Pregenomic and Genomic Effects of 24,25-Dihydroxyvitamin D3

Utah State University DigitalCommons@USU

All Graduate Theses and Dissertations Graduate Studies

5-2015

Yang Zhang Utah State University

Follow this and additional works at: https://digitalcommons.usu.edu/etd

Part of the Dietetics and Clinical Nutrition Commons, Food Science Commons, and the Nutrition Commons

Recommended Citation Zhang, Yang, "Pregenomic and Genomic Effects of 24,25-Dihydroxyvitamin D3" (2015). All Graduate Theses and Dissertations. 4551. https://digitalcommons.usu.edu/etd/4551

This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected].

PREGENOMIC AND GENOMIC EFFECTS OF 24,25-DIHYDROXYVITAMIN D3

Yang Zhang

A thesis submitted in partial fulfillment of the requirements for the degree

MASTER OF SCIENCE

Nutrition, Dietetics, and Food Sciences

Approved:

______Dr. Korry Hintze Dr. Heidi Wengreen Major Professor Committee Member

______Dr. Marie Walsh Dr. Mark R. McLellan Committee Member Dean of Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2015

iii

ABSTRACT

Pregenomic and Genomic Effects of 24,25(OH)2D3

Yang Zhang, Master of Science

Utah State University, 2015

Major Professor: Dr. Korry Hintze Department: Nutrition, Dietetics, and Food Sciences

Vitamin D is hydroxylated to form several active metabolites, of these, 1,25-

dihydroxyvitamin D3 [1,25(OH)2D3] is the most studied stimulatory product. It is now

accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate

homeostasis through its membrane receptor 1,25D3-MARRS (membrane associated rapid

response steroid binding) protein. Another metabolite, 24,25-dihydroxyvitamin D3

[24,25(OH)2D3] has been reported to be inhibitory with respect to calcium and phosphate

absorption in intestine. Previous work in this laboratory has indicated that 24,25(OH)2D3

inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops and in

vivo. This thesis further tested the hypothesis that the actions of 24,25(OH)2D3 on

phosphate homeostasis are physiologically important. Catalase has been identified as a

binding protein for 24,25(OH)2D3. We determined the localization of catalase in the

presence and absence of steroid, monitored catalase mRNA levels related to gene

24,25(OH)2D3 gene transcription regulation. We studied the effects of the two isomers of

24,25(OH)2D3 on localization of catalase in chicken enterocytes over a time course of 15

sec to 60 min. It was demonstrated that 24R,25(OH)2D3 is the effective metabolite for

catalase redistribution in vitro. We also studied the effects of vitamin D on catalase and

phosphate uptake in chicken intestinal cells. It was once again demonstrated that

24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake and catalase

gene expression. These combined results lead us to conclude that 24,25(OH)2D3 is an

important hormone in phosphate homeostasis in chick intestinal epithelial cells.

(101 pages)

PUBLIC ABSTRACT

Pregenomic and Genomic Effects of

24,25(OH)2D3

Yang Zhang

In the United States, the major dietary source of protein is poultry meat,

particularly chicken. Phosphorus absorption in chickens is a critical problem in poultry

production. It therefore is important to understand the cellular and molecular regulation

of phosphorous absorption in intestine to increase efficiency of the process. This would

provide the benefits of reducing feed costs and reducing phosphorous excretion, thereby

contributing to the sustainability of agriculture in the United States. Therefore, the focus

of this thesis is determining the effect of vitamin D metabolites involved in phosphate

homeostasis using the chicken as a model.

To Xiubin Zhang and Yao Zhang

vii

ACKNOWLEDGMENTS

I would like to express my gratitude to my major professors, Dr. Ilka Nemere and

Dr. Korry Hintze, for teaching me their experienced research skills and giving me the

valuable advice and comments. Without their assistance and understanding, I would not

have been able to complete my studies. I would especially like to thank my committee

members for their kindness and support and their helpful critical reviews and suggestions

for improving my thesis.

I would like to dedicate this thesis to my beloved parents, Xiubin Zhang and Yao

Zhang, who always love and understand and support me unconditionally.

I would also like to express my thanks to my friends Tremaine, Yu Meng and

others, for their sincere friendship. Thank you all.

Yang Zhang

viii

CONTENTS

Page ABSTRACT ...... iii

PUBLIC ABSTRACT ...... iv

ACKNOWLEDGMENTS ...... vii

LIST OF FIGURES ...... ix

LIST OF TABLES ...... xi

LIST OF ABBREVIATIONS AND DEFINITIONS ...... xii

CHAPTER

1. LITERATURE REVIEW ...... 1

2. EFFECTS OF 24,25-DIHYDROXYVITAMIN D3 ON LOCALIZATION OF CATALASE IN CHICK ENTEROCYTES ...... 36 Abstract ...... 36 Introduction ...... 37 Experimental ...... 39 Results ...... 42 Discussion ...... 57 References ...... 59

3. EFFECTS OF VITAMIN D ON CATALASE EXPRESSION AND PHOSPHATE UPTAKE IN CHICKEN INTESTINAL ...... 62 Abstract ...... 62 Introduction ...... 62 Experimental ...... 63 Results ...... 69 Discussion ...... 80 References ...... 82

4. SUMMARY AND CONCLUSIONS ...... 83 Summary ...... 83 Conclusions ...... 85 References ...... 87

LIST OF FIGURES

Figure Page

1.1 Schematic presentation of phosphate homeostasis………………………………... 9

1.2 Structure of vitamin D and its metabolites………………………………………… 10

1.3 Regulation of PTH and 1,25(OH)2D3 in calcium homeostasis…………………….. 11

1.4 Model for interaction between 1,25D3-MARRS receptor and catalase…………… 14

2.1 Time course study of catalase localization in isolated intestinal cells: ethanol control treatment…………………………………………………………………….47

2.2 Time course study of catalase localization in isolated intestinal cells: 6.5 nM 24R,25(OH)2D3 hormone treatment………………………………………………...48

2.3 Time course study of catalase localization in isolated intestinal cells: 200 pM 24S,25(OH)2D3 hormone treatment…………………………………………………49

2.4 Time course study of catalase localization in isolated intestinal cells: 360 pM 1,25(OH)2D3 hormone treatment……………………………………………………50

2.5 Time course study of catalase localization in isolated intestinal cells: 0.01% ethanol (final concentration) after prior incubation with antibody………....……….51

2.6 Time course study of catalase localization in isolated intestinal cells: 6.5 nM 24R,25(OH)2D3 hormone treatment after prior incubation with antibody………….52

2.7 Time course study of catalase localization in isolated intestinal cells: 200 pM 24S,25(OH)2D3 hormone treatment after prior incubation with antibody………….53

2.8 Time course study of catalase localization in isolated intestinal cells: 360 pM 1,25(OH)2D3 hormone treatment after prior incubation with antibody…………….54

2.9 Western analyses on effect of vitamin D and time on catalase expression in the nucleus…………………………………………………………..…………..55

2.10 Western blot of nuclear extract catalase band intensity quantified by ImageJ software…………………………………………………………………………….56

32 3.1 Time course of 1,25(OH)2D3, 24R,25(OH)2D3 or 24S,25(OH)2D3 – mediated P uptake in chicken intestinal cell suspensions, relative to vehicle controls………....71

Figure Page

32 3.2 Effect of vehicle, 1,25(OH)2D3, or 24,25(OH)2D3 on P uptake in cultured chicken intestinal cells…………………………………………..…………………73

3.3 Effect of vitamin D on gene expression for catalase in cultures of chicken intestinal cells…………………………………………..……………...... 75

32 3.4 Effect of vehicle, 1,25(OH)2D3, or 24R,25(OH)2D3 on P uptake in transfected cultured chicken enterocytes………………………………………..……………..77

3.5 Effect of vitamin D on protein levels of catalase in cultured chicken enterocytes………………………………………..……………………………….79

LIST OF TABLES

Table Page

1.1 Vitamin D metabolite action summary ...... 15

xii

LIST OF ABBREVIATIONS AND DEFINITIONS

µCi Microcurie, a unit of radiation

µg Microgram

µl Microliter

1,25(OH)2D3 1,25-dihydroxyvitamin D3

1,25D3-MARRS 1,25-dihydroxyvitamin D3

membrane associated, rapid response

steroid-binding receptors

24,25(OH)2D3 24,25-dihydroxyvitamin D3

32P radioisotope for phosphorous

BSA Bovine serum albumin

P1 fraction pellets contain nuclei, BBM and

large cell debris

P2 fraction Pellets contain BLM, Golgi,

mitochondria, lysosomes and

peroxisomes

CPM counts per minute, a measurement of

radioactivity

ECF substrate enhanced luminescence substrate

FBS Fetal bovine serum

GBSS Gey’s balanced salt solution

mM Millimolar

pM Picomolar

xiii

Min Minute

PBS Phosphate buffer saline

PKA Protein kinase A

PKC Protein kinase C

PTH Parathyroid hormone

P value Probability to test the hypothesis

PVDF Polyvinylidene difluoride

SDS-PAGE Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

Sec Second

SEM Standard error of mean

siRNA Small interfering RNA

TED Tris/EDTA/dithiothreitol

VDR Vitamin D receptor

CHAPTER 1

LITERATURE REVIEW

Outline

This literature review will begin with an overview of vitamin D history and background. Next, metabolic mechanisms of vitamin D will be discussed and introduced.

Then, a more focused look at different vitamin D metabolites and their receptors will be presented; phosphate and calcium homeostasis will also be addressed. Last, an overview of catalase metabolism and applications will be addressed.

Background

In the past several years, vitamin D has received enormous attention, specifically at the public health level, mainly because of the discovery of its many unexpected health benefits. It is now realized that vitamin D is important not only for mineralization and skeletal growth but also in regulation of the immune system, the parathyroid gland, the skin, and in cancer prevention. Since the initial discovery of vitamin D in the early 20th century, vitamin D was recognized not only for its major medical importance by eliminating rickets as a major medical problem, but also had received a bad reputation because of outbreak of idiopathic hypercalcemia after World War II attributed to food fortification with vitamin D [1]. In the United States and Canada, there is a campaign to increase the awareness of the benefits of vitamin D as well as to increase the recommended daily allowance of vitamin D [2]. However, vitamin D is potentially toxic when provided in large amounts [3]. A report in 2007 indicates 10,000 units of vitamin

D3 per day is safe for 6 months [4] (NOAEL: no observed adverse effects level). In 2011,

the Food and Nutrition Board set the upper limit (UL) for vitamin D3 to 4,000 IU per day,

simply calculated from the NOAEL by dividing by a safety factor (which is 2.5 in

vitamin D’s case). This is an important start, and the recommendation probably will be

changed in the years to come. It is important to note that vitamin D is fat-soluble so it is

stored in the adipose tissue and accumulation of vitamin D could possibly result in

toxicity.

History and Metabolic Mechanisms

Sir Edward Mellanby found the first evidence of the existence of vitamin D in

1919 [5]. He was able to produce rickets in dogs between 5 and 8 weeks by feeding them

one of four natural diets and keeping them indoors, and cure the disease with cod liver oil,

which is also rich in vitamin A. So there was uncertainty whether it was vitamin A that

treated rickets or some new factor. In the same year, Huldshinsky discovered that rickets

in chicken could be cured by ultraviolet light [6], which might be the first lead that

vitamin D is not really a traditional vitamin. In 1922, McCollum [7] demonstrated that

cod liver oil contained another vitamin that is responsible for bone mineralization, which

he named “vitamin D”. Scientists have demonstrated that ultraviolet light is capable of

converting an inactive substance into an active substance that could cure rickets [8, 9],

thus led to the conclusion that the skin is of major importance in the production of

vitamin D.

Increasing the amount of meat on chicken and turkeys, which requires sufficient

support of bone structure, has always been a goal in poultry production. To date,

strategies for improving bone strength have included increasing dietary calcium and

phosphate, as well as vitamin D (calciferol), which is not really a strict vitamin but the

pre-hormone that serves as the parent compound for the production of three active

metabolites: 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and 24,25-

dihydroxyvitamin D3. Discovered in 1919-1924, vitamin D is a fat-soluble antirachitic

factor, and it is naturally present in very few foods: a small amount in milk and eggs, and

relatively large amounts in fatty fish. The actual vitamin D content of fortified milk is not

the same as the stated 400 IU per liter [10]. Vitamin D is primarily produced when

ultraviolet rays (action spectrum 280-320 nM or UVB) in sunlight triggers vitamin D

synthesis endogenously under the skin. Today, many populations are still at risk of

vitamin D inadequacy despite the fortification of vitamin D in foods and the ability of the

body to synthesize the vitamin, these include breastfed infants [11], older adults [12],

people with limited sun exposure, people with dark skin [13], people with fat

malabsorption [14] and people who are obese [15] or who have undergone gastric bypass

surgery [16]. In the past, vitamin D was thought to only influence only bone diseases

such as rickets and osteoporosis through regulation of active transport of calcium in

enterocytes [17]. However, recently vitamin D has been accepted as a major player in

overall human health [18], playing a role in chronic diseases such as arthritis, obesity,

cancer, auto-immune diseases, cardiovascular diseases, and diabetes [19]. Vitamin D has

been shown to influence virtually every cell in the human body, and receptors that

respond to vitamin D have been found in nearly every cell type [19]. One recent study

has shown that a large cohort of women age 65 and older experience low vitamin D

levels, which possibly contributed to mild weight gain [20]. An association between

vitamin D and cancer was first proposed in 1980 based on an ecological study of colon

cancer mortality rates and annual sunlight levels in the United States [21]. Currently,

researchers have shown that higher vitamin D levels decrease the risk of 15 different

types of cancer [22]. In an effort to understand how to use vitamin D as a weapon to

combat chronic diseases, the underlying mechanisms involved with calcium and

phosphate homeostasis that is at the core of cell signaling, growth and proliferation must

be understood.

Our understanding of how vitamin D mediates biological responses has entered a

new era. Phosphorus and calcium are two of the most abundant trace elements in the

body and must be regulated in order to maintain proper cellular functions. Vitamin D

promotes calcium absorption and maintains adequate serum calcium and phosphate

concentrations to enable normal bone mineralization, growth and remodeling [23].

Vitamin D modulates cell growth, neuromuscular and immune function, and reduces

inflammation. It also regulates expression of many genes encoding proteins that regulate

cell proliferation, differentiation, and apoptosis [23].

Vitamin D Synthesis

The following steps occur in vitamin D synthesis [17]: in the epidermis there is

abundant 7-dehydrocholesterol, when the skin is exposed to the UVR, 7-

dehydrocholesterol is photolyzed to pre vitamin D3. Vitamin D3 (cholecalciferol, from

animal sources) is a secosteroid since the B ring of 7-dehydrocholesterol is broken from

photolysis, which in turn gives it a cis-triene structure [24]. The cis-triene structure gives

vitamin D3 its unique ultraviolet absorption at 265 nm with a minimum at 228 nm. With

continued UVR exposure, vitamin D3 is converted to biologically inactive lumisterol and

tachysterol [25]. The formation of lumisterol and tachysterol is reversible and can be

converted back to vitamin D3 if needed. This explains the lack of vitamin D3 toxicity

through prolonged sun exposure. After formation, vitamin D3 is transported to the liver

and is enzymatically hydroxylated to 25-hydroxyvitamin D3 [calcidiol, 25(OH)D3, the

major circulating form of the vitamin] after binding to its carrier protein-vitamin D-

binding protein (DBP) which does not bind pre vitamin D3 [26]. Hydroxylation is

catalyzed by a microsomal cytochrome P450 enzyme, type I mitochondrial cytochrome

P450 oxidases, CYP2R1 or the mitochondrial cytochrome P450 CYP27A1, which

utilizes NADPH and molecular oxygen to catalyze the hydroxylation reaction [27].

