Histone Deacetylase Inhibitors Trichostatin A

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Masters Theses 1911 - February 2014

2013 Histone Deacetylase Inhibitors Trichostatin A (tsa) And Sulforaphane (sfn) Modulate Vitamin D Responsive Cyp24 Gene Expression in 3t3-l1 Preadipocytes Eunjee Ahn University of Massachusetts Amherst

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HISTONE DEACETYLASE INHIBITORS TRICHOSTATIN A (TSA) AND SULFORAPHANE (SFN) MODULATE VITAMIN D RESPONSIVE CYP24 GENE EXPRESSION IN 3T3-L1 PREADIPOCYTES

A Thesis Presented

EUNJEE AHN

Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

September 2013

Department of Nutrition

HISTONE DEACETYLASE INHIBITORS TRICHOSTATIN A (TSA) AND SULFORAPHANE (SFN) MODULATE VITAMIN D RESPONSIVE CYP24 GENE EXPRESSION IN 3T3-L1 PREADIPOCYTES

A Thesis Presented

Eunjee Ahn

Approved as to style and content by:

______Richard Wood, Chair

______Yeonhwa Park, Member

______Nancy Cohen, Department Head Nutrition

ACKNOWLEDGMENTS

First and foremost I offer my sincerest gratitude to my advisor, Dr. Richard

Wood, who provided me with the opportunity to complete this project and guided me throughout my thesis with his patience and knowledge.

I would like to thank my committee member, Dr. Young-Cheul Kim, for his encouraging words and inspiring advice.

I would like to thank my committee member Dr. Yeonhwa Park, for her thoughtful comments and continuous support.

I would like to thank Jennifer Swick and Brianna Gray, with whom I worked together in the lab, for their support, help, and friendship.

I would like to give a special thanks to my family: my parents, brothers and sister for their unwavering love and support. Finally I would like to extend my deepest gratitude to my family: my husband, my son, Roy, and my daughter, Emily, for their love, patience, support and encouragement throughout my study. It was such a blessing to me to spend time together as a family over the last three years.

iv ABSTRACT

HISTONE DEACETYLASE INHIBITORS TRICHOSTATIN A (TSA) AND SULFORAPHANE (SFN) MODULATE VITAMIN D RESPONSIVE CYP24 GENE EXPRESSION IN 3T3-L1 PREADIPOCYTES

SEPTEMBER 2013

EUNJEE AHN, B.S., CHUNGNAM NATIONAL UNIVERSITY

M.S. UNIVERSITY OF MASSACHUSETTS, AMHERST

Directed by: Professor RICHARD WOOD

Vitamin D plays an important role in preserving healthy bones, and has additional roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function. The vitamin

D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development.

Histone acetylation weakens the association of histones with DNA, and increases the accessibility of transcriptional regulatory proteins to chromatin templates, thereby increasing transcriptional activity of gene expression. Histone deacetylases remove the acetyl groups and condense chromatin structure, thereby preventing transcription. TSA is a potent histone deacetylase inhibitor and can significantly enhance gene expression. Bioactive food component, sulforaphane (SFN) is found in cruciferous vegetables and is known to be a histone deacetylase inhibitor, leading to transcriptional activation of gene expression. The objective of this study is to demonstrate that the bioactive food components modulate vitamin D action in adipocytes. To investigate the effects of TSA and SFN on vitamin D response, 3T3L1

v mouse preadipocytes were treated with the combination of various concentrations of 1,25(OH)2 vitamin D, TSA, and SFN. Upon harvesting cells, the amounts of 24- hydroxylase mRNA, marker of vitamin D response, were measured by semiquantitative reverse transcriptase-PCR analysis. The results showed that the cells treated with 1μM TSA increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 3.5-fold (p < 0.05) at 1nM 1,25(OH)2 vitamin D and nearly 2.5-fold (p <

0.05) in 10 nM 1,25(OH)2 vitamin D, and the cells treated with 5μM SFN increased

1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 1.4-fold at 1nM 1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D.

vi TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iv

ABSTRACT ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

CHAPTER

1. LITERATURE REVIEW ...... 1

1.1 Introduction ...... 1 1.2 Vitamin D ...... 4

1.2.1 Production ...... 4 1.2.2 Absorption, transport and metabolism ...... 6 1.2.3 Genomic actions ...... 9

1.2.3.1 Calcemic functions ...... 9 1.2.3.2 Noncalcemic function ...... 10

1.2.4 Molecular mechanisms of genomic actions of vitamin D ...... 11 1.2.5 Molecular mechanisms of non-genomic effects of vitamin D ...... 15

1.3 Epigenetic modulation of gene expression ...... 16

1.3.1 Histone acetylation ...... 17 1.3.2 Histone deacetylase inhibitors (HDACIs) ...... 19

1.3.2.1 Trichostatin A (TSA) ...... 20 1.3.2.2 Sulforaphane (SFN) ...... 23

1.3.3 Epigenetic modulation of vitamin D-mediated gene expression by HDACI ...... 27

1.4 Vitamin D and adipogenesis in 3T3L1 Cells ...... 30

2. PURPOSE OF THE STUDY ...... 33

3. EXPERIMENTAL DESIGN AND METHODS ...... 36

vii 3.1 Experimental design ...... 36

3.2 Cell culture and treatments ...... 36 3.3 Gene expression analysis ...... 37

3.3.1 RNA isolation ...... 37 3.3.2 RNA quantification ...... 38 3.3.3 Reverse transcriptase PCR (RT-PCR) ...... 38 3.3.4 Agarose gel electrophoresis ...... 39

3.4 Statistical analysis ...... 39

4. RESULTS ...... 40

5. DISCUSSION ...... 46

6. FUTURE DIRECTIONS ...... 49

viii LIST OF TABLES

Table Page

1. TSA study experimental parameters ...... 22

2. SFN study experimental parameters ...... 25

3. Modulation of vitamin D-mediated gene expression by HDACIs ...... 28

ix LIST OF FIGURES

Figure Page

1. Modulation of chromatin conformation and transcriptional status by acetylation of lysine residues in histones ...... 2

2. Production of vitamin D2 and vitamin D3 ...... 4

3. Overview of vitamin D synthesis, intake, and activation ...... 5

4. Genomic actions of 1,25(OH)2D3 ...... 13

5. Regulations of chromatin structure and transcription by activities of HATs and HDACs ...... 17

6. Acetylation and deacetylation of the lysine residue ...... 18

7. Trichostatin A ...... 20

8. Sulforaphane ...... 23

9. Myrosinase-catalyzed conversion of glucosinolate to isothiocyanate ...... 23

10. Relative CYP 24 mRNA amounts measured by semiquantitative reverse transcription–PCR in 3T3L1 preadipocytes...... 40

11. Dose-dependent effect of 1,25(OH)2 vitamin D on CYP 24 mRNA expression in 3T3-L1 preadipocytes ...... 41

12. CYP 24 mRNA expressions at 3 different 1,25(OH)2 vitamin D concentrations in combination with either 1μM TSA (Trichostatin A) or 5μM SFN (Sulforaphane) in 3T3-L1 preadipocytes ...... 42

13. Dose-dependent effect of 1,25(OH)2 vitamin D in combination with SFN on CYP 24 mRNA expression in 3T3-L1 preadipocytes ...... 44

CHAPTER 1

LITERATURE REVIEW

1.1 Introduction

Vitamin D is produced in the skin in response to UVB radiation and is found in a limited number of foods. Vitamin D status plays an important role in maintaining healthy bones by promoting calcium absorption in the gut and maintaining adequate serum calcium and phosphate concentrations. Serum 25- hydroxyvitamin D concentration reflects vitamin D exposure. Low serum 25- hydroxyvitamin D concentrations are associated with low bone mineral density and fractures, while sufficient serum 25-hydroxyvitamin D concentrations can prevent rickets in children, osteomalacia in adults, and osteoporosis in older adults. Vitamin

D also has other roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function.

The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes.

Nuclear receptors act as ligand-inducible transcription factors and interact with

DNA response elements of target genes (Aranda and Pascual, 2001). Upon activation by the active 1,25-dihydroxyvitamin D metabolite, the VDR binds to vitamin D response elements (VDREs) on the DNA of target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development.

Figure 1 Modulation of chromatin conformation and transcriptional status by acetylation of lysine residues in histones. HDAC, histone deacetylase; HAT, histone acetyltransferase (Ho et al., 2009)

Within the nucleus, chromatin represents DNA that is wrapped around histone protein complexes to form nucleosomes (Figure 1). Chromatin structure can be modified by altering the binding affinity, which is controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), of

DNA to histone proteins. Histone acetylation weakens the association of histones with DNA, thereby altering nucleosomal conformation and increasing the accessibility of transcriptional regulatory proteins to chromatin templates. (Norton et al., 1989). As a consequence, transcriptional activity of gene expression may increase. Histone deacetylases remove the acetyl groups, thereby increasing the positive charge of histone tails and encouraging high-affinity binding between the histones and DNA backbone. The increased DNA binding to histone protein condenses chromatin structure and prevents transcription (Struhl, 1998). Certain

chemicals have been found that can alter HDAC activity, such as trichostatin A (TSA).

TSA is an organic antifungal antibiotic that is a potent inhibitor of the histone deacetylase (Yoshida et al., 1990) and can significantly enhance gene expression

(Nan et al., 2004). For example, a study by Luo et al. demonstrated that inhibition of histone deacetylases by TSA increases the expression of vitamin D 24-hydroxylase

(CYP24A1) in prostate cancer cells (Luo et al., 2010).

Certain plant foods contain bioactive food components that may be important for optimal health. Bioactive food components refer to nonessential biomolecules with biological activity that is predominantly found in plant foods such as whole grains, fruits, and vegetables. A diet, rich in nutrients and bioactive food components may play a vital role in improving health and reducing risk for chronic disease (Liu, 2003). In recent years, bioactive food components are being intensively studied to evaluate their effects on health. An underlying mechanism through which some bioactive food components may affect health is by influencing gene expression by altering epigenetic “marks” (methylation and acetylation) that control DNA access. For example, the bioactive food component sulforaphane (SFN) is found in cruciferous vegetables, such as broccoli and cauliflower, and is known to have histone deacetylase inhibitor activity (Myzak et al., 2004), which promotes hyperacetylation of chromatin and affects the expression of genes, leading to transcriptional activation of some genes (Struhl, 1998). Recent studies have found that epigenetic changes can lead to an increase in the expression of some genes and a decrease in the expression of others. Moreover, interestingly, only small subsets of genes are apparently affected by these epigenetic alterations, and these changes

appear also to be tissue specific. The identification of which tissues and genes are

affected by various bioactive food components and how these changes affect health

is a new and growing area of nutritional research.

1.2 Vitamin D

1.2.1 Production

Vitamin D is a fat-soluble vitamin that is naturally present in only a small

number of foods, but is produced endogenously in the skin by UVB irradiation

between 290-315 nm. The two major forms of vitamin D, which differ chemically in

their side chains, are cholecalciferol and ergocalciferol. Cholecalciferol is known as

vitamin D3 and is produced in animals from cutaneous 7-dehydrocholesterol (7-

DHC), while ergocalciferol is known as vitamin D2 and is produced in plants from

ergosterol, a plant sterol (Figure 2). Vitamin D3 is translocated from the skin to

bloodstream, bound in plasma to the vitamin D binding protein.

