 *** -a -!/) -a 2.0 -!/) CCI 30 CCI .0 .O· Q) Q) > 1.5 ~ 0 -0 20 "O 0 g ::::.. 1.0 C\I ..... CD C") 0:: 0 10 >- en C.) 0.5 J: 0.0 Basal Ang II ACTH Forskolin Basal Ang 11· ACTH Forskolin Progesterone 17a-Hydroxyprogesterone 6..------, CYP21 g"C 3 N 2 Cl. >- C.) 1 0 Basal Ang II ACTH Forskolin 11-Deoxycorticosterone 11-Deoxycortisol CYP11B1 cti CYP1182 -(/) l - 30321 CCI m .0- :j Q) 10 > ! 28 0 ~ -0 8 "C 0 :::. g 6 C\I 6 al· in 4 CL 4 >- CL 0 2 [) 2 0 0 Basal Ang II ACTH Forskolin Aldosterone Basal Ang 11 _ACTH Forskolin CortisoV 5 II. MANUSCRIPTS Invited Review Article Comparisons of adren·ocortical cell lines as in vitro test systems Jeniel Parmar and William E. Rainey Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912 Abbreviated Title: Adrenal glomerulosa targeting Key words: Cyp11 b2, adrenal cortex, zona glomerulosa, in vivo targeting Name/Address for Correspondence: Jeniel Parmar Department of Physiology Medical College of Georgia Augusta, Georgia 30912 Telephone: 1-706-721-8363 Fax: 1-706-721-8360 E-mail: [email protected] 76 I. BACKGROUND The adrenal gland is a compound endocrine organ separated into two distinct developmentally unrelated tissues; an adrenal medulla and an adrenal cortex. The adrenal medulla is the central core of the adrenal gland; the chromaffin cells of the medulla are the body's main source of the catecholamines. The cells comprising the adrenal cortex are derived from mesoderm of the dorsal coelomic wall and thus have common features to the steroidogenic cells within the gonads. In 1866, Arnold first described the histology of the adrenal cortex and noted that it was divided into three concentric zones which he named the zona glomerulosa, 43 zona fasciculata, and zona reticularis • While this description was based on the histological organization, it is now accepted that these zones have functionally distinct roles in steroid hormone production. Namely, the glomerulosa synthesizes mineralocorticoids, the fasciculata produces glucocorticoids, and in the human, the zona reticularis produces C19 steroids, including DHEA and \ DHEA-sulfate. Molecular mechanisms leading to zone specific expression of these steroids are yet to be defined. Each adrenocortical zone synthesizes its steroid products from the common substrate cholesterol. Steroidogenic cholesterol can arise from endogenous cholesterol stores, from serum derived lipoprotein or from de novo synthesis. Within the human adrenal cortex, steroid synthesis involves coordinated actions of five forms of cytochrome P450 and the enzyme 3~-hydroxysteroid dehydrogenase (Fig. 14}; these enzymes are 77 44 distributed between the mitochondria and the endoplasmic reticulum • It is the differential expression of these enzymes within the three adrenocortical zones that allows for the wide array of steroid hormones secreted by this gland. Although. the enzymes that are differentially expressed between the zones have been identified, the molecular mechanisms causing zone-specific expression patterns are yet to be fully understood. The mechanism regulating production of steroids from each distin_ct zone is quite different and therefore, extremely complex. Angiotensin II (ANG 11) and potassium (K+) are the major regulators of steroidogenesis in the zona glomerulosa while, ACTH is the principal hormone regulating steroidogenesis in the fasciculata and reticularis (Fig. 15). In each case, the rate-limiting step in steroid hormone biosynthesis is the translocation of substrate cholesterol from the outer mitochondrial membrane to cholesterol side chain cleavage (CYP11 A), the first enzyme in the steroidogenic pathway, which is located inside the mitochondrion. In addition, regulatory mechanisms control the bioavailability of substrate cholesterol, the synthesis of the enzymes required for steroidogenesis, and the size and structural integrity of the gland. Investigations into the complexity of the adrenal cortex have been hampered by the unavailability of adrenal tissue samples and cell cultures. This has made the development of viable in vitro cell models an attractive alternative to studies using whole animals. A number of in vitro systems have been investigated, 78 including;· cell suspensions from acutely dispersed tissue, primary cultures from 78 79 normal adrenal glands and adrenal tumors, • and established cell lines from 80 81 85 88 tumors • or from immortalized cells - • Primary cultures of adrenocortical cells have proven useful for examining mechanisms controlling the many aspects of adrenal physiology but the constant requirement for freshly acquired tissue and the difficul,ties associated with the isolation of c~lls has increased the demand for alternatives. Adrenal cell lines, which allow large numbers of functional• cells to be propagated without the need for animal sacrifice or acquisition of human tissue, have yielded considerable progress in defining basic mechanisms involved in adrenal function. Several issues become important for these adrenal model systems, including cell growth, response to agonists and maintenance of steroidogenic capacity. The ability of adrenal primary cultures and cell lines to produce steroids or to respond to ACTH, ANG U and K+ can change over time in culture. Therefore steroid synthesis and cellular responses to agonists must be monitored continuously. The expression of the enzymes involved in steroid hormone biosynthesis can also change under culture conditions. As a result, the steroids produced by cells in culture can be quite different from that seen in vivo. Thus, each of these criteria must also be considered when developing or evaluating cell lines_ to study the adrenal. 79 II. AVAILABLE RODENT ADRENOCORTICAL CELL LINES {see Table IV) A. The Y1 Adrenal Cell Line The Y1 adrenal cell_ line was derived from an adrenal tumor that developed in a LAF1 (C57L x A/HeJ) mouse following exposure -to an atomic blast; a direct relationship of tumor development to the radiation is not clear 143. Gordon Sato and colleagues 144 adapted the transplantable tumor to grow in vitro by . . alterna~ely propagating dispersed tumor cells as. mono'layer cultures and as tumors in mice. One of the clones developed from the mixed population of tumor cells was named Y1, and it was this clone that was deposited at the American Type Culture Collection (ATCC CCL-79)80. Major steroid pathways present in cultured Y1 cells are _shown in Figure 16. In contrast to the normal mouse adrenal gland that produces corticosterone as the major steroid product, Y1 cells in culture produce 20ct-hydroxy-~4-pregnen-3-one (20a-dihydroxyprogesterone) and 11 (l,20a-dihydroxy-~4 -pregnen-3-one (11 (l,20a-dihydroxyprogesterone}2°8· 209. This abnormal steroid profile relates to the deficiency in 21-hydroxylase (CYP21 )210 coupled with an increase in 20a hydroxysteroid dehydrogenase activity2°8. The ability of ACTH to regulate Y1 cell steroid synthesis is similar to that seen for normal mouse adrenal cells and other agents that raise intracellular levels of 80 cAMP, which leads _to an induction of a number of genes that are supportive for steroid hormone biosynthesis. Examples include genes encoding the ACTH receptor145• 206, Cyp11 a211 -214, 11 f3-hydroxylase (Cyp11 b1 )214-216·, steroidogenic acute regulatory · protein "(StAR)213· 217· 218, adrenodoxin211 · 212, adrenodoxin reductase212 and· the HDL receptor (SR-Bl} 219. As opposed to the robust stimulation of steroid hormone production by ACTH, Y1 cells do not respond to ANG II, although transgene expression of the Arig "II receptor in Y1 cells has been described 152. Yr cells are grown under a humidified atmosphere of 5 % CO2 and 95 % air in a variety of growth media supplemented with serum. The cells approximate doubling time· is 30-40 h220· 221 . Y1 cells, for the most part, grow in culture as flat, adherent cells with polyhedral shapes maintained by a network of stress fibers and focal adhesions near the cell surface. In response to ACTH or to the adenylyl cyclase activator Forskolin, which raises intracellular levels of cAMP, Y1 cells retract their extended plasma membranes, become spherical with short processes and detach easily from the substratum on which they are grown80· 222- 224. The effects of ACTH and cAMP on cell shape are closely linked to the effects of these agents on steroidogenesis and possibly reflect an involvement of the cytoskeleton in the delivery of cholesterol to the mitochondria225-228. 81 The Y1 ceH line, as maintained in continuous culture, started to accumulate in 220 clonal variants with markedly different steroidogenesis • Among the variants, 220 221 229 230 some appeared to be completely resistant to ACTH • • • and some were 231 resistant to ACTH-induced desensitization of adenylyl cyclases • Also, a family 145 232 of ACTH receptor deficient mutants were developed • , and some mutants 233 234 harbored a dominant inhibitory protein kinase A • • These mutants have proved to be very valuable in assessing the roles of cAMP, protein kinase A, adenylyl cyclases, ACTH, and ACTH receptor in adrenocortical study. B. Use of Viral Oncogenes to Develop Rodent Adrena_l Cell Lines 1. Ras transformed rat adrenal cells. Auersperg et al. explored the possibility of establishing rat adrenal cell lines by infection with Kirsten murine sarcoma 86 92 94 virus • - • Primary cultures of rat adrenal ·cells in early passage were infected with virus and allowed to overgrow the normal cell population. The transformants metabolized pregnenolone to progesterone and 20a.-dihydroxyprogesterone, indicating that they retained some steroidogenic potential. However, they did not synthesize 11- or 21-hydroxylated steroids nor was their capacity to form steroids from endogenous ·cholesterol measured. A similar strategy was used to establish 86 an adrenal cell line from rat zona glomerulosa cells • These cells produced basal levels of steroids and increased steroid production in response to ACTH or cAMP. However, the stimula~ed levels of steroid production as estimated by 82 radioimmunoassay were very low (8 ng per 105 cells per 24 hours}, and the identity of the steroid product was not rigorously identified. 2. Cell lines from tumors in transgenic animals. Several investigators established immortalized adrenal cell lines from adrenal tumors in mice carrying 87 88 96 98 the Simian Virus 40 T-antigen as a transgene • • - • Using a human CYP11A 87 96 promoter to target expression of T-antigen to the adrenal cortex~ Mellon et a/. • " generated adrenal tumors in female mice and used these tumors to establish cell lines that produced progesterone as the major steroid product. Progesterone synthesis was not responsive to ACTH or ANG II but was stimulated by 8-bromo cAMP. Progesterone was synthesiz~d at a rate of 100 ng/µg of DNA per 24 h under stimulated conditions. The cells expressed CYP11A and CYP11 B1 but did not synthesize 11-hydroxylated steroids. Although the cells · produced small amounts of deoxycorticosterone, they did not express measurable amounts of CYP21, suggesting that another enzyme carried out the steroid 21-hydroxylation 96 reaction. Curiously, these cells also expressed renin-1 mRNA • The mouse inhibin a-subunit promoter also was used to drive T-antigen expression and to generate adrenal tumors. These tumors were postulated to 97 98 originate from the X zone of the adrenal cortex • • A cell line derived from one of these tumors (clone Ca1} produced progesterone as the major steroid product. These cells expressed LH receptors, rendering them modestly responsive to 83 stimulation by hCG. Unstimulated Ca.1 cells produced progesterone at a relatively high rate (350 ng/106 cells per hour), and treatment with hCG produced a small but statistically significant (.::; 40%) increase in progesterone production. . Effects of ACTH on steroid production were not documented. Treatment of Ccx.1 cells with inhibin decreased T-antigen levels and slowed the rate of proliferation. Effects of inhibin on steroidogenesis were not reported although it would have been interesting to determine if suppression of T-antigen expression affected the rate or profile of steroid production. Several immortalized adrenocortical ceU lines were generated from mice bearing a transgene for a temperature-sensitive form of T-antigen under control of its own 88 promoter • At permissive temperatures, one group of cells expressed SF-1, CYP11A, StAR and CYP11 B1, suggesting that they were of fasciculata reticularis origin. A second group of' cells expressed SF-1, CYP11A, and StAR but not CYP11 B 1, suggesting that they had lost differentiated function or were derived from an undifferentiated · cell layer situated between the glomerulosa zone and the fasciculata zone. Mukai et al. suggested that these adrenal cell isolates may be useful in exploring the basis for functional zonation of the adrenal cortex. Major steroid products of these cells appeared to be pregnenolone and progesterone. None of the cells were responsive to ACTH or ANG II and none of the cells expressed aldosterone synthase (CYP11 B2). At permissive temperatures, the phenotypes of the cell lines remained stable for ~ 84 200 passages. Shifting cells to non-permissive temperatures or treating cells with dibutyryl cAMP had variable effects on gene expression; either treatment converted the undifferentiated cells to fasciculata-like phenotypes and also induced CYP21 and HSD3B expression. Perhaps the most successfully established mouse adrenocortical tumor cell lines 95 were recently described characterized