25(OH)D3 quickly enters the circulation with normal circulating levels between 25

nmol/L to 200 nmol/L, and it has a half-life of about 15 days [28]. 25(OH)D3, bound to

DBP, is then transported to the kidneys and is finally hydroxylated by 1α-hydroxylase

(also termed CYP27B1 or P450C1, an enzyme that is highly expressed in renal proximal

tubules [29]) at the C1 position to hormonally active 1,25(OH)2D3. This occurs when

serum levels of calcium and phosphate are low. The serum levels of 1,25(OH)2D3 range

from 75 pmol/L to 200 pmol/L and it has a serum half-life of 10-24 hours [30].

Regulation of CYP27B1 is controlled by PTH, and fibroblast growth factor (FGF)-23

which inhibits expression respectively [25]. By contrast, when serum levels of calcium

and phosphate are high, suppression of 1α-hydroxylase by high levels of circulating

calcium or phosphate stimulates another hydroxylase (CYP24 or P450C24) that converts

25(OH)D3 to 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Both positive (PTH, estrogen,

and growth hormone) and negative (1,25(OH)2D3, calcium) regulators of the renal

hydroxylases have been identified, and acute regulation of renal 1,25(OH)2D3 production

is mediated through the adenylate cyclase and protein kinase C pathways [31, 32].

Vitamin D and Calcium Transport

When intestinal calcium transport was measured in vitamin D-deficient rats given

a dose of vitamin D3, a 12-hour period was required for a response [33]. This coupled

with the observation that it took 24h before intestinal calcium absorption reached a

maximum level led to the belief that vitamin D3 needed to be activated before it could

carry out its physiological functions on calcium metabolism [34, 35]. In a study that

provided radiolabeled vitamin D3 to vitamin D-deficient rats caused the disappearance of

radiolabeled vitamin D3 and the appearance of polar metabolite fractions [34].

Researchers observed that the polar metabolite fraction stimulated intestinal calcium

transport much more rapidly than vitamin D3. In a subsequent study, pigs were given

vitamin D3, blood was collected and a lipid extraction followed by several

chromatography steps yielded a vitamin D3 metabolite that was about fourfold more

potent than vitamin D3 in stimulating intestinal calcium transport. It was structurally

identified as 25-hydroxyvitamin D3 [36]. This observation and the finding that the

hydroxylated metabolite took 12h to maximize intestinal calcium absorption in vitamin D

deficient rats [17] and supported the belief that 25(OH)D3 is the circulating active form of

vitamin D3. It was then reported [37] that a polar metabolite of vitamin D3 in the intestine

that was distinct from 25(OH)D3 also existed. The further modified metabolite 1,25-

dihydroxyvitamin D3 [1,25(OH)2D3, also known as calcitriol] was isolated from chicken

intestines, and the molecular attributes were revealed by mass spectrometry, ultraviolet

absorption spectrophotometry, and specific chemical reactions [38]. Once the structure

was discovered, it took 2 years and a 21 step synthesis to chemically synthesize

1,25(OH)2D3, yielding about 1mg final product out of 1kg starting materials [17]. The

configuration of the hydroxyl group on position 1 was discovered-1α,25(OH)2D3 (the

natural metabolite) and 1ß,25(OH)2D3 [39]. The hydroxylation of the 1α position is

strongly feedback-regulated and is one of the major endocrine systems regulating plasma

calcium and phosphorus concentrations.

Phosphate and Calcium Homeostasis

Homeostatic regulation of phosphate and calcium and their effects on the intestine,

kidneys and bone are maintained by several key hormones, which includes the vitamin D

metabolites, calcitonin, fibroblast growth factor-23 (FGF-23), and parathyroid hormone

(PTH) [40-42]. When serum phosphate or calcium levels are low, the parathyroid glands

synthesize PTH, which acts on the kidneys to decrease phosphate reabsorption, to

increase calcium reabsorption, and to stimulate synthesis of 1α-hydroxylase to convert

circulating 25(OH)D3 into 1,25(OH)2D3. Then 1,25(OH)2D3 travels through the blood

stream to enhance phosphate and calcium absorption in the intestine. Both PTH and

1,25(OH)2D3 simultaneously act on osteoclasts to induce bone resorption thereby

increasing circulating phosphate and calcium levels to normal ranges.

Abnormally high serum phosphate or calcium levels also trigger hormonal

regulation to maintain normal circulating ranges. Elevated phosphate levels stimulate

FGF-23 synthesis in bone [43, 44] which act on the kidneys to decrease 1,25(OH)2D3

synthesis by inhibiting 1α-hydroxylase activity, increasing phosphate excretion,

decreasing phosphate reabsorption which inhibits synthesis of phosphate transporters

[45]FGF-23 also acts by decreasing intestinal phosphate absorption which acts to

internalize phosphate transporters, and stimulate 24-hydroxylase activity to synthesize

24,25(OH)2D3 [46-49]. Elevated calcium levels stimulate calcitonin synthesis in the

thyroid glands [50, 51] which acts on bone to inhibit bone resorption [52, 53] and inhibit

PTH synthesis in the parathyroid glands. Inhibition of PTH synthesis allows increased

synthesis of 24-hydroxylase in the kidneys to convert 25(OH)D3 into 24,25(OH)2D3

which inhibits calcium transport in the intestine [40, 54]. Directly or indirectly,

25(OH)D3, calcitonin, FGF-23, and PTH act on 1,25(OH)2D3 and 24,25(OH)2D3

synthesis to regulate calcium or phosphate reabsorption and excretion in the kidneys,

resorption from bone, or absorption in the intestine to maintain homeostatic levels of both

microminerals.

Figure 1.1 Schematic presentation of phosphate homeostasis. The kidney converts 25- hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 when the organism is deficient in vitamin D, phosphate, and/or calcium. Hypocalcemia also signals the parathyroid glands to release parathyroid hormone (PTH), which stimulates 1α-hydroxylation of 25(OH)D3. 1,25(OH)2D3 stimulates absorption of minerals from the intestine and reabsorption from the kidney. Fibroblast growth factor 23 (FGF-23) inhibits the formation of 1,25(OH)2D3 and promotes phosphaturia. 24,25-dihydroxyvitamin D3, which blocks stimulated uptake of minerals from the intestine and PTH release from the parathyroid glands.

Figure 1. 1 Structure of vitamin D and its metabolites

Figure 1. 2 Regulation of PTH and 1,25(OH)2D3 in calcium homeostasis

25(OH)D3

25-hydroxyvitamin D3, a pre-hormone activated through the action of 25-

hydroxyvitamin D3 1 hydroxylase, was once thought to have no biological activity of its

own. However, evidence has been found to prove the opposite. Early reports suggested a

possible physiological role in egg hatchability [55, 56]. The metabolite is 1.4 times more

active than vitamin D3 in curing rickets in rats and chicks determined by the bone ash

assay [57]. It stimulates calcium transport in the intestine and bone resorption in vivo

more rapidly than cholecalciferol does [57]. A stimulation of bone resorption in tissue

culture in vitro was observed with 25(OH)D3 [58]. An enhancement of calcium transport

in isolated duodenal loops was observed after perfusion with 100 nM 25(OH)D3

compared with controls [59, 60]. 25(OH)D3 has also been observed to suppress

parathyroid hormone synthesis [61]. Gene expression analysis indicated that 25(OH)D3

induced 25-hydroxyvitamin D3 24-hydroxylase mRNA in a dose and time dependent

manner [62]. 25(OH)D3 has been shown to lower the risk of colorectal cancer [63], and

has been associated with increasing overall survival in early stage non-small cell lung

cancer (NSCLC) [64].

Effect of 1,25(OH)2D3 on Phosphate Absorption

1,25(OH)2D3, also known as calcitriol, stimulates the active transport of

phosphorus in the small intestine when plasma or serum phosphate levels are low. When

there is a vitamin D deficiency, phosphate transport is impaired and bone resorption

occurs. A continued deficiency results in hypophosphatemia, rickets or osteomalacia [45].

Studies [46, 65] have demonstrated that chick intestinal cells can be rapidly stimulated to

take up phosphate through a signal transduction pathway mediated by protein kinase C

(PKC). This mechanism can be activated by the 1,25D3-MARRS receptor, which is

identical to protein disulfide-isomerase A3 (PDIA3) [65]. In rats [66], membrane initiated

signaling relies on both cell surface VDR and the 1,25D3-MARRS receptor. 1,25(OH)2D3

was shown to enhance phosphate uptake within 1 min suggesting that stimulation of the

uptake process occurs through a pre-genomic pathway [67-70], not involving gene

regulation which usually occurs over a period of several hours to days. That led

researchers to identify a plasma membrane receptor to explain the rapid effects [71].

Membrane Receptors for 1,25(OH)2D3

In rat enterocytes, Lieberherr et al. [72] were the first to demonstrate the need for

a membrane receptor in mediating phosphoinositide metabolism. Traditionally,

1,25(OH)2D3 has been shown to act through a nuclear vitamin D receptor [73] (VDR).

Now, a membrane receptor called the 1,25(OH)2D3 membrane associated, rapid response

steroid-binding receptor (1,25D3-MARRS) has also been identified [40, 41, 71, 73]. The

role of this protein is to facilitate the rapid effects of 1,25(OH)2D3-mediated calcium and

phosphate absorption and uptake in isolated chick enterocytes and transport in perfused

duodenal loops [46, 74, 75]. The 1,25D3-MARRS receptor was subsequently reported to

be identical to ERp57/GR58/PDIA3 and has been characterized through prosite analysis

[74, 75] as containing several PKC and casein kinase phosphorylation sites, a Rel

homology domain (RHD), and two thioredoxin folds. The latter binding sites allow it to

interact with other proteins including catalase [65, 71].

Figure 1.4 Model for interaction between 1,25D3-MARRS receptor and catalase. Upon binding 1,25(OH)2D3, the 1,25(OH)2D3-MARRS receptor dimerizes and initiates signal transduction through PKA for enhanced calcium uptake and PKC for enhanced phosphate

uptake. Feedback inhibition of this stimulatory pathway is initiated when 24,25(OH)2D3

binds to catalase, thereby decreasing enzymatic activity. The increased H2O2 levels are known to inhibit stimulated ion uptake. Mechanistically, this occurs through oxidation of

the 1,25(OH)2D3-MARRS receptor to decrease 1,25(OH)2D3 binding, and subsequent signal transduction, as well as directly inhibiting hormone-stimulated PKC activity.

Metabolite Site of activation Proposed action Reference

Increased Ca2+ 25(OH)D3 Liver uptake in rat and [70, 76] chick duodena Increased phosphate uptake 1,25(OH) D Kidney [46] 2 3 and transport in intestine Decreased phosphate uptake 24,25(OH)2D3 Kidney in presence of [40] 1,25(OH)2D3 in intestine

Table 1. 1 Vitamin D metabolite action summary

Mechanism of action for 1,25(OH)2D3

The effects of 1,25(OH)2D3 are mediated through two mechanisms:

1.) Genomic actions – the cellular actions of this hormone are due to the binding of

the hormone to a nuclear vitamin D receptor (nVDR), and represents a slow

mechanism since it is involved with gene transcription and protein synthesis, for

example in bone matrix [77]. This ligand-receptor complex interacts with the

retinoid X receptor, and regulates the expression of approximately 500 genes [18].

The 1,25D3-MARRS receptor has also been found to translocate to the nucleus

upon binding hormone and associate with heterochromatin, suggesting gene

regulation [78].

2.) Pre-genomic or rapid response actions – there are some cellular responses that are

rapidly stimulated by 1,25(OH)2D3 and cannot be explained by the slow

mechanism via VDR. These actions are mediated through the 1,25D3-MARRS

receptor. The signal transduction pathway involved in the rapid stimulation of

mineral transport can be activated through protein kinase C, phospholipase C,

protein kinase A and mitogen activated protein kinase [79, 80].

Effect of 24,25(OH)2D3 on phosphate uptake

While 1,25(OH)2D3 stimulates the rapid transport of calcium and phosphate as

illustrated in Table 1, 24,25(OH)2D3 inhibits such stimulation [40, 46, 54]. Hormone-

stimulated phosphate uptake in chick intestinal cells is initiated by ligand binding to the

1,25D3-MARRS receptor and is inhibited by 24,25(OH)2D3. Since 24,25(OH)2D3 does

not compete for binding the 1,25D3-MARRS receptor [81], in order to elucidate how

24,25(OH)2D3 effects inhibition, a cellular binding protein with Kd = 7 nM for

24,25(OH)2D3 was purified [82] and sequenced and subsequently identified as catalase

[83]. The majority of binding material was vesicular [82] while specific binding was

found at the cell surface [84]. A functional consequence of 24,25(OH)2D3 binding to

catalase was found to be a decrease in enzyme activity, accompanied with an increase in

H2O2 production [85], beyond basal levels [86]. Oxidation of the 1,25D3-MARRS

3 receptor and inhibition of binding to [ H]1,25(OH)2D3 was found to be one possible

mechanism of inhibition [85]. Antioxidants were found to block the inhibitory actions of

24,25(OH)2D3, whereas pro-oxidants were found to mimic the inhibitory actions of the

steroid [48].

Membrane receptor for 24,25(OH)2D3

As stated previously, the metabolite 24,25(OH)2D3 is produced when an animal is

calcium, phosphate, and vitamin D replete, and represents an important feedback loop to

provide a protective mechanism against excess 1,25(OH)2D3 [40, 46, 87, 88]. Sequence

analysis of a 24,25(OH)2D3 binding protein was performed and five regions of homology

was found with enzyme catalase [83]. Studies were then undertaken to determine whether

24,25(OH)2D3 treatment would affect catalase activity. The results showed a time-

dependent decrease in catalase activity in isolated intestinal epithelial cells after the

addition of physiological levels of 24,25(OH)2D3 (6.5 nM), whereas 1,25(OH)2D3 did not

have a similar effect [85]. Another study was then performed to test whether pre-

incubation of cell suspensions with low concentration of hydrogen peroxide would inhibit

32 the rapid 1,25(OH)2D3-stimulated uptake of P. The results showed that after the

32 addition of 130 pM 1,25(OH)2D3, P uptake reached 135% of controls; whereas a 10-

min pre-incubation of cells with 50 µM H2O2 abolished the increase [85]. The effect of

hydrogen peroxide was shown to be reversible. The catalase inhibitor 3-amino-1,2,4-

triazole [89] mimicked the effects of 24,25(OH)2D3 since it blocked 1,25(OH)2D3-

stimulated phosphate uptake [90]. Pre-incubation of chicken enterocytes with either a

commercially available anti-catalase antibody [83] prevented the inhibitory actions of

24,25(OH)2D3 [48].

Mechanism of actions for 24,25(OH)2D3

One mechanism for the inhibitory effects of 24,25(OH)2D3 could be through

oxidation of the thioredoxin domains of the 1,25D3-MARRS receptor. The

3 24,25(OH)2D3-mediated decrease in [ H]1,25(OH)2D3 binding to MARRS was not

observed when incubated with the strong reducing agent dithiothreitol [65]. Nemere et al.

have previously reported that 24,25(OH)2D3 does not bind to the 1,25D3-MARRS

receptor [81]. Chicken enterocytes were incubated for 5, 10, 20 or 30 min with the

addition of a physiological level (6.5 nM) 24,25(OH)2D3. Cells were collected by

3 centrifugation, then homogenized, then incubated with [ H]1,25(OH)2D3 in the absence

(total binding) or presence (nonspecific binding) of a 200-fold molar excess of unlabeled

steroid. The result indicated no classical VDR binding detection [71]. Since hydrogen

peroxide could also influence the signal transduction pathway, isolated intestinal cells

were incubated with phorbol ester after pre-incubation with hydrogen peroxide. The

results showed that hydrogen peroxide inhibited the phorbol stimulation of 32P uptake and

PKC signaling in response to 1,25(OH)2D3 [91]. The ability of 24,25(OH)2D3 to decrease

catalase activity was determined by follow-up studies. A decrease in catalase specific

activity and catalase protein levels were observed after primary cultures of chick

enterocytes after exposure to 24,25(OH)2D3 for 5 h [92]. The model depicted in Figure

1.3 was formulated based on these observations and data.

Catalase

The discovery of catalase (H2O2: H2O2-oxidoreductase, EC 1.11.1.6) was first

hinted at 1811 when the French chemist who discovered hydrogen peroxide (H2O2),

Louis Jacques Thenard [93], suggested its breakdown is caused by an unknown substance.

