Probing the structural requirements for vitamin D3 inhibition of the hedgehog signaling pathway

A structure-activity relationship study to elucidate the structural basis for hedgehog (Hh) signaling inhibition by vitamin D3 (VD3) was performed. Functional and non-functional regions of VD3 and VD2 were obtained through straightforward synthetic means and their biological activity was determined in a variety of cell-based assays. Several of these compounds inhibited Hh signaling at levels comparable to the parent VD3 with no effects on canonical vitamin D signaling. Most notably, compounds 5 and 9, demonstrated potent inhibition of the Hh pathway, exhibited no binding affinity for the vitamin D receptor (VDR), and did not activate VDR in cell culture. In addition, several compounds exhibited anti-proliferative activity against two human cancer cell lines through a mechanism distinct from the Hh or VDR pathways, suggesting a new cellular mechanism of action for this class of compounds.     

Vitamin D receptor–retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation

The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines.     

Let-7d modulates the proliferation,migration, tubulogenesis of endothelial cells

Calcitriol, vitamin D3 (VD3), and structurally related VD3 analogues are inhibitors of Hh signaling in multiple contexts and are promising anti-cancer agents in Hh-dependent forms of cancer; however, the cellular mechanisms through which these compounds regulate Hh signal transmission are not clearly defined. Previous studies in this area have implicated both Smoothened, a key mediator of Hh signaling, and the vitamin D receptor (VDR) as potential mediators of Hh inhibition for this class of seco-steroids. We have performed a series of in vitro studies to more fully probe the cellular mechanisms that govern seco-steroid-mediated inhibition of Hh signaling. Our results support a role for both the Hh and VDR pathways in this process, as well as the possibility that other, as yet unidentified proteins, are also central to seco-steroid-mediated inhibition of Hh signaling.

     

Growth hormone induces mitotic catastrophe of glomerular podocytes and contributes to proteinuria

Glomerular podocytes are integral members of the glomerular filtration barrier in the kidney and are crucial for glomerular permselectivity. These highly differentiated cells are vulnerable to an array of noxious stimuli that prevail in several glomerular diseases. Elevated circulating growth hormone (GH) levels are associated with podocyte injury and proteinuria in diabetes. However, the precise mechanism(s) by which excess GH elicits podocytopathy remains to be elucidated. Previous studies have shown that podocytes express GH receptor (GHR) and induce Notch signaling when exposed to GH. In the present study, we demonstrated that GH induces TGF-β1 signaling and provokes cell cycle reentry of otherwise quiescent podocytes. Though differentiated podocytes reenter the cell cycle in response to GH and TGF-β1, they cannot accomplish cytokinesis, despite karyokinesis. Owing to this aberrant cell cycle event, GH- or TGF-β1-treated cells remain binucleated and undergo mitotic catastrophe. Importantly, inhibition of JAK2, TGFBR1 (TGF-β receptor 1), or Notch prevented cell cycle reentry of podocytes and protected them from mitotic catastrophe associated with cell death. Inhibition of Notch activation prevents GH-dependent podocyte injury and proteinuria. Similarly, attenuation of GHR expression abated Notch activation in podocytes. Kidney biopsy sections from patients with diabetic nephropathy (DN) show activation of Notch signaling and binucleated podocytes. These data indicate that excess GH induced TGF-β1-dependent Notch1 signaling contributes to the mitotic catastrophe of podocytes. This study highlights the role of aberrant GH signaling in podocytopathy and the potential application of TGF-β1 or Notch inhibitors, as a therapeutic agent for DN.Subject terms: Podocytes, Diabetic nephropathy     

Role of CD14 expression in the differentiation-apoptosis switch in human monocytic leukemia cells treated with 1alpha,25-dihydroxyvitamin D3 or dexamethasone in the presence of transforming growth factor beta1.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.     

Human periodontal ligament cells derived from deciduous teeth induce osteoclastogenesis in vitro

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins involved in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament cells derived from deciduous teeth (DPDL cells) and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated by application of 10(-8) M 1 alpha, 25(OH)2 vitamin D3 [1,25-(OH)2D3] and 10(-7) M dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated a decrease in OPG following application of 1, 25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) were induced when DPDL cells were co-cultured with mouse bone marrow cells in the presence of 1,25-(OH)2D3 and Dex. TRAP-positive MNCs increased significantly when the DPDL cells were co-cultured with bone marrow cells in the presence of anti-human OPG antibody together with 1, 25-(OH)2D3 and Dex. These results indicate that PDL cells derived from deciduous teeth synthesize both RANKL and OPG and could regulate the differentiation of osteoclasts.     

Cross-talk between 1,25-dihydroxyvitamin D3 and transforming growth factor-beta signaling requires binding of VDR and Smad3 proteins to their cognate DNA recognition elements

1,25-Dihydroxyvitamin D(3) (vitamin D) and transforming growth factor-beta (TGF-beta) regulate diverse biological processes including cell proliferation and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effectors of TGF-beta signaling pathways whereas the vitamin D receptor (VDR) confers vitamin D signaling. We investigated the molecular mechanisms by which TGF-beta and vitamin D signaling pathways interact in the regulation of the human osteocalcin promoter. Synergistic activation of the osteocalcin gene promoter by TGF-beta and vitamin D was observed in transient transfection experiments. However, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic activation was not detectable when the osteocalcin vitamin D response element (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: -1030 to -989 (SBE3) and -418 to -349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but not the distal SBE3 in this promoter reporter abolished TGF-beta responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-beta and vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.     

Repeated Haloperidol Administration has No Effect on Vitamin D Signaling but Increase Retinoid X Receptors and Nur77 Expression in Rat Prefrontal Cortex

Both vitamin D (VD) signaling and Nur77 are implicated in dopaminergic neurotransmission and dopamine-related neuropsychiatric disorders, such as schizophrenia and Parkinson’s disease. Developmental vitamin D (DVD) deficiency rats exhibit schizophrenia-like behaviors and disturbance of dopamine system, which could be partly normalized by haloperidol treatment. By blocking dopamine D₂ receptor, haloperidol induces Nur77 expression, suggesting a modulatory role of Nur77 in brain dopamine system. Rxr is the heterodimeric partner of both Nur77 and vitamin D receptor and also participates in homeostatic regulation of central dopamine neurotransmission. Although D₂ antagonist-induced Nur77 expression has been reported by several studies, the change of its active partner Rxr remains elusive. Here, we studied the impact of 2 weeks administration of haloperidol on VD signaling and Nur77/Rxr expression in rat prefrontal cortex. It was found that haloperidol has no effect on local VD signaling, but could significantly increase Nur77, Rxr_β, and Rxr_γ expression, which indicated that Nur77/Rxr, but not vdr/Rxr, was implicated in dopamine-related neuroadaptation. Given that VD deficiency is commonly observed in schizophrenia patients, the renal metabolism of VD was also examined.     

Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells

Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.