Minggu, 03 Juli 2016

Genomic Approaches to Understanding Vitamin D Action


Source: www,acemaxs31.com

1. INTRODUCTION
The molecular actions of vitamin D metabolites have been studied extensively over the
past 30 years. This has led researchers to recognize roles for vitamin D nutriture and
vitamin D metabolite action in a variety of physiological systems, e.g., calcium
homeostasis, immune function, and the control of cell proliferation, differentiation, and
apoptosis resulting in the prevention of various cancers. The following review is intended
to summarize our understanding of the molecular actions of vitamin D, to review the
limited approaches taken to date using genomic approaches to study vitamin D action,
and to identify issues that may benefit from a genomic approach to vitamin D action.

2. OVERVIEW OF VITAMIN D AND HEALTH
Vitamin D is a conditionally required nutrient.UV light-stimulated skin conversion of 7-
dehydrocholesterol to vitamin D can meet the physiological needs of most individuals.
However, low vitamin D status is a common condition during the winter months in
people who live in the Northern United States, Northern Europe, and in Canada, in
people who limit their sun exposure by wearing protective clothing and sunscreen, and in
the elderly (1). High vitamin D status has been associated with protection from
osteoporosis, through its traditional effects on calcium homeostasis (2), and protection
from cancer, due to its ability to suppress cellular proliferation, promote differentiation,
and activate apoptosis (3). These later features of vitamin D biology may also account for
the anti-inflammatory and immunoregulatory actions of vitamin D (4). Recent studies
suggest that current recommendations for vitamin D intake (400–600 IU per day) are not
sufficient to protect bone health, a classic role for vitamin D in the optimization of human
health (1,5,6).

2.1. Metabolism of Vitamin D
Vitamin D, whether from the diet or produced in skin, is hydroxylated in the liver to form
25 hydroxyvitamin D3 (25-OH D) (7), a marker of vitamin D status (8,9). The biological
actions of vitamin D require further activation of 25-OH D to 1α, 25 dihydroxyvitamin
D3 (1, 25(OH)2 D) by a la hydroxylase before the hormone is biologically active (10).
Alterations in renal la hydroxylase activity are responsible for changes in circulating 1,
25(OH)2 D levels associated with variations in dietary calcium intake (i.e., low calcium
intake increases renal lα hydroxylase activity through elevated parathyroid hormone
production). However, extra-renal lα hydroxlase has been documented in a variety of
tissues, including skin, prostate epithelial cells, colonocytes, and mammary epithelial
cells. Thus, 1, 25(OH)2D, which has traditionally been viewed as an endocrine hormone,
may also function as an autocrine- or paracrine-signaling molecule.

Vitamin D compounds can also be modified by the actions of cytochrome P-450
family member, 24-hydroxylase (CYP24). When 25-OH D is the substrate, 24, 25(OH)2
D results. This vitamin D metabolite has been implicated in chondrocyte biology and in
bone-fracture repair (11,12) Twenty-four hydroxylation of 1, 25(OH)2 D is the first step
in the metabolic degradation of the active hormone. CYP24 gene transcription and
activation is strongly activated by 1, 25(OH)2 D (13). Thus, CYP24 induction can be
viewed as a feedback mechanism to control the biological actions of 1, 25(OH)2D.

2.2. Vitamin D Mediated Gene Transcription
Classically, 1, 25(OH)2D alters cell biology by activating the nuclear vitamin D receptor
(nVDR), a member of the steroid hormone receptor superfamily, leading to the induction
of gene transcription (10). The nVDR is expressed in a wide variety of cell types, from
those that are involved in whole body calcium metabolism, i.e. enterocytes, renal tubule
epithelial cells, and osteoblasts, to nontraditional vitamin D target tissues, e.g. immune
cells, epithelial cells (mammary, prostate, colon, lung), pancreatic (β cells, and
adipocytes (14). The biological actions of 1, 25(OH)2 D depend upon the presence and
level of the nVDR. For example, vitamin D-mediated calcium absorption is increased in
nVDR-overexpressing Caco-2 cells (15) and lower in nVDR null mice (16,17) while
nVDR level is an important determinant of the growth inhibition in response to 1,
25(OH)2D in prostate cancer cells (18–21).