In 1900, the German chemist Oscar Loew found catalase in both plants and animals, and

was the first to name it catalase [94]. In 1937 ， catalase from bovine liver was

crystallized by James B. Sumner and Alexander Dounce, achieving one of the first

successful crystallizations of an intracellular enzyme [95], and the molecular weight was

determined in 1938, catalase is a tetramer with a total molecular weight of approximately

24kDa. [96].

Physiological importance and molecular mechanism

Oxygen is necessary for life but also produces free radicals that can damage cell

membranes, proteins and DNA. Hydrogen peroxide is a naturally occurring but

destructive waste product of all oxygen-dependent organisms. It is produced in the

human body when fatty acids are converted to energy, and when white blood cells attack

and kill bacteria. The optimum pH for human catalase is approximately 7.0 [97], and can

dispose of five times more H2O2 than glutathione peroxidase [98]. Hydrogen peroxide

can freely cross membranes and generate the most reactive free radicals, hydroxyl

radicals [99]. Hydrogen peroxide produced by the glucose oxidation system and can

damage nuclear and mitochondrial DNAs [100]. Catalase, which is normally found in the

peroxisomes [101] of nearly all aerobic cells, prevents this naturally occurring and

constantly generating hydrogen peroxide from harming the cells during these processes

[102-104]. Catalase is a biological bi-functional catalyst, like superoxide dismutase (SOD)

and glutathione peroxidase, is produced naturally within the body. Catalase works

flawlessly with superoxide dismutase system to prevent free radical damages to cells,

preventing cell death. SOD converts the dangerous superoxide radical to hydrogen

peroxide, which catalase then rapidly converts to harmless water and oxygen [98].

Catalase is a tetrametric enzyme consisting of four identical subunits of 60 kDa,

each containing a heme group and a NADPH binding site [105, 106]. The ferric oxidation

state allows the enzyme to react with H2O2, and four tightly bound NADPH molecules to

prevent the enzyme from inactivation by H2O2 [106] [107]. Catalase exerts its functions

in two ways depending on the concentration of H2O2: 1) catalytic reaction 2H2O2 à2H2O

+ O2: decomposition of H2O2 to give H2O and O2 if the concentration of H2O2 is high; 2)

peroxidatic reaction ROOH + AH2 à H2O + ROH + A: oxidization of substrate if the

concentration of H2O2 is low and in the presence of a suitable hydrogen donor (i.e.,

ethanol, methanol, phenol) [108]. The peroxidase function is probably the result of gene

duplication of an ancestral peroxidase gene [109] and is found in bacteria and fungi [110].

The concentration of catalase varies in different tissues, and differs from species to

species. The highest concentration from tissues is found in the liver. A higher level of a

catalase substrate in the cell leads to higher levels of catalase. This gene is conserved in

human, chimpanzee, dog, mouse, rat, chicken, fruit fly, C. elegans and rice. It has been

theorized that one of the primary reasons cells age is the damage to DNA caused by free

radicals and oxidizing agents [111-114] such as hydrogen peroxide, and that elevating

levels of the body’s endogenous antioxidants – SOD, catalase, and glutathione peroxidase

– could both improve human health and increase human lifespan [115-118].

Hypothesis and Objectives

The overall hypothesis is 24R,25(OH)2D3 is the active metabolite in phosphate

homeostasis by using immunological techniques to determine the localization of catalase

and by monitoring catalase mRNA levels to determine if 24,25(OH)2D3 regulates gene

transcription.

Part I – Effect of 24,25-dihydroxyvitamin D3 on localization of catalase in chick

enterocytes.

Hypothesis: 24R,25(OH)2D3 is the effective metabolite for catalase redistribution as

judged by confocal microscopy.

Objectives:

1.) To determine the localization of catalase;

2.) To evaluate whether punctuate staining in the nucleus arose from the

redistribution of cell surface catalase.

Part II – Effects of vitamin D on catalase expression and phosphate uptake in

chicken intestinal cells.

32 Hypothesis: 24R,25(OH)2D3 is the effective metabolite for decreasing P uptake and

catalase gene expression.

Objectives:

32 1.) To determine the effects of 24R,25(OH)2D3 on P uptake in chick enterocytes;

2.) To determine whether 24R,25(OH)2D3 has any effect on catalase gene expression;

3.) To evaluate whether 24R,25(OH)2D3 has effect on catalase protein expression.

References

1. SJ, M., Vitamin D and other calciferols. The Metabolic and Molecular Bases of

Inherited Disease, 1995. 2: p. 2.

2. Mullin, G.E. and A. Dobs, Vitamin d and its role in cancer and immunity: a

prescription for sunlight. Nutr Clin Pract, 2007. 22(3): p. 305-22.

3. Chaplin, H.J., L.D. Clark, and M.W. Ropes, VITAMIN D INTOXICATION. The

American Journal of the Medical Sciences, 1951. 221(4): p. 369-378.

4. Hathcock, J.N., et al., Risk assessment for vitamin D. Am J Clin Nutr, 2007.

85(1): p. 6-18.

5. Mellanby, E., An experimental investigation on rickets. 1919. Nutrition, 1989.

5(2): p. 81-6; discussion 87.

6. K., H., Heilung von rachitis durch kunstlich hohensonne. Deut Med Wochenschr.,

1919. 45: p. 2.

7. McCollum EV, S.N., Becker JE, Shipley PG, An experimental demonstration of

the existence of a vitamin which promotes calcium deposition. J Biol Chem.,

1922. 53: p. 5.

8. Steenbock, H., The introduction of growth promoting and calcifying properties in

a ration by exposure to light. Science, 1924. 60(1549): p. 224-5.

9. Hess, A.F. and M. Weinstock, Antirachitic properties imparted to lettuce and to

growing wheat by ultraviolet irradiation. Experimental Biology and Medicine,

1924. 22(1): p. 5-6.

10. Holick, M.F., et al., The vitamin D content of fortified milk and infant formula. N

Engl J Med, 1992. 326(18): p. 1178-81.

11. Balasubramanian, S., Vitamin D deficiency in breastfed infants & the need for

routine vitamin D supplementation. Indian J Med Res, 2011. 133: p. 250-2.

12. Gloth, F.M., J.D. Tobin, Vitamin D deficiency in older people. J Am Geriatr Soc,

1995. 43(7): p. 822-8.

13. Harris, S.S., Vitamin D and African Americans. The Journal of Nutrition, 2006.

136(4): p. 1126-1129.

14. Lo, C.W., et al., Vitamin D absorption in healthy subjects and in patients with

intestinal malabsorption syndromes. Am J Clin Nutr, 1985. 42(4): p. 644-9.

15. Wortsman, J., et al., Decreased bioavailability of vitamin D in obesity. The

American Journal of Clinical Nutrition, 2000. 72(3): p. 690-693.

16. Raman, M., et al., Vitamin D and gastrointestinal diseases: inflammatory bowel

disease and colorectal cancer. Therap Adv Gastroenterol, 2011. 4(1): p. 49-62.

17. Holick, M.F., Vitamin D: A millenium perspective. J Cell Biochem, 2003. 88(2):

p. 296-307.

18. Norman, A.W., From vitamin D to hormone D: fundamentals of the vitamin D

endocrine system essential for good health. The American Journal of Clinical

Nutrition, 2008. 88(2): p. 491S-499S.

19. Holick, M.F., Vitamin D: importance in the prevention of cancers, type 1

diabetes, heart disease, and osteoporosis. Am J Clin Nutr, 2004. 79(3): p. 362-71.

20. LeBlanc, E.S., et al., Associations between 25-hydroxyvitamin D and weight gain

in elderly women. J Womens Health (Larchmt), 2012. 21(10): p. 1066-73.

21. Garland, C.F. and F.C. Garland, Do sunlight and vitamin D reduce the likelihood

of colon cancer? Int J Epidemiol, 1980. 9(3): p. 227-31.

22. Grant, W.B., Ecological studies of the UVB-vitamin D-cancer hypothesis.

Anticancer Res, 2012. 32(1): p. 223-36.

23. Fenderson, B.A., Molecular biology of the cell, 5TH EDITION. Shock, 2008.

30(1): p. 100.

24. DeLuca, H.F., The transformation of a vitamin into a hormone: the vitamin D

story. Harvey Lect, 1979. 75: p. 333-79.

25. Bikle, D., Nonclassic actions of vitamin D. J Clin Endocrinol Metab, 2009. 94(1):

p. 26-34.

26. Holick, M.F. and M.B. Clark, The photobiogenesis and metabolism of vitamin D.

Fed Proc, 1978. 37(12): p. 2567-74.

27. Lehmann, B. and M. Meurer, Vitamin D metabolism. Dermatol Ther, 2010. 23(1):

p. 2-12.

28. Jones, G., Pharmacokinetics of vitamin D toxicity. Am J Clin Nutr, 2008. 88(2): p.

582s-586s.

29. Miller, W.L. and A.A. Portale, Vitamin D biosynthesis and vitamin D 1 alpha-

hydroxylase deficiency. Endocr Dev, 2003. 6: p. 156-74.

30. Levine, B.S., et al., Pharmacokinetics and biologic effects of calcitriol in normal

humans. J Lab Clin Med, 1985. 105(2): p. 239-46.

31. Welsh, J., V. Weaver, and M. Simboli-Campbell, Regulation of renal 25(OH)D3 1

alpha-hydroxylase: signal transduction pathways. Biochem Cell Biol, 1991.

69(12): p. 768-70.

32. Omdahl, J.L., H.A. Morris, and B.K. May, Hydroxylase enzymes of the vitamin D

pathway: expression, function, and regulation. Annu Rev Nutr, 2002. 22: p. 139-

66.

33. Morii, H., et al., Biological activity of a vitamin D metabolite. Archives of

Biochemistry and Biophysics, 1967. 120(3): p. 508-512.

34. Lund, J. and H.F. DeLuca, Biologically active metabolite of vitamin D3 from

bone, liver, and blood serum. J Lipid Res, 1966. 7(6): p. 739-44.

35. Norman, A.W., J. Lund, and H.F. Deluca, Biologically active forms of vitamin D3

in kidney and intestine. Arch Biochem Biophys, 1964. 108: p. 12-21.

36. Blunt, J.W., H.F. DeLuca, and H.K. Schnoes, 25-Hydroxycholecalciferol. A

biologically active metabolite of vitamin D3. Biochemistry, 1968. 7(10): p. 3317-

3322.

37. Lawson, D.E., et al., Isolation of chick intestinal nuclei. Effect of vitamin D3 on

nuclear metabolism. Biochem J, 1969. 115(2): p. 263-8.

38. Holick, M.F. and H.F. DeLuca, A new chromatographic system for vitamin D3

and its metabolites: resoluation of a new vitamin D3 metabolite. J Lipid Res,

1971. 12(4): p. 460-5.

39. Paaren, H.E., et al., Direct C-1 hydroxylation of vitamin D compounds:

convenient preparation of 1alpha-hydroxyvitamin D3, 1alpha, 25-

dihydroxyvitamin D3, and 1alpha-hydroxyvitamin D2. Proc Natl Acad Sci U S A,

1978. 75(5): p. 2080-1.

40. Nemere, I., Apparent nonnuclear regulation of intestinal phosphate transport:

effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25-

hydroxyvitamin D3. Endocrinology, 1996. 137(6): p. 2254-61.

41. Khanal, R.C., et al., Membrane receptor-initiated signaling in 1,25(OH)2D3-

stimulated calcium uptake in intestinal epithelial cells. J Cell Biochem, 2008.

105(4): p. 1109-16.

42. Carpenter, T.O., et al., 24,25 Dihydroxyvitamin D supplementation corrects

hyperparathyroidism and improves skeletal abnormalities in X-linked

hypophosphatemic rickets--a clinical research center study. J Clin Endocrinol

Metab, 1996. 81(6): p. 2381-8.

43. Mirams, M., et al., Bone as a source of FGF23: regulation by phosphate? Bone,

2004. 35(5): p. 1192-1199.

44. Yoshiko, Y., et al., Mineralized tissue cells are a principal source of FGF23.

Bone, 2007. 40(6): p. 1565-73.

45. Hruska, K.A., et al., Hyperphosphatemia of chronic kidney disease. Kidney Int,

2008. 74(2): p. 148-157.

46. Zhao, B. and I. Nemere, 1,25(OH)2D3-mediated phosphate uptake in isolated

chick intestinal cells: effect of 24,25(OH)2D3, signal transduction activators, and

age. J Cell Biochem, 2002. 86(3): p. 497-508.

47. Shimada, T., et al., Vitamin D receptor-independent FGF23 actions in regulating

phosphate and vitamin D metabolism. Am J Physiol Renal Physiol, 2005. 289(5):

p. F1088-95.

48. Peery, S.L. and I. Nemere, Contributions of pro-oxidant and anti-oxidant

conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-

dihydroxyvitamin D3 on phosphate uptake in intestinal cells. J Cell Biochem,

2007. 101(5): p. 1176-84.

49. Shimada, T., et al., FGF-23 is a potent regulator of vitamin D metabolism and

phosphate homeostasis. J Bone Miner Res, 2004. 19(3): p. 429-35.

50. Gropper SS, S.J., Groff JL, Advanced Nutrition and Human Metabolism. 4th ed.

2005, Belmont, CA: Wadsworth.

51. Widmaier EP, R.H., Strang KT, Vander, Sherman, & Luciano's Human

Physiology: The Mechanisms of Body Function. 9th ed. 2004, New York, NY:

McGrawHill.

52. Suzuki, H., et al., Calcitonin-induced changes in the cytoskeleton are mediated by

a signal pathway associated with protein kinase A in osteoclasts. Endocrinology,

1996. 137(11): p. 4685-90.

53. Chambers, T.J. and A. Moore, The sensitivity of isolated osteoclasts to

morphological transformation by calcitonin. J Clin Endocrinol Metab, 1983.

57(4): p. 819-24.

54. Nemere, I., 24,25-dihydroxyvitamin D3 suppresses the rapid actions of 1, 25-

dihydroxyvitamin D3 and parathyroid hormone on calcium transport in chick

intestine. J Bone Miner Res, 1999. 14(9): p. 1543-9.

55. Ameenuddin, S., et al., 24-hydroxylation of 25-hydroxyvitamin D3: is it required

for embryonic development in chicks? Science, 1982. 217(4558): p. 451-2.

56. Hart, L.E. and H.F. DeLuca, Effect of vitamin D3 metabolites on calcium and

phosphorus metabolism in chick embryos. Am J Physiol, 1985. 248(3 Pt 1): p.

E281-5.

57. Blunt, J.W., H.F. DeLuca, and H.K. Schnoes, 25-Hydroxycholecalciferol: a

biologically active metabolite of cholecalciferol. Chemical Communications

(London), 1968(14): p. 801-801.

58. Trummel, C.L., et al., 25-Hydroxycholecalciferol: stimulation of bone resorption

in tissue culture. Science, 1969. 163(3874): p. 1450-1.

59. Nemere, I., Y. Yoshimoto, and A.W. Norman, Calcium transport in perfused

duodena from normal chicks: enhancement within fourteen minutes of exposure to

1,25-dihydroxyvitamin D3. Endocrinology, 1984. 115(4): p. 1476-83.

60. Phadnis, R. and I. Nemere, Direct, rapid effects of 25-hydroxyvitamin D3 on

isolated intestinal cells. J Cell Biochem, 2003. 90(2): p. 287-93.

61. Ritter, C.S., et al., 25-Hydroxyvitamin D3 suppresses PTH synthesis and secretion

by bovine parathyroid cells. Kidney Int, 2006. 70(4): p. 654-9.

62. Lou, Y.R., et al., 25-hydroxyvitamin D3 is an active hormone in human primary

prostatic stromal cells. FASEB J, 2004. 18(2): p. 332-4.

63. Song, M., et al., Plasma 25-hydroxyvitamin D and colorectal cancer risk

according to tumour immunity status. Gut, 2015.