Figure 2 Production of vitamin D2 and vitamin D3: Photolysis of ergosterol in plants (a) and 7- dehydrocholesterol (b) in skin 7-DHC to previtamin D2 (pre-D2) and previtamin D3 (pre-D3) and their thermal isomerizations to vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), respectively. D2 and D3 differ only in the side chain in which D2 has a double bond between C22–C23 and a methyl group at C24 (Bikle, 2010).

Vitamin D synthesized from sun exposure, or obtained from dietary sources is biologically inert and must undergo two hydroxylations to become the active hormone form (Figure 3).

Figure 3 Overview of vitamin D synthesis, intake, and activation ( http://www.nap.edu /openbook.php?record_id=13050&page=76)

The first hydroxylation occurs in the liver due to the action of a P450 enzyme coded by the cyp2r1 gene and converts vitamin D to 25-hydroxyvitamin D

(25(OH)D3) known as calcidiol (Figure 3). The second hydroxylation occurs primarily in the kidney due to the action of the P450 enzyme (25-hydroxyvitamin D

1α-hydroxylase), coded by the cyp27b1 gene, and forms the physiologically active

1,25-dihydroxyvitamin D (1,25(OH)2D3) metabolite known as calcitriol. This vitamin

D metabolite is the hormonal form of vitamin D that is responsible for most of its biological functions (Figure 3). The production of 1,25(OH)2D3 in the kidney is stimulated and tightly controlled by parathyroid hormone (PTH) and plasma phosphate concentration, which enhances the activity of renal 1α-hydroxylase, and is inhibited by raised plasma calcium and phosphate concentration. The 1α- hydroxylase is also inhibited by fibroblast growth factor 23 (FGF23), which is produced in bone cells. CYP24A1 gene encodes 24-hydroxylase that catalyzes the conversion of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 into 24- hydroxylated products, which constitute the degradation of the vitamin D molecule

(Beckman et al., 1996, Holick, 2010).

1.2.2 Absorption, transport and metabolism

Being fat-soluble, dietary vitamin D (both D2 and D3) is mixed with other fats and incorporated with the aid of bile salts into micelles in the small intestine from which it is absorbed and enters mucosal enterocytes by passive diffusion. Once absorbed, it is incorporated into chylomicrons, which are released by enterocytes and enter the lymphatic system. Lymphatic circulation transfers vitamin D in

chylomicrons into the blood stream. After vitamin D in chylomicron reaches the liver, 25-hydroxylation can occur and the 25-hydroxyvitamin D metabolite produced is attached to plasma vitamin D binding protein (DBP). Vitamin D binding protein and albumin are the primary transporters of vitamin D metabolites in blood

(Wildman, 2011, SarDesai, 2012).

Since serum 25-hydroxyvitamin D (25(OH)D3) is the predominant circulating form of vitamin D in the blood and increases in proportion to vitamin D intake and sunlight exposure, it is measured in the blood to determine the vitamin D status (Anderson et al., 2003). The unbound form of 25(OH)D3 enters the kidney, where a second hydroxylation can occur by 1α-hydroxylase activity (25- hydroxyvitamin D-1α-hydroxylase, CYP27B1) forming the physiologically active

1,25-dihydroxyvitamin D (1,25(OH)2D3). Enzyme activity of CYP27B1 is predominant in the kidney, but is present in a number of non-renal tissues and cells, such as immune cells, pancreatic cells, muscle cells, adipocytes, etc. 1,25- dihydroxyvitamin D is the hormonal form of vitamin D responsible for most of its biological functions via its ability to activate the vitamin D receptor, a nuclear steroid hormone transcription factor. The production, which is controlled by parathyroid hormone, of 1,25(OH)2D3 from 25(OH)D3 in the kidney acts to raise the serum calcium level to normal physiological range by stimulating intestinal calcium absorption via an increased expression of calcium transport proteins, such as

TRPV6 and calbindin D. Low blood calcium level is associated with the increased release of parathyroid hormone from the parathyroid glands. In addition to its effect on 1α-hydroxylase to produce 1,25(OH)2D3, parathyroid hormone can stimulate the

release of calcium from bone and increase renal calcium reabsorption by increasing the activity of TRPV5 in the renal distal convoluted tubule.

The C-24 oxidation pathway plays a major role in the biodegradation of vitamin D metabolites in kidney (Figure 3) and other target tissues, and 24- hydroxylase (1,25(OH)2D3-24-hydroxylase, CYP24) functions in the catabolism of

1,25(OH)2D3 (Beckman et al., 1996, Holick, 2010). 24-hydroxylase converts

25(OH)D to 24,25 dihydroxyvitamin D (24,25(OH)2D) or 1,25(OH)2D to 1,24,25 trihydroxyvitamin D (1,24,25(OH)3D). This hydroxylation is the first step of the C-24 oxidation pathway, which catabolizes vitamin D metabolites to the water-soluble calcitroic acid for its excretion by the kidney (Makin et al., 1989, Reddy and Tserng,

1989). Although CYP24 is highly expressed in the kidney tubule, it is also distributed in tissues including the intestine, osteoblasts, placenta, keratinocytes, and prostate.

In general, CYP24 appears to be found wherever there is vitamin D receptor (VDR), which elicits the action of 1,25(OH)2D3. Overproduction of 1,25(OH)2D3 is regulated by a negative feedback mechanism, in that 1,25(OH)2D3 inhibits PTH synthesis and induces 24-hydroxylase (CYP24A1) activity, which catabolizes 1,25(OH)2D3 by hydroxylation on C24. Thus the biological activity of 1,25-dihydroxyvitamin D

(1,25(OH)2D3) in a cell is determined mainly by the combination of the level of VDR expression and the local 24-hydroxylase activity (Anderson et al., 2003). However, other cellular factors may subsequently modulate vitamin D action by altering VDR genomic activity in the nucleus, such as epigenetic factors that influence chromatin structure.

1.2.3. Genomic actions

1.2.3.1. Calcemic functions

Production and accumulation of 1,25(OH)2D3 is regulated either directly or indirectly by serum calcium concentration, and the major function of 1,25(OH)2D3 is to increase the efficiency of intestinal calcium absorption, thereby increasing the availability of dietary calcium. As mentioned above, parathyroid hormone is the primary hormonal factor in determining synthesis of 1,25(OH)2D3 in response to the concentration of free circulating calcium concentration (Garabedian et al., 1972). A drop in serum calcium concentration leads to induction of synthesis and secretion of parathyroid hormone, which then acts in the kidney to stimulate transcellular calcium reabsorption and in bone to stimulate osteoclast biogenesis (Thomas et al.,

2001) and osteoclast bone-resorptive function. Calcium homeostasis in the blood is maintained by the coordinated regulation of intestinal calcium absorption, mobilization of calcium from bone, and renal reabsorption of calcium (DeLuca,

1981). Once these functions return serum calcium to normal, then 1,25(OH)2D3 exerts a negative feedback signal on the parathyroid glands to suppress further synthesis and secretion of PTH (Cantley et al., 1985, Szabo et al., 1989). 1,25(OH)2D3 also stimulates production of fibroblast growth factor (FGF23), produced primarily by bone by osteoblasts and osteocyte, and negatively control its own levels by reducing renal1α-hydroxylase (Kolek et al., 2005) (Figure 3).

1.2.3.2. Noncalcemic functions

The noncalcemic functions of 1,25-dihydroxyvitamin D include regulation of cell proliferation and differentiation, hormone secretion, and immunomodulation. A wide number of tissues and cells including the brain, gonads, pituitary gland, thymus, pancreas, stomach, breast, skin, adipocytes and lymphocytes possess vitamin D receptor (VDR) (Holick, 1995). The expression of VDRs in these tissues and cells suggests that they must serve functions there.

Proliferation and differentiation of normal and cancerous cells are affected by 1,25(OH)2D3 through directly or indirectly altering the transcription of cell regulatory genes, as the inhibition of the growth and differentiation of cancer by actions of 1,25(OH)2D3 was shown in leukemia and melanoma cells (Colston et al.,

1981). Studies have shown that vitamin D inhibits adipogenesis by playing an important role in the cell growth and differentiation (Ishida et al., 1988, Kong and Li,

2006, Wood, 2008).

1,25(OH)2D3 has direct effects on the pancreatic islet cells and stimulates insulin secretion (Lee et al., 1994). VDR and calbindin-D28k, which was the first known target of vitamin D action and is a member of a family of high affinity calcium-binding proteins, are found in pancreatic beta cells (Morrissey et al., 1975).

Calbindin-D28k can modulate depolarization-stimulated insulin release (Sooy et al.,

1999) and protect beta cells against cytokine mediated destruction. A number of studies have suggested that vitamin D deficiency contributes to increased risk for type 2 diabetes mellitus (Bikle, 2009, Baz-Hecht and Goldfine, 2010).

1,25(OH)2D3 is a potent immune system modulator and can help in suppression of certain autoimmune diseases and cancers (DeLuca, 2004). It can induce phagocytic activity, cell surface antigen expression, and monocyte differentiation (Abe et al., 1981), and enhance lysosomal activity and interleukin 1 production. (Bar-Shavit et al., 1981, Holick, 1995). 1,25(OH)2D3 inhibits interleukin

2 production in activated T lymphocytes, and DNA synthesis and immunoglobulin

(IgM and IgG) production in activated B lymphocytes (Provvedini et al., 1986).

At the molecular level, it has been shown that 1,25(OH)2D3 can inhibit adipogenesis through a vitamin D receptor (VDR)-dependent inhibition of CCAAT enhancer binding protein-alpha (C/EBPα) and peroxisome proliferator-activated receptor-gamma (PPARγ) expression in a dose-dependent and time-sensitive manner, a decrease in PPARγ transactivating activity, and a stabilization of the inhibitory VDR protein in the pre-adipocyte. Treatment of the 3T3-L1 mouse preadipocytes cell line with 1,25(OH)2D3 stabilizes the inhibitory VDR protein levels, which are normally increased during the early phase of adipogenesis, peaking at 4 – 8 hours and subsiding afterward throughout the rest of the differentiation

(Kong and Li, 2006).

1.2.4. Molecular mechanisms of genomic actions of vitamin D

Nuclear receptors are a class of proteins found within cells and can act as ligand-inducible transcription factors that specifically regulate the expression of target genes involved in metabolism, development, and reproduction. Their primary function is to mediate the transcriptional response in target cells to steroid

hormones or other lipid soluble compounds. More than 100 nuclear receptors are known to exist and these proteins comprise the nuclear receptor superfamily

(McKenna et al., 1999). All of the nuclear receptors have common structural features and are composed of several independent, but interacting, functional modules. These are an amino-terminal regulatory domain (A/B domain containing the activation function 1 (AF-1)), a DNA-binding domain (DBD; region C), a hinge region (region D), a ligand-binding domain (LBD; region E containing the activation function 2 (AF-2)), and a carboxyl-terminal domain (region F) (Giguère, 1999,

Olefsky, 2001).