by Ragazzon et a/., • Using genetically targeted oncogenesis, the first immortalized cell lines, ATC1 and ATC7-L, were developed. Both ATC1 and ATC7-L have retained a complete ZF phenotype. This phenotype was manifested at multiple levels: steroidogenic capacity, ACTH responsiveness, and expression profile of specific genes. The two cell lines were established' from adrenal tumors of two transgenic mice harboring the large T antigen of SV40 under the control of adrenocortical-specific promoter of Akr1 b7. Both lines showed a significant stimulation of corticosterone secretion upon treatment with 10-11 M ACTH and then increased in a dose-dependent manner, reaching the highest values at 10-7 M ACTH. Detailed examination of expression of hormonal responsiveness of the mRNAs for the genes involved in the different steps in glucocorticoid synthesis revealed detectable levels of Mc2r (ACTH receptor mRNA) and Cyp21a1 mRNAs but were unresponsive to ACTH in both cell lines. By contrast, mRNA levels for Sr-b1, StAR, Cyp11 a 1, Cyp11 b1 and Akr1 b7 were strongly induced in a time-dependent manner by ACTH treatment, and these inductions were not abolished by the protein synthesis inhibitor, 85 cycloheximide. Thus, the effect of ACTH on accumulation of these mRNAs is essentially transcriptional. The ATC cell lines have been developed. as novel in vitro models, maintaining differentiated endocrine functions of ZF adrenocytes. They have proven very useful in investigating the mechanisms of steroidogenic gene regulation in response to the physiological activator of glucocorticoid synthesis ACTH. Ill. Bc;>VINE ADRENOCORTICAL CELL LINES (see Table IV) Primary cultures of bovine adrenal cells have been a favored model to study adrenal cell function. This is likely due to the fact that bovine adrenal glands are readily available at abattoirs. and the large size of the gland allows for isolation of sufficient cells for large experiments. Bovine adrenal cells were the first adrenal model system to be used for the development of cells Unes through viral gene transformation. Hornsby and colleagues used SV40 to create a series of clonal 153 bovine adrenal cell lines • The addition of SV40 to the cells greatly enhanced the cell culture lifespan of the cells. The cells continued to respond to cAMP related agonists by induction of 17a-hydroxylase 17, 20-lyase (CYP17) and CYP11A. However, cellular expression of CYP21 and CYP11 B were found to 154 require specialized growth conditions • 86 More recent studies by Hornsby and colleagues have combined human telomerase reverse transcriptase with SV 40 T antigen- and ras oncogene to 235 immortalize bovine adrenocortical cells • The immortalized bovine adrenal cells have been used in vivo to rescue adrenalectomized SCID (Severe combined immunodeficient) mice from glucocorticoid insufficiency through the production of cortisol. These data suggest that such strategies could be used in the future as a means of adrenocortical replacement. IV. HUMAN ADRENOCORTICAL CELL UNES (see·Table V} A. Development of Human Adrenal Cell Lines Using Viral Oncogenes In a strategy similar to that used for bovine adrenal cells, Hornsby and colleagues used the SV40 T-antigen to isolate clonal populations of proliferating human fetal adrenal cells. These human adrenal clones responded to cAMP by increasing both CYP17 and CYP11A, but levels of CYP21 and CYP1181 were low. Transformed cells could be maintained in culture for 30 to 40 population doublings after isolation, but would then enter "crisis". and stop dividing. The cause of this late passage crisis is the result of telomere shortening that occurs with each population doubling and is normally not corrected by SV40 236 expression • B. The NCI-H295 Cell Line and Related Strains 87 · 1. Origins and Steroid Synthesis. A 48-year-old African American female from whom cell line, NCI-H295, was established, had the typical clinical, biochemical, and pathological profile of adrenocortical carcinoma. She was diagnosed· with acne, facial hirsutism, edema, diarrhea, weight loss, and a recent cessation of 84 menses • Computer assisted tomography revealed a large locally invasive malignant tumor, which later metastasized to the lungs and liver, had clinical and. biochemical evidence of excessive secretion of glucocorticoids and ketosteroids. The excised tumor was 14- x 13- x 11-cm and was used to establish the original NCI-H295 cell line. Tumor tissue was finely minced and the resulting suspension was maintained in various serum-containing and serum-free culture media for a 84 one year period • Gas chromatography / mass spectroscopy (GCMS) and radioimmunoassay were used to identify the production of steroids by these cells. Of the 30 steroids detected, approximately 20 were identified. Based on secreted steroids, these cells appeared to contain all of the adrenocortical enzyme systems which presumably were present in the original tumor, including CYP11 A, HSD3B2, CYP11 B1, CYP21, CYP17, CYP11 B2, 3(3-hydroxystJlfotransferase, and low levels of aromatase (CYP19). Cytogenetically the H295 cell is highly aneuploid and hypertriploid, with 30% of the cells containing a modal chromosome number of 62. This cell line is presently available from the American Type Culture Collection as ATCC CRL-10296 and will grow in suspension. 88 2. Growth and Morphology. Initial tumor cell growth in conventional serum containing medium was unsuccessful due to fibroblast over-growth. The culture was later adapted to grow in such media. Initial or continued growth of NCl~H295 cells did not require fibroblast growth factor, a potent stimulator of normal and cultured adrenocortical cells. Due to continuous growth and development, multiple sub-strains have been adapted from the NCI-H295 cell line using alternative growth conditions to encourage substrate attachment and shorter cell cycle times. The cell culture medium growth supplement, Ultroser G (2% - BioSepra SA, Villeneuve la Garenne Cedex, France) is a relatively defined bovine derived serum substitute which was used to increase cell growth rate. This supplement was selected due to previous reports suggesting that it helped 237 238 retain steroidogenic cell function , • NCI-H295 cells grown in Ultroser supplemented medium demonstrated increased growth rate, but the cells continued to grow as floating aggregates or loosely attached cells. Over a three- · month period, changing culture medium every three days and discarding unattached cells selected cells that maintained attachment to plastic culture dishes. After characterization, it was subsequently designated as H295R to differentiate this strain from the original cells. In comparison to the parent H295 ceH line, H295R cells grow as an adherent monolayer, and population doubling time was reduced from five to two days. This substrain is propagated in Dulbecco's modified Eagle's and Ham's F12 medium, 1% ITS plus (Collaborative 89· Biomedical Products, Bedford, MA, USA, 1% penicillin/streptomycin (Gibco}, 0.01 % gentarnicin, and 2% Ultroser G (BioSepra SA, Villeneuve la Garenne Cedex, France). Because of the difficulty in the importation of the Ultroser G serum substitute required for maintaining the NCI-H295R,. a population of these cells was selected to grow in a commercially available serum substitute (Nu Serum type I - Collaborative Biomedical Products, Bedford, MA, USA). The strain that can be grown in Nu-Serum is available from the American Type Culture Collection as ATCC CRL-2128. These cells have decreased responses to ANG It and K+ but continue to respond to agonists of the protein . kinase A signaling pathways. In an attempt to develop a strain of H295 cells that would grow in co.mmerci~I serum and retain ANG II and K+ treatment, a series of sera were tested. Growth of cells in DME/F12 medium with 5' % supplementation with Cosmic Calf Serum (CCS) (Hyclone, Logan, UT) was found to maintain cell growth and responses to ANG II and K+. This strain of H295R is advantageous because the growth medium is considerably less expensive and no supplementation with ITS-plus is needed. Also, in comparison to the original H295 cells that grew in suspension, these also grow as a monolayer. Subsequently, another strain of cells, designated NCI-H295A, have been 81 described which also grow as a monolayer • The method for isolation of the NCI H295A strain is similar to that described above, relying on the removal of non attached cells with medium changes and, therefore, selecting a population of 90 cells that grow as a monoJayer. It has been recently proposed that NCI-H295A 136 ceHs produce more mineralocorticoids than NCI-H295R cells ._ The same study also showed a lack of ANG II stimulated steroidogenesis in NCI-H295A and the angiotensin type 1 receptor was highly expressed in NCI-H295R strain compared to NCI-H295A. This study did not show any K+ (one of the primary mineralocorticoid regulators) regulated steroidogenesis nor did it show any · CYP1182 expression by_ NCI-H295A. The study failed to show any legitimate conclusive evidence about NCI-H295A strain's steroidogenesis. 3. Hormonal Response and Expression of Hormone Receptors on H295 cells. Peptide hormones, ANG 11 and ACTH are the primary ·physiologic regulators of steroid hormone production in the human adrenal gland, along with circulating levels of K+ (Fig. 15). The original description of NCI-H295 cells did 84 not report on the hormonal responsiveness of these cells • Subsequently, the responses to ANG II, K+, and ACTH treatment, as well as expression of trophic hormone receptors, were characterized for the NCI-H295R cell strain. In vivo ANG II acts on the adrenal zona · glomerulosa to increase production of . aldosterone through type 1 ANG II (AT1) receptors. Studies of [1 25 1] radiolabeled ANG 11 binding to H295R cells in the presence of antagonists to the AT1 and AT2 receptors established that the H295R cells express AT1 receptors almost 103 104 exclusively • • The AT1 receptor is coupled to phosphoinositidase C and 105 increases the production of inositol phosphates in H295R cells • ANG II, 91 through the AT1 receptor, also increases H295R cell production of aldosterone. - ANG II-mediated production of aldosterone has been shown to be differentially regulated by the dopamine receptors D2 and D4. Both receptors are found in H295 cells with D2 inhibiting and D4 activating aldosterone production via ANG 11239_ Under appropriate culture conditions, as described above, the H295R cell line has shown sustained regulation of AT1 receptor expression and activity. Regulation of aldosterone production in H295R cells is mediated via the transcription factors Sp 1 and Sp3, which are required for AT1 receptor 240 expression • These cells readily exhibit detectable. levels of AT1 receptor 125 103 104 241 rnRNA, as well as [ 1] ANG II binding • • • Expression of transcript and 104 241 binding appears to be affected in parallel • • Thus, the H295R cell line may be useful to define the mechanisms regulating adrenal cell responsiveness to ANG II. Extracellular K+ is the other major physiologic regulator of adrenal aldosterone production. K+ increases intracellular calcium levels in H295R cells, which appears to be the mechanism to increase aldosterone biosynthesis. Recent studies have also shown that K+ stimulation increases the production of both ANG I and ANG 11, suggesting that these cells may provide a model to study the 242 hypothesis that an intra-adrenal renin/angiotensin system may exist • 92 An alternate pathway for the regulation of adrenal aldosterone production is through the action of parathyroid hormone (PTH) and parathyroid hormone related peptides· (PTHRP), which have been shown to regulate aldosterone 243 production in freshly isolated glomerulosa cells • PTH and PTHRP also 244 stimulate H295R cells to increase aldosterone synthesis • PTH and PTHRP activate steroidogenesis in a cAMP-dependent manner in normal cells and the H295R cell line. Considering these observations, the H295R cell strain appears to be an appropriate model to study several of the major and minor physiologic regulators of aldosterone biosynthesis. The primary hormonal regulator of adrenal cortisol production is ACTH. The H295R ceH line is only mildly responsive to ACTH, and most strains are completely resistant. While ACTH treatment did cause an acute increase in 244 aldosterone synthesis, long-term stimulation could not be maintained • The low response to ACTH _may reflect the low level of ACTH receptor expression in 206 the H295R cell • Therefore, most experiments designed to examine the cAMP dependent pathway requires the addition of either forskolin (to activate adenylyl 104 105 113 115 127 241 cyclase) or cAMP analogues • • • • • • The low response to ACTH is therefore, a drawback of this cell model. Studies directed toward ACTH action would need to be pursued using either primary cultures of adrenal cells or the Y1 220 mouse adrenal cell line, which retains ACTH responsiveness • An alternate 93 strategy would be to use transgenic technology to reinstate ACTH receptor expression in the H295 cell line. The ·responsiveness of the H295R cell strain is highly dependent on growth conditions. Thus, when adapting the cells to alternate growth conditions, a characterization of responsiveness may be necessary. Because of the bovine components of Ultroser G, its importation into the USA from France requires a specific permit from the U.S. Department of Agriculture. For that reason, the · selection of a strain of H295R cells that grow in CCS (commercially available from Hyclone) has made it very easy to maintain the cells. 