64. Verone, A.R., et al., Abstract 4751: 25-hydroxyvitamin D3 induces vitamin D

signaling independent of CYP27B1 in non-small cell lung cancer cells. Cancer

Research, 2014. 74(19 Supplement): p. 4751.

65. Nemere, I., et al., Identification and characterization of 1,25D3-membrane-

associated rapid response, steroid (1,25D3-MARRS) binding protein. J Steroid

Biochem Mol Biol, 2004. 89-90(1-5): p. 281-5.

66. Nemere, I., The 1,25D3-MARRS protein: contribution to steroid stimulated

calcium uptake in chicks and rats. Steroids, 2005. 70(5-7): p. 455-7.

67. Nemere, I. and C.M. Szego, Early actions of parathyroid hormone and 1,25-

dihydroxycholecalciferol on isolated epithelial cells from rat intestine: II.

Analyses of additivity, contribution of calcium, and modulatory influence of

indomethacin. Endocrinology, 1981. 109(6): p. 2180-7.

68. Nemere, I. and C.M. Szego, Early actions of parathyroid hormone and 1,25-

dihydroxycholecalciferol on isolated epithelial cells from rat intestine: I. Limited

lysosomal enzyme release and calcium uptake. Endocrinology, 1981. 108(4): p.

1450-62.

69. Lieberherr, M., et al., Phosphatase content of rat calvaria after in vivo

administration of vitamin D3 metabolites. Calcified Tissue Research, 1977. 23(1):

p. 235-239.

70. Sterling, T.M. and I. Nemere, 1,25-dihydroxyvitamin D3 stimulates vesicular

transport within 5 s in polarized intestinal epithelial cells. J Endocrinol, 2005.

185(1): p. 81-91.

71. Nemere, I., et al., Ribozyme knockdown functionally links a 1,25(OH)2D3

membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal

cells. Proc Natl Acad Sci U S A, 2004. 101(19): p. 7392-7.

72. Lieberherr, M., et al., A functional cell surface type receptor is required for the

early action of 1,25-dihydroxyvitamin D3 on the phosphoinositide metabolism in

rat enterocytes. J Biol Chem, 1989. 264(34): p. 20403-6.

73. Haussler, M.R., et al., The nuclear vitamin D receptor: biological and molecular

regulatory properties revealed. J Bone Miner Res, 1998. 13(3): p. 325-49.

74. Larsson, B. and I. Nemere, Effect of growth and maturation on membrane-

initiated actions of 1,25-dihydroxyvitamin D3. I. Calcium transport, receptor

kinetics, and signal transduction in intestine of male chickens. Endocrinology,

2003. 144(5): p. 1726-35.

75. Larsson, B. and I. Nemere, Effect of growth and maturation on membrane-

initiated actions of 1,25-dihydroxyvitamin D3-II: calcium transport, receptor

kinetics, and signal transduction in intestine of female chickens. J Cell Biochem,

2003. 90(5): p. 901-13.

76. Olson, E.B. and H.F. DeLuca, 25-hydroxycholecalciferol: direct effect on calcium

transport. Science, 1969. 165(3891): p. 405-7.

77. Christakos, S., et al., Vitamin D and Intestinal Calcium Absorption. Molecular

and Cellular Endocrinology, 2011. 347(1-2): p. 25-29.

78. Nemere, I. and K. Campbell, Immunochemical studies on the putative

plasmalemmal receptor for 1, 25-dihydroxyvitamin D3. III. Vitamin D status.

Steroids, 2000. 65(8): p. 451-7.

79. Farach-Carson, M.C. and I. Nemere, Membrane receptors for vitamin D steroid

hormones: potential new drug targets. Curr Drug Targets, 2003. 4(1): p. 67-76.

80. de Boland, A.R. and A.W. Norman, 1alpha,25(OH)2-vitamin D3 signaling in

chick enterocytes: enhancement of tyrosine phosphorylation and rapid stimulation

of mitogen-activated protein (MAP) kinase. J Cell Biochem, 1998. 69(4): p. 470-

82.

81. Nemere, I., et al., Identification of a specific binding protein for 1 alpha,25-

dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium

and relationship to transcaltachia. J Biol Chem, 1994. 269(38): p. 23750-6.

82. Nemere, I., et al., Biochemical characterization and purification of a binding

protein for 24,25-dihydroxyvitamin D3 from chick intestine. J Endocrinol, 2002.

172(1): p. 211-9.

83. Larsson, D., et al., 24,25-dihydroxyvitamin D3 binds to catalase. J Cell Biochem,

2006. 97(6): p. 1259-66.

84. Watanabe, N., et al., Bio-effectiveness of Tat-catalase conjugate: a potential tool

for the identification of H2O2-dependent cellular signal transduction pathways.

Biochem Biophys Res Commun, 2003. 303(1): p. 287-93.

85. Nemere, I., et al., Mechanism of 24,25-dihydroxyvitamin D3-mediated inhibition

of rapid, 1,25-dihydroxyvitamin D3-induced responses: role of reactive oxygen

species. J Cell Biochem, 2006. 99(6): p. 1572-81.

86. Ha, E.M., et al., A direct role for dual oxidase in Drosophila gut immunity.

Science, 2005. 310(5749): p. 847-50.

87. Sundell, K. and B.T. Bjornsson, Effects of vitamin D3, 25(OH) vitamin D3,

24,25(OH)2 vitamin D3, and 1,25(OH)2 vitamin D3 on the in vitro intestinal

calcium absorption in the marine teleost, Atlantic cod (Gadus morhua). Gen

Comp Endocrinol, 1990. 78(1): p. 74-9.

88. Larsson, D., B.T. Bjornsson, and K. Sundell, Physiological concentrations of

24,25-dihydroxyvitamin D3 rapidly decrease the in vitro intestinal calcium uptake

in the Atlantic cod, Gadus morhua. Gen Comp Endocrinol, 1995. 100(2): p. 211-

89. Chuang, S.M., et al., Short-term depletion of catalase suppresses cadmium-

elicited c-Jun N-terminal kinase activation and apoptosis: role of protein

phosphatases. Carcinogenesis, 2003. 24(1): p. 7-15.

90. Seo, E.G. and A.W. Norman, Three-fold induction of renal 25-hydroxyvitamin

D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3

levels are correlated with the healing process after chick tibial fracture. J Bone

Miner Res, 1997. 12(4): p. 598-606.

91. Nemere, I., et al., Intestinal cell phosphate uptake and the targeted knockout of

the 1,25D3-MARRS receptor/PDIA3/ERp57. Endocrinology, 2012. 153(4): p.

1609-15.

92. Khanal, R.C., N.M. Smith, and I. Nemere, Phosphate uptake in chick kidney cells:

effects of 1,25(OH)2D3 and 24,25(OH)2D3. Steroids, 2007. 72(2): p. 158-64.

93. J.W.Mellor, A Comprehensive Treatise on Inorganic and Theoretical Chemistry.

Longmans Green, London, 1924. Vol.1: p. 878.

94. Loew, O., A new enzyme of general occurrence in organisms. Science, 1900.

11(279): p. 701-2.

95. Sumner, J.B. and A.L. Dounce, Crystalline catalase. Journal of Biological

Chemistry, 1937. 121(2): p. 417-424.

96. Sumner, J.B. and N. Gralén, The molecular weight of crystalline catalase. Journal

of Biological Chemistry, 1938. 125(1): p. 33-36.

97. Maehly, A.C., The Assay of Catalases and Peroxidases, in Methods of

Biochemical Analysis. 2006, John Wiley & Sons, Inc. p. 357-424.

98. Gaetani, G.F., et al., Predominant role of catalase in the disposal of hydrogen

peroxide within human erythrocytes. Blood, 1996. 87(4): p. 1595-9.

99. Halliwell, B. and J.M. Gutteridge, Oxygen toxicity, oxygen radicals, transition

metals and disease. Biochemical Journal, 1984. 219(1): p. 1-14.

100. Salazar, J.J. and B. Van Houten, Preferential mitochondrial DNA injury caused

by glucose oxidase as a steady generator of hydrogen peroxide in human

fibroblasts. Mutat Res, 1997. 385(2): p. 139-49.

101. Alberts B, J.A., Lewis J, Raff M, Roberts K, Walter P, Peroxisomes. Molecular

Biology of the Cell (4th ed), 2002.

102. Derevianko, A., et al., Endogenous PMN-derived reactive oxygen intermediates

provide feedback regulation on respiratory burst signal transduction. J Leukoc

Biol, 1997. 62(2): p. 268-76.

103. Chelikani, P., I. Fita, and P.C. Loewen, Diversity of structures and properties

among catalases. Cell Mol Life Sci, 2004. 61(2): p. 192-208.

104. Evans, C.A.L., On the Catalytic Decomposition of Hydrogen Peroxide by the

Catalase of Blood. Biochemical Journal, 1907. 2(4): p. 133-155.

105. Scibior, D. and H. Czeczot, [Catalase: structure, properties, functions]. Postepy

Hig Med Dosw (Online), 2006. 60: p. 170-80.

106. Kirkman, H.N. and G.F. Gaetani, Catalase: a tetrameric enzyme with four tightly

bound molecules of NADPH. Proc Natl Acad Sci U S A, 1984. 81(14): p. 4343-7.

107. Kirkman, H.N., et al., Mechanisms of protection of catalase by NADPH. Kinetics

and stoichiometry. J Biol Chem, 1999. 274(20): p. 13908-14.

108. Aebi, H., Catalase in vitro. Methods Enzymol, 1984. 105: p. 121-6.

109. Welinder, K.G., Bacterial catalase-peroxidases are gene duplicated members of

the plant peroxidase superfamily. Biochim Biophys Acta, 1991. 1080(3): p. 215-

20.

110. Klotz, M.G. and P.C. Loewen, The Molecular Evolution of Catalatic

Hydroperoxidases: Evidence for Multiple Lateral Transfer of Genes Between

Prokaryota and from Bacteria into Eukaryota. Molecular Biology and Evolution,

2003. 20(7): p. 1098-1112.

111. Rao, K.S., Free radical induced oxidative damage to DNA: relation to brain

aging and neurological disorders. Indian J Biochem Biophys, 2009. 46(1): p. 9-

15.

112. Dizdaroglu, M., et al., Free radical-induced damage to DNA: mechanisms and

measurement. Free Radic Biol Med, 2002. 32(11): p. 1102-15.

113. Dizdaroglu, M. and P. Jaruga, Mechanisms of free radical-induced damage to

DNA. Free Radic Res, 2012. 46(4): p. 382-419.

114. Floyd, R.A. and J.M. Carney, Free radical damage to protein and DNA:

mechanisms involved and relevant observations on brain undergoing oxidative

stress. Ann Neurol, 1992. 32 Suppl: p. S22-7.

115. Harman, D., Free radical theory of aging: an update: increasing the functional

life span. Ann N Y Acad Sci, 2006. 1067: p. 10-21.

116. Harman, D., Free radical involvement in aging. Pathophysiology and therapeutic

implications. Drugs Aging, 1993. 3(1): p. 60-80.

117. Taub, J., et al., A cytosolic catalase is needed to extend adult lifespan in C.

elegans daf-C and clk-1 mutants. Nature, 1999. 399(6732): p. 162-166.

118. Hackenberg, T., et al., Catalase and no catalase activity 1 Promote Autophagy-

Dependent Cell Death in Arabidopsis. The Plant Cell, 2013. 25(11): p. 4616-

4626.

CHAPTER 2

EFFECT OF 24,25-DIHYDROXYVITAMIN D3 ON LOCALIZATION OF CATALASE IN CHICK ENTEROCYTES

Abstract

The vitamin D metabolite, 24,25(OH)2D3 has been reported to have hormonal

activity. Catalase has been reported to be a binding protein for 24,25(OH)2D3, based on

sequence analysis of the protein isolated on the basis of specific binding of the

metabolite. In the current work, we report that 24R,25(OH)2D3, not 24S,25(OH)2D3 is the

effective metabolite for catalase redistribution as judged by confocal microscopy. We

have used male chick intestinal cells treated with either vehicle, 24R,25(OH)2D3,

24S,25(OH)2D3 or 1,25(OH)2D3 to determine the localization of catalase. Confocal

microscopy analyses showed punctate staining, on the cell surface and in the cytoplasm

of cells treated with vehicle, 24R,25(OH)2D3, 24S,25(OH)2D3 or 1,25(OH)2D3 for all

time points tested. Cells treated with 24R,25(OH)2D3 showed punctuate staining of

catalase inside the nucleus. Western analysis confirmed that the punctuate staining in the

nucleus arose from the redistribution of cell surface catalase. Western analysis also

indicated 24S,25(OH)2D3 treatment resulted in redistribution of catalase to the nucleus,

but to a lesser extent than treatment with 24R,25(OH)2D3. By understanding the

molecular and cellular actions of 24,25(OH)2D3 in chick intestine, progress will be made

in enhancing phosphate and calcium absorption in animals to supply the minerals for

adequate bone growth, and phosphate in manure of production animals could be

diminished.

Introduction

Vitamin D was discovered in 1922 and has been categorized as a pre-hormone

ever since, based on the fact that the utilization of most vitamin D in higher animals

undergoes a photochemical process. Activation of vitamin D starts in the liver with the

production of major circulating metabolite 25-hydroxyvitamin D3 [25(OH)D3], followed

by hydroxylation in the kidneys to yield two metabolites, either 1,25-dihydroxyvitamin

D3 [1,25(OH)2D3] made when phosphate and calcium levels are low or 24,25-

dihydroxyvitamin D3 [24,25(OH)2D3] made when phosphate and calcium levels are high.

24,25-dihydroxyvitamin D3 is no longer considered an inactive hormone. Earlier studies

showed that in order to reach normal chick hatchability [1] and bone formation [2], both

1,25(OH)2D3 and 24,25(OH)2D3 were necessary. While 1,25-dihydroxyvitamin D3

stimulates the rapid transport of calcium and phosphate in both perfused chick duodenal

loops and isolated enterocytes, 24,25-dihydroxyvitamin D3 inhibits such stimulation [3,

4]. In osteoblasts and osteosarcoma cells, 1,25(OH)2D3 has been found to have a rapid

effect on calcium-channel-opening, while 24,25(OH)2D3 was found to inhibit this non-

nuclear effect [5-7]. In chick intestinal cells, 1,25(OH)2D3 has been found to have an

acute, non-nuclear effect on phosphate transport, and 24,25(OH)2D3 has been found to

inhibit the reaction [8], with no effect on parathyroid hormone (PTH) stimulated

phosphate transport [9]. In perfused chick duodenal loops, 24,25(OH)2D3 inhibits the

rapid stimulation of phosphate transport [8] mediated by 1,25(OH)2D3, as well as calcium

transport [10].

In chick intestinal cells, hormone-stimulated phosphate uptake is initiated by

ligand binding to the 1,25D3-membrane associated, rapid response steroid-binding

receptor – 1,25D3-MARRS [11], also known as ERp57/GR58/PDIA3. The ability of

24,25(OH)2D3 to inhibit 1,25D3-MARRS receptor activation of protein kinase A and C

activities was found in chick intestine [12] and kidney [4], which indicates the existence

of a specific binding protein for 24,25(OH)2D3. Percoll gradient analysis revealed

3 lysosomal fractions to have the highest [ H]24,25(OH)2D3 binding activity [10]. In order

to explain how 24,25(OH)2D3 works to affect inhibition, a cellular binding protein (66

kDa) was isolated, purified [10] and sequenced [13]. It was found to have a binding

constant of 7 nM for 24,25(OH)2D3, and identified as catalase using Edman degradation

techniques [13]. The enzyme catalase is sensitive to cell signaling molecules [14]. It was

found that the inhibitory action of 24,25(OH)2D3 is caused by a decrease in catalase

activity in both chick intestine [15, 16] and kidney [12], accompanied by an increase in

H2O2 production [13]. Thus, one possible mechanism for the inhibitory action would be

the oxidation of thioredoxin domains in 1,25D3-MARRS receptor that occurred after 10

min of exposure [15], accompanied by a loss of binding activity. However, studies [15]

showed a time-dependent decrease with either 24,25(OH)2D3 or H2O2 treatments after 5

minutes of incubation, indicating another mechanism because 24,25(OH)2D3 does not

compete with 1,25(OH)2D3 for MARRS binding.

