Milan R. Uskokovic

Vitamin D: a negative endocrine regulator of the renin-angiotensin system and blood pressure

The renin-angiotensin system (RAS) plays a central role in the regulation of blood pressure, volume and electrolyte homeostasis. Inappropriate activation of the RAS may lead to hypertension. Clinical and epidemiological studies have suggested a correlation between Vitamin D-deficiency and high blood pressure. Our recent studies demonstrate that Vitamin D is a potent endocrine suppressor of renin biosynthesis to regulate the RAS. Mice lacking the Vitamin D receptor (VDR) have elevated production of renin and angiotensin (Ang) II, leading to hypertension, cardiac hypertrophy and increased water intake. These abnormalities can be prevented by treatment with an ACE inhibitor or AT(1) receptor antagonist. Vitamin D repression of renin expression is independent of calcium metabolism, the volume- and salt-sensing mechanisms and the Ang II feedback regulation. In normal mice, Vitamin D-deficiency stimulates renin expression, whereas injection of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] reduces renin synthesis. In cell cultures, 1,25(OH)(2)D(3) directly suppresses renin gene transcription by a VDR-dependent mechanism. Furthermore, we have found that Gemini compounds have more potent renin-suppressing activity than 1,25(OH)(2)D(3). Collectively, our studies reveal a critical role of the Vitamin D endocrine system in the regulation of blood pressure and volume homeostasis, and suggest that low calcemic Vitamin D analogs may potentially be developed into a new class of anti-hypertensive agents to control renin production and blood pressure.

Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL‐12 and enhanced IL‐10 secretion. We have found that a short treatment with 1,25(OH)2D3 induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor‐type spleen cells and allows acceptance of donor‐type vascularized heart grafts. This effect is enhanced by co‐administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)2D3 on DCs. Graft acceptance is associated with an increased percentage of CD4+CD25+ regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4+CD25+ cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4+CD25− cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25‐dihydroxy‐16,23Z‐diene‐26,27‐hexafluoro‐19‐nor vitamin D3 (BXL‐698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non‐hypercalcemic doses. The enhancement of CD4+CD25+ regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach. J. Cell. Biochem. 88: 227–233, 2003.

Direct α-iodination of cycloalkenones

Abstract The direct preparation of α-iodoenones by treatment of enones with I 2 /pyridine in CCl 4 is described.

Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands.

Vitamin D receptor (VDR) ligands, in addition to controlling calcium metabolism, exert important effects on the growth and differentiation of many cell types and possess pronounced pro-tolerogenic immunoregulatory activities. VDR ligands can act directly on T cells, but antigen-presenting cells (APCs), and in particular dendritic cells (DCs), appear to be primary targets for their tolerogenic properties. The capacity of VDR ligands to target APCs and T cells is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kB that is down-regulated in APCs and in T cells. VDR ligands can induce in vitro and in vivo tolerogenic DCs able to enhance CD4(+)CD25(+) suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR ligands, and are potentially relevant for the treatment of Th1-mediated autoimmune diseases and allograft rejection.

Synergistic growth inhibition of prostate cancer cells by 1α, 25 dihydroxyvitamin D3 and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1α,25 Dihydroxyvitamin D3 (1α,25(OH)2D3) in regulating the growth and differentiation of the normal prostate gland yet prostate cancer cells appear significantly less sensitive to this action. Vitamin D3 receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative effects of 1α,25(OH)2D3 and therefore it is unclear why prostate cancer cell lines are significantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1α,25(OH)2D3 are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 μM and 15 nM respectively), which alone were relatively inactive, synergized with 1α,25(OH)2D3 in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D3 hexafluoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclin-dependent kinase inhibitor, p21(waf1/cip1) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1α,25(OH)2D3 during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D3 analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgen-dependent and independent prostate cancer.

1α,25-Dihydroxyvitamin D3 inhibits phorbol ester-dependent chemical carcinogenesis in mouse skin

The effect of topical application of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) on the promotional phase of skin tumor formation in mice was evaluated using 7,12-dimethylbenz [a] anthracene as the tumor initiator and 12-0-tetradecanoylphorbol-13-acetate (TPA) as the tumor promoter. Fifteen weeks of twice weekly topical application of 1 alpha, 25-(OH)2D3 1 hour prior to topical treatment with 16 nmol of TPA inhibited tumor formation in a dose-dependent manner. Doses of 0.25-0.50 nmol of the vitamin D3 metabolite inhibited tumor formation approximately 50% and had no significant effect on the survival or weight gain of the mice. These results indicate that in addition to maintaining calcium homeostasis and affecting the growth and differentiation of certain neoplastic cells, 1 alpha, 25-(OH)2D3 can also suppress the formation of chemically induced tumors.