4. Steroid biosynthesis. As previously noted, adrenocortical steroid hormone biosynthesis is complex and varies dramatically between the glomerulosa, fasciculata, and reticularis zones. The clinical features of the patient with the adrenal tumor that gave rise to the H295 cell line indicated that steroids which normally arise from each of the adrenal zones were produced by the tumor. The steroids produced by the original H295 cell line, as well as the H295R strains, maintain this diversity. The ability of the cells .to produce steroids which originate from multiple zones of the adrenal suggests that the H295R cell line remains pluripotent with regard to adrenocortical differentiation. In Gazdar's initial report84 concerning these cells, a broad spectrum of basal steroid hormone synthesis was noted (30 steroids}. The steroid profile was greatly influenced by serum 94 conditions. Using the H295R cell line we have shown that these cells also 105 127 produce an array of steroids even under basal. conditions • • However, treatment with agonists appears to selectively prom 103 113 245 wm promote the cells to produce aldosterone • - • While aldosterone does not constitute the major product, even under these conditions, the H295R cell can be used to study aldosterone synthesis and factors which preferentially activate its synthesis. This is particularly important when one considers the difficulty in obtainin·g primary cultures of aldosterone-producing cells. Treatment of the H295R cell strain with agonists working through the cAMP pathway produce a pattern of steroids approaching those of the zona fasciculata and reticularis. Steroids produced during treatment with forskolin include -cortisol, 11 f3-hydroxyandrostenedione, DHEA, DHEA-sulfate, corticosterone, 11- 127 deoxycortisol, and androstenedione • These data support the proposition that the H295R cell line can act as an appropriate model for adrenocortical steroid hormone biosynthesis and may be useful in defining the various mechanisms causing the synthesis of the "zone-specific" steroids. 5. Expression of steroid-metabolizing enzymes in H295 cells. The original NCI-H295, as well as the H295R and H295A cell strains, have been used as -genetic models for studying steroidogenic enzyme gene expression. The NCI H295 adrenocortical cells express all of the enzymes participating in normal 95 197 human adrenal steroidogenesis • In addition, the genes that encode these enzymes respond to the same second messengers controlling normal human adrenocortical function. Expression of genes encoding CYP11A, CYP17, and CYP21, together with CYP11 B1 and CYP11 B2, were first studied in the NCI 197 246 H295 cells • • The transcripts encoding these enzymes accumulated in response to agonists (8-bromo-cAMP, forskolin, cholera toxin, and 3~isobutyl-1- methylxanthine) which activate the protein kinase-A pathway. Consistent _with normal adrenocortical tissue, stimulating the protein kinase-C pathway. using phorbol esters resulted in decreased CYP11A1 and CYP17, but accumulation of CYP21. In a similar manner, 8-bromo-cAMP increased CYP11 B1, CYP11 B2 and unexpectedly, aromatase (CYP19) mRNA levels. Recent studies have demonstrated that mRNAs encoding the StAR gene as well as the five forms of cytochrome P450 known to be involved in adrenal steroidogenesis (CYP11 A 1, CYP17, CYP21, CYP11B2 and CYP11B1) are also detectable in the NCI-H295R cell sub-strain 103-1os, 112-116, 121,241, 244_ 247 248 Similar to other mammalian adrenal cells • , the levels of mRNA encoding 249 CYP17 and HSD3B2 appear to be differentially regulated in NCI-H295R cells • ANG II promoted a significant increase in mRNA level for l-:fSD3B2 and CYP11 A 1 but only marginally increased the mRNA levels of CYP17. Potent activators of the protein kinase A pathway (forskolin and dbcAMP), while increasing the message for HSD3B2, have a greater effect on levels of CYP17 mRNA. Thus, 96 activation of the protein kinase C and- calcium signaling- pathways tend to support a glomerulosa-like steroidogenic enzyme expression, while -activation of the protein kinase A pathway promotes fasciculata-like enzyme expression. Another pair of steroidogenic enzymes that exhibit an adrenal zone-specific distribution includes CYP1182 and CYP1181. CYP1182 is expressed only in the zona glomerulosa and is essential for corticosterone conversion to aldosterone (Fig. 14). On the other hand, CYP1181 is expressed at higher levels in the zona fasciculata/reticularis and is essential for the production of cortisol from deoxycortisol (Fig. 14 ). The transcripts encoding these two enzymes are increased by tre?ttment of H295R cells with activators of the protein kinase-A pathway, although the effect on CYP11 B1 mRNA levels is greater.- In addition, levels of CYP1182 mRNA are increased in H295R cells by treatment with ANG 103 116 250 251 105 116 250 11 , • • or K+ • • • The effects of ANG II on the expression of CYP1181 were not as pronounced as was· observed for CYP1182, suggesting that the protein kinase-A and calcium pathways differentially regulate the expression of CYP1182 and CYP1181. The H295R and H295A cell lines have also been shown to be useful model systems to define the mechanisms controlling transcription of the steroid metabolizing genes. Fusion genes containing the 5'-flanking DNA from CYP1181, CYP1182, CYP17 and HSD382 have been studied using these cell 97 81 117 118 lines • - • Further regulati_on of genes in the steroidogenic pathway has been 119 121 demonstrated by a number of transcription factors identified in H295 celfs - • The H295R cell line is the first steroidogenic cell line to maintain the full complement of adrenal steroid-metabolizing enzymes and therefore may continue to be useful in defining the elements in the 5'-flanking region of these genes that regulate transcription. C. The SW13 human adrenal carcinoma-derived cell line. These cells were derived from a human adrenal carcinoma 142 and are available from the American Type Culture Collection (CCL-105). The SW13 is an interesting cell culture model that has a mosaic pattern of vimentin expression and is deficient in the 252 mammalian homologues of Brahma genes, Brm and BRG1 • Although adrenal in origin, SW13 cells produce no steroids and it is not clear that it derived from steroidogenic adrenal cells. Therefore its usefulness as an adrenocortical model system is limited. D. Human adrenocortical carcinoma (HAC) cell line and related clones. The above mentioned NCI-H295 cell line and its strains have proven useful due to the retention of ANG II dependent aldosterone production; however, they lack ACTH response. In addition, the H295 models are not clonal but represent a mixture of cells isolated from primary cultures of the original tumor. In an attempt to develop a HAC cell line, our group recently isolated clonal populations of cells from an 98 adrenal tumor. Primary adrenocortical carcinoma cells were isolated from a · tumor removed from a child diagnosed with hypertension, obesity, and hi~sutism. Cells were maintained in DME/F12 medium supplemented with 1Qc % CCS, ITS+ premix, penicillin, streptomycin, and gentamicin. Parental tumor cells were used to develop forty seven discrete clones, six of which grew well enough for further characterization. Preliminary results suggest that ACTH increased cortisol 253 production in two of the clones • The cells also retained responsiveness to Ang 254 II and K+ through the production of aldosterone • Further characterization is ·ongoing, but these cell lines promise to provide an improvement in the available systems to study human adrenal steroid production. V. SUMMARY The mechanisms involving hormonal regulation of adrenocortical cell growth, morphology, and steroidogenesis remain a topic of research. The murine Y1 and human H295 adrenocortical cell· lines are the most widely used adrenal cell culture models. Together, they provide unique systems to study both the molecular and biochemical characteristics ·of adrenal function. The Y1 cell line has provided a unique experimental system to dissect the mechanisms of ACTH action on steroidogenesis, cell proliferation, morphology, and the factors that govern the expression of genes required for steroidogenesis. The ability of H295 cells to produce mineralocorticoids, glucocorticoids, and C19 steroids based on 99 culture conditions has provided a valuable model to study the processes involved in adrenocortical differentiation. However, both model systems have limitations. The Y1, due to the deficiency of 21-hydroxylase, cannot produce corticosteroids. The H295 models have low or no ACT~ response. The recent development of a corticosterone-producing mouse adrenal cell line and an ACTH responsive human adrenal cell line should improve our ability to better study adrenal toxicology and cellular physiology. 100 Table IV: Summary of Information on Rodent and Bovine Adrenal Cell Lines Cell Line Name ACTH K+ ANG II cAMP Steroids produced Availability Reference Cited: 220 Y1 and its mutant strains + - - + Progesterone metabolites ATCC and Schimmer Lab 255 196,' 256, ' 257 258 86 Rat adrenal cells: 2FASC and + - - + Progesterone metabolites Roskelley and 7GLOM Auersperg ' Auersperg 93 94 Rat adrenocortical line: TRA + + Progesterone metabolites Lab - - . ' 95 Mouse adrenal cell-lines: ATC1 + ND - + Reported glucocorticoids Martinez lab and ATC7-L 88 Immortalized mouse adrenal cell - - - + Progesterone metabolites lshimura lines: AcA201, AcE60 and AcA 101 lab Immortalized mouse adrenal cell lines: ST2, ST5-R, and ST5-L - ND ND + Progesterone metabolites Mellon and Weiner Lab 87 r Immortalized mouse adrenal cell 96 line $T5Lc - ND ND + Progesterone metabolites Mellon Lab SBAC and other Bovine Adrenocortical Cell Lines ND None reported ATCC 2~9 260 - + + ' .. (+)Positive;(-) Negative; ND: Not documented 101 Table V: Summary of lnformatio'n on Human Adrenal Cell Lines Cell Line ACTH K+ ANG II cAMP Steroids produced Availability Reference Name.. Cited: Mineralocorticoids ' NCI-H295 - ND ND ND Glucocorticoids ATCC 84 Adrenal Androgens Mine'ralocorticoids 113 NCI-H295A - ND - + Glucocorticoids Miller Lab Adrenal Androgens 261 262' ' NCI-H295R -/+ + + + Mineralocorticoids ATCC 105 113 245. 114-116' ' Strains Glucocorticoids And ' Adrenal Androgens Rainey Lab Mineralocorticoids · HAC15 + + + + Glucocorticoids Rainey Lab 253,254 Adrenal Androgens (+)Positive;(-) Negative; ND: Not documented 102 -\ Fig. 14: Human adrenal steroid biosynthetic pathways illustrating the three main products of the adrenal: aldosterone, cortisol, and DHEA/S and the enzymes that synthesize these products. Classification for the enzymes in the P450 263 superfamily follow the guidelines previously reported • Nomenclature for the enzymes is as _followed: StAR: Steroidogenic acute regulatory protein; CYP11 A 1: cholesterol side-chain cleavage; CYP17: 17a hydroxylase 17, 20 lyase; SULT2A 1: DHEA-sulfotransferase; HSD3B2: 3P hydroxysteroid dehydrogenase type 11; CYP21 : 21-hydroxylase; CYP 11 B 1: 11 p hydroxylase; _CYP 11 B2: aldosterone synthase. Fig. 15: Signaling pathways that regulate steroid production in human adrenal cells. The primary regulators of adrenal steroid hormone biosynthesis are ACTH, Angiotensin 11 and potassium, which work at the level of the cell membrane. Both the calmodulin-dependent protein kinases (CaM Kinases) and protein kinase A pathways are able to activate acute and chronic steroid hormone production. Fig. 16:. Rodent adrenal steroid biosynthetic pathways for the production of aldosterone and corticosterone. Also illustrated are the variations seen Y-1 adrenal cell steroid production, which produces primarily 20a dihydroprogesterone and 11 J3,20a-dihydroxyprogesterone. Abbreviations are as described in Figure 14. 103 Fig. 14: Cholesterol StAR CYP11A1 SULT2A1 7aOH DHEA sulfate 17aOH Progesterone Progesterone CYP21 Deoxycorticosterone Deoxycortisol CYP11B1 . CYP11B2 Corticosterone - Cortisol (CYP11B2] Aldosterone 104 Fig. 15: ACTH ANG II Calcium ATP cAMP i Protein Kinase A ~ Cam Kinases ! ~ ! Increased StAR ~iat~ :,. , Cholesterol StAR with the ,_ J/,. ~ / surface of ~ mitochondria facilitates cholesterol entry release of steroids Chronic Effects Acute Effects 105 Fig.. 16: Cholesterol StAR CYP11A1 HSD382 Progesterone ~-CY-P2-1 Deoxycorticosterone--. CYP.1182 Corticosterone 11 ~ 20a-dihydroxyprogesterone (CYP11B2] Aldosterone Corticosterone . 106 II. MANUSCRIPTS · Book Chapter Adrenocortical Cell Lines Jeniel Parmar, Anita Kulh~rya, and William E. Rainey Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912 Abbreviated Title: Adrenal glomerulosa targeting Key words: Cyp11 b2, adrenal cortex, zona glomerulosa, in vivo targeting Name/Address for Correspondence: Jeniel Parmar Department of Physiology Medical College of Georgia Augusta, Georgia 30912 Telephone: 1-706-721-8363 Fax: 1-706-721-8360 E-mail: [email protected] . 