In intestinal cells, studies showed 1,25D3-stimulated phosphate uptake is

mediated by PKC signaling [3], and 24,25(OH)2D3 seems to abolish that effect [10].

Thus, another possible mechanism would be the affecting the signal transduction pathway

[17].

In this study, chick enterocytes were used to determine the localization of catalase

in response to different vitamin D steroid hormones. Western analysis was performed to

verify the results from confocal microscopy.

Experimental

Animals

All surgical procedures were approved by Utah State University Institutional

Animal Use and Care Committee. White leghorn cockerels were obtained on the day of

hatch (Privett Hatchery, Portales, NM) and raised on a commercially available vitamin D-

replete diet (Nutrena Feeds, Murray, UT) generally for 3-7 weeks prior to

experimentation. On the day of use, chicks were anesthetized with anhydrous ethyl ether

(Fisher Scientific, Fair Lawn, NJ). The abdominal cavity was surgically opened and the

duodenal loop was removed to ice-cold 0.9% saline solution and chilled for 15 min. The

pancreas was excised from the duodenal loop and discarded. The duodenal loop was

everted and rinsed three times in chilled saline solution.

The chick intestinal cells were isolated with citrate chelation media (96 mM

NaCl, 27 mM Citrate Anhydrous, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na2HPO4, pH

5.0 - the acidic pH promotes viability and retention of morphology) [3, 18]. The cells

were collected by low speed centrifugation (500 x g, 5 min, 4ºC), and cell pellets were

resuspended in a small volume of Gey’s balanced salt solution (GBSS, containing 119

mM NaCl, 4.96 mM KCl, 0.22 mM KH2PO4, 0.84 mM NaHPO4, 1.03 mM MgCl2•6H2O,

0.28 mM MgSO4•7H2O, 0.9 mM CaCl2, pH 7.3). Aliquots of the cell suspension (0.4 ml)

were pipetted into 35 mm-plastic Petri dishes (Falcon, Scientific Products; Franklin

Lakes, NJ) containing 1.5 ml of RPMI 1640 medium and antibiotics (100 units/ml

penicillin, 100 mg/ml streptomycin, Sigma Chemical Co, St. Louis, Mo). The cells were

incubated overnight in the absence of serum at 37ºC with 5% CO2/ 95% air to promote

cell adhesion.

Confocal Microscopy

The following morning, media were replaced with 0.1% BSA in GBSS (GBSS-

BSA, Bovine Serum Albumin, Sigma, St. Louis, MO) and cells treated either with

vehicle (0.01% ethanol) or hormone for 15 sec to 60 min (15s, 30s, 7 min, 10 min, 15

min, 25 min, 30 min, 40 min, 50 min, 60 min). At the end of each time point, media were

replaced with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), 3%

sucrose in PBS (phosphate buffered saline) and fixed for 30 min. After washing with

0.1% PBS-BSA, cells were incubated with 0.05% NaBH4 in PBS for 5 min to eliminate

auto-fluorescence in the brush border, then washed and permeabilized with 0.15% Triton

X-100 in PBS for 5 min for intracellular catalase immunoreactivity, or without Triton X-

100 for surface catalase immunoreactivity. After washing with 0.1% PBS-BSA, cells

were overlaid with normal rabbit serum (JacksonImmuno Research, West Grove, PA) for

5 min and then washed. Coverslips were then overlaid with primary antibody Ab365 (a

highly specific polyclonal antibody, Center for Integrated BioSystems, Logan, UT) for 30

min (1/1000 in PBS-0.1% BSA), and then incubated for another 30 min (after addition of

more PBS-BSA to prevent drying), washed, and then overlaid with fluorescently-tagged

secondary antibody Alexa Fluor 594 (with excitation at 591 nm and emission at 614 nm)

(JacksonImmuno Research, West Grove, PA) and Phalloidin (Sigma-Aldrich, St. Louis,

MO) labeled with fluorescein isothiocyanate (with excitation at 495 nm and emission at

513 nm) for 30 min. Coverslips were then washed three times. After the final wash, the

coverslips were placed over mounting media (10% 1 M Tris, 80% glycerol) on a

microscope slide, and sealed for subsequent confocal microscopy analysis. A Nikon TE-

200 microscope (BioRad) was used for confocal imaging. Images were collected with

ZEN software, using a 60x oil immersion objective and further processed with ImageJ

and Adobe Photoshop CS5.

Western Blots

The isolated intestinal cells described above were collected by centrifugation at

500 x g, 5 min (4ºC), and resuspended in 30 ml of GBSS. 5 ml of the cell suspensions

were combined with test substance in GBSS to give a final concentration of 0.01%

ethanol, 6.5 nM 24R,25(OH)2D3 or 200 pM 24S,25(OH)2D3. The cells were incubated for

10 minutes and 25 minutes, then 1 ml were removed to 10 ml of ice-cold PBS for

cytoplasmic and nuclear extraction. The extraction procedure used a nuclear extract kit

(Active Motif, Carlsbad, CA) according to the instruction manual, which involves mixing

with a series of detergents in the presence of protease inhibitors.

SDS-PAGE and western blot analyses were used to determine immunoreactive

levels of catalase in control and vitamin D treated cells. Protein was determined with the

Bradford reagent (Bio-Rad, Hercules, CA) and then samples (5-30µg/well) were

separated on 8% (wt/vol) SDS-PAGE gels with 5% stacking gels. After separation on

SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes

(Immobilon-P, Fisher Scientific) by the use of a TransBlot SD Semidry transfer cell (Bio-

Rad.) and western analyses were performed. To avoid nonspecific binding, the membrane

was soaked for 1 hr at 37ºC in blocking solution (0.5% nonfat dry milk in phosphate

buffered saline (PBS; 0.9% NaCl and 10 mM Na2HPO4, pH 7.4), followed by washing

three times for 5 min each time with washing solution (0.1%(wt/vol) BSA in Tris-

buffered saline (TBS; 0.9% NaCl in 20 mM Tris-HCl, pH 7.4), and incubation with

primary antibody (Abcam Inc., Cambridge, MA) overnight at 4ºC in1%BSA and 0.05%

Tween 20 in TBS. After three additional washes, the membrane was incubated with

secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG) in the

antibody incubation buffer described above for 2 hr at room temperature and then washed

as previously indicated. Immunoreactive bands were visualized with the chromogens 5-

bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium, and relative amounts of

catalase were quantitated using Adobe Photoshop.

Results

Effect of 24,25(OH)2D3 on catalase redistribution in isolated chick enterocytes

In isolated chick enterocytes, 130 pM 1,25(OH)2D3 has been shown to stimulate

phosphate uptake after 5 minutes [3] and 24,25(OH)2D3 was reported to abolish that

effect [19]. In this study, time course experiments on catalase localization in isolated cells

were compared among ethanol control, 1,25(OH)2D3 and 24,25(OH)2D3 treatments. In

initial experiments, catalase localization by confocal microscopy was determined by

adding hormones before primary antibody Ab365 to allow potential redistribution of the

receptor. Chick intestinal cells were cultured in petri dishes, treated with 0.01% ethanol,

6.5nM 24R,25(OH)2D3, 200pM 24S,25(OH)2D3, or 360pM 1,25(OH)2D3, concentrations

that have been shown to be equivalent to circulating levels[20], for selected times (15 sec,

30 sec, 7 min, 10 min, 15 min, 25 min, 30 min, 40 min, 50 min, 60 min), and fixed for

confocal microscopy. Red staining depicts Alexa Fluor 594 fluorescence, which is

indicative of catalase localization on the cell surface. Figure 2.1A shows shorter

hormone/ethanol incubation time points and Figure 2.1B shows longer hormone/ethanol

incubation time points. Control cells treated with 0.01% ethanol for selected times

revealed obvious surface staining, with a low level inside the nucleus. These observations

in this and subsequent experiments were reproduced in triplicate experiments. These

results are shown quantitatively in Figure 2.1C, and indicate no significant differences

with time or a slight increase in redistribution was seen with time. Figure 2.2 depicts

results from experiments in which cells were treated with 24R,25(OH)2D3 for shorter

(Figure 2.2A) and longer (Figure 2.2B) time points, in which there were obvious

increases in nuclear staining (indicated by yellow arrows) as well as surface staining

relative to controls. Quantification of nuclear staining is depicted in Figure 2.2C.

Significant differences were observed at 15 seconds after hormone treatment. Figure 2.3

depicts results from experiments in which cells were treated with 24S, 25(OH)2D3 for

shorter (Figure 2.3A) and longer (Figure 2.3B) time points, in which there were slight

increases in nuclear staining relative to controls, but to a much lesser extent compared to

24R,25(OH)2D3 treatment. Figure 2.3C provides quantification of the data, and illustrates

that 24S,25(OH)2D3 treatment caused 100%-111% change in nuclear staining, relative to

cells treated with 24R,25(OH)2D3. Figure 2.4 depicts results from experiments in which

cells were treated with 1, 25(OH)2D3 for shorter (Figure 2.4A) and longer (Figure 2.4B)

time points, in which there were no increases in nuclear staining nor surface staining

relative to controls, since 1,25(OH)2D3 does not compete with 24R,25(OH)2D3 for

binding to catalase. These results are shown quantitatively in Fig. 2.4C. In a previous

study, it was found that 24R,25(OH)2D3 is capable of decreasing phosphate absorption

after a 1-h injection in vivo and 24S,25(OH)2D3 is capable of increasing phosphate

absorption after a 5-h injection in vivo, suggesting that the inhibitory effect might be

mainly performed by 24R,25(OH)2D3 [21]. One possible gene regulatory mechanism

could be catalase binding to STAT3 [22]. Further study is required to see if STAT3 is a

binding partner of catalase.

The question was raised as to the origin of the nuclear staining. In order to answer

that, additional confocal microscopy experiments were undertaken to determine whether

cell surface catalase was the source of steroid-mediated nuclear redistribution. In these

experiments, primary antibody Ab 365 was first added to cells for 30 min, and

subsequently incubated with ethanol vehicle, 1,25(OH)2D3, 24S,25(OH)2D3, or

24R,25(OH)2D3. Figure 2.5 depicts control cells treated with 0.01% ethanol for shorter

(Figure 2.5A) and longer (Figure 2.5B) time points revealed obvious surface staining, but

little inside the nucleus. As shown in Figure 2.6A and B, nuclear redistribution after

24R,25(OH)2D3 treatment did not occur, suggesting that ligand binding to cell surface

catalase induced redistribution to the nucleus, but was blocked by the primary antibody.

Similarly, treatment of cells with addition of primary antibody before hormone treatment

inhibited the effects of 24S,25(OH)2D3 (Figure 2.7A, and 2.7B), and as expected, no

effect of 1,25(OH)2D3 treatment on catalase localization was found (Figure 2.8A and

2.8B).

To further elucidate the observations above, catalase-staining intensity inside the

nucleus was quantified with ImageJ and Adobe Photoshop CS5 software. Figure 2.9

depicts comparisons among ‘hormones first treatments’—in which cells were treated with

hormones/ethanol vehicle prior to primary antibody Ab 365. There was a significant

increase (P<0.01) in the intensity within the 24R,25(OH)2D3 treated group, a smaller

increase (P<0.05) in the intensity within the 24S,25(OH)2D3 treated group was also

observed, while the other groups stayed the same. Figure 2.10 depicts a comparison

among cells treated with primary antibody Ab 365 prior to hormones/ethanol, or

‘antibody first treatments’. While blocking the cell surface catalase, there was no obvious

increase in the nuclear staining intensity among the four groups, indicating that the

nuclear redistribution mediated by 24R,25(OH)2D3 is indeed from cell surface catalase.

An independent approach was taken to verify these findings. In these experiments,

cells were treated with either ethanol vehicle, 24S,25(OH)2D3, or 24R,25(OH)2D3 for 10

and 25 min, collected by centrifugation, and resuspended in homogenization buffer for

subcellular fractionation. Aliquots of P1 (nuclei, brush borders, and unbroken cells), P2

(peroxisomes, lysosomes, mitochondria, Golgi, and basal lateral membranes, and S2

(microsomes and cytosol), were subjected to SDS-polyacrylamide gel electrophoresis,

followed by western blotting. The results reproducibly showed more nuclear

redistribution after 24R,25(OH)2D3 treatment compared to 24S,25(OH)2D3 (Figure 2.11).

One possible explanation might be there was a higher concentration of 24R,25(OH)2D3 in

the incubations than 24S,25(OH)2D3. Catalase bands intensity were then quantified using

ImageJ software. Figure 2.12 indicates a significant increase (P<0.01) in catalase band

intensity among 24R,25(OH)2D3 treatments. However, the fact that redistribution from

cell surface and organelles to the nucleus following 24S,25(OH)2D3 was detected by

western analysis indicates that confocal microscopy is the less sensitive method. The

results shown in Figure 2.11 also indicate that the commercially available antibody is less

specific than Ab 365 [17].

Cells were incubated with vehicle or steroid (24R or 24S) for the indicated

lengths of time. The cells were then collected by centrifugation and the pellets

resuspended in homogenization medium. After disruption in a homogenizer, the cells

were subjected to centrifugation at 1000 x g for 20 min to yield pellet 1 (P1 containing

nuclei, brush borders, and unbroken cells). And the supernatant again centrifuged to yield

pellet 2 (P2 containing basal lateral membranes, Golgi, mitochondria, and lysosomes.

Western analyses were performed with primary antibody from Abcam.

Figure 2.1 Time course study of catalase localization in isolated intestinal cells: ethanol control treatment. Figure 2.1A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.1B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Cells were isolated by citrate chelation, resuspended in Gey’s balanced salt solution, and pipeted into petri dishes containing a coverslip and RPMI 1640. Cells were allowed to adhere overnight in the absence of serum at 37ºC with

5% CO2/95% air. The following day, media were replaced with GBSS-0.1% BSA. Ethanol was then added for the indicated times, after which cells were fixed and stained with anti-catalase antibody and Alexa Fluor 594 and fluorescein conjugated Phalloidin (not shown). Cell images were observed and taken using Zeiss Confocal Microscope LSM 710, 60 x oil objective. Images were edited with ImageJ and Adobe Photoshop CS5.

Figure 2. 2 Time course study of catalase localization in isolated intestinal cells: 6.5 nM

24R,25(OH)2D3 hormone treatment. Cells were isolated, cultured, and stained as described in the legend to Figure 2.1. Catalase punctate staining inside nucleus is indicated using a yellow arrow. Figure 2.2A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.2B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Figure 2.2C presents a bar graph of intensity of nuclear staining vs. time. Values represent means ± S.E.M for n =7 independent experiments. Significant differences (**P<0.01) were determined using the student t-test, are relative to corresponding controls.

Figure 2.3 Time course study of catalase localization in isolated intestinal cells: 200 pM

24S,25(OH)2D3 hormone treatment. Figure 2.3A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.3B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Figure 2.3C presents a bar graph of intensity of nuclear staining vs. time. Values represent means ± S.E.M for n =7 independent experiments. Significant differences (**P<0.01) (*P<0.05) were determined using the student t-test, are relative to corresponding controls, are relative to corresponding controls.

Figure 2.4 Time course study of catalase localization in isolated intestinal cells: 360 pM

1,25(OH)2D3 hormone treatment. Figure 2.4A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.4B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Figure 2.4C indicates catalase nuclear

punctate staining intensity between ethanol control treatment and 1,25(OH)2D3 hormone treatment. Values represent means ± S.E.M for n =7 independent experiments.

Figure 2.5 Time course study of catalase localization in isolated intestinal cells: 0.01% ethanol (final concentration) after prior incubation with antibody. Figure 2.5A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.5B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Cells were isolated, cultured, and stained as describe in the legend to Figure 2.1.