The vitamin D receptor is a member of the nuclear receptor superfamily that regulates the expression of vitamin D responsive gene and mediates the biological activity of its ligand 1,25(OH)2D3. The liganded VDR can form a VDR-RXR heterodimer that is able to enter the deep groove of DNA and recognize vitamin D responsive elements (VDREs) in promoter region of vitamin D target genes. Binding of 1,25(OH)2D3 to the VDR appears to cause a conformational change at the COOH terminus of the VDR, permitting the AF-2 domain (ligand-dependent transcriptional activation domain) to interact with other transcription factors including coactivator proteins that lead to activation of vitamin D target gene transcription (Masuyama et al., 1997). Some of these coactivator proteins are known to possess intrinsic histone acetylase activity and are capable of chromatin modification by histone acetylation

(Jones et al., 1998, Haussler et al., 2008). Therefore, a large multiprotein coactivator complex, which assembles on the heterodimeric VDR-RXR complex, functions to remodel condensed chromatin and can interact with RNA polymerase II located at

the transcriptional start site (Näär et al., 2001). These interactions are necessary to alter the rate of gene expression (Figure 4).

Figure 4 Genomic actions of 1,25(OH)2D3. 1,25(OH)2D3 molecules pass through the plasma membrane with the help of vitamin D binding protein (DBP). Ligand binding to the VDR induces a conformational change in the receptor and subsequent heterodimerization with RXR on vitamin D responsive element (VDRE). Corepressor (CoR) proteins are released from the surface of the VDR, allowing interaction with coactivator (CoA) proteins. These molecules modulate chromatin structure and allow the interaction of the receptor with the RNA polymerase II transcriptional complex (POL II), thus activating transcription of the target gene ( Seshadri et al., 2011).

The VDR interacts with other transcription factors, such as transcription factor IIB (TFIIB), and coactivator proteins, such as SRC-1 and TIF-1. Nuclear receptor coactivator protein (ACTR) has been shown to enhance transcription from a vitamin D target gene (Chen et al., 1997, McKenna and O'Malley, 2002). Several corepressor proteins, NCoR, SMRT, and Alien, have been shown to interact with the

VDR and block VDR-mediated transcriptional activities (Polly et al., 2000). The hairless gene product HR is another protein that binds directly to VDR and VDR- mediated transactivation is strongly inhibited by HR in a manner relieved by

1,25(OH)2D3 (Xie et al., 2006, Bikle, 2009, Malloy et al., 2009). These corepressors typically bind VDR in the absence of 1,25(OH)2D3 and are displaced when

1,25(OH)2D3 binding recruits the coactivators to the VDR. Corepressors recruit histone deacetylases that repress transcription by removing acetyl groups from histone proteins in the nucleosome and stabilizing chromatin (Marks et al., 2001).

Various genes have been shown to be responsive to vitamin D and most of these genes play a direct role in calcium endocrinology or bone formation including osteocalcin, osteopontin, PTH, the hydroxylases cyp24 and cyp27b1, and the calbindin genes. Expression of the bone protein osteocalcin, which is secreted by osteoblasts and play a significant role in maintaining bone integrity, is upregulated by 1,25(OH)2D3, whereas several genes have been shown to be downregulated by

1,25(OH)2D3, including the gene for PTH, which plays a critical role in controlling the levels of 1,25(OH)2D3 in serum. Calbindins are another set of genes that have been shown to be regulated by vitamin D. Calbindin D9K, which plays a role in absorption of calcium from the mammalian gut, is expressed primarily in upper small intestine, whereas the calbindin D28K, which may be involved in calcium reabsorption from glomerular filtrate, is expressed in mammalian kidney and other tissues (Jones et al., 1998). 1,25(OH)2D3 stimulates the differentiation of many cell types by through induction of arrays of genes and stimulation of signal transduction pathways (Marks et al., 2000, Samuel and Sitrin, 2008). Phospholipase C (PLC)

activity is upregulated by 1,25(OH)2D3 at the transcriptional level in normal human keratinocytes via a DR6 type vitamin D responsive element (VDRE) in the PLC-1 promoter (Xie and Bikle, 1997).

The CYP24 hydroxylase, an immediate target gene of VDR, is one of the most highly regulated genes that respond to vitamin D. In the presence of ligand, it is a sensitive marker for VDR function, because its promoter region contains multiple

VDREs and its transcription is very responsive to 1,25(OH)2D3 treatment (Chen and

DeLuca, 1995, Alimirah et al., 2010). 1,25(OH)2D3 is thought to be the sole physiological inducer of 24-hydroxylase activity (Shinki et al., 1992). In the proposed studies, I plan to use cyp24 gene expression as a measure of VDR transcriptional activity to assess to what extent histone deacetylase inhibitors

(trichostatin A and sulforaphane) alter cellular vitamin D action.

1.2.5. Molecular mechanisms of non-genomic actions of vitamin D

1,25(OH)2D3 also exerts biologic effects that do not appear to require gene regulation and may work through a membrane receptor rather than its nuclear hormone receptor. 1,25(OH)2D3 has been shown to be a biological regulator of calcium channels (Yukihiro et al., 1994) by acting as a potent modulator of calcium channel function in osteosarcoma cells and affecting the intracellular Ca2+- dependent signaling processes (Caffrey and Farach-Carson, 1989). It regulates chloride channel activity by promoting the rapid enhancement of outwardly rectifying Cl− currents in a concentration-dependent manner in osteoblasts (Zanello and Norman, 1997), and plays a role in protein kinase C activation and distribution

by increasing the breakdown of membrane phosphoinositides, raising intracellular calcium concentration, and translocating protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells (Bissonnette et al., 1994). These rapid nongenomic effects occurring in minutes could be elicited by the classical nuclear

VDR acting at the cytoplasmic membrane or by a separate membrane VDR, and have been most extensively studied in the intestine (Bikle, 2009). Transcaltachia, which describes the rapid hormonal stimulation of intestinal calcium transport, occurs via nongenomic mechanisms, which involve a plasma membrane receptor (Nemere et al., 1984). The physiological role of Transcaltachia is unknown.

1.3 Epigenetic modulation of gene expression

Conrad Waddington originally defined epigenetics as ‘‘the study of the causal interactions between genes and their products, which bring the phenotype into being”, referring to all molecular pathways modulating the expression of a genotype into a particular phenotype. Today, epigenetics has been generally accepted as ‘‘the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence, including histone variants, posttranslational modifications of amino acids on the amino-terminal tail of histones, and covalent modifications of DNA bases.’’ (Dupont et al., 2009). Epigenetic modifications altered by external or internal environmental factors can change gene expression and maintain the acquired phenotype permanently. Thus, epigenetics can provide a new model for the search for etiological factors of many diseases, in particular environment-associated diseases known to be affected by many

environmental factors (Choi and Friso, 2010). Epigenetic modification includes DNA methylation, histone modifications, and chromatin remodeling.

1.3.1 Histone acetylation

Histone proteins and their posttranslational modifications have an important role in chromatin structure, nucleosome assembly, and regulation of gene transcription. Histone acetylation is one of the most extensively studied histone modifications associated with transcriptional activation (Figure 5) (Dupont et al.,

2009).

Figure 5 Regulations of chromatin structure and transcription by activities of HATs and HDACs. The amino termini of histones, which are called histone tails, can be post- translationally modified. HAT acetylates lysine residues (K) of the histones and HDAC removes the acetyl group (http://www.biotek.com/resources/articles/cellular-lantha- screen-histone.html).

A nucleosome is a building block of chromatin and consists of double- stranded DNA and an octamer of four different (H2A, H2B, H3, H4) histones.

Chromatin can regulate transcriptional processes through modifications of DNA and the histone. The location of the reversible histone modifications is at the N-terminal histone tails, where the lysine residues are on the surface of the nucleosome. (Choi and Friso, 2010). Acetylation of the lysine residues neutralizes the positive charges on the lysine amino groups and decreases their affinity for DNA, thus weakening the association between histones and DNA and altering the nucleosomal conformation

(Figure 6. (Hong et al., 1993). This chromatin conformational change can increase the accessibility of transcriptional regulatory proteins to chromatin templates, leading to increased transcriptional activity (Struhl, 1998). Histone acetylation status is modulated by histone acetyltransferase (HAT) and histone deacetylase

(HDAC) activity (Figure 5, (Zhang et al., 2002)).

Figure 6 Acetylation and deacetylation of the lysine residue. A HAT catalyzes the transfer of an acetyl group from acetyl CoA to the amino group of the lysine residue. HAT, histone acetyltransferase; HDAC, histone deacetylase (Yang and Seto, 2007).

1.3.2 Histone deacetylase inhibitors (HDACIs)

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression (Figure 5). Histone deacetylase inhibitors (HDACIs) inhibit the activity of HDAC and induce hyperacetylation of core histones, thereby modulating the chromatin structure and affecting the gene expression (de Ruijter et al., 2003)

Inhibitors of HDAC activity can induce upregulation and downregulation of a small subset of cellular genes, suggesting that HDACIs act selectively on specific genes.

In fact, HDACIs alter the transcription of only approximately 2% of expressed genes with a variable transcriptional outcome (Van Lint et al., 1996, Marks et al., 2001,

Moreira et al., 2003). Furthermore, chromatins of normal and transformed cells are differentially affected by histone deacetylase inhibitors. Normal cells are up to ten times more resistant to histone deacetylase inhibitor (HDACI)-induced cell death compared with transformed cells due to the difference in chromatin conformation and accessibility between normal and transformed cells (Kim et al., 2003). The transformed cells have different nuclear sizes and cellular shapes compared with the normal cells (Zink et al., 2004). In transformed cells, acetylation of K16 on histone H4 (H4-K16Ac) is markedly reduced (Fraga et al., 2005), and levels of histone acetylation can directly impact chromatin accessibility at specific loci

(Ehrenhofer-Murray, 2004). Altered chromatin structure may increase the accessibility to the histone tails, indicating that the structural characteristic of transformed cells predispose them to HDACI sensitivity. However, the mechanisms

that affect chromatin structure and the molecular processes underlying this selectivity for transformed cells are still not well understood (Nalabothula and

Carrier, 2011).

Sodium butyrate, phenylbutyrate, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), trapoxin (TPX), MS-27–275, apicidin, oxamflatin, and

FR901228 are the known compounds that inhibit HDAC activity and cause a variety of effects, including cell growth inhibition, cell differentiation and apoptotic cell death (Moreira et al., 2003).

1.3.2.1 Trichostatin A (TSA)

Figure 7 Trichostatin A (TSA, 7-[4-(dimethylamino)phenyl]-N- hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide)

There have been many histone deacetylase inhibitors discovered, but the most potent is trichostatin A (TSA, 7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6R- dimethyl-7-oxo-2E,4E-heptadienamide, Figure 7). TSA is a fermentation product of

Streptomyces and belongs to the hydroxamic acid family. Originally it was used as an anti-fungal agent, but later it was found to have potent proliferation-inhibitory

properties with cancer cells. TSA is effective at nanomolar concentrations in vitro

(Yoshida et al., 1990, de Ruijter et al., 2003). It interacts reversibly with the HDAC catalytic site to prevent binding of the substrate and leads to histone acetylation

(Finnin et al., 1999). It has been known to induce cell cycle arrest, differentiation, and apoptosis. (Finnin et al., 1999, Marks et al., 2000, Tóth et al., 2004). Table 1 provides information on the experimental parameters tested in published TSA studies. These studies show that TSA modified histone acetylation status and gene expression levels of some genes at nano- or micromolar concentrations. TSA at concentration of 1 μM was used in 3T3L1 cells in my study.