107 4.2.1 Introduction Initially, primary cultures of adrenocortical cells have traditionally been utilized to study the mechanisms controlting adrenocortical steroidogenesis. However, the eventual onset of senescence in culture· creates a recurring need for the costly and difficult isolation of fresh cultures, and subsequently increases the risk of contamination. For these reasons, the use of primary cultures has been increasingly replaced by immortalized cell lines. This chapter describes the major adrenocortical cell lines that are available for the study of physiologic and pathologic adrenocortical functions. Adrenocortical cell lines· that have been derived from adrenal tumors that have developed spontaneously or experimentally from a variety of species will be discussed. These in vitro models have allowed a multitude of studies that have broadened our understanding of normal adrenoco·rtical endocrine function as well as the properties of adrenal tumor cells. The adrenal gland is an integral part of the endocrine system, as it regulates essential physiological functions in all vertebrates. It is noteworthy, however, tha_t considerable variations exist with regard to the anatomical locations of the adrenal and its significance as an endocrine organ among different animals. In mammals, the gland is subdivided into two different compartments-the outermost cortex and the innermost medulla. This subdivision does not exist in amphibians or fish; in fact, adrenal cells of some fish are found within the 108 kidneys, where they are collectively termed the "intrarenal" gland. During mammalian embryonic adrenocortica-t development, the adrenocortical mesenchymal cells originate from the coelomic cavity near the urogenital ridge. These cells go on to form first the adrenogonadal primordium, which is the precursor of the adrenal cortex and the somatic cells of the ovaries. Later the· adrenocortical primordium separates to form the fetal adrenal gland, which becomes encapsulated by surrounding mesenchymal cells. With ongoing development, adrenal medullary cells start to migrate into the fetal adrenal gland, eventually forming the adrenal medulla. In a last step the adult adrenal cortex ·arises with its classical zonation and the fetal adrenal cortex regresses. The histologic zonation of the cortex in three functionally distinct regions (zona glomerulosa, zona fasciculata, and zona reticularis) was first described by Arnold 43 in 1866 • For all adrenocortical steroidogenesis, the rate-limiting step is the translocation of cholesterol from the outer mitochondrial membrane into the mitochondrion for cholesterol side-chain cleavage by the cytochrome P450 44 CYP11 A 1, the first enzyme in the steroidogenic pathway (Fig. 17) • In the zona glomerulosa cholesterol is used to synthesize mineralocorticoids in response to Angiotensin II (ANG II}. and potassium (K+) stimulation (Fig. 18). In the zona fasciculata cholesterol is used for glucocorticoid synthesis under the regulation of ACTH. In humans, the zona reticularis metabolizes cholesterol to 19-carbon (C 19} steroids, including DHEA and DHEA-sulfate, a process that is 109 r regulated by ACTH and yet-to-be determined factors. There is no physical barrier separating the three different adrenocortical zones. The molecular mechanisms that cause the zone-specific expression pattern of these enzymes are yet to be resolved. However, it has been established that these regulatory mechanisms control the relative expression of the enzymes required for steroidogenesis as well as the size and structural integrity of each of the zones. There are still numerous molecular and cellular studies needed to better define the mechanisms regulating adrenal cell steroid production, prompting the need for further studies both in vivo and in vitro. Appropriate, well-characterized adrenal model systems are critical for-these studies. Initially, primary cultures of adrenocortical cells proved useful for examining the mechanisms controlling many aspects of adrenal physiology. However, several issues limit the use of the primary adrenal cell culture models, including cell growth in culture, responses to agonists and maintenance of steroidogenic capacity. It is well-documented that the ability of adrenal primary cultures and cell lines to produce steroids and/ or to respond to ACTH, ANG II and K+ can change over time. The expression of the enzymes involved in steroid hormone biosynthesis can also change under in vitro culture conditions. As a result, the steroids produced by cells in culture can be quite different from those produced in vivo. Perhaps the most practical limitation to the use of primary adrenal cell cultures is the constant requirement for freshly acquired tissue and the difficulties associated with the isolation of cortical cells in the numbers needed to accomplish in vitro studies. Taken together the problems 110 and limitations of primary cultures have caused many groups to attempt the use of adrena~ tumors cells as model systems. Moreover, these cell lines can· be used in xenotransplant models and in vitro studies to- research adrenocortical cancer. A number of in vitro tumor cell models have been investigated, including 77 79 cell suspensions from acutely dispersed adrenal tumors, - , established cell 80 84 lines from tumors - and the isolation of immortalized cells from transgenic 85 88 animals - . Herein, we provide a summary of the adrenoco_rtical cell lines and. provide some information as to their development and 'utility. 111 4.2.2 Human Adrenocortical Cell Lines (see Table VI) 4.2.2.1 The NCI-H295, H295R and H295A Adrenocortical Carcinoma Cell Lines · Origin and development The NCI-H295 cell line was established from a female patient diagnosed with an 84 adrenocortical carcinoma • A large 14- x 13- x 11-cm adrenocortical invasive tumor was detected by CT imaging and was later reported to have metastasized to the lungs and liver. In the initial process of developing the original NCI-H295 cell line, the tumor tissue was finely minced, defragmented and maintained in 84 various serum-containing and serum-free cult_ure media for a one year period • Monoclonal cell lines were not established and therefore, the NCI-H295 cell line represents a mixed population of tumor cells from the original carcinoma. Cell line growth and characterization Since parental NCI-H295 cells sustained a slow rate of growth in the initially described culture media, alternative growth conditions were sought in order to segregate a population of cells with better attachment and growth. Initially, the cell culture medium growth supplement, Ultroser G, a relatively defined bovine derived serum substitute, was used to increase cell growth rate. Previous 112 assessments. of this media supplement reported increased adrenal cell growth 237 238 and retention of steroidogenic function • • Despite the increased growth rate · of NCI-H295 cells grown in Ultroser supplemented medium, the cells continued to grow as floating aggregates or loosely attached cells, making it difficult to experimentally treat and manipulate these cells in culture. In an attempt to establish a superior cell model, the parental NCI-H295 cells were continuously flushed ·with fresh growth medium for a period of three months to deliberately remove the floating suspended cells and keep the viable attached subtype. The newly selected population of cells was subsequently designated as H295R to differentiate this strain from the original cells. H295R cells grow as an adherent monolayer (Fig. 18A). The population doubling time of the new H295R c~lls was approximately 2 days in comparison to the original parental NCI-H295 cell line which grows in suspension and takes approximately 5 days to double in population. Unfortunately, the success of the H295R was challenged by the limited availability of the Ultroser-G medium in select countries. Consequently, a series of sera were tested in an attempt to develop a population of H295R cells that would grow in a relatively inexpensive commercial serum and retain ANG II and K+ responsiveness. Growth of cells in DME / F12 medium supplemented with 5 % Cosmic Calf Serum (CCS) (Hyclone, Logan, UT) was found to maintain cell growth and responsiveness ·to ANG 11 and K+. Another strain of H295R cells was also selected to grow in commercially available Nu- 113 Serum type I (10 % - Collaborative Biomedical Products, Bedford, MA). This strain is available from .the American Type Culture Collection· as ATCC CRL- . 2128._ These cells however have a decreased response to ANG II and K+, though the cell doubling time· is relatively similar to those grown in other sera. Since their first establishment, H295R cells have been cultured with variety of media in which they have shown a range of steroidogenic responses and adherent properties. As a result the agonist responses and growth characteristics of H295H cells vary depending on individual laboratory conditions. As with H295R, a second population of the parental NCI-H295 cell line was 81 se~ected to grow as a monolayer, yielding another strain of the original cell line , designated H295A. The method for isolation of the H295A strain was similar to that described for H295R, relying on the removal of non-attached cells with medium changes and, therefore, selecting a population. of cells that grow as a monolayer. Expression of hormone receptors and hormonal responsiveness Although ANG 11, K+ and ACTH are the primary hormonal regulators of steroid hormone production in the human adrenal cortex (Fig. 17), there is no description 84 of hormonal responsiveness in the original description of the NCI-H295 cells . Subsequently, the responses to ANG 11, K+, and ACTH treatment, as well as expression of hormone receptors, were cha~acterized for the H295R cell strain. 114 ANG H is the primary hormonal. regulator within the renin-angiotensin-aldosterone system (RAAS}; upon conversion from its precursor by. angiotensin converting enzyme, it acts on the adrenal glomerulosa by binding to type 1 ANG II (AT1) receptors to increase the production of aldosterone. H295R cells have proven to be a useful model to study ANG II regulated aldosterone production. Only AT1 receptor antagonists have a significant inhibitory effect while type 2 ANG 11 (AT2) receptor antagonists have little impact on ANG II stimulation, demonstrating that 103 104 H295R cells respond almost exclusively to the AT1 receptors • • Subsequent studies also revealed AT1 receptor couple with phosphoinositidase 105 C and increased production of inositol phosphates in H295R cells • Circulating extracellular K+ is the other major physiologic regulator of adrenal aldosterone production. An increase in intracellular calcium levels in response to elevated K+ mediates an increase in aldosterone biosynthesis. In addition, there 242 is evidence of an intra-adrenal renin / angiotensin system , in which K+ stimulation increases the production of both ANG I and ANG II. The H295R cells are used by many laboratories as a model to study the mechanisms of K+ 106 111 regulation of adrenal steroid production - • ACTH is the primary hormonal regulator of adrenal cortisol production. The H295H cell line is only mildly responsive to ACTH, while most other 82 adrenocortical cell lines are completely unresponsive • ACTH treatment of 115 H295~ cells does cause an acute increase in aldosterone biosynthesis, though 83 244 the ceH line lacks long term responsiveness • • The low response to-ACTH is therefore, a drawback of this cell- model. Poor ACTH response is in part a reflection of low levels of ACTH receptor expression in the H295R cell, however even after induction of receptor expression the cells still exhibit a poor 83 206 response • • Since ACTH primarily regulates cortisol production through cAMP signaling, the addition of either forskolin (to activate adenylyl cyclase) or 104 105 113 115 127 241 cAMP analogues • • • • • can be used to overcome this dilemma. An alternate strategy could involve the use of transgenic technology to reinstate ACTH receptor expression in the H295R cell line; however, attempts to do so have increased receptor expression but not response to ligand (unpublished observation). At present, investigations of_ ACTH responsiveness rely heavily on 220 the mouse adrenal cell line Y1, which retains ACTH responsiveness • Steroidogenesis Most research focuses. on adrenal production of aldosterone, cortisol and DHEA for which the steroidogenic pathways are well defined (Fig. 19). After isolation and establishment of the parental NCI-H295, the cell line was found to produce an array of steroids, including those characteristic of the adrenal cortex and some unique to tumor cells. Primary assessment of steroidogenic capacity was performed using gas chromatography / mass spectroscopy (GCMS) and radioimmunoassay. Of a total of 30 different steroids reportedly synthesized and 116 secreted by NCI-H295, approximately 20 were formally identified as known steroid hormones. The production of these steroids suggested the presence of all of the adrenocortical steroidogenic enzymes including CYP11 A 1, HSD382, CYP1181, CYP21, CYP17, CYP1182, and 3(3-hydroxysulfotransferase. The H295R strain also maintains this steroidogenic profile; however, it is greatly influenced by serum conditions. The ability of these cells to produce steroids which originate from multiple zones of the adrenal suggests that the H295R cell line remains pluripot.ent with regard to adrenocortical differentiation. These cells produce an array of steroids even 105 127 under basal conditions • • This is particularly important when one considers the difficulty in obtaining primary cultures of aldosterone-producing cells. It is noteworthy that treatment with agonists appears to selectively promote the 103 113 245 synthesis of certain zone-specific steroid hormone groups in H295R • • • For instance, production of aldosterone drastically increases following ANG II and K+ stimulation, while treatment with forskolin induces cortisol, 