Figure 2. 6 Time course study of catalase localization in isolated intestinal cells: 6.5 nM

24R,25(OH)2D3 hormone treatment after prior incubation with antibody. Figure 2.6A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.6B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Cells were isolated and cultured as described in Fig. 2.1 The following day, media were replaced with primary anti-catalase antibody for 30 min prior to addition of steroid and the indicated incubation periods. Cells were fixed and stained as described in Figure 2.1. Figure 2.6C indicates catalase nuclear punctate staining intensity between ethanol control

treatment and 24R,25(OH)2D3 hormone treatment. Values represent means ± S.E.M for n =7 independent experiments.

Figure 2. 7 Time course study of catalase localization in isolated intestinal cells: 200 pM

24S,25(OH)2D3 hormone treatment after prior treatment with antibody. Figure 2.7A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.7B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Figure 2.7C presents a bar graph with quantification of the observed nuclear fluorescence. The following day, media were replaced with primary anti-catalase antibody for 30 min prior to addition of steroid for the indicated times. Cells were processed for microscopy as describe in the legend to Figure 2.1. Figure 2.7C indicates catalase nuclear punctate staining intensity between ethanol control treatment and

24S,25(OH)2D3 hormone treatment. Values represent means ± S.E.M for n=7 independent experiments.

Figure 2.8 Time course study of catalase localization in isolated intestinal cells: 360 pM

1,25(OH)2D3 hormone treatment after prior treatment with antibody. Figure 2.8A indicates shorter incubation time points (15 sec, 30 sec, 7 min, 10 min, 15 min); Figure 2.8B indicates longer incubation time points (25 min, 30 min, 40 min, 50 min, 60 min). Figure 2.8C presents a bar graph depicting quantification of nuclear staining intensity for the various time points. Cells were isolated and cultured as described in Figure 2.1. The following day, media were replaced with primary anti-catalase antibody for 30 min. Cells

were fixed and stained with Alexa Fluor 594 and Phalloidin. 1,25(OH)2D3 hormone was then added for the indicated times. Figure 2.8C indicates catalase nuclear punctate

staining intensity between ethanol control treatment and 1,25(OH)2D3 hormone treatment. Values represent means ± S.E.M for n = 7 independent experiments.

Figure 2. 9 Western analyses on effect of vitamin D and time on catalase expression in the nucleus. Cells were isolated and nuclei extracted using nuclear extract kit. Anti- catalase was used as the primary antibody. 10µg of protein was loaded for each lane.

Figure 2.10 Western blot of nuclear extract catalase band intensity quantified by ImageJ software. Values represent means ± S.E.M for n =3 independent experiments. Significant differences (*P<0.05; ** P<0.01) were determined using Student’s t-test, are relative to corresponding ethanol controls, and are indicated by an asterisk.

Discussion

In this study, localization of catalase inside chick intestinal cells was determined

using confocal microscopy. Figure 2.1 depicts no redistribution of catalase in vehicle

ethanol control treatments after 15 sec to 60 min incubation. Figure 2.2 indicates an

obvious catalase redistribution to nucleus at every indicated incubation time point in

24R,25(OH)2D3 treatments. Figure 2.3 depicts no obvious redistribution of catalase in

24S,25(OH)2D3 treatments, the same observation can be found in Figure 2.4, where no

redistribution of catalase was found after 1,25(OH)2D3 treatment. In order to test whether

the observed redistribution of catalase to the nucleus after 24R,25(OH)2D3 treatments are

from the cell surface, another set of experiments were undertaken. On the day of the

experiments, primary antibody was added to the cells before treatment with vehicle or

hormones to block internalization of cell surface catalase. Figure 2.5 depicts no

redistribution of catalase in vehicle control treatments after 15 sec to 60 min of

incubation. In Figure 2.6, no catalase redistribution is observed at any indicated

incubation time point in 24R,25(OH)2D3 treatment, which indicates that adding primary

antibody prior to 24R,25(OH)2D3 treatment completely abolishes the effect. In Figure 2.7

and 2.8, no redistribution of catalase was observed either after 24S,25(OH)2D3 or

1,25(OH)2D3 treatment. In order to quantify the catalase punctate staining intensity, each

image was edited and quantified using Adobe Photoshop CS5. As indicated in with each

figure, when vehicle control or hormones were added prior to primary antibody, a

significant increase (200%, P<0.01) in the nuclear punctate staining intensity is observed

in 24R,25(OH)2D3 treatments, compared to vehicle control treatments as well as

1,25(OH)2D3 treatment [190%, increase of 24R,25(OH)2D3 relative to 1,25(OH)2D3]. An

increase in the punctate staining intensity is also observed in 24S,25(OH)2D3 treatments,

but to a lesser extent (130% relative to ethanol control; 110% relative to 1,25(OH)2D3

treatment; P<0.05). When vehicle control or hormones are added after primary antibody

addition, as indicated in Figure 2.10, all four treatments (vehicle control, 24R,25(OH)2D3,

24S,25(OH)2D3, and 1,25(OH)2D3 treatment) share the same pattern. No significant

difference is observed among the four treatment groups, indicating cell surface catalase is

required as the receptor for redistribution. Western analyses were undertaken to further

prove the hypothesis tested above. In Figure 2.11, a denser band was observed in the

nuclear extract sample in both 24R,25(OH)2D3 and 24S,25(OH)2D3 treatments, relative to

vehicle controls. As indicated in Figure 2.12, catalase band intensity was quantified by

ImageJ software. There is a significant increase in the band intensity after

24R,25(OH)2D3 treatment (300%, P<0.01) and a slight increase in the band intensity after

24S,25(OH)2D3 treatment (140%, P<0.05), which is in agreement with the results from

experiments above.

The combined data indicate that 24,25(OH)2D3 treatment of intestinal cells results

in the migration of cell surface catalase to the nucleus. This in turn strongly suggests that

24R,25(OH)2D3 has effects on the genome in addition to the reported rapid effects on

phosphate transport. Microarray analyses may in the future identify particular genes or

groups of genes that are modulated by 24,25(OH)2D3.

Taken together, these results suggest that the changes in catalase localization that

occur during the time points tested with 24R,25(OH)2D3 treatment may have potential

clinical significance in phosphate homeostasis and in the development of the

development of pharmacological therapies.

References

1. Henry HL, Norman AW (1978) Vitamin D: Two dihydroxylated metabolites are

required for normal chicken egg hatchability. Science 201: 835-837.

2. Ono T, Tanaka H, Yamate T, Nagai Y, Nakamura T, et al. (1996) 24R,25-

dihydroxyvitamin D3 promotes bone formation without causing excessive

resorption in hypophosphatemic mice. Endocrinology 137: 2633-2637.

3. Zhao B, Nemere I (2002) 1,25(OH)2D3-mediated phosphate uptake in isolated

chick intestinal cells: effect of 24,25(OH)2D3, signal transduction activators, and

age. J Cell Biochem 86: 497-508.

4. Nemere I (1999) 24,25-dihydroxyvitamin D3 suppresses the rapid actions of 1,25-

dihydroxyvitamin D3 and parathyroid hormone on calcium transport in chick

intestine. J Bone Miner Res 14: 1543–1549.

5. Yukihiro S, Posner GH, Guggino SE (1994) Vitamin D3 analogs stimulate

calcium currents in rat osteosarcoma cells. J Biol Chem 269: 23889-23893.

6. Khoury RS, Weber J, Farach-Carson MC (1995) Vitamin D metabolites modulate

osteoblast activity by Ca2+ influx-independent genomic and Ca2+ influx-dependent

nongenoomic pathways. J Nutr 125: 1699S-1703S.

7. Takeuchi K, Guggino SE (1996) 24R,25(OH)2D3 inhibits 1α,25(OH)2D3 and

testosterone potentiation of calcium channels in osteosarcoma cells. J Biol Chem

271: 33335-33345.

8. Nemere I (1996) Apparent non-nuclear regulation of intestinal phosphate

transport: Effects of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and

25-hydroxyvitamin D3. Endocrinology 137: 2254-2261.

9. Nemere I (1996) Parathyroid hormone rapidly stimulates phosphate transport in

perfused duodena: Lack of modulation by vitamin D metabolites. Endocrinology

137: 3750-3755.

10. Nemere I, Yazzie-Atkinson D, Johns DO, Larsson D (2002) Biochemical

characterization and purification of a binding protein for 24,25-dihydroxyvitamin

D3 from chick intestine. J Endocrinol 172: 211-219.

11. Nemere I, Farach-Carson MC, Rohe B, Sterling TM, Norman AW, et al. (2004)

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding

protein (1,25D3-MARRS) and phosphate uptake in intestinal cells. Proc Natl

Acad Sci U S A 101: 7392-7397.

12. Khanal RC, Smith NM, Nemere I (2007) Phosphate uptake in chick kidney cells:

effects of 1,25(OH)2D3 and 24,25(OH)2D3. Steroids 72: 158-164.

13. Larsson D, Anderson D, Smith NM, Nemere I (2006) 24,25-dihydroxyvitamin D3

binds to catalase. J Cell Biochem 97: 1259-1266.

14. Yano S, Yano N (2002) Regulation of catalase enzyme activity by cell signaling

molecules. Mol Cell Biochem 240: 119-130.

15. Nemere I, Wilson C, Jensen W, Steinbeck M, Rohe B, et al. (2006) Mechanism of

24,25-dihydroxyvitamin D3-mediated inhibition of rapid, 1,25-dihydroxyvitamin

D3-induced responses: role of reactive oxygen species. J Cell Biochem 99: 1572-

1581.

16. Sven, P (2006) Catalase activity mediates the inhibitory actions of 24,25-

dihydroxyvitamin D3. Master's Thesis, Utah State University.

17. Peery SL, Nemere I (2007) Contributions of pro-oxidant and anti-oxidant

conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-

dihydroxyvitamin D3 on phosphate uptake in intestinal cells. J Cell Biochem 101:

1176-1184.

18. Nemere I, Campbell K (2000) Immunochemical studies on the putative

plasmalemmal receptor for 1, 25-dihydroxyvitamin D3. III. Vitamin D status.

Steroids 65: 451-457.

19. Nemere I (2002) Biochemical characterization and purification of a binding

protein for 24,25-dihydroxyvitamin D3 from chick intestine. J Endocrinol 172:

211-219.

20. Ishizuka S, Takeshita T, Norman AW (1984) Naturally occuring 24,25-

dihydroxyvitamin D3 is a mixture of both C-24R and C-24S epimers. Arch

Biochem Biophys 234: 97-104.

21. Meng Y (2011) The Effects of 24R,25-dihydroxyvitamin D3 and 24S,25-

dihydroxyvitamin D3 on Phosphate Transport in Vivo. Master's Thesis, Utah State

University.

22. Ndubuisi MI, Guo GG, Fried VA, Etlinger JD, Sehgal PB (1999) Cellular

physiology of STAT3: Where's the cytoplasmic monomer? J Biol Chem 274:

25499-509.

CHAPTER 3

EFFECTS OF VITAMIN D ON CATALASE EXPRESSION AND PHOSPHATE UPTAKE IN CHICKEN INTESTINAL CELLS

Abstract

Many researchers now recognize that the steroid 24,25-dihydroxyvitamin D3

[24,25(OH)2D3] has biological activity. In previous work from our lab, it has been

reported that 24,25(OH)2D3 decreases catalase protein levels in cultured intestinal cells

over a period of several hours. An effect on catalase gene expression combined with

rapid signaling would conclusively demonstrate that 24,25(OH)2D3 is not a ‘scrap’

metabolite. In the current work, we report that 24R,25(OH)2D3 is the effective metabolite

for decreasing 32P uptake and catalase gene expression, but not for protein regulation. We

have used male chick intestinal cells treated with either vehicle, 24,25(OH)2D3 or

32 1,25(OH)2D3 for each condition. P uptake assays showed a decrease in uptake after

24R,25(OH)2D3 treatment in both suspension cells and cultured cells. RT-PCR further

indicated a down-regulation of catalase gene expression after treatment with

24R,25(OH)2D3 compared to vehicle control and 1,25(OH)2D3. Protein expression was

then checked using western blotting, with a result of no change in protein expression

levels. By understanding molecular and cellular actions of 24,25(OH)2D3 in chick

intestine, progress will be made in enhancing phosphate and calcium absorption in

animals to supply the minerals for adequate bone growth, and cellular metabolism.

Introduction

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most studied vitamin D metabolite, exerts

its effect through both genomic and non-genomic (more accurately termed pre-genomic)

mechanisms. Non-genomic actions are rapid, regulated by membrane-associated

receptors [1] and signal transduction pathways, while genomic actions take longer, and

are exerted largely through transcription family factors to control gene expression [2].

Another vitamin D metabolite, 24,25(OH)2D3, is produced mainly when an animal is

calcium, phosphate, and 1,25(OH)2D3 replete. The inhibitory action of 24,25(OH)2D3 on

phosphate absorption is observed across species. It has been reported that 24,25(OH)2D3

32 inhibits the rapid 1,25(OH)2D3-mediated stimulation of P transport in the perfused chick

duodenal loop [3, 4], as well as 45Ca transport [5]. Catalase, which has been identified as

an endogenous 24,25(OH)2D3 binding protein [6], is in part bound to the cell surface. It

has been reported that 24R,25(OH)2D3, not 24S,25(OH)2D3, is the effective metabolite

for catalase redistribution [7]. In earlier work we also found that 24,25(OH)2D3 decreases

catalase protein levels in cultured intestinal cells over a period of several hours [8].

32 In the current study we report that in intestinal cells 24R,25(OH)2D3 decreased P

uptake and catalase gene expression after 7 min treatment at the mRNA level. As mRNA

levels cannot be used as surrogates for corresponding protein levels without verification

[9], we performed western blotting to confirm the relation, with a result of no change in

protein expression.

Experimental

Animals

All surgical procedures were approved by the Institutional Animal Use and Care

Committee at Utah State University (Logan, UT). White leghorn cockerels (Privett

Hatchery; Portales, NM) were obtained on the day of hatch and raised for 3-7 weeks on a

commercially available vitamin D-replete diet (Nutrena Feeds; Murray, UT). On the day

of use, chicks were anesthetized with anhydrous ethyl ether (Fisher Scientific, Fair Lawn,

NJ). The abdominal cavity was surgically opened and the duodenal loop was removed to

ice-cold 0.9% saline solution and chilled for 15 min. The pancreas was excised from the

duodenal loop and discarded. The duodenal loop was everted and rinsed three times in

chilled saline solution.

Cell Isolation and Cell Culture

The chick enterocytes were isolated with citrate chelation media (96 mM NaCl,

27 mM sodium citrate, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na2HPO4, pH 5.0 – (the

acidic pH promotes viability and retention of morphology[3, 10-12]). The cells were

collected by low speed centrifugation (500 x g, 5 min, 4ºC), and cell pellets were

resuspended in a small volume of Gey’s balanced salt solution (GBSS, containing 119

mM NaCl, 4.96 mM KCl, 0.22 mM KH2PO4, 0.84 mM NaHPO4, 1.03 mM MgCl2•6H2O,

0.28 mM MgSO4•7H2O, 0.9 mM CaCl2, pH 7.3). Aliquots of the cell suspension were

pipetted into 35 mm plastic Petri dishes (Falcon, Scientific Products, Franklin Lakes, NJ)

containing 2 ml RPMI-1640 (Hyclone; Logan, UT) and antibiotics (100 units/ml

penicillin, 100mg/ml streptomycin; Sigma-Aldrich Chemical Company, St. Louis, MO).

The cells were incubated overnight or longer at 37ºC with 5% CO2/ 95% air in the

absence of serum to promote cell adhesion.