Table 1 TSA study experimental parameters

TSA Exposure Cell Type Dosage Time Outcome Measure Effect Reference Human breast 0.1, 0.3, 24 h Histone p21, Hep27, (Glaser et carcinoma 1 (μM) hyperacetylation, p21, α-Fucosidase, al., 2003) cells MDA 468 Hep27, α-Fucosidase, Histone H2B, TRPM- and 435, Histone H2B, TRPM-2, 2, human bladder α-Tubulin, α-Tubulin, carcinoma Glutaredoxin, Glutaredoxin, cells T24 Metallothionein 1L, Metallothionein 1L Thymidylate were up-regulated synthetase, TRP, by 2.8 - 48.3 folds, Importin β, APRIL, CTP Thymidylate synthase expression, synthetase, TRP, acetylation of α-tubulin Importin β, APRIL, in T24 cells and CTP synthase were expression of a core set down-regulated by of genes 3.7 – 7.4 folds. HeLa-H2A-YFP 12.5, 25, 2, 6, 12, Histone acetylation on Cell density was (Tóth et al., cell line 50, 100, 24 h the interphase reduced to about 2004) 200 chromatin 50% (ng/ml) conformation Human 100, 200 24 h Expression of Either up-regulated (Mogal and prostate (ng/ml) housekeeping genes (LNCaP and ZR-75-1 Abdulkadir, cancer cell line including 18S rRNA, β at 100 ng/ml TSA) 2006) LNCaP, human actin, GAPDH and or down-regulated breast cancer ribosomal highly-basic (prostate explants, cell lines T- 23-kDa protein (rb 23- T-47D, LNCaP and 47D, ZR-75-1 kDa, RPL13A) ZR-75-1 at 200 ng/ml TSA) Human 100 48 h P21Cip1/Waf1 and Bax Lowered HDAC (Myzak et prostate BPH- ng/ml protein expression, activity by 30% al., 2006) 1 cells, HDAC activity level, prostate acetylated histone cancer PC-3 levels, p21 expression, and LNCaP multi-caspase activity cells Human 50, 100, 8 h Expression of CYP24A1 CYP24A1 enzyme (Luo et al., prostate 200, 300, mRNA activity was 2010) cancer cell 400 increased by 1.6 – 4 lines; DU145, (nM) fold LNCaP, PC3 Mouse 3T3-L1 100 nM 1, 2, 4, 6, 8 Expression of HDACIs block (Haberland cells days adipogenic marker adipogenesis. et al., genes: LIPE, AdipoQ, 2010) and aP2, expression of PPARγ and Fabp4, and SREBP-1c or C/EBPβ Human lung 1, 2 (µM) 12, 24 h Changes of HBP1 Transcriptional (Wang et fibroblasts acetylation, p16INK4A activity of p16INK4A al., 2012) 2BS, Human expression promoter was embryonic increased to about kidney 10-fold HEK293T

1.3.2.2 Sulforaphane (SFN)

Figure 8 Sulforaphane (SFN, 1-Isothiocyanato-4-methylsulfinylbutane)

Bioactive food components are defined as a constituent in foods or dietary supplements, other than those needed to meet basic nutritional needs that may be responsible for changes in health status. Sulforaphane (1-isothiocyanato-4-

(methylfulfinyl)-butane or SFN, Figure 8) is a bioactive food component found in cruciferous vegetables, such as broccoli, cauliflower, brussels sprouts, cabbages or kale. It is a naturally occurring sulfur-containing isothiocyanate derivative produced during the hydrolysis of glucosinolates. When cruciferous vegetables are chopped or chewed, myrosinase interacts with glucosinolates and releases isothiocyanates from their precursors (Figure 9. (Zhang, 2004)).

Figure 9 Myrosinase-catalyzed conversion of glucosinolate to isothiocyanate

Sulforaphane has multiple biological effects, including an ability to affect xenobiotic metabolism by inhibiting and inducing various biotransformation enzymes, and to alter gene expression by inducing epigenetic modifications (Mahéo et al., 1997, Basten et al., 2002, Myzak et al., 2004). It also exhibits many other biological activities such as preservation of normal cell cycle regulation(Chiao et al.,

2002), inhibition of proliferation and induction of apoptosis (Singh et al., 2009), anti-inflammatory activity (Heiss et al., 2001), antibacterial activity (Fahey et al.,

2002), and antidiabetic activity (Song et al., 2009). Furthermore, sulforaphane is known to be a histone deacetylase inhibitor, which induces acetylation of chromatin protein and affects the expression of genes by modulating transcriptional activation of the genes. Myzak et al. reported that SFN acted as HDACIs with evidence of altered histone acetylation status and increased p21Cip1/Waf1 expression in human embryonic kidney 293 cells and HCT116 colon cancer cells (Myzak et al., 2004).

Table 2 provides information on the experimental parameters tested in published

SFN studies. These studies show that SFN modified histone deacetylase activity level or histone acetylation status, and gene expression levels of some genes at various micromolar concentrations. SFN at concentration of 5 μM was used in

3T3L1 cells in my study.

Table 2 SFN study experimental parameters

SFN Exposure Outcome Cell Type Dosage Time Measure Effect Reference Human 3, 9, 15 47 h HDAC activity; SFN alone (Myzak et al., embryonic (μM) activity of a β- increased 2004) kidney 293 catenin- reporter activity (HEK293) responsive by 3.9-fold and cells, Human reporter >8-fold in HCT116 (TOPflash) combination colorectal with TSA (100 cancer cells ng/ml). About 4-fold p21Cip1/Waf1 increase in expression p21Cip1/Waf1 expression Human 3, 9, 15 48 h P21Cip1/Waf1 and SFN (15 μM, for (Myzak et al., prostate BPH- (µM) Bax protein 48 h) inhibited 2006) 1 cells, expression, HDAC activity by prostate HDAC activity 40, 30 and 40%, cancer PC-3 level, acetylated p21 protein and LNCaP histone levels, expression was cells p21 expression, increased by 50– multi-caspase 100%, and pro- activity apoptotic protein Bax was increased by 50% Human 1, 10, 20 24 and 48 h Expression of SFN (20 μm) (Schwab et al., colorectal (μM) HBD-2, VDR, increased β- 2008) cancer Caco- PPARγ, ERK1/2, defensin-2 2, HT-29 and phospho- mRNA SW480 cells ERK1/2, p38 expression up to MAPK and 5.5-fold (HT-29) phospho-p38 and HBD-2 MAPK protein expression up to 104% (Caco-2) Human 10, 20 or 24 h Levels of 40–80% (Kim and prostate 40 (μM) androgen decrease in AR Singh, 2009) cancer LNCaP receptor (AR) promoter and C4-2 cells protein, activity. Ser210/213- Phosphorylat-ed phosphorylat-ed AR, PSA protein, AR, prostate and AR mRNA specific antigen levels in both cell (PSA) protein lines were and AR mRNA decreased

Sulforaphane can readily permeate the enterocytes in the perfused human jejunal segment. At a physiologically relevant concentration 11.0 ± 2.7 μM, the percentage of absorption (± S.D.) is 74 ± 29% (Petri et al., 2003). Chewing of raw cruciferous vegetables may increase glucosinolate contact with plant myrosinase and increases the amount of isothiocyanates absorbed, and the myrosinase activity of human intestinal bacteria also allows for some formation and absorption of isothiocyanates (Shapiro et al., 1998). It has been estimated that the amount of SFN in 1 g of dry broccoli florets ranges from 507 to 684 μg (Campas-Baypoli et al.,

2010) and approximately 20 μmol/l SFN can be derived from consumption of 50 g of broccoli (Chuang LT. et al., 2013). The bioavailability study showed that higher amounts of SFN were found in the blood and urine when 200 g broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%). Absorption of SFN was delayed when cooked broccoli was consumed (peak plasma time of 6 h) compared to raw broccoli (1.6 h). Half-lives of excretion were comparable, 2.6 hours and 2.4 hours on average, for raw and cooked broccoli, respectively (Vermeulen et al., 2008).

Glucosinolates are hydrolyzed by myrosinase, an enzyme found in plants and bowel microflora and completely inactivated by heat, to form isothiocyanates. In vivo, isothiocyanates are conjugated with glutathione and then sequentially metabolized to mercapturic acids, which are found in the urine (Shapiro et al.,

2001). These isothiocyanate metabolites are highly correlated with dietary intake of cruciferous vegetables (Seow et al., 1998).

1.3.3 Epigenetic modulation of vitamin D-mediated gene expression by HDACI

Various studies (Gommersall et al., 2004b, Banwell et al., 2006, Wang et al.,

2008, Luo et al., 2010, Schwab et al., 2008) have revealed that the function of

1α,25(OH)2D3 can be enhanced in combination with HDACIs, thereby modulating the expressions of vitamin D-mediated genes. Table 3 shows the studies that demonstrated the modulated effects of vitamin D-mediated gene expression by

HDACIs.

TSA activity on CYP24A1 expression was tested in prostate cancer cells and the result showed that the treatment with trichostatin A (TSA) enhanced the expression of CYP24A1 in LNCaP and PC3 cells and induced its expression in a dose- dependent and 1,25(OH)2D3-dependent manner (Luo et al., 2010). Furthermore, the epigenetic effect of HDACI activity on the binding of the VDR to the CYP24A1 promoter region was also tested in PC3 cells treated with TSA and a significant increase in binding of VDR to the CYP24A1 promoter with TSA treatment was observed (Luo et al., 2010).

Table 3 Modulation of vitamin D-mediated gene expression by HDACIs

Dosage of Cell Type HDACI and Exposure Gene/ Effect Reference 1α,25(OH)2D3 Time Protein Prostate 15 nM TSA 7 h GADD45α Induction of gene (Gommers cancer cell 100 nM1α,25- expression was increased all et al., lines; PC-3 (OH)2D3 by 4.3-fold 2004a) PZ-HPV-7 10 μM SAHA 24 h 1α-OHase 2-fold increase in 1α- (Wang et cell line 50 nM 25- OHase promoter activity al., 2008) (OH)D3 and more than a 30-fold stimulation in 1α-OHase mRNA expression Human 50, 100, 200, 8 h for CYP24A1 CYP24A1 enzyme activity (Luo et al., prostate 300, and 400 TSA was increased by about 2010) cancer cell nM TSA, 100 followed 1.6 – 4 fold and CYP24A1 lines; DU14, nM by 24h mRNA expression was LNCaP, PC3 1α,25(OH)2D3 for 1α,25- increased by about 4 – 10 (OH)2D3 fold Human 10 - 20 μM SFN 24 and 48 VDR Increased the production (Schwab et colorectal h al., 2008) cancer Caco- 2, HT-29 and SW480 cells SCC4, MDA- 100 nM 8 h 1,25D3- Gene regulatory profile is (Tavera- MB231 Triciferol dependent distinct from that of Mendoza gene 1,25D et al., (cdkn1c/ki 2008) p2, alox12, and pex) VDR Enhanced markedly VDR binding to the cyp24 promoter Cyp24 Induced strong expression within a factor of ≈10 as potent as 1,25D p57KIP2(cdk Markedly up-regulated n1c/kip2) col13a1 Induced VDR binding to the VDRE P160 Induced its recruitment coactivaor to VDR-bound target AIB1 genes

TSA can upregulate the expressions of target genes of VDR and combined use of 1α,25(OH)2D3 and TSA may be an effective therapeutic for some cancers

(Gommersall et al., 2004b). Combinations of 1α,25(OH)2D3 compounds

(1α,25(OH)2D3 or its analogue 1α,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19- nor 1α,25(OH)2D3 (RO-26-2198), 100 nmol/L) with TSA (15 nmol/L) restored VDR antiproliferative signaling in breast cancer cell lines T-47D, ZR-75-1, MCF-7, and

MDA-MB-23. Cotreatment with RO-26-2198 and TSA showed significant enhancement of mRNA accumulation of target genes GADD45α, VDUP-1 and CYP24 in MDA-MB-231 cells notably at 12 hours (Banwell et al., 2006).