The steps leading to vitamin D-mediated gene transcription are summarized in Fig. 1.
Ligand binding promotes heterodimerization of the nVDR with the retinoid X receptor
(RXR) and is required for migration of the RXR-nVDR-ligand complex from the
cytoplasm to the nucleus (22–25) where it then regulates gene transcription by interacting
with specific vitamin D response elements (VDRE) in the promoters of vitamin Dresponsive
genes (14). Although the consensus is that only a direct repeat with a 3 base
spacing (DR3)-type VDRE is functional in vivo (14,26), Makishima et al., (27) recently
found that both 1, 25(OH)2 D and lithocholic acid bind to the nVDR and induces CYP3A
gene transcription through a nontraditional ER6 (everted repeat with a 6 base spacing)
element. This suggests that the promoter elements conferring molecular regulation of
gene expression by 1, 25 (OH)2 D may be more diverse than researchers have
traditionally considered.

FIGURE 1 Steps required for activation of gene transcription by 1, 25(OH)2 D.

Access to VDREs in their chromosomal context may be limited (28) and may require
the release of constraints imposed by chromosomal structure through phosphorylation of
histone H3, acetylation of histones H3 and H4, and SWI/SNF complex-mediated
phosphorylation events (29–31). Protein-protein interactions mediated by the nVDR are
critical for chromosomal unwinding. The nVDR-RXR dimer recruits a complex with
histone acetyl transferase (HAT) activity (e.g., CBP/p300, SRC-1 (32,33)) and the
BAF57 subunit of mammalian SWI/SNF directly interacts with p160 family members
like SRC-1 as well as steroid hormone receptors (34). After chromosomal unwinding, the
nVDR-RXR dimer recruits the mediator D complex (DRIP) and utilizes it to recruit and
activate the basal transcription unit containing RNA polymerase II (35,36). It is known
that the composition of the mediator complex can vary depending upon the anchoring
transcription factor (31). Thus, mediator D complex contains 16 proteins, only 14 of
which are a part of the 18 protein mediator T/S complex involved in thyroid hormone
receptor gene transcription. Further examination of coactivator complexes associated
with nVDR-mediated gene transcription may be warranted. Preliminary evidence from
kerotinocytes indicates that the major anchoring protein in the mediator complex,
DRIP205, is replaced by the the steroid receptor coactivator (SRC) family members
SRC-2 and SRC-3 in differentiated cells (37). Several smaller members of the mediator
complex were still present in the complex. This suggests that there may be cell stagespecific
(and perhaps cell type-specific) differences in the coactivator complexes that
drive vitamin D-mediated gene expression.

2.3. Rapid Actions of 1, 25(OH)2 D
There is now compelling evidence for the existence of 1, 25(OH)2D-inducible signal
transduction pathways within various cell types (38) that includes the rapid (within
seconds and minutes) activation of phospholipase C (PLC), protein kinase C (PKC), and
the MAP kinases JNK and ERK (39–42). Figure 2 summarizes the pathways that have
been shown to be activated through rapid 1, 25(OH)2 D-mediated signaling. While
activation of these pathways by 1, 25(OH)2 D is now generally accepted, it is not clear
whether these actions require the activation of a unique membrane vitamin D receptor, as
suggested by Nemere et al. (43), or whether these rapid actions reflect a unique,
nonnuclear function of the traditional nVDR. Using cells isolated from mice expressing a
mutant nVDR lacking a DNA binding domain, Erben et al. (44) found that rapid calcium
signaling was dependent upon a functioning nVDR. This hypothesis is also supported by
recent work in myocytes, where nVDR binds to, and is a target for src kinase (45) and in
the enterocyte-like cell line, Caco-2, where 1, 25(OH)2 D binding to nVDR induces an
interaction between a ser/thr phosphatase that results in cell cycle arrest (46). In contrast,
Wali et al. (47) found that in osteoblasts from nVDR null mice, rapid increases in
calcium fluxes and PKC translocation did not require the presence of the nVDR.