32P uptake in suspension and cultured intestinal cells

Chick intestinal cells were isolated by citrate chelation as described above and

centrifuged at low speed (500 x g, 5 min, 4ºC). Cell pellets were resuspended in room

temperature Gey’s Balanced Salt Solution. 2.2 mL of cell suspensions were pipetted into

32 a polypropylene tube along with 1 µCi/ml (1 Ci = 37 GBq) H3 PO4, final concentration

(Perkin Elmer, Boston, MA). 100 µl of cell suspensions were removed at T = -5 and -1

min to establish basal uptake. At T = 0 min, cells were treated with either vehicle (0.01%

ethanol, final concentration), 300 pM 1,25(OH)2D3, 6.5 nM 24R,25(OH)2D3 or 200 pM

24S,25(OH)2D3, and incubated at room temperature. At T = 1, 3, 5, 7, 10 min, 100 µl of

cell suspension aliquots were transferred to 900 µl ice-cold GBSS in 1.5 ml

microcentrifuge tubes to stop phosphate uptake. Samples were put on ice until

centrifuged at 1000 x g for 5 min, 4ºC. Supernatant was decanted and any residual liquid

was carefully swabbed with Kimwipes while still in the inverted position. Pellets were

resuspended in 500 µl double distilled water, aliquots were used for protein determination

using a UV spectrophotometer (Beckman Coulter) and radionuclide measurement using a

multi-purpose scintillation counter (Beckman) [8]. Values obtained for the treated phase

were normalized to average basal phase, and represent means ± S.E.M for n independent

experiments. Time course data indicated that optimal responsiveness to 24,25(OH)2D3

occurred at 7 min of steroid treatment.

Chicken intestinal cells were isolated by the citrate chelation protocol as

previously described. Under sterile conditions, 200 µl aliquots of cell suspension were

pipetted into 35 mm plastic Petri dishes (Falcon, Scientific Products) containing 3 ml of

RPMI-1640 medium and antibiotics [100 units/ml penicillin, 100 mg/ml streptomycin

(Sigma Chemical Co, St. Louis, MO)]. The cells were incubated overnight with 5%

CO2/95% air at 37ºC. No serum was added to promote better cell adherence.

The next day, media were aspirated and cells were pre-incubated at room

temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of GBSS-0.1%BSA

32 containing 4 µCi of H3 PO4 with either 300 pM 1,25(OH)2D3, 200 pM 24S,25(OH)2D3

or 6.5 nM 24R,25(OH)2D3 were added and incubated for an additional 7 min. Adherent

cells were washed three times with 4 ml ice-cold GBSS and lysed with 0.5 ml 0.1% (v/v)

Triton X-100 in TED. Finally, cells were collected by scrapping and aspirating. Aliquots

were used for protein determination using a UV spectrophotometer (Beckman Coulter,

City State) and radionuclide measurement using a multi-purpose scintillation counter

(Beckman) [8]. Values represent means ± S.E.M for N independent experiments.

siRNA transfection in cultured intestinal cells

Chicken intestinal cells were isolated by the citrate chelation protocol as

previously described. Under sterile conditions, 200 µl aliquots of cell suspension were

pipetted into 35 mm plastic Petri dishes (Falcon Scientific Products; Franklin Lakes, NJ)

containing 2 ml of RPMI-1640 medium and antibiotics [100 units/ml penicillin, 100

mg/ml streptomycin (Sigma Chemical Co; St. Louis, MO)]. The cells were incubated

overnight with 5% CO2/95% air at 37ºC prior to transfection. No serum was added to

promote better cell adherence.

The next day, cells were transfected with 100 nM scrambled siRNA (Santa Cruz

Biotechnology; Santa Cruz, CA), or 80 pmols siRNA against catalase, under RNase-free,

sterile conditions. Cells were then incubated 5-7 hours at 37ºC in a CO2 incubator. 1 ml

of normal growth medium containing 2 times the normal serum and antibiotic

concentration (2x normal growth medium) were added without removing the transfection

mixture. After another additional 18-24 hours incubation, media were aspirated and

replaced with RPMI-1640 with 10% FBS. The following procedures were conducted

after 24 hours of addition of fresh medium.

Western Blots

The isolated intestinal cells described above were collected by centrifugation at

500 x g, 5 min (4ºC), and resuspended in 30 ml of GBSS. Cells were then cultured

overnight, then transfected the next day according to the siRNA transfection protocol

(Santa Cruz). On the day of use, cells were treated with 0.01% ethanol, 6.5 nM

24R,25(OH)2D3 or 300 pM 1,25(OH)2D3 for 7 min at room temperature. Media was

removed and cells were lysed with 0.5 ml 0.1% (v/v) Triton X-100 in TED. 10µg of total

protein were loaded in each lane.

SDS-PAGE and western blot analyses were used to determine immunoreactive

levels of catalase in controls and cells treated with vitamin D metabolites. Protein was

determined with the Bradford reagent (Bio-Rad, Hercules, CA) and then samples (5-

30µg/well) were separated on 8% (wt/vol) SDS-PAGE gels with 5% stacking gels. After

separation on SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF)

membranes (Immobilon-P, Fisher Scientific) by the use of a TransBlot SD Semidry

transfer cell (Bio-Rad) and western analyses were performed. To avoid nonspecific

binding, the membrane was soaked for 1 hr at 37ºC in blocking solution (0.1% wt/vol

BSA) in Tris-buffered saline (PBS; 0.9% NaCl in 20 mM Tris-HCl, pH 7.4), and

incubation with primary antibody (Abcam Inc., Cambridge, MA) in 1% BSA and 0.05%

Tween 20 in PBS overnight at 4ºC. After three additional washes, the membrane was

incubated with secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit

IgG) in the same incubation buffer for 2 hr at room temperature and then washed as

previously indicated. Immunoreactive bands were visualized with the chromogens 5-

bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium, and relative amounts of

catalase were quantitated using ImageJ software.

RNA isolation and RT-PCR using SYBR green

On the day of use, total RNA was isolated with TRIzol reagent (Sigma) using

glycogen as a carrier because of low cell numbers (<104). For RT-PCR, 100 ng of total

RNA was used for RT reactions to make cDNA. The primer for the 259-bp catalase

product in the forward direction was a 23-mer (5’-CTG GCA ACC CAA TAG GAG

ATA AG-3’), and for the reverse direction, (5’-GCA ACA GTG GAG AAC CGT ATA

G-3’), a 22-mer was used. The primer for the 545-bp GAPDH product in the forward

direction was a 20-mer (5’-AGT CGG AGT CAA CGG ATT TG-3’), and for the reverse

direction, (5’-TCT TCT GTG TGG CTG TGA TG-3’), a 20-mer was used. The primers

were validated using end point PCR, with subsequent experiments using real time PCR.

Power SYBR Green PCR Master Mix (Applied Biosystems, UK) was used for RT-PCR.

The RT-PCR detection procedure was the MiniOpticon System (Bio-Rad) and results

were analyzed with the company’s software.

Determination of Protein Concentration

The Bradford assay was performed to determine sample protein concentrations.

After homogenization of cells, 10 µl of sample was added to 790 µl double distilled water

followed by 200 µl of Bradford reagent (Bio-Rad,). The contents were mixed, set at room

temperature for 10 min for color development, and analyzed by spectrophotometry at 595

nm. Bovine gamma globulin (Sigma) was used as the standard.

Statistical Analysis

Values are expressed as the means ± S.E.M for the number of independent

experiments indicated in the figure legends. The data were analyzed for significance

using Student’s t-test and significant and differences were determined with a 95%

confidence level (P<0.05).

Results

Time course study of 32P uptake after hormone treatments in intestinal cells suspensions

Uptake studies in suspension were first undertaken to determine an optimal time

point of hormone effect prior to studies using cultured adherent cells, which are

technically more difficult. Chick intestinal cells were isolated with citrate chelation media

described above and resuspended in 40 mL GBSS, aliquoted, and then treated with 0.01%

32 ethanol, 130 pM 1,25(OH)2D3, or 6.5 nM 24,25(OH)2D3 with 1 µCi H3PO4/1 ml of cell

suspension at room temperature. The selected steroid concentrations have been shown to

be equivalent to circulating levels [13]. Incubations were for selected times (1min, 3min,

5min, 7min, 10min), and uptake stopped by transferring aliquots of cell suspension into

ice-cold GBSS at the indicated times. Cells were pelleted by low speed centrifugation

(1000 x g, 5 min, 4ºC), supernatants decanted, and any residual GBSS was carefully

swabbed with Kimwipes while the tubes were still in an inverted position. Cells were

resuspended in 0.5 ml reagent grade water, and aliquots were taken for determination of

protein and measurement of radionuclide. Figure 3.1 illustrates 32P uptake levels with

different hormone treatment conditions. As shown, 1,25(OH)2D3 had an overall

32 32 stimulatory effect on P uptake, 24R,25(OH)2D3 had an inhibitory effect on P uptake,

while 24S,25(OH)2D3 had no change relative to the ethanol control treatment. As

illustrated in Figure 3.1, both 1,25(OH)2D3 and 24R,25(OH)2D3 hormone treatments led

to significant changes: 1,25(OH)2D3 at T = 7 min with a 140% increase (P < 0.01) and

for 24R,25(OH)2D3 at T = 7 min with an 136 percent decrease, each relative to vehicle

controls (P < 0.01). The 7 min time point was then chosen for the rest of the study.

Figure 3.1 - Time course of 1,25(OH)2D3, 24R,25(OH)2D3 or 24S,25(OH)2D3 - mediated 32P uptake in chicken intestinal cell suspensions, relative to vehicle controls. Cells were isolated with citrate chelation media, and collected by low speed centrifugation (500 x g, 5 min, 4ºC), cell pellets were resuspended in room temperature Gey's Balanced Salt

Solution (GBSS). Cells were treated with 360 pM 1,25(OH)2D3, 6.5 nM 24,25(OH)2D3 or 32 vehicle (0.01% ethanol, final concentration) with 1 µCi H3PO4/1 ml of cell suspension at room temperature and stopped by transferring aliquots of cell suspension into ice-cold GBSS at the indicated times. Cells were pelleted by low speed centrifugation (1000 x g, 5 min, 4ºC), supernatants decanted, and any residual GBSS was carefully swabbed with Kimwipes while the tubes were still inverted. Cells were resuspended in 0.5 ml reagent grade water, and aliquots were taken for determination of protein and measurement of radionuclide. Data were calculated as cpm/µg protein and then related to corresponding basal levels. Values represent mean ± S.E.M for n=5 independent experiments. Significant differences (*P<0.05, **P<0.01) were determined using Student’s t-test, are relative to corresponding controls.

Effect of 32P uptake after different hormone treatments at 7 min in cultured intestinal cells

Chick intestinal cells were isolated with citrate chelation media and resuspended

in 40 mL GBSS and cultured overnight as described above. The next morning, cells were

pre-incubated at room temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of

32 GBSS-0.1%BSA containing 4 µCi of H3 PO4 with one of three hormones was added and

the cells incubated for an additional 7 min. Adherent cells were washed three times with

4 ml ice-cold GBSS and lysed with 0.5 ml 0.1% (v/v) Triton X-100 in TED. Finally, cells

were collected by scrapping and aspirating, and aliquots were taken for determination of

protein and measurement of radionuclide. Figure 3.2 shows 32P uptake after treatments

with different hormones or vehicle. According to Figure 3.2, a similar pattern is observed

32 as in Figure 3.1: 1,25(OH)2D3 had a stimulatory effect on P uptake to 220 % of vehicle

controls (P<0.05), 24R,25(OH)2D3 had an inhibitory effect to 140% of vehicle controls

(P<0.05), while 24S,25(OH)2D3 had no effect under these conditions. Thus treatment of

adherent cells in culture gave results equivalent to those in suspension, allowing further

studies with siRNA.

32 Figure 3.2 Effect of vehicle, 1,25(OH)2D3 or 24,25(OH)2D3 on P uptake in cultured chicken intestinal cells. On the days of experiments, cells cultured overnight were pre- incubated at room temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of GBSS- 32 0.1%BSA containing 4 µCi of H3 PO4 with vehicle, 1,25(OH)2D3, 24S,25(OH)2D3 or

24R,25(OH)2D3 were added and incubated for an additional 7 min. Adherent cells were washed three times with 4 ml ice-cold GBSS and lysed with 0.5 ml 0.1% (v/v) Triton X- 100 in TED. Finally, cells were collected by scraping and aspirating, and aliquots were taken for determination of protein and measurement of radionuclide. Data were calculated as cpm/µg protein and then related to corresponding vehicle controls. Values represent means ± S.E.M for n = 8 independent experiments. Significant differences were determined using the student t-test, and are indicated by an asterisk (*P<0.05, relative to vehicle control; ** P<0.01, relative to vehicle control).

The lack of effect by 24S,25(OH)2D3 suggested that no further testing with this metabolite need be performed.

Effect of vitamin D on cultured chicken enterocytes on catalase gene expression

Chick enterocytes were isolated, cultured, and transfected as described above.

Assays were performed 24 hours after the addition of fresh medium. Total RNA was

prepared with TRIzol reagent, 100 ng was used for cDNA synthesis and 100 ng was used

for RT-PCR with primers specific for catalase or GAPDH as described above. Figure 3.3

provides data as a fold ratio normalized to GAPDH expression. Cells were treated with

test substance for 7 min. siRNA transfection had no effect on gene transcription levels in

cells treated with ethanol or 1,25(OH)2D3 compared to those treated with scramble RNA

(Figure. 3.4). This finding suggests that catalase siRNA treatment was not effective in

reducing catalase expression. However, comparisons between the scrambled siRNA

treatments reveal that 1,25(OH)2D3 increased catalase gene expression relative to ethanol

treatment controls (P<0.01). Conversely, 24R,25(OH)2D3 decreased catalase gene

expression (P<0.01) compared to the ethanol control. When treated with scrambled

control siRNA, the 1,25(OH)2D3 treatment group showed an increase in catalase mRNA

expression relative to ethanol controls, while the 24R,25(OH)2D3 treatment group had a

decrease in catalase mRNA expression, relative to ethanol controls (Figure 3.4).

Figure 3.3 Effect of vitamin D on gene expression for catalase in cultures of chicken intestinal cells. Cells were transfected with scrambled siRNA or catalase siRNA according to siRNA transfection protocol (Santa Cruz, CA). After transfection, cells were incubated with ethanol control or test hormones at room temperature for 7 min. Total RNA was prepared with the TRIzol reagent, and 100 ng was used for RT-PCR with primers specific for catalase or GAPDH. Values represent means ± S.E.M for n=6 independent experiments. Significant differences were determined using Student's t-test (**P<0.01).

Effect of 32P uptake after different hormone treatments at 7 min in cultured transfected

enterocytes

Chick enterocytes were isolated with citrate chelation media and resuspended in

40 mL GBSS cultured overnight at 37ºC with 5% CO2/ 95% air to promote cell adhesion.

The next day, cells were transfected with either scrambled control siRNA or siRNA

directed against catalase according to the siRNA transfection protocol, and were

incubated for an additional 20 hours at 37ºC with 5% CO2/ 95% air. Assays were

performed 24 hours after the addition of fresh medium. Cells were pre-incubated at room

temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of GBSS-0.1%BSA

32 containing 4 µCi of H3 PO4 with vehicle or hormones were added and incubated for an

additional 7 min. Adherent cells were washed three times with 4 ml ice-cold GBSS and

lysed with 0.5 ml 0.1% (v/v) Triton X-100 in TED. Finally, cells were collected by

scraping and aspirating, and aliquots were taken for determination of protein and

measurement of radionuclide. Although as demonstrated in Figure 3.4, catalase siRNA

was ineffective in reducing catalase expression; comparisons between the scramble

32 treated cells revealed that treatment with 1,25(OH)2D3 increased P uptake (P<0.01) and

32 24R,25(OH)2D3 reduced P uptake (P<0.01) relative to ethanol controls.

32 Figure 3.4 Effect of vehicle, 1,25(OH)2D3 or 24R,25(OH)2D3 on P uptake in transfected cultured chicken enterocytes. Cells were transfected with scrambled control siRNA or catalase siRNA according to the siRNA transfection protocol (Santa Cruz Biotechnology, CA). On the day of the experiments, transfected cells were pre-incubated at room temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of GBSS-0.1%BSA 32 containing 4 µCi of H3 PO4 and either vehicle or 1,25(OH)2D3, 24S,25(OH)2D3 or

24R,25(OH)2D3 were added and incubated for an additional 7 min. Adherent cells were washed three times with 4 ml ice-cold GBSS and lysed with 0.5 ml 0.1% (v/v) Triton X- 100 in TED. Finally, cells were collected by scraping and aspirating, and aliquots were taken for determination of protein and measurement of radionuclide. Data were calculated as cpm/µg protein and then related to corresponding basal levels. Open bars represent data from scrambled siRNA treatments; Filled bars represent data from siRNA treatments. Values represent means ± S.E.M for n =4 independent experiments. Significant differences (**P<0.01) were determined using Student’s t-test, are relative to corresponding treatments also transfected with scramble RNA.