Triciferol, a hybrid molecule in which the 1α,25(OH)2D3 side chain is replaced with TSA, binds directly to the VDR ligand binding domain and functions as an agonist with 1α,25(OH)2D3-like potency on several 1α,25(OH)2D3 target genes.

Triciferol (100 nM) induced strong cyp24 expression and exhibited more efficacious antiproliferative and cytotoxic activities than 1α,25(OH)2D3 (100 nM) alone in human squamous carcinoma cells SCC4 and SCC25, and human breast cancer cells

MCF-7 and MDA-MB231 (Tavera-Mendoza et al., 2008).

Suberoylanilide hydroxamic acid (SAHA) increases the expression of genes that modulate cell cycle progression, tumor suppression, differentiation and apoptosis in PZ-HPV-7 prostate cells. SAHA upregulates the expression of the 1α- hydroxylase gene (CYP27B1) that controls the synthesis of 1α,25(OH)2D3 regulating prostate growth and differentiation in an autocrine/paracrine fashion (Wang et al.,

2008).

Schwab et al. demonstrated that sulforaphane (SFN, 10–20 μmol/L), a dietary HDACI, increased the production of VDR protein in HT-29 and Caco-2 cells

(Schwab et al., 2008) after 24-hour treatment.

1.4 Vitamin D and adipogenesis in 3T3L1 Cells

Adipocyte differentiation, adipogenesis, is the process of development of fat cells from preadipocytes, and has been one of the most intensely studied models of cellular differentiation. To investigate the regulatory mechanism of adipocyte differentiation, many studies have utilized the well-established mouse 3T3-L1 preadipocyte experimental systems as a cell culture model (Ishida et al., 1988,

Lagace and Nachtigal, 2004, Kong and Li, 2006). The 3T3-L1 cells are fibroblastic cells already determined to the adipocyte lineage and originally derived from mouse embryos. Fully differentiated 3T3-L1 adipocytes possess most of the morphological, biochemical, and hormonal response characteristics of adipocyte (Gregoire, 2001).

Peroxisome proliferator-activated receptor-gamma (PPARγ) and CCAAT enhancer binding protein-alpha (C/EBPα) are essential in adipocyte differentiation.

PPAR family and the CCAAT/enhancer-binding proteins are important transcriptional factors involved in the regulation of differentiation. PPARs are members of the nuclear-receptor superfamily and nuclear transcription factors, and form heterodimers with RXRs prior to binding of DNA in the promoter of PPAR- responsive genes (Chu et al., 1995). Permanent exit from the cell cycle with the coexpression of PPARγ and C/EBPα establishes the irreversible commitment to adipocyte differentiation (Shao and Lazar, 1997). C/EBPα terminates the mitotic clonal expansion preceding entry into the terminally differentiated state (Umek et al., 1991) and activates several adipogenic genes that create and maintain the adipocyte phenotype by autoactivating transcription of its own gene (Lin et al.,

1993).

1α,25(OH)2D3 inhibits differentiation of preadipocyte to adipocyte (Ishida et al., 1988) and reduces triacylglycerol accumulation by 50% of that of fully differentiated control cells through a vitamin D receptor (VDR)-dependent inhibition of C/EBPα and PPARγ expression (Kong and Li, 2006, Wood, 2008). Kong et al. demonstrated the molecular mechanism whereby 1α,25(OH)2D3 inhibited 3T3-

L1 cell adipogenesis by inhibiting only the early events of adipogenesis in which

1α,25(OH)2D3 could effectively inhibit 3T3-L1 cell adipogenesis within the first 48 hours after the differentiation program was initiated in a dose-dependent manner

−11 −7 (from 10 to 10 M). The blockade of 3T3-L1 cell differentiation by 1α,25(OH)2D3 involves direct suppression of C/EBPα and PPARγ upregulation, antagonization of

PPARγ activity, and stabilization of the inhibitory VDR protein (Kong and Li, 2006).

Vitamin D receptor (VDR) mediates 1α,25(OH)2D3-mediated inhibition of adipogenesis in 3T3-L1 cells. The study by Kong et al. demonstrated that the level of

VDR protein was increased during this early phase of adipogenesis, peaked at 4-8 hours after the cells were switched to differentiation media (hormonal cocktail containing insulin, dexamethasone, and isobutyl methylxanthine) containing 10−8 M

1α,25(OH)2D3, and subsided afterward along the progression of the differentiation so that the VDR protein became barely detectable in mature adipocytes at day 8. The study also showed that overexpression of VDR in 3T3-L1 cells blocked adipogenesis.

These results indicates that 1α,25(OH)2D3 treatment increased and stabilized the

VDR levels and confirms that VDR is inhibitory on 3T3-L1 cell adipogenesis (Kong and Li, 2006). Given that VDR and PPARγ share the same heterodimeric partner

RXR, Kong et al. suggested that VDR directly suppress the transacting activity of

PPARγ by sequestering the limited amount of RXR in 3T3-L1 cells. (Kong and Li,

2006).

CHAPTER 2

PURPOSE OF THE STUDY

Although some studies have examined the effects of TSA on 1,25- dihydroxyvitamin D mediated gene expression in various cell types including human prostate cancer cells (Luo et al., 2010), human lung fibroblasts, human embryonic kidney cells (Wang et al., 2008), and human colorectal cancer cells

(Schwab et al., 2008), to my knowledge, no studies have investigated the role of TSA in murine 3T3-L1 in preadipocytes or other fat cell lines. In addition, the ability of the dietary histone deacetylase inhibitor SFN to alter VD/VDR–mediated gene expression has not been explored in any cell types.

The long-term objective of this line of research is to determine to what extent bioactive food components can be used to promote health. The objective of this study is to determine whether epigenetic modification can increase vitamin D-induced gene expression in adipocytes. This hypothesis will be tested by successfully completing the following Specific Aim:

Specific Aim: Enhancement of vitamin D cellular action by epigenetic modification

Changes in gene expression can drive changes in cellular function. Recently, there has been increased appreciation of the role of epigenetic modification in the control of gene expression. Moreover, some bioactive food components, such as SFN, have been shown to cause epigenetic changes in nuclear chromatin due to histone

deacetylase inhibitor activity. The working hypothesis tested in this specific aim is that treatment of 3T3-L1 mouse preadipocytes with the histone deacetylase inhibitors (tricostatin A or sulforaphane) will increase 1,25(OH)2 vitamin D-induced gene expression, as evidenced by an increase in CYP24 mRNA levels following

1,25(OH)2 vitamin D and HDACI treatment compared to 1,25(OH)2 vitamin D alone.

This hypothesis is based on strong support in the published literature showing that the expression of certain genes are increased following treatment with histone deacetylase inhibitors, and a specific report that vitamin D-induced CYP24 gene expression in human squamous carcinoma cell lines and human breast cancer cell lines are increased by treatment with triciferol, a hybrid 1,25(OH)2 vitamin D molecule with a modified side chain containing tricostatin A (Tavera-Mendoza et al.,

2008). To our knowledge, modulation of vitamin D-induced gene expression by sulforaphane, a natural histone deacetylase compound found in cruciferous vegetables such as broccoli and cabbage, has not been investigated, nor has the effects of any HDACIs in preadipocytes.

The rationale for performing this research is that since 1,25(OH)2 vitamin D has been shown to influence gene expression and adipogenesis in 3T3-L1 preadipocytes and other adipocyte cell lines, the findings of this research will be significant in advancing our understanding of how altered intake of bioactive food components that cause specific epigenetic changes in cells may modulate the action of the vitamin D hormone in adipose tissue. These findings could have future implications in the design of vitamin D intervention studies to influence adipose

metabolism and help combat obesity, or other diseases in which vitamin D may be involved.

CHAPTER 3

EXPERIMENTAL DESIGN AND METHODS

3.1. Experimental design

My experiment investigated the effect of two histone deacetylase inhibitors,

TSA and SFN, on 24-hydroxylase CYP24 gene expression levels. The 3T3-L1 preadipocytes cell culture system was used for the study due to their well- documented response to vitamin D (Sato and Hiragun, 1988, Kamei et al., 1993,

Kong and Li, 2006). I measured CYP24 expression levels by PCR-based quantitation of CYP24 mRNA following incubation of cells with 1,25(OH)2 vitamin D alone and

1,25(OH)2 vitamin D in combination with either 1µM TSA or 5µM SFN. CYP 24 induction is a well-known measure of 1,25(OH)2 vitamin D responsiveness in cells.

In order to confirm the cells’ dose-dependent responses to 1,25(OH)2 vitamin D, three different 1,25(OH)2 vitamin D concentrations of 0, 1, and 10 nmol/L were used. The incubation time of 24 hours was kept as a constant. Concentrations of TSA and SFN and incubation period were set in accordance with literature values

(Schwab et al., 2008, Wang et al., 2012).

3.2. Cell culture and treatments

Mouse 3T3-L1 preadipocytes cells were grown in 6-well culture plates to

100% confluence in growth media (89% DMEM, 10% calf serum, 1% pen-strep).

The media was changed with fresh growth media every 48 hours. At confluence, cells were treated with 3 different concentrations of hormonal form of vitamin D

(1,25-dihydroxycholecalciferol 0 nM, 1 nM, or 10 nM) alone the 3 different

concentrations of 1,25(OH)2 vitamin D in combination with either 1μM TSA

(Trichostatin A, Sigma-Aldrich) or 5μM SFN (Sulforaphane, Sigma-Aldrich) for 24 hours. An experiment consisted of 6 different treatment combinations that were performed in three 6-well plates, and so there were 2 replicate wells for each treatment combination including vehicles (no vitamin D treatment (control); 2 different concentrations of 1,25(OH)2 vitamin D; 1 and 10 nmol/L with either 1μM

TSA or 5μM SFN). The experiment was repeated 7 times (n = 7). In additional experiments, 1,25(OH)2 vitamin D dose-response experiment at 0, 1, 2, 5 and 10 nM were also conducted in the presence and absence of 5μM SFN. These experiments were repeated 3 times (n = 3).

3.3. Gene Expression Analysis

3.3.1. RNA isolation

After 24 hours treatment incubation, the media was discarded and cells were harvested to assess 24-hydroxylase mRNA expression. Cells were scraped in 1 mL

Trizol reagent (Invitrogen) and RNA was isolated from the treated cells according to the manufacturer’s protocol. In brief, after collecting Trizol-treated cells in the tube,

200μL chloroform was used for phase-separation. To get the total RNA pellet, RNA obtained from the upper aqueous phase layer was precipitated in 100% isopropanol

(1:1), centrifuged to obtain an RNA pellet, and then washed with 75% ethanol followed by centrifugation. After discarding the supernatant, the washed RNA pellet was resuspended in DEPC-treated water (20μl) and then heated to 55oC for 10 min.

3.3.2 RNA Quantification

2μl of the RNA sample in DEPC-treated water was diluted (1:500) in 998μl

DEPC-treated water and read at OD of 260nm on a spectrophotometer to determine

RNA concentration.

