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There are three major projects in the laboratory.

(1) Molecular genetics of signaling by the nuclear vitamin D receptor and identification of vitamin D target genes implicated in (i) its capacity to stimulate innate immune responses, and (ii) its function as an anticancer agent. Full project description

(2) Chemical biology of vitamin D analogs. In collaboration with Dr. Jim Gleason in the Chemistry Department we are developing a series of bifunctional vitamin D analogs that combine agonism for the vitamin D receptor with inhibition of histone deacetylase activity. Full project description

(3) Characterization of novel factor identified in the lab called ligand-dependent corepressor (LCoR). LCoR was identified as a repressor of signaling by nuclear receptor transcription factors. Full project description

Nuclear receptors such as the vitamin D receptor are ligand-regulated transcription factors whose activities are controlled by a range of lipophilic extracellular signals, such as steroid hormones and thyroid hormone and vitamin D. They directly regulate transcription of genes whose products control many aspects of physiology and metabolism. As such, ligand-regulated nuclear receptors function as gene switches and control the expression of specific target genes by interacting with specific DNA sequences known as response elements (RE; see Figure 1)

Figure 1

Background: Vitamin D and the vitamin D receptor.
Vitamin D was first identified as a cure for nutritional rickets, a disease of bone growth caused by an inadequate uptake of dietary calcium. Cod liver oil was discovered as an excellent source of anti-rachitic activity in 1827, although it wasn’t until several decades later that the active ingredient was identified as vitamin D3. Even earlier, in 1822, a Polish physician experimenting with children reached the remarkable conclusion that sunlight cured rickets after noting that rickets was relatively rare in unpolluted rural areas. Almost 100 years later, in 1919, it was shown that artificial UV light cured rickets. Indeed, it is now known that secosteroidal vitamin D3 is produced in skin via photochemical and thermal conversion of 7-dehydrocholesterol in the presence of UVB light. UVB irradiation is absorbed by atmospheric ozone; consequently, surface UVB varies markedly in intensity with latitude and time of year. Moreover, as vitamin D intake is generally inadequate in most diets (29-31), vitamin D insufficiency or deficiency rises in frequency with increasing latitude.

Biologically active vitamin D (Figure 2) is generated via largely hepatic 25-hydroxylation catalyzed by CYP27A1 and CYP2R1 to produce 25-hydroxvitamin D (25D), followed by 1α-hydroxylation catalyzed by CYP27B1 in the kidneys and peripheral tissues.

Figure 2.

Much of the action of 1,25D can be explained by its binding to and activation of the vitamin D receptor (VDR). The VDR is a ligand-activated transcription factor composed of a highly conserved DNA binding domain, and an α-helical ligand binding domain. The ligand-bound VDR activates transcription by heterodimerization with retinoid X receptors (RXRs), which is essential for high affinity DNA binding to cognate vitamin D response elements (VDREs) located in the regulatory regions of 1,25D target genes.

VDREs are composed of direct repeats of PuG(G/T)TCA motifs separated by 3bp (DR3) or everted repeats with 6bp spacing (ER6). We discovered that ER8 motifs can also function as response elements for the VDR and related retinoic acid receptors (Tavera-Mendoza et al, 2006 [view PDF format]), thus partially integrating 1,25D and retinoid signaling. DNA-bound VDR/RXR heterodimers act to recruit numerous so-called coregulatory proteins, which control histone modifications, chromatin remodeling and RNA polymerase II binding and transcriptional initiation.

To find out more about vitamin D and the VDR, read our recently published article in Scientific American [view PDF format], and our reviews in BioEssays [view PDF format] and Infection and Immunity [view PDF format].

Background: Vitamin D insufficiency/deficiency and disease.
While there is no strict definition, vitamin D deficiency is widely defined as circulating 25D levels of less than 20ng/ml (50nM), whereas one is generally considered to be vitamin D sufficient with circulating 25D concentrations of greater than 30ng/ml (75nM). While vitamin D intoxication can occur, it is not observed until 25D levels reach 150ng/ml (375nM) or more, and is associated with hypercalcemia, which if chronic can result in urinary calculi (renal or bladder stones) and renal failure.

While cases of vitamin D toxicity do occur, vitamin D insufficiency/deficiency is far more prevalent. In temperate regions, solar UVB is insufficient to induce cutaneous vitamin D3 synthesis for periods around the winter solstice of up to 6 months or more at higher latitudes a period that is known as vitamin D winter (Figure 3). For obvious reasons, cutaneous vitamin D synthesis is also strongly influenced by skin colour. Lack of cutaneous vitamin D synthesis, coupled with vitamin D-poor diets, has contributed to high levels of vitamin D insufficiency or deficiency in European and North American populations.