Effect of vitamin D on protein expression of catalase in cultured chicken enterocytes

Chick enterocytes were isolated, cultured, and transfected as described above.

Assays were performed 24 hours after the addition of fresh medium. Cells were pre-

incubated at room temperature for 5 min in 1 ml GBSS-0.1%BSA. Then 1 ml of GBSS

containing vehicle or hormones were added and incubated for additional 7 min. Adherent

cells were washed, lysed, and collected as described. 10 µg of protein for each treatment

was used for SDS-PAGE and western blotting. Protein band intensity was determined

using ImageJ software. Figure 3.5 shows no change in protein expression among control

treatments. In contrast, 1,25(OH)2D3 increased catalase protein levels, relative to ethanol

treated controls transfected with scramble siRNA (P<0.01), while 24R,25(OH)2D3,

decreased catalase expression compared to 1,25(OH)2D3 (P<0.05) and ethanol controls.

This result indicated that protein levels are not proportional to mRNA in cultured chicken

enterocytes after 7 min treatment with vitamin D.

Figure 3.5 Effect of vitamin D on protein levels of catalase in cultured chicken enterocytes. Cells were transfected with scrambled siRNA or catalase siRNA according to siRNA transfection protocol (Santa Cruz, CA). Treatment with vehicle or hormones was for 7 min. Cells were lysed with 0.5 ml 0.1% (v/v) Triton X-100 in TED, 10 µg of proteins were used for SDS-PAGE and western blotting. Figure 3.5A shows a western blot; Figure 3.5B depicts densitometric analyses of the bands shown. Values represent means ± S.E.M for n=3 independent experiments. Significant differences were determined using the student t-test, are relative to corresponding controls, and are indicated by asterisks (**P<0.01) (*P<0.05).

Discussion

Previous experiments with primary cultures of chick intestinal cells exposed to

24,25(OH)2D3 for 5 h have demonstrated that 24R,25(OH)2D3 is capable of decreasing

both catalase specific activity and catalase protein levels [8]. In this study with suspended

chick enterocytes treated with 24R,25(OH)2D3, a decrease in phosphate uptake occurs as

early as 1 min and is significantly lower than ethanol controls at 7 min. Although the

treated/average basal values increase in both 24S,25(OH)2D3 and 24R,25(OH)2D3 with

time, 24S,25(OH)2D3 remained more closely to basal values compared to

32 24R,25(OH)2D3. In contrast, the decrease in P uptake is sustained at an average of

150% below controls from 1 to 10 min in 24R,25(OH)2D3 treatments. A similar result is

observed with primary cultured chick enterocytes. After a 7 min incubation with

hormones or the vehicle ethanol (a time point determined by previous study in cell

suspension), a significant decrease in phosphate uptake is observed in 24R,25(OH)2D3

treatments (at an average of 140% below ethanol controls).

With equivalent results observed in both suspension and cultured chick

enterocytes, further siRNA study was undertaken to determine whether this manipulation

affects 24R,25(OH)2D3 mediated changes in catalase gene expression and/or protein

expression. Unfortunately, catalase siRNA treatment was not effective in reducing

catalase gene expression. Nonetheless, several important observations were made. A

decrease in 1,25(OH)2D3-mediated phosphate uptake is observed with a 7 min after

24R,25(OH)2D3 treatment combined with scrambled siRNA (P<0.01). An increase in

1,25(OH)2D3-mediated phosphate uptake is observed with 7 min after 1,25(OH)2D3

treatment combined with scrambled siRNA (P<0.01). Catalase gene expression was

determined using RT-PCR, and a significant 281% decrease is observed in

24R,25(OH)2D3 treated cells with scramble siRNA relative to the corresponding ethanol

control (P<0.01). The inhibitory effect was again diminished with catalase siRNA.

Protein levels of catalase were determined by western blots after 7 min of steroid

treatment. With catalase antibody directed against chick catalase as primary antibody,

fainter bands were observed in cultured cells treated with 24R,25(OH)2D3, while

1,25(OH)2D3 treatment produced denser bands (Figure 3.5A). Densitometric analysis

(Figure 3.5B) indicated a significantly lower level of immunoreactive protein in cultured

intestinal cells treated for 7 min with 24R,25(OH)2D3, relative to either corresponding

control or 1,25(OH)2D3 treatments (P<0.01). This observation is in agreement with

similar effect observed in 24 h samples for chick intestinal cells and kidney cells [8].

These combined data indicated that 24R,25(OH)2D3 has significant physiological

consequences in maintaining phosphate homeostasis even though the siRNA did not

work in manipulating catalase gene expression in cultured chick enterocytes.

References

1. Rohe, B., Safford, S.E., Nemere, I. and Farach-Carson, M.C. 2004, J. Steroid

Biochem. Mol. Biol., 89-90(1-5): 281-5.

2. Klaus, G., von Eichel, B., May, T., Hugel, U., Mayer, H., Ritz, E. and Mehls, O.

1994, Endocrinology, 135(4): 1307-15.

3. Bin, Z. and Nemere, I. 2002, J. Cell Biochem., 86(3): 497-508.

4. Nemere, I. 1996, Endocrinology, 137(6): 2254-61.

5. Nemere, I. 1999, J. Bone Miner. Res., 14(9): 1543-9.

6. Larsson, D., Anderson, D., Smith, N.M. and Nemere, I. 2006, J. Cell Biochem.,

97(6): 1259-66.

7. Zhang, Y. and Nemere, I. 2014, Integrative Molecular Medicine, 2, 5.

8. Khanal, R.C., Smith, N.M., and Nemere, I. 2007, Steroids, 72(2): 158-64.

9. Nancy Kendrick, K.L., Inc., A gene's mRNA level does not usually predict its

protein level. 2014.

10. Nemere, I., Farach-Carson, M.C., Rohe, B., Sterling, T.M., Norman, A.W.,

Boyan, B.D. and Safford, S.E. 2004, Proc Natl Acad Sci U S A, 101(19): 7392-7.

11. Nemere, I. and Campbell, K. 2000, Steroids, 65(8): 451-7.

12. Caldwell, D.J., Droleskey, R.E., Elissalde, M.H., Kogut, M.H., DeLoach, J.R.,

and Hargis, B.M. 1993, Journal of tissue culture methods, 15(1): 15-18.

13. Ishizuka, S., Takeshita, T. and Norman, A.W. 1984, Arch Biochem Biophys,

234(1): 97-104.

CHAPTER 4

SUMMARY AND CONCLUSIONS

Summary

1,25-dihydroxyvitamin D3 has proven to be the up-regulating steroid hormone for

phosphate homeostasis. This secosteroid hormone exerts its effect through two different

mechanisms, either via a nuclear vitamin D receptor (nVDR) to express a slow action to

generate genomic responses or through the more rapid action of 1,25D3-MARRS receptor

to generate pre-genomic actions of ion transport and signal transduction inside cells [1-6].

From previous studies, it was demonstrated in chick enterocytes that the signal

transduction mechanism of 1,25(OH)2D3-mediated rapid uptake of phosphate was

mediated by PKC [7, 8].

Catalase has been proven to be the cellular binding protein for 24,25-

dihydroxyvitamin D3 [24,25(OH)2D3] [9]. Both 24,25(OH)2D3 and the production of

H2O2 have been demonstrated to inhibit catalase activity [10]. Somjen et al. [11] reported

that 24R,25(OH)2D3, not 24S,25(OH)2D3 was the active metabolite in bone. However, a

previous study found that the two isomers of 24,25(OH)2D3 had different effects on

phosphate transport in vivo [12]. The inhibitory effect is mainly performed by

24R,25(OH)2D3 since it was capable of decreasing phosphate absorption after a 1h

injection in vivo, while 24S,25(OH)2D3 was found to be increasing phosphate transport at

5h in chicks in vivo but the latter effect was diminished after 10h. This suggests another

receptor may be involved, or that the steroid may stimulate other pathways to mediate

phosphate absorption in vivo [12]. Previous work done in the perfused duodenal loop

system has proved that the antagonism of the stimulatory effect of 1,25(OH)2D3 by

24,25(OH)2D3 was seen 10 min after the onset of perfusion [13]. In these studies, the

main focus was on how 24,25(OH)2D3 effects phosphate uptake in chick enterocytes in

vitro.

The first series of experiments were performed to determine the effects of the two

isomers of 24,25(OH)2D3 on localization of catalase in chick enterocytes. The present

work demonstrated that 24R,25(OH)2D3 is the effective metabolite for catalase

redistribution in vitro, not 24S,25(OH)2D3. Male chick intestinal cells were treated with

either vehicle, 24R,25(OH)2D3 or 1,25(OH)2D3, and punctate staining was observed on

the cell surface and in the cytoplasm for all time points tested. Figure 2.2 depicts results

from 24R,25(OH)2D3 treatments, in which there were obvious increases in nuclear

staining as well as surface staining relative to controls (Figure 2.1). Figure 2.3 shows

results from 24S,25(OH)2D3 treatments, in which there was an obvious decrease in

punctuate nuclear staining compared to 24R,25(OH)2D3 treatments. Additional western

analyses were performed to confirm the punctate staining inside the nucleus was from the

redistribution of cell surface catalase.

Further experiments were undertaken to examine the origin of the nuclear staining

by confocal microscopy experiments. Primary antibody was first added to cells for 30

min in order to block surface staining, and subsequently incubated with vehicle,

1,25(OH)2D3, 24R,25(OH)2D3 or 24S,25(OH)2D3. This work demonstrated that ligand

binding to cell surface catalase induced redistribution to the nucleus, but was blocked by

the antibody. Figure 2.6 indicates nuclear redistribution after 24R,25(OH)2D3 treatment

did not occur, indicating that the cell surface catalase induced redistribution was blocked

by the antibody. A similar effect was not found in either 24S,25(OH)2D3 nor control

treatments. Catalase punctate staining intensity was also quantified by densitometry.

The second series of experiments were performed to determine the relationship

between vitamin D metabolites, catalase expression and phosphate uptake in chicken

intestinal cells. It has been reported that 24,25(OH)2D3 decreases catalase protein levels

in cultured intestinal cells over a period of several hours [14], an effect on catalase gene

expression combined with rapid signaling would conclusively demonstrate that

24,25(OH)2D3 is not a ‘scrap’ metabolite. The present work demonstrated that

24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake and catalase

32 gene expression, not 24S,25(OH)2D3. Figure 3.1 and Figure 3.2 illustrates P uptake

levels with different hormone treatment conditions in normal enterocytes, in which

24R,25(OH)2D3 has a inhibitory effect on phosphate uptake, while 24S,25(OH)2D3 does

not. Figure 3.3 demonstrates the effect of different hormone treatments on 32P uptake in

32 transfected cultured enterocytes. A decrease in P was observed only in 24R,25(OH)2D3

treatment (P<0.05), indicating an elimination of the steroid-catalase effect on decreasing

phosphate uptake. As shown in Figure 3.4, only 24R,25(OH)2D3 treatment led to a

decrease in catalase mRNA expression. It was also determined that 24R,25(OH)2D3 had

no effect on catalase protein expression.

Conclusions

From these recent results, we can then conclude that:

1. 24R,25(OH)2D3, not 24S,25(OH)2D3, is the effective metabolite for catalase

redistribution in chicken enterocytes in vitro.

2. Western analysis and additional confocal microscopy experiments confirmed that the

punctate staining inside the nucleus arose from the redistribution of cell surface

catalase.

3. 24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake as well as

catalase gene expression determined by RT-PCR, but did not have an effect on protein

expression as determined by western analysis.

Future Study

1. Determination of the possible mechanism involved in catalase binding to STAT3 [15];

2. Evaluation of whether 24R,25(OH)2D3 mediates the production of separate hormones

that regulate phosphate transport.

Benefit

This study provides some basic framework of cellular and molecular mechanism

of 24,25(OH)2D3 regulated phosphate homeostasis, and increases our understanding of

the effects of 24,25(OH)2D3 on phosphate transport in vitro. The results from this

research may open new ways for drugs or gene therapies to regulate phosphate

homeostasis. Furthermore, this research also has implications in agriculture. Since the

production of manure high in phosphate in poultry farms is a serious problem, phosphate

in manure of production animals could be diminished.

References

1. de Boland, A.R. and Norman A.W. 1alpha,25(OH)2-vitamin D3 signaling in chick

enterocytes: enhancement of tyrosine phosphorylation and rapid stimulation of

mitogen-activated protein (MAP) kinase. J Cell Biochem, 1998. 69(4): p. 470-82.

2. Fleet, J.C., Rapid, Membrane-Initiated Actions of 1,25 Dihydroxyvitamin D: What

Are They and What Do They Mean? The Journal of Nutrition, 2004. 134(12): p.

3215-3218.

3. Nemere, I., et al., Identification of a specific binding protein for 1 alpha,25-

dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium

and relationship to transcaltachia. J Biol Chem, 1994. 269(38): p. 23750-6.

4. Nemere, I., et al., Identification of a membrane receptor for 1,25-

dihydroxyvitamin D3 which mediates rapid activation of protein kinase C. J Bone

Miner Res, 1998. 13(9): p. 1353-9.

5. Khanal, R.C., et al., Membrane receptor-initiated signaling in 1,25(OH)2D3-

stimulated calcium uptake in intestinal epithelial cells. J Cell Biochem, 2008.

105(4): p. 1109-16.

6. Nemere, I., et al., Intestinal cell calcium uptake and the targeted knockout of the

1,25D3-MARRS (membrane-associated, rapid response steroid-binding)

receptor/PDIA3/Erp57. J Biol Chem, 2010. 285(41): p. 31859-66.

7. Zhao, B. and I. Nemere, 1,25(OH)2D3-mediated phosphate uptake in isolated

chick intestinal cells: effect of 24,25(OH)2D3, signal transduction activators, and

age. J Cell Biochem, 2002. 86(3): p. 497-508.

8. Peery, S.L. and I. Nemere, Contributions of pro-oxidant and anti-oxidant

conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-

dihydroxyvitamin D3 on phosphate uptake in intestinal cells. J Cell Biochem,

2007. 101(5): p. 1176-84.

9. Larsson, D., et al., 24,25-dihydroxyvitamin D3 binds to catalase. J Cell Biochem,

2006. 97(6): p. 1259-66.

10. Nemere, I., et al., Mechanism of 24,25-dihydroxyvitamin D3-mediated inhibition

of rapid, 1,25-dihydroxyvitamin D3-induced responses: role of reactive oxygen

species. J Cell Biochem, 2006. 99(6): p. 1572-81.

11. Somjen, D., I. Binderman, and Y. Weisman, The effects of 24R,25-

dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine

decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat

bone and in the duodenum. Biochem J, 1983. 214(2): p. 293-8.

12. Yu Meng, I.N., The Effects of 24R,25-Dihydroxyvitamin D3 and 24S,25-

Dihydroxyvitamin D3 on Phosphate Transport in Chicks in Vivo. Utah State

University Thesis, 2012.

13. Nemere, I., Apparent nonnuclear regulation of intestinal phosphate transport:

effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25-

hydroxyvitamin D3. Endocrinology, 1996. 137(6): p. 2254-61.

14. Khanal, R.C., N.M. Smith, and I. Nemere, Phosphate uptake in chick kidney cells:

effects of 1,25(OH)2D3 and 24,25(OH)2D3. Steroids, 2007. 72(2): p. 158-64.

15. Ndubuisi, M.I., et al., Cellular physiology of STAT3: Where's the cytoplasmic

monomer? J Biol Chem, 1999. 274(36): p. 25499-509.