11 (3- hydroxyandrostenedione, DHEA, DHEA-sulfate, corticosterone, 11-deoxycortisol, 127 and androstenedione.production • While H295R cells do not represent a single zone of the adrenal cortex, the cell line provides ample flexibility for studies on adrenocortical steroidogenesis. As stated above, with a wide variety of effective agonists, a mosaic of unique steroid expression profiles are possible in H295R cells, thus making this cell line a potential model for steroid hormone 61 128 135 biosynthesis in all adrenocortical zones • - • 117 · Steroidogenic enzyme expression -The original NCI-H295 adrenocortical cells express all of the enzymes 99 101 participating in normal human adrenal steroidogenesis • • Along with the parental NCI-H295, the H295R and H295A cell strains have also been used as genetic models for studying steroidogenic enzyme gene expression. Transcripts encoding· StAR, as well as the five forms of cytochrome P450 . known to be involved in normal adrenal steroidogenesis (CYP11A1, CYP17, CYP21, 103 105 112 116 CYP1182 and CYP-1181), are detectable in the H295R cell strains • • - • 127 241 244 • • • Moreover, as with primary cultures and other mammalian adrenal 247 248 cells • , the levels of mRNA encoding CYP17 and HSD382 appear to be 249 differentially regulated in H295R cells • Furthermore, the transcripts encoding CYP1181 and CYP1182 enzymes are increased by treatment of H295R cells with activators of the protein kinase-A pathway, although the effect on CYP1181 mRNA levels is greater (Fig. 17). In addition, ANG II and K+ cause a drastic 103 105 116 250 251 increase in CYP1182 mRNA encoding aldosterone synthase • , • , • The original NCI-H295 and its strains have proven to be useful models for defining the mechanisms controlling adren_ocortical steroidogenesis. The basal - promoter and enhancer elements of the genes encoding CYP1181, CYP1182, 81 117 118 CYP17 and HSD382 . • - together with_the transcription factors mediating the 118 expression of these genes has been studied in great detail using these cell lines11e-121; 111, 122;.12a, 1~. 131-141_ NCI-H295 cell line· as adrenal cancer therapy tool In vitro cancer therapy screening represents an important mechanisms of drug testing264. The National Cancer Institute has made wide use of. neoplasms and -carcinoma cell lines for anticancer drug screening and drug discovery265-268 . Below is a brief description of the use of the NCI-H295 and its sub-strains as model systems for the study of cancer therapies. NCI-H295 has been widely used as a model for screening anti-cancer drugs. -Suramin, one of the first anti-cancer drugs tested in the NCI-H295 cells reduced the production of glucocorticoids, mineraloco~icoids and adrenal androgens in this cell line269. Schteingart and colleagues screened mitotane and mitometh, two distinct anti-cancer drugs in NCI-H295. Mitotane strongly suppressed cell growth and mitometh was not effective270. Fallo et al. tested the cytotoxic/ anti proliferation drugs, - taxol and paclitaxel, which effectively exhibited dose dependent inhibition of cellular growth and reduced steroidogenesis in NCI H295271· 272. Adrenostatic compounds aminoglutethimide (AG), metyrapone (MTP) and etomidate (ETO} were also tested in NCI-H295 for their antiproliferative property. AG and ETO inhibited cell proliferation and ETO was much more potent273. Thiazolidinediones (TZDs) are specific peroxisome 119 proliferator-activated receptor (PPAR)-gamma ligands. Examination in NCI-H295 suggest that TZDs might have favorable effects in the treatment of a variety of 274 tumors as differentiation-inducing agents • NCI-H295 cells have also been utilized to assess the role of. certain interferons, chemokines and growth factors 215 2 in adrenocortical ~ancers - n. Preclinical assessment of anti-cancer compounds with potent antiproliferative activity in NCl-295 cells is an essential first step before any clinical evaluation in Phase I, II and Ill programs could be commenced. Recent studies have utilized NCI-H295 cells in cell culture and as a xenograft model to evaluate the efficacy of a variety of new potential targeted therapies. T cell factor / J3-catenin antagonists, PKF115-584278 and type I insulin-like growth 279 factor receptor inhibitors have been recently tested in the NCI-H295 • These studies are aimed at a limited number of -known genetic mutations in adrenocortical carcinoma that are present in the NCI-H295 cells. 4.2.2.2 Human Adrenocortical Carcinoma, Clone 15 (HAC15) Cell Line and Related Clones The above mentioned NCI-H295 cell line and its strains have proven useful due to the retention of ANG ll~dependent aldosterone production. However, as previously discussed, they lack significant ACTH responsiveness. In addition, the NCI-H295 models are polyclonal, as they represent a mixture of cells isolated 120 from primary cultures of the original tumor. In an attempt to develop a new human adrenocortical carcinoma (HAC) cell line, the authors recently isolated 82 83 monoclonal populations of cells from an adrenal tumor • • Primary adrenocortical carcinoma cells were isolated and used to develop 47 discrete clones, six of which grew well enough for further characterization. Initially, the ceHs were maintained in DME / F12 based growth medium supplemented with 10 % Cosmic Calf Serum (CCS), penicillin, streptomycin, gentamicin and ITS+ Premix (a solution containing human recombinant insulin, human transferrin, selenious acid, BSA, and linoleic acid). Preliminary results suggested that ACTH . stimulation increased cortisol production in two of the clones. Same clones also retained responsiveness to ANG II and K+ through the production of aldosterone. Further analyses of these cells will judge, whether they are an improvement over the currently available systems for the study of human adrenocortical steroid 83 production • HAC15 origin and development HAC15 was established from an adrenocortical carcinoma removed from an 11 month old female who presented with hypertension, hirsutism, and associated obesity. The carcinoma was initially fragmented and tissue was finely minced into small pieces and incubated in DME / F12 medium containing 0.1 % collagenase. For a final digestion and dispersion, the cells were further incubated with the addition of 0.01 % deoxyribonuclease (DNase I). Once finely 121 dispersed, parental HAC cells were plated at approximately 1000 cells per 100 mm petri-dish. Sterilized cloning rings were placed around each of the clones and trypsin was used to remove the cells (Fig•. 20). After a close assessment of several steroidogenic clones, the most responsive clone to ANG II, K+ and ACTH 83 treatments-HAC15-was further characterized • HAC15 cell line growth, steroidogenesis and enzyme expression HAC15 ·cells demonstrate structural features previously observed for the H295R carcinoma cells, including the loss of contact inhibition which leads to cells growing as attached clumps (Fig 18B). As with H295R, HAC15 cells grow as attached cells; however, unlike H295R, they seem to aggregate and grow as clusters of cells in a staggered-stacking pattern. Upon stimulation with ANG II and K+, a robust increase in aldosterone level is observed in the HAC15 cell line. These cells respond to ACTH as it causes a modest yet significant increase in cortisol and DHEA. Other HAC clones have reportedly responded with increased aldosterone and cortisol production to ANG 11 but have lost ACTH responsiveness. Bypassing the ACTH receptor by treating with forskolin significantly increases both aldosterone and cortisol production in HAC15. Other clones also respond to forskolin, with a similar increase in both steroids. 122 The HAC15 cell line has proven practical for steroidogenic studies as it expresses all of the genes involved in the adrenocortical pathway including- StAR, CYP11A1, CYP17, CYP21, HSD3B2, CYP11B1, CYP11B2, and SULT2A1. Treatment with ANG II, K+, ,ACTH and forskolin causes significant increase in StAR, CYP11A1, CYP17, CYP21, CYP11 B1, and CYP11 B2 mRNA as compared 83 to untreated cells • The melanocortin 2 receptor (MC2R) is expressed in HAC15 cells with mRNA levels that are significantly higher than in the H295R cell line. However, the expression of the MC2R and response of the HAC clones is not as robust as that seen in the Y1 adrenal cell line. In summary, the HAC15 cell line exhibits hormonal response, steroidogenesis and expression of steroid-metabolizing enzymes and improved ACTH response compared to the H295R. 4.2.2.3 Cytogenetic Analysis of H295R and HAC15 Cell Lines The HAC15 and H295R cell lines were karyotyped using standard trypsin Giemsa banding methods. Briefly, each cell type was exposed to colcemid for 2 h at 37 °C. In HAC15, the chromosome_ number ranged from 68-74 chromosomes with modal chromosome number of 70. It appeared that there were two populations in the HAC15 cells that exhibited different karyotypes. One population exhibited a near pseudotriploid karyotype with a modal chromosome 123 number of 70 (Fig. 21A). This was not a true triploid state because of the loss of certain chromosomes and gain of two copies of other chromosomes. A true triploid has three copies of each chromosome as compared to the two copies of each chromosome seen in a normal diploid female. The other population of HAC cells exhibited a hyperdiploid karyotype with a modal chromosome number of 52. This HAC15 cell population demonstrated the same abnormalities as seen in the near triploid cell population but had a loss of several of the abnormal and normal chromosomes. No new abnormalities were present in the hyperdiploid clone. It is likely that the hyperdiploid clone evolved from the near triploid cell population. In the H295R cell strain used in our laboratory, the chromosome number ranged from 52 to 58 with a modal chromosome number of 55 (Fig. 21 B). This · represents a shift from the modal chromosome number of 62 observed in the 84 original NCI-H295 cell population described in 1990 by Gazdar and colleagues • A composite description of the H295R and HAC15 cells with each structural and numerical chromosome abnormality, as well as chromosome markers have been summarized in Table VII. It is interesting that both the H295R and HAC15 cell lines demonstrated some similar chromosome abnormalities that include abnormal chromosome 3 with interstitial deletion, chromosome 9 with additional material on 9p, deleted 11 p, deleted 12p, chromosome 16 with additional 124 material on the distal end of the long arm, and chromosome 21 with additional material on distal 21 q. Also, there were at least three marker chromosomes in both cell lines that appear to be similar by morphology and banding analysis. However, it is likely that the karyotype pattern will change as the cells are maintained in culture for extended periods and moved from laboratory to laboratory. 4.2.2.4 Pediatric Adrenocortical Adenoma Derived Cell Line Origin and development Primary adrenocortical adenoma cells were isolated from a non-hemorrhagic region of a functional tumor from a 1 year old female that presented with 275 virilization and Cushing syndrome . This pediatric adrenocortical cell line was last reported to have sustained growth· and viability after eight passages. Sections. of tumor were initially fragmented and subjected to collagenase digestion. Fine tumor fragments were then segregated and plated in two different media; DMEM and Opti-MEM 1 (reduced serum medium of MEM) and supplemented with 1O and 2 % fetal bovine serum (FBS) and 1 % penicillin / streptomycin maintained under standard culture conditions (37 °C in 95 % air-5 %CO2). 125 Growth and steroidogenesis The cell line iniUally grew at a slow rate with a spindle ~ike morphology in both of the media. As with the HAC15 cell line, a flattened homogeneous morphology, characteristic of a single cell-type origin was noted. Melan-A, a melanocytic differentiation marker, which is expressed in steroid hormone producing adrenal 280 adenomas and carcinomas , was seen in all examined cells. All steroidogenic studies were performed during the ·fifth .passage in medium from 5 day experiments. Biosynthesis of cortisol, aldosterone, androstenedione, and 17-0H progesterone was observed along with the expression of steroidogenic enzymes namely; HSD382, CYP1181 and CYP21 were reported. Taking into consideration that this newly developed pediatric cell line has last reported to have only reached eight passages, the likelihood of the cell line's viability remains to be established. As these cells are part of a mixed tumor cell population further clonal selection may be necessary. 4.2.2.5 The SW13 Human Adrenal Carcinoma Derived Cell Line Although adrenal in origin, SW13 cells produce no steroids and it is unclear whether the cell li~e was derived from steroidogenic adrenal cells. These cells were derived from a human adrenal· carcinoma 142 and are available from the 126 American Type Culture Collection (CCL-1_05). ·The SW13 is an interesting cell culture model th~t has a mosaic pattern of vimentin ~xpression and is deficient in the mammalian homologues of Brahma genes, Brm and BRG1 252. Due to the lack of steroidogenesis, its usefulness as an adrenocorticaJ model system is limited. 