3.3.3 Reverse transcriptase PCR (RT-PCR)

First-strand cDNA was synthesized from isolated RNA by reverse transcriptase with SuperScript III First-Strand Synthesis System for RT-PCR kit

(Invitrogen) and then followed by PCR amplification to obtain the specific mRNA of interest. cDNAs were synthesized from RNA in a final volume of 20μl using 2 μg of total RNA as input. , PCR reactions were performed with 1μl of cDNA per reaction according to the protocol provided by manufacturer. Semiquantitative RT-PCR method was used to assess relative 24-hydroxylase (CYP24) and GAPDH gene expression. The sequences for 24-hydroxylase and GAPDH primers used for PCR are as follows (Shao et al., 2001).

Gene Primer Sequence

Cyp24 Forward 5’-CTCATGCTAAATACCCAGGTG-3’

Reverse 5’-TCGCTGGCAAAACGCGATGGG-3’

GAPDH Forward 5’-CCATGGAGAAGGCTGGGG-3’

Reverse 5’-CAAAGTTGTCATGGATGACC-3’

PCR was performed for 30 cycles, which falls within the linear range of amplification for both 24-hydroxylase and GAPDH. The annealing temperature that was used for PCR for both genes is 55oC.

3.3.4 Agarose Gel Electrophoresis

5 μl of loading dye was added to each 25ul of PCR product. Then, 6 μl of the

PCR sample of CYP24 mRNA, a gene of interest, and GAPDH mRNA, a housekeeping gene, were loaded into wells on a 2.5% agarose gel made of Tris acetate-EDTA (TAE) buffer and containing ethidium bromide. The bands obtained after electrophoresis were visualized under UV light and a digital picture of the gel with the PCR product bands was obtained. DNA band intensity was quantified using Image J software

(NIH) and relative band intensity of 24-hydroxylase was calculated against the respective samples GAPDH housekeeping gene. The fold-change in expression of the cyp24 mRNA compared to control (no treatment with either 1,25(OH)2 vitamin D or

HDACI) was calculated.

3.4 Statistical Analysis

Relative mRNA expression was analyzed in Prism version 5.0 (GraphPad

Software) and, when appropriate, the difference between treatment groups for 24- hydroxylase mRNA expression was assessed by post-hoc comparison of treatment means. Main effects of treatments on 24-hydroxylase mRNA expressions was analyzed using two-way ANOVA to test the effects of 1,25(OH)2 vitamin D and

HDACI (TSA or SFN), and interaction at P < 0.05.

CHAPTER 4

RESULTS

CYP24 mRNA Expression is Dependent on 1,25(OH)2 Vitamin D Dose in 3T3-L1

Mouse Preadipocytes

In the absence of 1,25(OH)2 vitamin D there was no evidence of CYP24 mRNA by PCR for 30 cycles (representative PCR bands shown in Figure 10); however, there was an evident induction of CYP24 mRNA expression at 1 nM and 10 nM

1,25(OH)2 vitamin D. The response of CYP24 mRNA level to 1,25(OH)2 vitamin D treatment was dependent on the dose (0, 1, 2, 5 and 10 nM for 24h) of 1,25(OH)2 vitamin D administered (Figure 11), which increase by 6.3-fold at 10 nM 1,25(OH)2 vitamin D, the highest vitamin D dose investigated compared to 1 nM 1,25(OH)2 vitamin D, the lowest dose to evoke an apparent increase in CYP24 expression

(Figure 10). However, the greatest relative response of CYP24 mRNA to vitamin D was apparent at the lower doses between 1 and 2 nM 1,25(OH)2 vitamin D, suggesting an approaching saturation of the CYP24 response.

1,25 Vitamin D 0 1 10 0 1 10 0 1 10 (nM) SFN 5uM - - - + + + - - - TSA 1uM ------+ + +

24-hydroxylase

Figure 10. Relative CYP 24 mRNA amounts measured by semiquantitative reverse transcription–PCR in 3T3L1 preadipocytes. CYP 24 mRNA expressions at 3 different 1,25(OH)2 vitamin D (nM) concentrations in combination with either 1μM TSA (Trichostatin A) or 5μM SFN (Sulforaphane) in 3T3-L1 preadipocytes.

1,25 (OH)2 Vitamin D Dose-response 25 P < 0.05 20 n = 3

Relative 24-hydroxlyase mRNA 24-hydroxlyase Relative 0

1nM 2 nM 5 nM Vehicle 10 nM

1,25 (OH)2 Vitamin D Concentrations

Figure 11. Dose-dependent effect of 1,25(OH)2 vitamin D on CYP 24 mRNA expression in 3T3- L1 preadipocytes. With increasing doses of 1,25(OH)2 vitamin, CYP24 mRNA expression increased (p < 0.05). The sharpest increase in CYP24 mRNA appeared to occur at the lower doses with tapering of response at the higher vitamin D treatment levels. Results indicate mean ± SEM, n = 3 independent experiments.

TSA Histone Deacetylase Inhibitor Treatment Increases Vitamin D-induced

CYP24 mRNA Expression

To examine the effects of the potent fungus-derived histone deacetylase inhibitor TSA on vitamin D-induced CYP24 gene expression in mouse preadipocytes, preadipocytes were treated with 3 different concentrations of 1,25(OH)2 vitamin D

(0, 1 and 10 nM for 24h) in combination with a high dose (1μM) of TSA. The denser

PCR bands evident in Figure 10 following 1,25(OH)2 vitamin D and TSA treatment compared to 1,25(OH)2vitamin D alone (far right set of bands compared to far left set of bands in the figure) indicate an apparent TSA-induced enhancement of CYP24

41 VD + SFN 7 rep

1.5 Vehicle 1nM 1,25 Vitamin D 1.0 10nM 1,25 Vitamin D

P < 0.05 n = 7 0.5

Relative 24-hydroxlyase mRNA 24-hydroxlyase Relative 0.0

VD + TSA 7 rep Vehicle 5uM SFN

1,25 (OH)2 Vitamin D Concentrations

2.5 Vehicle 2.0 1nM 1,25 Vitamin D 10nM 1,25 Vitamin D 1.5 P < 0.05 1.0 n = 7

0.5

Relative 24-hydroxlyase mRNA 24-hydroxlyase Relative 0.0

Vehicle 1uM TSA 1,25 (OH) Vitamin D Concentrations 2

Figure 12. CYP 24 mRNA expressions at 3 different 1,25(OH)2 vitamin D concentrations in combination with either 1μM TSA (Trichostatin A) or 5μM SFN (Sulforaphane) in 3T3-L1 preadipocytes. Relative CYP 24 mRNA amounts measured by semiquantitative reverse transcription–PCR in 3T3L1 preadipocytes. TSA treatment had a significant effect on CYP24 mRNA expression. SFN treatment also increased CYP24 mRNA expression, but to a lesser extent. Data are shown as means ± SEM of n = 7 separate experiments.

mRNA expression in the presence of 1,25(OH)2vitamin D. Note also that TSA alone had no effect on CYP24 mRNA.

Cells treated with TSA in 7 independent experiments (Figure 12) had higher mean vitamin D-induced CYP24 mRNA expression, by 3.8-fold (p < 0.05) at a low

(1nM) 1,25(OH)2 vitamin D dose and by 2.4-fold (p < 0.05) at a high (10 nM)

1,25(OH)2 vitamin D dose.

SFN Increases Vitamin D-induced CYP24 mRNA Expression

To test whether the bioactive food component SFN, which has been shown to have HDACI activity, could also influence vitamin D-induced CYP24 gene expression in 3T3-L1 preadipocytes, cells were treated with 3 different concentrations of

1,25(OH)2 vitamin D (0,1 and 10 nM for 24h) with or without 5μM SFN, followed by measurement of vitamin D-induced CYP24 mRNA expression. The apparently denser PCR bands, shown in the middle set of bands in Figure 10, following SFN and

1,25(OH)2 vitamin D treatment compared to 1,25(OH)2vitamin D treatment alone

(far left set of bands in the figure) suggest an apparent up regulation of CYP24 mRNA due to SFN treatment. This notion would be supported by the data, shown in

Figure 13, in which a vitamin D-induced dose-response of CYP24 expression was compared with and without concomitant SFN treatment. Over 3 independent experiments, cells treated with 5μM SFN had increased mean vitamin D-induced

CYP24 mRNA, by 3.6-fold at 1nM 1,25(OH)2 vitamin D, 6% at 2 nM, 74% at 5 nM, and

45% at 10 nM 1,25(OH)2 vitamin D. However, the main effect of SFN on CYP24 mRNA was not statistically significant by ANOVA (P = 0.0876). The main effect

difference in CYP24 mRNA between treatments without and with SFN was 2842 ±

2908. The reason for the apparent lack of response to SFN at 2 nM 1,25(OH)2 vitamin D in these experiments is unknown, but may reflect a possible experimental 1,25 (OH) Vitamin D Dose-response anomaly given the apparent effect of SFN at the both lower and higher vitamin D 2 doses.

30 No SFN 5µM SFN

20 P < 0.05 n = 3

Relative 24-hydroxlyase mRNA 24-hydroxlyase Relative 0

1nM 2nM 5nM 10nM Vehicle

1,25 (OH)2 Vitamin D Concentrations

Figure 13. Dose-dependent effect of 1,25(OH)2 vitamin D in combination with SFN on CYP 24 mRNA expression in 3T3-L1 preadipocytes. The main effect of SFN on CYP24 mRNA was not statistically significant by ANOVA (P = 0.0876). Results indicate mean ± SEM, n = 3 independent experiments.

Overall, our findings suggest that CYP24 mRNA expression is a highly dependent upon the presence of 1,25(OH)2 vitamin D in 3T3-L1 mouse preadipocytes. In addition, treatment with a HDACI can increase the responsiveness of 3T3-L1 cells to the vitamin D hormone, at least as judged by increased CYP24

mRNA expression. Based on our findings, it appears that the ability of SFN to increase CYP24 induction by 1,25(OH)2 vitamin D is not as effective as TSA.

CHAPTER 5

DISCUSSION

1,25(OH)2 vitamin D has been known for a long time to inhibit the differentiation of preadipocytes to adipocytes (Ishida et al., 1988) in a vitamin D receptor (VDR)-dependent manner. In this study, we used the mouse 3T3-L1 preadipocyte experimental system, which is a well-established cell culture model to study the mechanism of adipocyte differentiation, to investigate the effects of HDACI on vitamin D-induced gene expression. To study how histone deacetylase inhibitor influences VD/VDR–mediated gene expression, and to what extent histone deacetylase inhibitor can modulate the VD/VDR–mediated gene expression in preadipocytes, we examined the individual effect of two HDACIs, TSA and SFN, on

CYP24 mRNA expression. Our central hypothesis was that treatment of 3T3-L1 mouse preadipocytes with either histone deacetylase inhibitor (tricostatin A or sulforaphane) would increase 1,25(OH)2 vitamin D-induced CYP24 mRNA levels.