Figure 3. Vitamin D winter (see Tavera-Mendoza and White, Scientific American, Nov. 2007).

Epidemiological studies link vitamin D deficiency to increased rates of cancer, as well as autoimmune and infectious diseases. U.S. rates of bladder, breast, colon, ovary and rectal cancer increase 2-fold from south to north and north-south gradients of autoimmune conditions such as multiple sclerosis, Crohn’s disease, and type 1 diabetes and have been documented.

Connections between vitamin D insufficiency and infectious diseases go back over 100 years, with the recognition in the 19th century that solar radiation was beneficial for patients suffering from tuberculosis (TB). Associations between vitamin D deficiency and TB susceptibility were made over 20 years ago. In addition, we have known for over 20 years that 1,25D inhibits the growth of M. tuberculosis in cultured human macrophages.

Project 1(i). Regulation of antimicrobial innate immunity by vitamin D.

Given that the VDR is a transcription factor and acts as a ligand-regulated gene switch, its signaling is ideally suited to analysis using genomic approaches. We have used a combination of microarrays and in silico screens for VDREs to identify several hundred 1,25D target genes We have performed extensive microarray analyses in my laboratory (Akutsu et al, Mol. Endocrinol. 15, 1127-39, 2001 [view PDF format]; Lin et al, Mol. Endocrinol., 16, 1243-56, 2002 [view PDF format]; Wang et al, Mol. Endocrinol. 19, 2005 [view PDF format], Tavera-Mendoza et al, EMBO Rep. 2006 [view PDF format] and have identified ~1000 novel target genes of 1,25D in head and neck squamous carcinoma cells. Our early studies were the first of their kind with vitamin D and among the first with nuclear receptor ligands in general.

In the course of in silico screening for VDREs, we noted that two genes encoding antimicrobial peptides (AMPs) CAMP (cathelicidin antimicrobial peptide, hCAP18, LL37) and DEFB2 (DEFB4, β-defensin 2) contained promoter-proximal consensus DR3-type response elements (Wang et al, 2004 [view PDF format]; Figures 4 and 5). AMPs are vanguards of innate immune responses against bacterial, fungal and viral attack, and many act directly by disrupting the integrity of pathogen membranes. These studies demonstrated for the first time that vitamin D is a direct inducer of antimicrobial innate immunity in humans. Since then we have found that 1,25D regulates other genes that control innate immune responses, and are actively studying the mechanisms and physiological consequences of their regulation.

These studies demonstrated for the first time that vitamin D is a direct inducer of antimicrobial innate immunity in humans. Since then we have found that 1,25D regulates other genes that control innate immune responses, and are actively studying the mechanisms and physiological consequences of their regulation.

Project 1(ii). Investigating the anticancer properties of vitamin D.

Identifying 1,25D target genes underlying its anticancer effects.
For the last several years, we have been analyzing the potential of analogs of vitamin D3 as potential agents of cancer chemoprevention. Vitamin D3 signals through a nuclear receptor, and is thus a regulator of gene transcription. They have provided several insights into how vitamin D signaling controls cell proliferation, and showed that vitamin D can induce mechanisms of DNA repair in target cells in the absence of a DNA damage signal. This includes the induction in vitro and in vivo of GADD45α, whose activity is essential for maintenance of global genomic stability.

In addition, we have found treatment of squamous carcinoma cells with a vitamin D analog induces expression of several markers of squamous cell differentiation, and suppresses markers of squamous carcinoma progression. We also showed that EB1089 had significant antitumor activity in a mouse model of head and neck squamous carcinoma in the absence of detectable hypercalcemia (Prudencio et al, J. Nat. Cancer Inst. 93, 745-53 [view PDF format]). The antiproliferative and “genoprotective” effects of EB1089 bode well for the potential of vitamin D analogs as chemoprevention agents.

We showed that 1,25D signaling induces expression of the cyclin-dependent kinase inhibitor p27KIP1 by reducing its turnover. Stablization of p27KIP1 protein arise from inhibition of expression by 1,25D of p45SKP2, a protein that regulates p27KIP1 proteasomal turnover (Lin et al 2003 [view PDF format].)