2.4. Do Rapid and Nuclear Signaling Pathways Interact?
Several studies support the hypothesis that signal transduction pathways are important
regulators of nVDR-mediated gene expression. For example, suppression of PKC activity
with staurosporine or H7 inhibited 1, 25(OH)2 D-regulated 25-hydroxyvitamin D 24-
hydroxylase (CYP24) gene expression in proliferating, small intestine crypt-like, rat IEC-
6 cells (48) and activation of PKC with phorbol esters enhanced 1, 25(OH)2 D-regulated
CYP24 gene transcription in IEC-6 and IEC-18 cells (49). Similar findings have been
observed for 1, 25(OH)2 D-mediated osteocalcin gene expression in the osteoblast-like
ROS 17/2.8 cell (50), CYP24 gene induction in COS-1 cells (51), c-myc activation in
proliferating skeletal muscle (52) and CYP3A4 gene regulation in proliferating Caco-2
cells (53). Specific cross-talk between rapid, membrane initiated vitamin D actions and
nVDR-mediated genomic actions are supported by the observation that an antagonist of
the nongenomic pathway, 1β, 25(OH)2 D, blocks 1α, 25(OH)2 D-mediated osteocalcin
gene transcription in osteoblasts (54).

4. GENOMIC EVALUATION OF VITAMIN D ACTION

The application of genomic technology to the study of vitamin D action has been
relatively limited to date. Like most of the work that has been conducted using arrays, the
full power of the technology has not been applied. This is because until very recently,
arrays capable of profiling the entire transcriptome of 30,000–40,000 transcripts did not
exist.The studies that do exist have examined gene expression profiles in both classical
(e.g., bone, kidney, intestine, Caco-2, ROS/17/2.8) cells and nonclassical (e.g., HL-60,
squamous cell carcinoma, B) cells and used a variety of platforms (e.g., filters, spotted
Genomics and proteomics in nutrition 218
cDNA arrays, Affymetrix Genechips), sometimes with a limited number of highly
focused transcripts (e.g., 406 transcripts related to human hematology), and rarely with a
significant transcript target overlap with other platforms. This lack of consistency makes
it very hard to compare the results of one experiment to the next. However, even with this
caveat, the few studies available have been very promising. This section will review the
available array studies on vitamin D action.
4.1. Preliminary Reports in Classical Vitamin D Target Tissues
Surprisingly, a genomic examination of classical vitamin D target tissues is not yet
available as a peer reviewed report. However, several preliminary reports are available,
although caution should be used when interpreting these reports due to the lack of
experimental description (e.g., replicates, validation, statistical analysis, number of genes
represented on array that are present). Henry et al. (63) used the Affymetrix U74B
Genechip (12,000 targets; 6000 named genes) to compare the response of nVDR null
mice and wild-type mice to a single i.p. injection with 1, 25(OH)2 D (250 ng, 8 h). Using
a two-fold cut off, they identified 43 bone transcripts, 20 intestinal transcripts, and 98
kidney transcripts as 1, 25 (OH)2 D regulated in wild-type, but not in nVDR null, mice.
Peng et al. (64) injected vitamin D depleted mice with 1, 25(OH)2 D three times over 48 h
(30 ng per injection at 48, 24, and 6 h prior to the end of the experiment) and examined
the induction of renal transcripts using the U74B chip. They found only 57 genes
increased by 50% or greater and they confirmed vitamin D regulation of two of them,
C/EBP β and FK506. C/EBP β was subsequently shown to be involved in the regulation
of another 1, 25(OH)2 D-inducible gene, CYP24.
Megalin is a protein involved in the renal reabsorption of fat soluble vitamins like
vitamin D; as such, the megalin null mouse is somewhat equivalent to a vitamin D
depleted animal (plasma 1, 25(OH)2 D and 25-OH D are 60% lower in these mice). When
Hilpert et al. (65) examined renal gene expression in megalin knockout mice using the
Affymetrix MullK B chip (6,000 known transcripts), they found that the level of only six
transcripts fell and 13 transcripts increased. Finally, Wood et al. (66) examined gene
expression in the enterocyte-like Caco-2 cells after treatment with 100 nM 1, 25(OH)2 D
for 24 h using the Affymetrix U95A chip (12,000 targets). Using a two-fold cut off, 25
genes were upregulated (including amphiregulin, alkaline phosphatase, carbonic
anhydrase XII, and CYP 24) and five genes were downregulated (including dihydrofolate
reductase and a Ras-like protein). While these preliminary reports are interesting, the
genomic analysis of classical vitamin D target tissues clearly requires additional
examination.