Our observations are consistent with the idea that both TSA and SFN can increase 1,25(OH)2 vitamin D-induced CYP24 mRNA expression in 3T3-L1 preadipocytes (Figure 12, 13). This finding of up regulation of CYP24 gene expression by TSA in preadipocytes is consistent with previous studies, which tested TSA in different cell lines, such as various prostate cancer cell lines

(Gommersall et al., 2004b, Luo et al., 2010). To our knowledge, our observations concerning the effect of TSA in 3T3-L1 mouse preadipocytes are novel and support the view that concomitant vitamin D and HDACI treatment could influence the

effects of the vitamin D hormone on adipocyte biology. The dietary histone deacetylase inhibitor (HDACI) sulforaphane (SFN) is one of the biologically active compounds in the human diet and is present at high levels in some cruciferous vegetables, such as broccoli. SFN has received a great deal of attention because of its ability to inhibit histone deacetylase enzymes, which was identified in human embryonic kidney 293 (HEK293), HCT116 human colon cancer cells (Myzak et al.,

2004), and prostate epithelial cells (BPH-1, LnCaP and PC-3) (Myzak et al., 2006), and its potential use in cancer chemoprevention or treatment.

Our novel observation that SFN increased vitamin D-induced CYP24 mRNA level in our 3T3-L1 preadipocytes model system supports the idea that dietary consumption of foods containing bioactive food components with HDACI activity, such as certain cruciferous vegetables, may be able to affect adipogenesis by altering vitamin D-induced gene expression. Adipogenesis in human primary adipocytes studied in culture has been recently reported to be increased by 1,25(OH)2 vitamin

D [add reference from the BU group here]. It is thus possible that HDACI could further increase the adipogenic response in humans to vitamin D compounds. On the other hand, there are studies that showed the inhibitory effect of TSA and SFN on adipogenesis in murine 3T3-L1 preadipocytes (Lagace and Nachtigal, 2004,

Haberland et al., 2010, Choi et al., 2012).

Additional studies are needed to investigate the influence of HDACIs on cell function because of the potential complex effects of these compounds. For example, an increase in CYP24 expression induced by HDACIs could reduce the intracellular bioavailability of vitamin D metabolites because its main biochemical function is to

initiate vitamin D catabolism and thereby reduce VDR signaling. The extent to which

HDACIs, including SFN, affect other vitamin D-dependent genes that may be involved with regulating adipocyte biology needs further exploration, as well as the net effect of HDACI-induced effects on complex physiological processes, such as adipogenesis.

CHAPTER 6

FUTURE DIRECTIONS

Although SFN treatment increased CYP24 mRNA expression 1.4-fold at 1nM

1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D in 3T3-L1 mouse preadipocytes, the main effect of SFN on CYP24 mRNA was not statistically significant. Testing different concentrations of SFN would be helpful to understand the effect of SFN on CYP24 mRNA expression.

REFERENCES

Abe E, Miyaura C, Sakagami H, Takeda M, Konno K, Yamazaki T, Yoshiki S, Suda T (1981) Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25- dihydroxyvitamin D3. Proceedings of the National Academy of Sciences of the United States of America 78:4990-4994.

Alimirah F, Vaishnav A, McCormick M, Echchgadda I, Chatterjee B, Mehta R, Peng X (2010) Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription. Molecular and Cellular Biochemistry 342:143-193.

Anderson P, May B, Morris H (2003) Vitamin D metabolism: new concepts and clinical implications. The Clinical Biochemist Reviews / Australian Association of Clinical Biochemists 24:13-39.

Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiological Reviews 81:1269-1573.

Banwell C, MacCartney D, Guy M, Miles A, Uskokovic M, Mansi J, Stewart P, O'Neill L, Turner B, Colston K, Campbell M (2006) Altered nuclear receptor corepressor expression attenuates vitamin D receptor signaling in breast cancer cells. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 12:2004-2017.

Bar-Shavit Z, Noff D, Edelstein S, Meyer M, Shibolet S, Goldman R (1981) 1,25- dihydroxyvitamin D3 and the regulation of macrophage function. Calcified Tissue International 33:673-679.

Basten G, Bao Y, Williamson G (2002) Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells. Carcinogenesis 23:1399-1803.

Baz-Hecht M, Goldfine A (2010) The impact of vitamin D deficiency on diabetes and cardiovascular risk. Current Opinion in Endocrinology, Diabetes, and Obesity 17:113-119.

Beckman M, Tadikonda P, Werner E, Prahl J, Yamada S, DeLuca H (1996) Human 25- hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme. Biochemistry 35:8465- 8537.

Bikle D (2009) Nonclassic actions of vitamin D. The Journal of Clinical Endocrinology and Metabolism 94:26-60.

Bikle DD (2010) Vitamin D: newly discovered actions require reconsideration of physiologic requirements. Trends in Endocrinology & Metabolism 21:375-759.

Bissonnette M, Tien X, Niedziela S, Hartmann S, Frawley B, Roy H, Sitrin M, Perlman R, Brasitus T (1994) 1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i. The American Journal of Physiology 267: 465-75.

Caffrey J, Farach-Carson M (1989) Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells. The Journal of Biological Chemistry 264:20265-20339.

Campas-Baypoli O, Sánchez-Machado D, Bueno-Solano C, Ramírez-Wong B, López- Cervantes J (2010) HPLC method validation for measurement of sulforaphane level in broccoli by-products. Biomedical Chromatography 24:387-92.

Cantley L, Russell J, Lettieri D, Sherwood L (1985) 1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture. Endocrinology 117:2114-2123.

Chen H, Lin R, Schiltz R, Chakravarti D, Nash A, Nagy L, Privalsky M, Nakatani Y, Evans R (1997) Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569-649.

Chen K, DeLuca H (1995) Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24- hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochimica et Biophysica Acta 1263:1-10.

Chiao J, Chung FL, Kancherla R, Ahmed T, Mittelman A, Conaway C (2002) Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. International Journal of Oncology 20:631-637.

Choi K-M, Lee Y-S, Sin D-M, Lee S, Lee M, Lee Y-M, Hong J-T, Yun Y-P, Yoo H-S (2012) Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest. Obesity (Silver Spring, Md) 20:1365-1371.

Choi S-W, Friso S (2010) Epigenetics: A New Bridge between Nutrition and Health. Advances in Nutrition (Bethesda, Md) 1:8-24.

Chu R, Lin Y, Rao M, Reddy J (1995) Cooperative formation of higher order peroxisome proliferator-activated receptor and retinoid X receptor complexes on the peroxisome proliferator responsive element of the rat hydratase-dehydrogenase gene. The Journal of Biological Chemistry 270:29636-29639.

Chuang L, Nagarsheth N, Hayes M, Zakashansky K, Gretz H, Nezhat F, Wu X, Fishman D, Chiao J (2013) Sulforaphane induces cell cycle arrest, migration, invasion, and apoptosis in epithelial ovarian cancer cells. American Journal of Cancer Review 1:9- 24.

Colston K, Colston M, Feldman D (1981) 1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture. Endocrinology 108:1083-1089. de Ruijter A, van Gennip A, Caron H, Kemp S, van Kuilenburg A (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. The Biochemical Journal 370:737-786.

DeLuca H (1981) Recent advances in the metabolism of vitamin D. Annual review of physiology 43:199-408.

DeLuca H (2004) Overview of general physiologic features and functions of vitamin D. The American Journal of Clinical Nutrition 80:1689-96.

Dupont C, Armant D, Brenner C (2009) Epigenetics: definition, mechanisms and clinical perspective. Seminars in Reproductive Medicine 27:351-358.

Ehrenhofer-Murray A (2004) Chromatin dynamics at DNA replication, transcription and repair. European Journal of Biochemistry / FEBS 271:2335-2384.

Fahey J, Haristoy X, Dolan P, Kensler T, Scholtus I, Stephenson K, Talalay P, Lozniewski A (2002) Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene- induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America 99:7610-7615.

Finnin M, Donigian J, Cohen A, Richon V, Rifkind R, Marks P, Breslow R, Pavletich N (1999) Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401:188-281.

Fraga M, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K, Iyer N, Pérez-Rosado A, Calvo E, Lopez J, Cano A, Calasanz M, Colomer D, Piris M, Ahn N, Imhof A, Caldas C, Jenuwein T, Esteller M (2005) Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nature Genetics 37:391-791.

Garabedian M, Holick M, Deluca H, Boyle I (1972) Control of 25- hydroxycholecalciferol metabolism by parathyroid glands. Proceedings of the National Academy of Sciences of the United States of America 69:1673-1679.

Giguère V (1999) Orphan nuclear receptors: from gene to function. Endocrine Reviews 20:689-1414.

Glaser K, Staver M, Waring J, Stender J, Ulrich R, Davidsen S (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Molecular Cancer Therapeutics 2:151-163.

Gommersall L, Khanim F, Peehl D, Doherty A, Campbell M (2004a) Epigenetic repression of transcription by the Vitamin D3 receptor in prostate cancer cells. The Journal of Steroid Biochemistry and Molecular Biology 89-90:251-256.

Gregoire F (2001) Adipocyte differentiation: from fibroblast to endocrine cell. Experimental Biology and Medicine 226:997-1002.

Haberland M, Carrer M, Mokalled M, Montgomery R, Olson E (2010) Redundant control of adipogenesis by histone deacetylases 1 and 2. The Journal of biological Chemistry 285:14663-14670.

Haussler M, Haussler C, Bartik L, Whitfield G, Hsieh J-C, Slater S, Jurutka P (2008) Vitamin D receptor: molecular signaling and actions of nutritional ligands in disease prevention. Nutrition Reviews 66:98-112.

Heiss E, Herhaus C, Klimo K, Bartsch H, Gerhäuser C (2001) Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. The Journal of Biological Chemistry 276:32008-32023.

Ho E, Clarke J, Dashwood R (2009) Dietary sulforaphane, a histone deacetylase inhibitor for cancer prevention. The Journal of Nutrition 139:2393-2396.

Holick M (1995) Noncalcemic actions of 1,25-dihydroxyvitamin D3 and clinical applications. Bone 17:107-111.

Holick M (2010) Vitamin D: physiology, molecular biology and clinical applications.

Hong L, Schroth G, Matthews H, Yau P, Bradbury E (1993) Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. The Journal of Biological Chemistry 268:305-319.

Hu R, Hebbar V, Kim R, Chen C, Winnik B, Buckley B, Soteropoulos P, Tolias P, Hart RP, Kong N (2004) In vivo pharmacokinetics and regulation of gene expression profiles by isothiocyanate sulforaphane in the rat. Journal of Pharmacology and Experimental Therapeutics 310:263-71.

Ishida Y, Taniguchi H, Baba S (1988) Possible involvement of 1 alpha,25- dihydroxyvitamin D3 in proliferation and differentiation of 3T3-L1 cells. Biochemical and Biophysical Research Communications 151:1122-1129.

Jones G (2010) Metabolism and Catabolism of Vitamin D, Its Metabolites and Clinically Relevant Analogs In: Holick M, ed. Vitamin D: Physiology, Molecular Biology, and Clinical Applications. New York, NY: Humana Press, 2010: 99-134.

Jones G, Strugnell S, DeLuca H (1998) Current understanding of the molecular actions of vitamin D. Physiological Reviews 78:1193-1424.

Kamei Y, Kawada T, Kazuki R, Ono T, Kato S, Sugimoto E (1993) Vitamin D receptor gene expression is up-regulated by 1, 25-dihydroxyvitamin D3 in 3T3-L1 preadipocytes. Biochemical and Biophysical Research Communications 193:948- 955.

Kim M, Blake M, Baek J, Kohlhagen G, Pommier Y, Carrier F (2003) Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Research 63:7291-7591.

Kim S-H, Singh S (2009) D,L-Sulforaphane causes transcriptional repression of androgen receptor in human prostate cancer cells. Molecular Cancer Therapeutics 8:1946-1954.