4.2.2.6 The ACT-1 Human Adrenal Carcinoma Derived Cell Line Tumor cells were isolated from a 62 year old male who was initially diagnosed with a left adrenocortical carcinoma. After a surgical removal of tumor tissue, aggregated pieces were excised, minced and passed through a cell strainer to dissociate the cells. Cells were supplemented with 10 % FBS, insulin-transferrin selenium solution and appropriate antibiotics maintained at 5 % CO2 and 95 % air. In its initial description, ACT-1 cells were only shown to express HSD3B2 enzymes and were devoid of any adrenocortical steroids281 . Hence, ACT-1 has limited adrenocortical function and therefore serves a restricted role for normal adrenal function. However these cells may prove useful to screen adrenocortical carcinoma therapies. 4.2.2.7 The RL-251 Human Adrenal Carcinoma Derived Cell Line An adrenocortical carcinoma was identified by CT scan in a 75 year old male who presented with high blood pressure (219 / 107 mm Hg} and multiple spells of chills, warmth, sweating and flushing. A 5 x 6 x 7 right adrenal mass was removed by laparotomy and placed in cell suspension. Initially, tumor sample was treated with collagenase and plated in fibronectin-coated plastic with RPMI 1640 medium containing 1O % FBS and fibroblast growth factors with a population doubling time of 36 h. As with H295R and HAC15 cell lines, RL-251 also exhibits abnormal karyotypic profile with diploid karyotype consisting of numerous deletions and translocations. Assessments of adrenocortical function showed atypical steroidogenesis and hormonal response with insignificant 277 cortisol production and no ACTH stimulation • To date, there is no description of any other corticosteroid production by the RL-251 cell line. Intriguingly, in its initial report, RL-251 cells expressed ample amounts of interleukin-a, epithelial cell-derived neutrophil-activating peptide 78, growth regulated oncogene-a and growth regulated oncogene-y. These are CXC chemokine family cytokines that have potent angiogenic activity essential for tumor growth. It is speculated that these chemokines may play a role in enhancement of carcinoma growth and proliferation by an autocrine or paracrine manner. A lack of reported steroidogenesis and hormonal responses do not make these cells an appropriate model to study normal adrenocortical function, however, these cells may prove useful in identifying the role of chemokines in adrenocortical carcinomas. 128 4.2.2.8 Human Adrenal Cell Lines from Viral Oncogenes Simian virus 40 (SV40) T-antigen is a hexamer protein that is an oncogene which is capable of transforming many cell types. SV40 T-antigen was used by 282 Hornsby and colleagues to transform human fetal adrenal cells • The strategy yielded human fetal adrenal cell clones that responded to cAMP with an increase in both CYP 17 and CYP 11 A, but no · change in CYP21 and CYP 11 B 1. Transformed cells were maintained in culture for 30 to 40 population doublings after isolation, but then entered ''crisis" and stopped dividing. The cause of this late passage "senescence" is the result of telomere shortening that occurs with 236 each population doubling and is normally not corrected by SV 40 expression • 4.2.3 Rodent Adrenocortical Cell Lines (see Table VI) 4.2.3.1 The Y1 Adrenal Cell Line · Presumably following. exposure to radiation during nuclear weapon testing, an 143 adult LAF1 {C57L x A/HeJ) mouse developed an adrenal tumor • Gordon Sato and colleagues 144 adapted the transplantable tumor to grow in vitro by alternately propagating dispersed tumor cells as monolayer cultures and as tumors in mice. The most robust clones developed from the mixed population of tumor cells was 129 named Y1, and ·it· was this clone that was deposited at the American Type Culture Collection (ATCC CCL-79)8°. It is noteworthy that the major steroid pathways present in cultured Y1 cells are unlike the normal physiologic mouse adrenocortical steroid pathway. For instance, Y1 cells in culture produces 2Oa-hydroxy-~4-pregnen-3-one (2Oa - dihydroxyprogesterone} and 11 J3,2Oa-dihydroxy-~4-pregnen-3-one (11 J3,2Oa dihydroxyprogesterone)208· 209 . (Fig 22). This abnormal steroid profile relates to the deficiency in 21-hydroxylase (Cyp21 )210 coupled with. an increase in 2Oa hydroxysteroid dehydrogenase activity208. Y1 cells could be grown under a humidified atmosphere of 5 % CO2 and 95 % air in a variety of growth media supplemented with serum. Doubling time for this cell line is merely 30-40 h220· 221 . These cells grow in culture as flat, adherent cells with polyhedral shapes maintained by a network of stress fibers and focal adhesions near the cell surface. Upon stimulation with any of the cAMP inducing agonists, Y1 cells retract their extended plasma membranes, become spherical with short processes and detach easily from the .substrate on which they are grown so, 222-224. The ability of ACTH to stimulate Y1 cell steroidogenic activity and enzymes is similar to that seen in primary mouse adrenal cells and other agents that raise intracellular levels of cAMP. For instance genes encoding the ACTH receptor145· 130 206 211 214 214 216 13 217 218 211 212 , Cyp11 a • , Cyp11 b1 - , star2 • • , adrenodoxin • , adrenodoxin 212 219 reductase and the HDL receptor (SR-BI) • A wealth of information has been gained through studying this cell line and several of its sub-clones which have proved to be invaluable in assessing the roles of cAMP, protein kinase A, adenylyl cyclases, ACTH, and ACTH receptor in adrenocortical cell physiology. Y1 cells do not respond to ANG II, although transgene expression of the ANG II 152 receptor in Y1 cells has been described . 4.2.3.2 Experimentally Induced Rodent Adrenal Cell Lines . Kirsten murine sarcoma virus (MSV) transforms cells resulting in reduced requirement for attachment, density and reliability of other cells in terms of growth and development. Auersperg et al. took primary cultures of rat adrenal cells in early passage and infected them with MSV and allowed them to overgrow 86 92 94 the normal cell population • • _ The clones exhibited little steroidogenic potential and only produced progesterone and 20a-dihydroxyprogesterone. Similarly, an adrenal cell line from rat zona glomerulosa cells86 was created but found to produce negligible steroids (8 ng per 105 cells per 24 h), despite responsiveness to ACTH and cAMP agonists. An SV40 T-antigen strategy was also attempted in the quest of developing immortal· rodent cell lines. Several investigators established immortalized adrenal cell lines from adrenal tumors in mice carrying the Simian Virus 40 T- 131 87 88 96 98 antigen as a transgene • • - • Mellon et al. used a human CYP11A promoter 87 96 to target expression of T-antigen to- the adrenal cortex • and generated adrenal tumors in female mice and used these tumors to establish cell lines. These cell lines were not responsive to ·either ANG II or ACTH and only produced progesterone following treatment with cAMP analogues. Progesterone was synthesized at a rate of 100 ng / µg of DNA per 24 h under stimulated conditions. 96 Noteworthy, these cells reportedly produced renin-1 mRNA • In terms of developing immortalized rodent adrenocortical cell lines, several others have utilized similar transgenic approaches such as the use ·of mouse inhibin a-subunit promoter to drive T-antigen expression to generate adrenal 97 98 tumors • . As with others, these cells only produced progesterone. · Subsequently, another group used a strategy with mice bearing a transgene with 88 a- temperature-sensitive form of T-antigen under control of T-antigen promoter . At the permissive temperatures, one group of cells expressed Sf 1, Cyp11 a 1, Star and Cyp11 b1, suggesting that they were of fasciculata-reticularis origin. A second cell line expressed Sf1, Cyp11 a 1 and Star but not Cyp11 b1, suggesting that they had lost differentiated function or were derived from an undifferentiated cell layer situated between the glomerulosa zone and the fasciculata zone. None of the cells were responsive to ACTH or ANG II and none of the cells expressed · aldosterone synthase (Cyp11 b2). 132 Perhaps the most successful oncogene targeted mouse adrenocortical tumor cell 95 lines are described and characterized by Ragazzon et a/. • Using genetically targeted oncogenesis, adrenocortical immortalized cell lines, ATC1 and ATC7-L, were developed. AKr1 b7 gene promoter was used to drive the expression of SV40 T-antigen. The two cell lines were established from adrenal tumors of two transgenic mice harboring the large T-antigen.of SV40. Both ATC1 and ATC7-L have retained a zona fasciculata phenotype evident by their robust steroidogenic capacity, ACTH responsiveness and expression profile of steroid-metabolizing genes._ Both cell lines responded well to ACTH stimulation with a concentration dependent increase in corticosterone production. Examination of hormone responsiveness revealed detectable levels of Mc2r and Cyp21 mRNAs but these transcripts were not responsive to ACTH in either cell line. By contrast, mRNA levels for Srb1, Star, Cyp11 a 1, Cyp11 b1 and Akr1 b7 were strongly induced in a time-dependent manner by ACTH treatment, and these inductions were not abolished by the protein synthesis inhibitor, cycloheximide. The ATC cell lines represent novel in vitro models, maintaining differentiated endocrine functions of zona fasciculata. They may prove to be very useful in investigating the mechanisms of steroidogenic gene regulation in response to the physiological activator of glucocorticoid synthesis, ACTH. 133 4.2.4 Bovine Adrenocortical Cell Lines (see Table VI) In terms of primary cultures, due to the size and availability of bovine adrenals, bovine adrenocortical cells have remained a critical resource for the study of adrenocortical biology. Hornsby and colleagues have used SV40 to create a 153 series of clonal bovine adrenal cell lines • The introduction of SV40 greatly enhanced the lifespan of the cells in culture. A population of cells continued to respond to cAMP-related agonists by induction of CYP17 and CYP11A. However, cellular expression of CYP21 and CYP11 B was found to require 154 specialized growth conditions • Subsequently, Hornsby et al. have combined expression of human telomerase reverse transcriptase with SV40 T antigen and 235 ras oncogene to immortalize bovine adrenocortical cells • The immortalized bovine adrenal cells have been used in vivo to rescue adrenalectomized SCIO (Severe combined immunodeficient) mice from glucocorticoid deficiency through the production of cortisol. 4.2.5 Summary Adrenal cell- lines represent a crucial tool for molecular and cellular studies that cannot practically be done in animal models. The murine Y1 and ·human H295 adrenocortical cell lines are currently the most widely used adrenal cell culture models. The Y1 adrenal cell line has long provided a unique experimental system to dissect the mechanisms of ACTH action on steroidogenesis, cell 134 proliferation, morphology, and the factors that govern the expression of genes required for steroidogenesis. The additions of the human H295R and HAC15 adrenal carcinoma cells that produce mineralocorticoids, glucocorticoids, and C19 steroids has provided valuable models to study the processes involved in adrenocortical differentiation. However, both the human and murine adrenal model systems have limitations. The Y1 cell, due to the deficiency of 21- hydroxylase, cannot produce corticosteroids. On the other hand, the H295 cell · models have low or no ACTH response. The recently described ACTH responsive murine ATC1 / ATC7-L and human HAC15 cell lines offer new opportunities to study the regulation of adrenal steroid production. In addition, the HAC15 offers a new model to test human adrenal carcinoma therapies. Together, they provide unique model systems to study both the molecular and biochemical characteristics of adrenal function. 135 Table VI. Summary of Information on Adrenocortical Cell Lines Ang Cell Line Name Species . II K+ ·ACTH .cAMP Steroids produced Reference Mineralocorticoids NCI-H295 ND ND ND Glucocorticoids 84 Adrenal Androgens Mineralocorticoids NCI-H295A ND +- Glucocorticoids 261 262 Adrenal Androgens Mineralocorticoids NCI-H295R 105 113 245. -/+ Glucocorticoids 114-116' ' Strains + + + ' Adrenal Androgens RL-251 ND ND ND None reported 277 ACT-1 ND ND ND ND None reported 281 SW13 ND ND ND ND None reported 142 Pediatric ad renocortical Mineralocorticoids ND ND ND ND 275 adenoma derived Glucocorticoids cell-line Mineralocorticoids HAC15 + + + + Glucocorticoids 82,83 Adrenal Androgens 220 Y1 and its mutant Progesterone 255 196,' 256, ~ + + strains metabolites ' 257 Rat adrenal cells: Progesterone 2FASC and 258 86 + + metabolites 7GLOM Rat Progesterone 93 94 ad renocortical -' . + + metabolites line: TRA Mouse adrenal - 95 cell-lines: ATC1 v•r··--· ND + + Glucocorticoids and ATC7-L 136 - - Immortalized mouse adrenal Progesterone cell lines: ....._ + 88 - - - metabolites AcA201,AcE60 andAcA101 Immortalized mouse adrenal Progesterone cell lines: ST2, ..