4.2. Nonclassical Cells
1, 25(OH)2 D action has been studied in a number of nonclassical cell systems due to its
ability to initiate growth arrest and differentiation—characteristics that may be useful for
the prevention and treatment of cancer. A short report by Savli et al. (67) used the Atlas
hematology spotted filter array (406 genes) to examined the impact of 1, 25(OH)2
treatment (5nM, 24 or 72 h) on HL-60 leukemia cell gene expression. At 24 h 7 transcript
levels were upregulated and 25 transcript levels were downregulated. Twelve of these
Genomic approaches to understanding vitamin D action 219
transcripts were also downregulated at 72 h, including c-myc and 3 other oncogenes,
providing a glimpse into the mechanisms of chemop-revention by 1, 25(OH)2 D.
The most extensive genomic profiling of vitamin D action has been reported in
squamous cell carcinoma cell lines (68, 69). Akutsu et al. (68) found that 24 h of
treatment with 100 nM EB 1089 (a 1, 25(OH)2 D analog that is resistant to 24-
hydroxylation) increased 38 transcript levels (1.5-fold cut off) based on a combined
screening with an Atlas spotted cDNA filter array (588 genes) and a Research Genetics
GF211 spotted cDNA filter array (4000 named genes). This is likely a conservative
estimate due to problems the authors encountered with filter-to-filter, and hybridization
variability (a common problem with spotted filter arrays). Still, this analysis identified
up-regulation of several interesting transcripts that were validated by Northern blot
analysis: gadd45α, a p53 target gene that is involved in DNA repair, components of
various signal transduction pathways like the growth factor amphiregulin and
transcription factors AP-4, STAT3, and fra-1, and cell adhesion proteins like integrin
α7B. Six transcripts continued to be regulated in subsequent experiments even in the
presence of cycloheximide (e.g., p21, amphiregulin, VEGF, fra-1, gadd45α, and integrin
α7B). The mode of vitamin D regulation was not explored.
Lin et al. (69) conducted a time course of response to 100 nM 1, 25(OH)2 D and
EB1089 in squamous cell carcinoma cells (SCC25). Using the Affymetrix FL array and a
2.5-fold cut-off, 152 genes were identified as vitamin D regulated (89 up, 63 down).
Where overlap occurred, the results from Akutsu et al. (68) were validated and a number
of expected changes in transcripts previously shown to be vitamin D regulated were also
seen (e.g., CYP24, osteopontin, TGF β, PTHrp, CD14, VDUP1, carbonic anhy-drase II).
Clustering was done based upon the pattern of expression or the functional classification
of the transcripts. Figure 5 shows the diversity of the vitamin D responses in these cells.
Even within the category of genes with documented, functional VDREs, there was
heterogeneity in the response. For example, the CYP24 transcript level was rapidly
increased (significantly increased in 1 h) and was placed in group 1 (U1) while
osteopontin transcript levels increase more slowly (maximum expression at 12 h). This
suggests that similar DR3-type VDREs are differentially regulated depending upon the
promoter context, a finding that is consistent with studies by Toell (26). Another
interesting finding from this study is that the vitamin D-induced responses were much
more diverse than one might have previously predicted. For example, a number of
transcripts encoding for proteins involved in the protection from oxidative stress were
gradually up-regulated by EB1089 (falling into class U4 and U5); these include glucose 6
phosphate dehydrogenase (NADPH generation), glutathione peroxidase, and
selenoprotein P. In addition, the thioredoxin reductase transcript was increased by 1 h
after treatment with a peak induction by 6 h. Rapid suppression of a transcripts for a
variety of signaling peptides (e.g., PTHrp, galanin) and induction of intracellular cell
signaling proteins (e.g., cox-2, PI3K p85 subunit) was also seen after treatment. It is not
clear which of these responses is primary; none of these genes has previously been shown
to be vitamin D regulated or contain a functional VDRE. However, since 1, 25(OH)2 D
promotes cellular differentiation, the up regulation of some transcripts may represent a
vitamin D-induced shift to a more differentiated phenotype. Regardless, these data
suggest that the traditional approach of examining cell cycle proteins alone provides only
a limited

Author: James C.Fleet
Purdue University, West Lafayette, Indiana, U.S.A.

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