Kolek O, Hines E, Jones M, LeSueur L, Lipko M, Kiela P, Collins J, Haussler M, Ghishan F (2005) 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. American Journal of Physiology Gastrointestinal and Liver Physiology 289:42.

Kong J, Li Y (2006) Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells. American Journal of Physiology Endocrinology and Metabolism 290:E916-E924.

Lagace D, Nachtigal M (2004) Inhibition of histone deacetylase activity by valproic acid blocks adipogenesis. The Journal of Biological Chemistry 279:18851-18860.

Lee S, Clark S, Gill R, Christakos S (1994) 1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion. Endocrinology 134:1602-1612.

Lin F, MacDougald O, Diehl A, Lane M (1993) A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity. Proceedings of the National Academy of Sciences of the United States of America 90:9606-9610.

Liu R (2003) Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. The American Journal of Clinical Nutrition 78:517-520.

Luo W, Karpf A, Deeb K, Muindi J, Morrison C, Johnson C, Trump D (2010) Epigenetic regulation of vitamin D 24-hydroxylase/CYP24A1 in human prostate cancer. Cancer Research 70:5953-6015.

Mahéo K, Morel F, Langouët S, Kramer H, Le Ferrec E, Ketterer B, Guillouzo A (1997) Inhibition of cytochromes P-450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Research 57:3649- 3701.

Makin G, Lohnes D, Byford V, Ray R, Jones G (1989) Target cell metabolism of 1,25- dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation. The Biochemical Journal 262:173-253.

Malloy P, Wang J, Jensen K, Feldman D (2009) Modulation of vitamin d receptor activity by the corepressor hairless: differential effects of hairless isoforms. Endocrinology 150:4950-4957.

Marks P, Richon V, Breslow R, Rifkind R (2001) Histone deacetylase inhibitors as new cancer drugs. Current Opinion in Oncology 13:477-560.

Marks P, Richon V, Rifkind R (2000) Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. Journal of the National Cancer Institute 92:1210-1216.

Masuyama H, Brownfield C, St-Arnaud R, MacDonald P (1997) Evidence for ligand- dependent intramolecular folding of the AF-2 domain in vitamin D receptor- activated transcription and coactivator interaction. Molecular Endocrinology (Baltimore, Md) 11:1507-1524.

McKenna N, Lanz R, O'Malley B (1999) Nuclear receptor coregulators: cellular and molecular biology. Endocrine Reviews 20:321-365.

McKenna N, O'Malley B (2002) Minireview: nuclear receptor coactivators--an update. Endocrinology 143:2461-2466.

Mogal A, Abdulkadir S (2006) Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes. Molecular and Cellular Probes 20:81-86.

Moreira J, Scheipers P, Sørensen P (2003) The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses. BMC Cancer 3:30.

Morrissey R, Bucci T, Richard B, Empson N, Lufkin E (1975) Calcium-binding protein: its cellular localization in jejunum, kidney and pancreas. Proceedings of the

Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine (New York, NY) 149:56-116.

Myzak M, Hardin K, Wang R, Dashwood R, Ho E (2006) Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells. Carcinogenesis 27:811-819.

Myzak M, Karplus P, Chung F-L, Dashwood R (2004) A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase. Cancer Research 64:5767-5841.

Näär A, Lemon B, Tjian R (2001) Transcriptional coactivator complexes. Annual Review of Biochemistry 70:475-976.

Nalabothula N, Carrier F (2011) Cancer cells' epigenetic composition and predisposition to histone deacetylase inhibitor sensitization. Epigenomics 3:145- 200.

Nan X, Hyndman L, Agbi N, Porteous D, Boyd A (2004) Potent stimulation of gene expression by histone deacetylase inhibitors on transiently transfected DNA. Biochemical and Biophysical Research Communications 324:348-402.

Nemere I, Yoshimoto Y, Norman A (1984) Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25- dihydroxyvitamin D3. Endocrinology 115:1476-1559.

Norton V, Imai B, Yau P, Bradbury E (1989) Histone acetylation reduces nucleosome core particle linking number change. Cell 57:449-506.

Olefsky J (2001) Nuclear receptor minireview series. The Journal of Biological Chemistry 276:36863-36867.

Petri N, Tannergren C, Holst B, Mellon F, Bao Y, Plumb G, Bacon J, O'Leary K, Kroon P, Knutson L, Forsell P, Eriksson T, Lennernas H, Williamson G (2003) Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo. Drug metabolism and disposition: the biological fate of chemicals 31:805-813.

Polly P, Herdick M, Moehren U, Baniahmad A, Heinzel T, Carlberg C (2000) VDR- Alien: a novel, DNA-selective vitamin D(3) receptor-corepressor partnership. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14:1455-1518.

Provvedini D, Tsoukas C, Deftos L, Manolagas S (1986) 1 alpha,25-Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production. Journal of Immunology (Baltimore, Md : 1950) 136:2734-2774.

Reddy G, Tserng K (1989) Calcitroic acid, end product of renal metabolism of 1,25- dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry 28:1763-1772.

Samuel S, Sitrin M (2008) Vitamin D's role in cell proliferation and differentiation. Nutrition Reviews 66:116-124.

SarDesai V (2012) Fat-Soluble Vitamins In: SarDesai V, ed. Introduction to clinical nutrition. Boca Raton, FL: CRC Press, 2012:175-185.

Sato M, Hiragun A (1988) Demonstration of 1 alpha,25-dihydroxyvitamin D3 receptor-like molecule in ST 13 and 3T3 L1 preadipocytes and its inhibitory effects on preadipocyte differentiation. Journal of Cellular Physiology 135:545-550.

Schwab M, Reynders V, Loitsch S, Steinhilber D, Schröder O, Stein J (2008) The dietary histone deacetylase inhibitor sulforaphane induces human beta-defensin-2 in intestinal epithelial cells. Immunology 125:241-292.

Seow A, Shi C, Chung F, Jiao D, Hankin J, Lee H, Coetzee G, Yu M (1998) Urinary total isothiocyanate (ITC) in a population-based sample of middle-aged and older Chinese in Singapore: relationship with dietary total ITC and glutathione S-transferase M1/T1/P1 genotypes. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 7:775-781.

Seshadri K, Tamilselvana B, Rajendrana A, (2011) Role of vitamin D in diabetes. Journal of Endocrinology and Metabolism 1:47-56.

Shao A, Wood R, Fleet J (2001) Increased vitamin D receptor level enhances 1,25- dihydroxyvitamin D3-mediated gene expression and calcium transport in Caco-2 cells. Journal of bone and mineral research: the Official Journal of the American Society for Bone and Mineral Research 16:615-624.

Shao D, Lazar M (1997) Peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha, and cell cycle status regulate the commitment to adipocyte differentiation. The Journal of Biological Chemistry 272:21473-21481.

Shapiro T, Fahey J, Wade K, Stephenson K, Talalay P (1998) Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 7:1091-1100.

Shapiro T, Fahey J, Wade K, Stephenson K, Talalay P (2001) Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in

humans. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 10:501-508.

Shinki T, Jin C, Nishimura A, Nagai Y, Ohyama Y, Noshiro M, Okuda K, Suda T (1992) Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha, 25-dihydroxyvitamin D3 in rat kidney but not in intestine. The Journal of Biological Chemistry 267:13757-13819.

Singh S, Warin R, Xiao D, Powolny A, Stan S, Arlotti J, Zeng Y, Hahm E-R, Marynowski S, Bommareddy A, Desai D, Amin S, Parise R, Beumer J, Chambers W (2009) Sulforaphane inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice in association with increased cytotoxicity of natural killer cells. Cancer Research 69:2117-2142.

Song M-Y, Kim E-K, Moon W-S, Park J-W, Kim H-J, So H-S, Park R, Kwon K-B, Park B- H (2009) Sulforaphane protects against cytokine- and streptozotocin-induced beta- cell damage by suppressing the NF-kappaB pathway. Toxicology and Applied Pharmacology 235:57-124.

Sooy K, Schermerhorn T, Noda M, Surana M, Rhoten W, Meyer M, Fleischer N, Sharp G, Christakos S (1999) Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines. The Journal of Biological Chemistry 274:34343-34352.

Struhl K (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes & Development 12:599-1205.

Szabo A, Merke J, Beier E, Mall G, Ritz E (1989) 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney International 35:1049-1105.

Tavera-Mendoza L, Quach T, Dabbas B, Hudon J, Liao X, Palijan A, Gleason J, White J (2008) Incorporation of histone deacetylase inhibition into the structure of a nuclear receptor agonist. Proceedings of the National Academy of Sciences of the United States of America 105:8250-8255.

Thomas G, Baker S, Eisman J, Gardiner E (2001) Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts. The Journal of Endocrinology 170:451-511.

Tóth K, Knoch T, Wachsmuth M, Frank-Stöhr M, Stöhr M, Bacher C, Müller G, Rippe K (2004) Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin. Journal of Cell Science 117:4277-4364.

Umek R, Friedman A, McKnight S (1991) CCAAT-enhancer binding protein: a component of a differentiation switch. Science (New York, NY) 251:288-292.

Van Lint C, Emiliani S, Verdin E (1996) The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expression 5:245- 298.

Vermeulen M, Klöpping-Ketelaars I, van den Berg R, Vaes W (2008) Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli. Journal of Agricultural and Food Chemistry. 56:10505-9.

Wang L, Persons K, Jamieson D, Flanagan J, Hsu H, Holick M, Luo Z, Sakaki T, Urushino N, Faller D, Chen T (2008) Prostate 25-hydroxyvitamin D-1alpha- hydroxylase is up-regulated by suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. Anticancer Research 28:2009-2022.

Wang W, Pan K, Chen Y, Huang C, Zhang X (2012) The acetylation of transcription factor HBP1 by p300/CBP enhances p16INK4A expression. Nucleic Acids Research 40:981-1076.

Wildman REC (2011) Advanced human nutrition. Jones & Bartlett Publishers. Wood R (2008) Vitamin D and adipogenesis: new molecular insights. Nutrition Reviews 66:40-46.

Xie Z, Bikle D (1997) Cloning of the human phospholipase C-gamma1 promoter and identification of a DR6-type vitamin D-responsive element. The Journal of Biological Chemistry 272:6573-6580.

Xie Z, Chang S, Oda Y, Bikle D (2006) Hairless suppresses vitamin D receptor transactivation in human keratinocytes. Endocrinology 147:314-337.

Yang XJ, Seto E (2007) HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene 26:5310-5318.

Yoshida M, Kijima M, Akita M, Beppu T (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. The Journal of Biological Chemistry 265:17174-17183.

Yukihiro S, Posner G, Guggino S (1994) Vitamin D3 analogs stimulate calcium currents in rat osteosarcoma cells. The Journal of Biological Chemistry 269:23889- 23982.

Zanello L, Norman A (1997) Stimulation by 1alpha,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells. A structure-function study. The Journal of Biological Chemistry 272:22617-22639.

Zhang K, Williams K, Huang L, Yau P, Siino J, Bradbury E, Jones P, Minch M, Burlingame A (2002) Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. Molecular & Cellular Proteomics: 1:500-508.

Zhang Y (2004) Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutation Research 555:173-263.

Zink D, Fischer A, Nickerson J (2004) Nuclear structure in cancer cells. Nature Reviews Cancer 4:677-764. http://www.biotek.com/resources/articles/cellular-lantha-screen-histone.html http://www.nap.edu/openbook.php?record_id=13050&page=76