Altered Nuclear Receptor Corepressor Expression Attenuates Vitamin D Receptor Signaling in Breast Cancer Cells

Purpose: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1α,25-dihydroxyvitamin D3. Experimental Design: Profiling, transcriptional, and proliferation assays were undertaken in 1α,25(OH)2D3-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21). Results: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G1 of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor α–negative (n = 7) tumors. 1α,25(OH)2D3 resistance in cancer cell lines was targeted by cotreatments with either 1α,25(OH)2D3 or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D3 compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells. Conclusions: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1α,25(OH)2D3 resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D3 compounds and low doses of TSA.

Analogs of 1α,25-dihydroxyvitamin D3 as novel inhibitors of renin biosynthesis

The renin-angiotensin system (RAS) plays a central role in the pathogenesis of hypertension. Recently, we discovered that 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions as a negative endocrine regulator of renin biosynthesis, which provides a molecular basis to explore the potential of Vitamin D analogs as renin inhibitors to control the RAS. To search for renin-inhibiting Vitamin D analogs, we screened 20 Vitamin D analog compounds using As4.1-hVDR cell (a juxtaglomerular cell line) culture by Northern blot and luciferase reporter assays. We found that the Gemini compounds, which have two side-chains at carbon-20 position, were particularly active in suppressing renin expression. Eight Gemini compounds were identified that were equally or 10- to 100-times more potent than 1,25(OH)(2)D(3) in renin inhibition. These Gemini compounds also potently stimulate 25-hydroxyvitamin D 24-hydroxylase expression in As4.1-hVDR cells. Administration of compound RO-27-5646 [1,25-dihydroxy-21-(3-methyl-3-hydroxy-butyl)-19-nor-cholecalciferol] in mice caused a marked reduction in renal renin mRNA expression without invoking severe hypercalcemia as seen in 1,25(OH)(2)D(3) treatment. These data establish in principle that Vitamin D analogs can indeed inhibit renin expression in vitro and in vivo, and support the notion that low calcemic Vitamin D analogs can potentially be used as therapeutic agents to control the RAS.

Total synthesis of acosamine and daunosamine utilizing a diastereoselective intramolecular [3+2] cycloaddition

Abstract A total synthesis of daunosamine (3) and acosamine (4) has been accomplished via a diastereoselective intramolecular nitrone-olefin cyclization. In the key step the chiral nitrone 12a cyclized to give two isoxazolidines 13a and 14a in an 82:18 ratio. Further elaboration of 13a led to daunosamine and acosamine. The effects of olefin substitution on the diastereoselectivity of the cycloaddition was also examined.

Combined effect of vitamin D3 analogs and paclitaxel on the growth of MCF-7 breast cancer cells in vivo

Vitamin D3 analogs and paclitaxel (Taxol) are able to inhibit the in vitro growth of a variety of malignant cells including breast cancer cells. These two compounds decrease growth by different mechanisms and they have non-overlapping toxicities. We examined the abilities of three vitamin D3 compounds to inhibit growth of a human mammary cancer (MCF-7) in BNX triple immunodeficient mice either alone or with Taxol. Vitamin D3 analogs were 1,25(OH)2D3 (code name, Compound C), 1,25(OH)2-16-ene-23-yne-19-n or-26,27-F6-D3 (Compound LH), and 24a,26a,27a,-trihomo-22,24-diene-1,25(OH)2D3 (EB1089). At the doses chosen, the antitumor effect of vitamin D3 analogs alone was greater than that of Taxol alone, and an additive effect was observed when a vitamin D3 analog and Taxol were administered together. EB1089 was the most potent compound, and the EB1089 plus Taxol was the most active combination, decreasing the tumor mass nearly 4-fold compared to controls. Weight-gain in each of the experimental cohorts at the end of the study was less than the control group, but the gain was significantly less in only two experimental groups (those receiving either EB1089 or Compound C plus Taxol). None of the animals became hypercalcemic, and their complete blood counts, serum electrolyte analyses, and liver and renal functions were all fairly similar and within the normal range. In summary, this combination of a vitamin D3 analog and Taxol has the potential to be a therapy for breast cancer.