___ ND ND 87 - + metabolites ST5-R, and ST5- L Immortalized Progesterone mouse adrenal ND ND - metabolites and 96 ceU-line ~ + Glucocorticoids ST5Lc SBAC and other Bovine .. ... + ND + None reported 259 Adrenocortical ' - Cell Lines Abbre v·IatIon. ND, Not determined 137 Table VII. Comparison of cytogenetic abnormalities in HAC15 and-H295R cell lines. HAC15 H295R Modal number of chromosomes: 68-74 Modal number of chromosomes: 52-58 -X -X add (2)( q37) -2,del(2}( q35) del(3)(p13p21) del(3}( q27) -4,-4 del(4)(q31.3) +5,+5,del(5(p13)x2 add(5)(p15) +6,add(6}( q27) +6 del(8)(p21) -8,add(8}(q24) +del(9)(p22},add(9)(p2?)x2 +9,+9,del(9)(p13},add(9)(q24} + 10,add(10)(p13} add(10)(p13) add(11)(p13),+add(11)(p15.5},del(11 }(p11.2) + 11,del(11 )(p11.2),add(11 )(p11.2) + 12,del(12)(p11.2)x2 del(12)(p11.2} +13 -13 +15 -14 add(16}(q24}x2 +15 add(17)(q25} + 16,add(16}(q24} +20,+20 -18 add(21 )(q22} -21,add(21 ):(q22) +9~19 mar +8~13 mar For chromosome analysis, HAC15 and H295R cells were treated with colcemid for 2 h at 37 °C, followed by trypsin/EDTA treatment. Loose cells were subjected to hypotonic conditions for 15 minutes then fixed (3: 1 methanol: glacial acetic acid) and metaphase chromosomes stained with Trypsin/Giemsa stain .. The slides were analyzed under an Olympus BX61 microscope and the images were captured with an Applied Imaging Automated Karyotyping System. Abbreviations used in the table include: add (additional material of unknown origin-unidentifiable); del (deletion of part of a chromosome); - (loss of an entire chromosome); + (gain of an entire chromosome); mar (marker chromosomes of unknown origin-unidentifiable}. 138 Figure Legends: Fig. 17: Intracellular adrenocortical cell signaffng and mechanisms leading to adrenocortical steroid synthesis upon stimulation with the physiological agonists Ang II, K+ and ACTH. Fig. 18: Panel A: Phase contrast photomicrograph of cultured H295R cells, demonstrating celi growth attached patches of· cells. · Panel B: Phase contrast photomicrograph of cultured HAC15 cells, demonstrating cell growth as clumps or clusters of cells. Fig. 19: Human adrenocortical steroid biosynthetic pathway. Fig. 20: Development and isolation- of HAC clones with cylindrical cloning rings. Fig. 21: Cytogenetic profiles depicted through karyotypes. Difference in chromosome size, position of centromeres, in total number of chromosomes and presence of ambiguous marker chromosomes were apparent when comparing the two adrenocortical cell lines. Panel A: Representative HAC15 karyotype and Panel B: Representative of H295R karytoype. Fig. 22: Murine adrenocortical steroid biosynthetic pathway including modifications seen in some mouse cell lines. 139 Fig. 17: 140 Fig,. 18: 141 Fig. 19: Cholesterol CYP11A1SIAR i HSD382 Pregnenolori Progesteto Demfycortloosteron~eoo~osteron,~ ~ Aldoste,ooo 1 Nomenclature for·the enzymes DHEA StAR: ster;pldogenlc acute regulatory protein. p 142 Fig-. 20: . Examine Select and circle viable Remove medium and place clones clones to be isolated rings over desired clones ...... l Harvest each clone in separate Trypsinize cells from within the flask rings • 143 Fig. 21: A 12 A 144 Fig. 22: 20a- ~--1.. , 20ij,. ----i~. 11~20a ditiyd(oxypl;Oge.st_ero_lje· dihydroxyprogesterope 145 Ill. DISCUSSION A. Specific aim I: CYP1182 Gene Regulation 283 The regulation of mineralocorticoid biosynthesis and secretion is complex • In the present investigation we sought to extend our understanding of the molecular mechanisms regulating the production of the principal mineralocorticoid- aldosterone. It is well documented that CYP1182 conducts the late rate-limiting 61 111 284 step in aldosterone biosynthesis • • • Since the CYP11B2 gene in humans and mice is predominantly expressed in the ZG of the adrenal cortex, we proposed to define the regions responsible for controlling in vivo ZG-specific expression. We demonstrated that a 985 bp promoter of the Cyp11 b2 gene is sufficient to target the adrenal ZG with expression of a Lacz reporter in mice. The 985 bp Cyp11 b2 promoter-driven Lacz was detected solely in the ZG of the _adrenal cortex. Upon qPCR examination of 16 key mouse tissues, no aberrant extra-adrenal Lacz transcript was found. Hence, the major findings of this project are that Cyp11 b2 gene expression is controlled by its 985 bp 5' flanking promoter region and that the expression of the Cyp11 b2 gene is primarily confined to the adrenal cortex under normal physiological conditions in mice. 146 In terms of the history of ZG-specific targeting with the Cyp11 b2 promoter, others have previously utilized a bacterial artificial chromosome (BAC): containing, the entire Cyp11 b2 gene (unpublished data)- or a large segment (>5 kb} of the 176 Cyp11 b2 gene promoter • Morley et al generated 7 independent Tg lines with a construct harboring 5.4 kb of mouse Cyp 11 b2 5' flanking region and the lacZ reporter gene, which resulted in no, detectable lacZ expression. In contrast, we used less than a 1 kb region from the same gene to successfully target the adrenal ZG layer. These results suggest that the additional 4.4 kb region many contains further regulatory element(s) that have silenced or limited lacZ reporter expression in the 5.4 kb Tg mice. The additional 4.4 kb sequence, however, does not show any homology to the corresponding human CYP1182 region and, in fact, most of this sequence is repetitive. Because our NS adrenals show faint X gal staining that could go unnoticed without meticulous inspection, it is also possible that the 5.4 kb Tg mice .may have expressed low levels under NS condition which could have been amplified by the administration of LS diet. There is a substantial amount of evidence to support the contention that changes in dietary Na+ and K+ have a direct effect on Cyp11 b2 expression and serum aldosterone levels. Rats given a LS diet or a high K+ diet respond with a profound 285 287 increase in Cyp11 b2 levels - • In-vitro analyses using human· and rodent 57 58 141 288 adrenocortical cell lines have also shown matching results • • • • Our results complement these previous reports with robust increases in endogenous Cyp11 b2 and Lacz mRNA, as well as serum aldosterone levels noted in the LS groups compared to NS control groups. To some extent, we owe our successful 147 characterization of the Tg mouse to the LS diet and its strong activation of the RAAS. It is noteworthy that_part of the success may be attributed to the SIB cassette that we used as our reporter gene in the development of Tg mice. The SIB cassette contains a splice donor / splice acceptor (SD/SA) sequence, which allows the spliceosome to bring together neighboring exons and increase transgene 89 stability2 • The SIB cassette also includes an internal ribosomal entry site (IRES) sequence, located between _-985 bp Cyp11 b2 promoter and the Lacz reporter gene, which allows ribosomal subunits to bind our Tg mRNA independently of the 290 291 5'-cap initiation of translation, enhancing the stability of our construct • • Lastly, the SIB cassette harbors a long poly A tail, which should further stabilize the transgene in the mouse genome. Several reports have shown targeting of the adrenal cortex utilizing other 163 172 177 182 adrenocortical-specific gene promoters • - • • Morley et al have also utilized a 5.0- kb Cyp11 b1 promoter fragment that failed to target the adrenal 177 gland • In another study, however, the same group successfully targeted adrenal cortex with a 6.4 kb regulatory fragment of the Cyp21 promoter. This Cyp21 promoter driven Tg construct exhibited scattered-inconsistent targeting thro~ghout the adrenal cortex and hence ·no further analyses have been 182 conducted using this strategy • Martinez et al. reported adrenal targeting by 17 174 - using regulatory regions from aldo-keto reductase 1B7 (Akr1 b7) ~· • The 148 Akr1 b7 Tg construct contained 5'-flanking and intragenic sequences of the Akr1 b7 gene which targeted vas deferens and adrenal glands in the reported Tg mice. Adrenal targeting has allowed examination of genes believed to control adrenal development and function. The transcriptional coactivator beta-catenin, along with SF-1, reportedly regulates Wnt family members, which play a crucial role in the development and maintenance of the adrenal gland. The role of beta-catenin as part of the Wnt signaling pathway has been assessed through the use of Tg mice in terms of both embryonic development of the . adrenal cortex and in 163 172 183 maintenance of the adult organ • • • Hammer et a.I. also utilized SF-1/Cre Tg mice to conditionally inactivate beta-catenin alleles and showed adrenal aplasia in newborn Tg mice. Although adrenocortical-specific, all of the above mentioned Tg strategies have limited efficacy since they also permit extra-adrenal expression. Our current 985 bp Cyp11 b2 sequence is unique and distinct as it induces transgene expression selectively and exclusively in the adrenal ZG layer. Non-steroidogenic gene promoters have also been .used to target the adrenal gland. Jorgez et al. utilized 6 kbp of the promoter of mouse transforming growth factor ·~ superfam1ly member inhibin a subunit promoter, which targeted the 184 gonads, pituitary, and the adrenal gland • The 6 kbp a subunit promoter-driven Cre construct targeted both the adrenal cortex and medulla in three separate Tg lines and only the medulla in two other Tg lines. Hence, these animals may be 149' useful for recombination of flexed alleles, specif~cally in the adrenal medulla. Tg studies have also shown telomerase function in adrenocortical physiology, as 292 telomeres reportedly operate to replenish the cells within the adrenal cortex - 294. While important for their respective analyses and applicable to adrenal cortex, these Tg studies also allowed extra-adrenal expression. Hence, any further genetic manipulation studies utilizing these strategies would affect not only the adrenal cortex, but also all other cell types that are targeted. Nevertheless, since our transgene solely targeted adrenal ZG cells, it will allow tissue-specific downstream operations such as ZG-specific knock-in and_ knock out. A substantial amount of evidence supports the contention -that low levels of Cyp11 b2 gene are expressed in extra-adrenal tissues such as the heart, brain, 187 195 295 304 and vasculature - • - • The function of locally produced extra-adrenal aldosterone remains unclear; however, it is well understood that cardiovascular and CNS-derived aldosterone does not contribute to the. total pool of circulating aldosterone. Nonetheless, in order to assess the extra-adrenal expression of the endogenous Cyp11 b2 gene, as well as our transgene, we -investigated the ex-pression profile of Cyp11 b2 in 16 key organs in mice. As we anticipated, exceptionally low levels Cyp11 b2 mRNA in most of the extra-adrenal tissues, we therefore, designed primer sets that were highly specific and sensitive to extremely low mRNA lev~ls. We began by designing a DNA template for Cyp11 b2 that was identical to the target mRNA sequences _to be amplified. 150 Previous qPCR methods measured product relative to the adrenal Cyp11 b2 mRNA levets or relative·to a housekeeping gene, such as 18S ribosomal RNA; however, by using known quantities of the DNA transcripts as standards, we were able to express the number of Cyp11b2 mRNA copies in absolute terms. Expressing mRNA levels in terms of copy number provides sustained reproducibility and permits quantification of the low levels of Cyp11 b2 mRNA present in extra-adrenal tissues. In order to eliminate concerns regarding cross reactivity between the two isozymes, we tested ·our. in-house designed primer and probe sets for Cyp11 b1 and Cyp11 b2 against their respective transcripts and found little or no cross-reactivity. Under a NS diet, we detected Cyp11 b2 transcript in all organs tested, however, the levels of mRNA in extra-adrenal organs were extremely low compared to the adrenal gland. Intriguingly, a LS diet induced Cyp11 b2 mRNA up-regulation in thoracic aorta, thymus, and kidney, although their fold changes were small compared to the adrenal glands. While this extra-adrenal Cyp11 b2 expression may have little impact on systemic plasma aldosterone levels, it may activate the local aldosterone-mineralocorticoid receptor system, and hence it should not be overlooked. Nonetheless, qPCR analysis of transgene showed that our Tg mice have no detectable reporter gene expression in any of the extra-adrenal tissues tested under NS or LS diet. This phenomenon may be due to the limitations in our qPCR assay sensitivity as the NS adrenals in Tg mice showed minimal transgene expression. However, even under a LS diet none of the extra-adrenal 151 organs exhibited transgene expression. Further analyses under various pathological conditions are necessary to shed more light on this issue. Subsequently, because transgene expression was solely localized to the adrenal glands, even under a LS diet, we investigated the expression of the 13- galactosidase reporter protein. X-gal staining was performed for Tg and wild-type littermate mice on all extra-adrenal tissues aforementioned, on LS and NS diets. Besides the apparent (3-galactosidase staining in the Tg adrenals, as compared to the wild-type, we found no specific staining in any other tissue. The uniform 13- galactosidase staining seen in the Tg whole mount adrenals and immunohistochemistry (IHC) slides did not appear to be due to artifacts of endogenous lacZ staining because we eliminated that possibility. As described by Bolon et al., the complexity involved in background (3-galactosidase staining 305 was taken into consideration • X-gal staining time points including 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and overnight were assessed utilizing Tg and wild-type control adrenals on LS and NS diets. Little or no background staining was observed up un~il 8 h of X-gal staining. Hence, for all the mounting and IHC experiments, we assigned 8 h staining time as the standard. 