In addition, we identified a novel binding site of the VDR, an ER8 element, which is also recognized by related retinoic acid receptors (RARs), thus partially integrating vitamin D and retinoic acid (RA) signaling. We found that both 1,25D and RA induce expression of the gene encoding the cyclin-dependent kinase inhibitor p19^INK4D through an ER8 element (Tavera-Mendoza et al, 2006  [view PDF format])

Combined effects of 1,25D and histone deacetylase (HDAC inhibitors on cancer cell proliferation and survival.
More recently, we have been studying the combined effects of 1,25D and HDAC inhibitors on the proliferation and survival of 1,25D-resistant cancer cells. Histones are proteins that oligomerize to form nucleosomes, forming the structural backbone of chromatin. Interaction of DNA with histones is modulated by the acetylation state of exposed lysine residues. Acetylation is controlled by two complementary enzymes, histone acetyl-transferases (HATs) and HDACs. During gene transcription, HAT-catalyzed acetylation results in a reduction of charge on the histone surface, loosening the interaction with DNA and allowing access of transcription machinery. HDAC catalyzed deacetylation restores the charge interaction and thus inhibits transcription.

While HDACs were initially characterized for their capacity to deacetylate histones, they are probably better known as protein deacetylases as some HDACs, notably HDAC6, are cytoplasmic and can deacetylate cytoplasmic proteins such as HSP90 and tubulin.

Histone deacetylase inhibitors (HDACi's) have been investigated for application in treatment of cancer and recent preclinical studies have suggested that they may have therapeutic utility in treatment of immune system disorders. The most studied of these compounds are trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). HDACi's block cell cycle progression and induce apoptosis or differentiation depending on the cell type. The anti-proliferative activities of HDACi's have been demonstrated notably in breast, endometrial and ovarian cancer cells.

Thus, like 1,25D, HDACi inhibit cell proliferation and modify gene expression. We have found a very strong synergistic effect between 1,25D and TSA on 1,25D-resistant squamous carcinoma cells, and have been studying the mechanisms of this effect (Tavera-Mendoza et al, 2008 submitted for publication).

Triciferols: Bifunctional vitamin D analogues combining VDR agonism with HDAC inhibition.
Following up on our observations (above) that 1,25D and HDAC inhibitor TSA combined synergistically to block the proliferation of cancer cells, we collaborated with Dr. Jim Gleason in the Chemistry Department to develop and characterize a series of analogues of 1,25D, called triciferols (Figure 6) that combine VDR agonism and HDAC inhibition. The parent compound displayed more efficacious antiproliferative activity in a series of cancer cell lines. In addition, it was more efficacious than 1,25D at inducing autophagic cell death in the MCF-7 breast cancer cell line (Figure 7).

The bifunctionality of triciferols is notable for two reasons: (i) the HDACi activity is generated by modifying the 1,25D side chain without resorting to linker technology and (ii) 1,25D and HDACi have sympathetic, but very distinct biochemical targets; the hydrophobic VDR ligand binding domain and the active sites of HDACs, which are zinc metalloenzymes. These studies demonstrate the feasibility of combining HDAC inhibition with nuclear receptor agonism to enhance their therapeutic potential. In addition to the recently published parent compound ([view PDF format], Figure 6), we have now generated a series of triciferols that combine VDR agonism with HDACi activity with varying HDAC specificities. We are actively investigating their therapeutic potential.

Project 3. Structure/function studies of LCoR, a corepressor of gene expression.

We have recently identified a previously uncharacterized nuclear factor that we are calling ligand-dependent corepressor (LCoR; Fernandes et al, Mol. Cell, 11, 139-50, 2003). LCoR was identified as a protein that interacted with the hormone-bound estrogen receptor. This finding was remarkable as agonist-bound nuclear receptors usually recruit coactivators of transcription. LCoR represses ligand-dependent transcription by several steroid and non-steroid nuclear receptors. It binds to ligand-bound steroid receptors in vitro via a single “nuclear receptor” (NR) box, an amphipathic alpha helix containing the motif LXXLL. Mutation of this motif abolishes binding of LCoR to ligand-bound receptors, and disrupts its repressor activity. Mutagenesis of the estrogen receptor ligand binding has identified a broad surface of interaction with LCoR centered on the coactivator binding pocket (Figure 8)

Figure 8.

LCoR is widely expressed in the adult and throughout fetal development. Highest levels are found in the placenta, where most of LCoR is expressed in the syncytiotrophoblast layer, a critical interphase of maternal and fetal circulation and an important site of steroid hormone signaling.

Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and –independent modes of action. LCoR binds directly to specific class I and II HDACs in vitro and in vivo through a series of distinct binding domains.

This page was last edited on 04 July, 2008