185 186 In agreement with pervious reports • , a LS diet in both wild-type C57BU6 and Tg mice triggered ZG cell proliferation. The ZG layer expanded from 3-4 layers to a 12-15 layered zone. ZG cells under LS conditions began to proliferate and migrate down towards the ZF, which has a distinctively straight-cords-like 152 _ appearance. Co-staining experiments, consisting of X-gal staining, followed by primary Cyp11 b2 antibody staining of the Tg adrenocortical tissue sections, show· ZG-specific co-localization of both endogenous Cyp 11 b2 and '3-galactosidase proteins under NS ~nd LS diets. Although both proteins were co-localized in ZG layer in LS adrenals, there was a slight difference in their staining patterns. Within the expanded the ZG layer, the X-gal staining was more prominent on the outer (capsule) side, whereas the Cyp11 b2 immunostaining was more intense on the inner (medulla) side· of the ZG. This difference may be explaine.d by the current hypotheses that adrenocortical stem cells in the capsule or sub-capsular 175 proliferate, differentiate, and move inward to generate adrenal cortical cells • We believe that upon a LS diet administration, the ZG cells start inward proliferation and simultaneous activation of Cyp11 b2 promoter. This would lead to Cyp11 b2 and '3-galactosidase protein production throughout the· ZG layer, which would result in increased aldosterone production and intense X~gal staining, respectively. Because Cyp11 b2 enzyme is required for aldosterone synthesis it would presumably be stablilized in the rapidly dividing ZG cells as compared to ~-galactosidase protein. Hence we observe the accumulated Cyp11 b2 enzyme but not the ~-galactosidase protein in the inner-most ZG or outer ZF cells. Cumulatively, this suggests that the 985 bp consensus promoter fragment exclusively targets the ZG and responds to RAAS activation; however, regions required for extra-adrenal expression of Cyp11 b2 are not present in this promoter 153 fragment. This phenomenon is consistent with the literature, as there are abundant reports that show varied lengt~s of gene promoters capable of driving· 164 165 306 308 tissue and cell-specific expression of transgenes • • - • Alternatively, we must acknowledge that our transgene does not exactly emulate the endogenous Cyp11 b2 gene, as it is solely expressed in the ZG cells. Perhaps other regulatory regions exist outside of 985 bp promoter region or within the Cyp11 b2 gene that allow its expression in extra-adrenal organs. Indeed, the 985 bp promoter of Cyp11 b2 has experimental limitations due to the absence of appropriate enhancers that would allow us to study Cyp11 b2 gene expression as it occurs normally. Conversely however, this unique promoter fragment will allow several possibilities in terms of ZG-specific genetic manipulations. The characterization of this sequence should facilitate further experiments designed to specifically activate or sitence desired genes linked to human adrenocortical diseases, such as hyperaldosteronism. Conditional Cre/loxP system could be utilized to understand adrenocortical zonation . especially during development and throughout adulthood. Nevertheless, this regulatory fragment will be an invaluable tool for the advancement of res.earch associated with adrenal development, physiology, and pathology.· 154 B. Specific aim II: A Paucity of Adrenal Cell Lines The purpose of this study was to develop a well-characterized human adrenocortical cell line that would be appropriate for many in vitro analyses. Previously Gazdar et al. demonstrated that an adrenocortical cell line could be established from a heterogeneous population of cells from an adrenocortical carcinoma. Our study confirms-the work of Gazdar and colleagues, as a human adrenocortical carcinoma was utilized in development of the HAC15 cell line. The major findings of this project are that carcinomas provide a viable population of immortalized cells and that these cells can be utilized in the development of cell lines. Data from our current study indicate that a mixed p9pulation of adrenocortical carcinomas could be subjected to a series of cloning experiments in the development of cell lines from a single-cell origin. Moreover, along with HAC15, several other clones have been isolated from carcinomas. Although the HAC clones share the same origin, the adrenocortical steroidogenesis and responsiveness to physiological agonists by each clone is quite different. According to our numerous repeated analyses, the most dominant clone overall, exhibiting the best steroidogenesis and the best responsiveness to physiological and pharmacological agonists proves to be HAC15. From a clinical standpoint, our collaborative work with St. Jude's Children's Research Hospital (SJCH} (Zambetti laboratory) has allowed the assessment of chemotherapy agents on HAC15 cells in culture. In addition, at SJCH, HAC15 155 cells have been injected into immunodeficient mice, which led to tumor formation, allowing exploration .of the efficacy of chemotherapy agents in vivo~ Moreover, HAC15 gene expression profiling via DNA microarray analysis confirmed high 309 310 expression of marker genes to be expressed in adrenal carcinomas • • Collectively, results from the HAC15 cell line suggest it to be a good· tool for studying chemotherapy ·agents both in vitro and in vivo and perhaps changes · associated with carcinomas. As aforementioned, HAC15 cells were developed from a naturally occurring carcinoma, and these cells have been growing steadily in standard culture conditions for over two years. Tumors, naturally occurring or experimentally generated, have been utilized to characterize and develop viable adrenal cell lines. Several cell lines of different origins, cell type- and organ-specific, have been established that are extremely useful for in vitro analyses. Hela cells were the first immortalized human cells derived from a cervical carcinoma. Since its establishment, the Hela cell line has been a great resource for the scientific community, as it represented the prototype for the establishment of stable 311 immortalized cell lines • Moreover, recent advances in cell line development have revealed other techniques for the isolation of immortalized cells by · utilization of oncogenesis and Tg animals to artificially induce tumors. There have been a number of rodent adrenal cell lines developed using Tg mice with 82 176 196 281 adrenal-targeted oncogenes • • • • In terms of success rate for the development of cell lines and their viability as cell culture models, the newer 156 techniques appear to be inferior to naturally occurring tumors. Because cell lines developed from oncogene simulated tumors have a tendency to reach senescence, as is the case with primary cultures. In this regard, our HAC15 cell line is superior . because it has been effectively psssaged for numerous generations and has entered crisis. Taking long-term steroidogenesis and hormonal response into consideration, artificially generated tumor-derived cell lines are also inferior, as they tend to lose responsiveness to agonists and synthesize less steroid hormones with time. Evidence suggests that rarely occurring tumors may represent the best source for the development of cell lines. The Y1 mouse adrenal cell line originated from a tumor that presumably occurred due to an atomic blast. This cell line has been widely used for studying acute regulation of steroidogenesis, as well as the mechanisms controlling transcription of genes . encoding steroidogenic 210 220 312 313 enzymes • • • • The Y1 cell line has a number of positive attributes, including rapid growth, ACTH response, and easy trasfection with plasmid DNA. Our HAC15 cells have a much slower doubling time as compared to the Y1 cells. In terms of ACTH responsiveness, the youngest population of HAC15 cells exhibits an excellent and significant response to ACTH stimulation; however, after extended population doublings, the ACTH response tends to decrease. The lessening trend of ACTH responsiveness seen in HAC15 cells is not uncommon; the human adrenocortical H295R cells have also shown a similar trend. 157 Nevertheless, in our laboratory, we have also seen a time/passage-dependent decline in Y1 cell, responsiveness to ACTH_ stimulation. The reduction in response to ACTH by these cell lines is likely due to the down- regulation of its 83 205 314 receptor, melanocortin type 2 receptor (MC2R) • • • ACTH i$ a unique ligand for its receptor, as it has been shown to maintain the expression of MC2R in vivo 315 316 and in vitro • • Certainly, the lack of ACTH in culture media may somewhat explain the phenomenon of reduction in ACTH responsiveness exhibited by cell lines over time. The greatest drawback of the Y1 cells is their inability to produce 196 mineralocorticoids, glucocorticoids, or adrenal androgens • Two other mouse adrenocortical cell lines, ATC1 and ATC7-L, have been 95 produced using genetically targeted oncogenesis • Both cell lines represent a ZF phenotype, which is manifested at multiple levels: ·corticosterone secretion, ACTH responsiveness, and expression profile of specific genes. Corticosterone secretion is increased by ACTH in both cell lines in a dose-dependent manner; consequenUy, in this regard, these cells may prove superior to the Y1 cells. As with the Y1 cells, the ATC cells do not respond to Ang II nor do they secrete significant amounts of aldosterone. Hence, while these cell lines will be useful for certain adrenal research projects, they, like other mouse adrenal cell lines, will not be useful as a human model for most studies because they do not produce aldosterone, cortisol or adrenal androgens, as is seen in the H295R and HAC15 cell lines. 158 In terms of the availability of human adrenocortical cell lines, for several years, 84 13 the NCI-H295 and its substrains were the only option • t _ One substrain; H295R, is immensely useful due the retention of Ang II- responsiveness. The H295R cell line has also been ext~nsively utilized as a model to define the mechanisms controlling transcription of the steroid-metabolizing genes. Promoter constructs containing the 5'-flanking DNA from CYP11A 1, CYP11 B1, CYP11 B2, 57 81 CYP17, CYP21, and HSD3B2, have been studied ustng this strain • • Although the H295R cell line express low levels of MC2R, this cell line _has been used by a number of investigators to study regulation of receptor expression, as well as 206 207 acute actions of ACTH • • Along. with H295R, another substrain, designated H295A, was developed from the original NCI-H295. This substrain has also proven superior to the original NCI-H295, as it has been characterized and 81 199 shown to grow as a monolayer • • However, these cells reportedly lack Ang II responsiveness. Our study has put forth HAC15 cells as an alternative to the currently available H295 cell models. As with the H295R cells, our recurrent characterization of HAC15 cells is promising, as the cell line expresses all of the genes involved in -the adrenocortical pathway, including StAR, CYP11A1, CYP17, CYP21, HSD3B2, CYP1181, CYP1182, and SUL T2A 1. Upon stimulation with Ang II and K+, an increase in aldosterone levels and the expression levels for all of ·the enzymes involved in its production is observed. ACTH causes a modest, but significant, increase in aldosterone, cortisol, and DHEA, along with enhanced 159 expression of all of the enzymes in their respective pathways. As with H295R, HAC15- cells grow as attached cells; however, unlike H295R, they seem to· aggregate and grow as clusters of cells in a staggered-stacking pattern. HAC15 cells demonstrate structural features previously observed for the H295R cells, including the loss of contact inhibition, which leads to cells growing as attached clumps. It is noteworthy that the H295 cell models represent a mixture of cells isolated from primary cultures of the original tumor. On the other hand, HAC15 ceHs possess a single cell origin, as they have been derived from a series of 82 83 clonal experiments • • A. considerable body of evidence now exists that supports the contention that gene · expression profiling through microarray analysis can assist in tumor 317 322 classification and cancer outcome prediction - • Over the past decade, genome-wide microarray analyses have been actively explored, as adrenocortical carcinomas have increasingly risen to clinical attention. We have assessed the role of the two available adrenocortical carcinoma-derived cell lines, HAC15 and H295R, in DNA microarray analysis. Overexpression in adrenocortical carcinomas of a number of genes that have been designated as biomarkers for malignancy or survival have been concurrently· identified in the HAC15 and H295H cell lines. Further studies are needed in which objective measurements of genome-wide expression profiles are elucidated so that the associations found in this study, and in the · studies of others, can be substantiated. 160 We demonstrated that the HAC15 cells secrete . mineralocorticoids, glucocorticoids, and adrenal androgens and express all of the enzymes involved in the generation of these hormones. In addition, the levels of these enzymes are shown to increase upon treatment with ACTH, Ang II, and K+. In summary, we show that under conventional culture conditions, HAC15 cells exhibit hormonal responses, steroidogenesis, and expression of steroid-metabolizing enzymes. This cell line represents only the second human adrenocortical cell model and the first that has retained significant ACTH responsiveness. HAC15 cells have thus proven to be a stable cell model for studies directed towards understanding physiological, as well as pathophysiological, adrenocortical functions. 161 IV. 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