Beyond the established role of OH D in VDR

Beyond the established role of 1,25(OH)D3 in VDR signaling, the biological function of vitamin D precursors in mediating non-VDR dependent events remains controversial. Mutations in 7-dehydrocholesterol reductase are associated with Hh defects in mammals (Koide et al., 2006). Cell culture studies designed to isolate cholesterol derivatives affecting Hh signaling identified 3beta-hydroxysteroids, including D3, as negative Hh regulators in C3H/10T1/2 fibroblasts (Bijlsma et al., 2006). This study and others indicated exogenous addition of D3 may block Hh activity in a VDR-independent manner, potentially at the level of Smoothened (Bijlsma et al., 2006; Tang et al., 2011). Beyond these preliminary analyses, the mechanism of action of potential D3-mediated Hh regulation remains unclear and in vivo physiological relevance has not been elucidated. Here, we demonstrate that exposure to elevated D3 (12–24 hours post gli protein [hpf]) negatively regulates hematovascular niche specification, decreasing HSPC numbers while sparing the gross development of the dorsal aorta. D3 inhibits the Hh signaling axis via binding in vivo to the recently described extracellular sterol-binding domain of Smoothened. Likewise, physiological accumulation of D3 mediated by inhibition of cyp2r1 significantly reduced Hh activity and disrupted hemogenic niche specification, leading to low HSPCs. Together, these in vivo studies indicate inefficient vitamin D synthesis affects cellular specification and tissue maturation in the embryo, independent of reductions in 1,25(OH)D3 content, via negative regulation of the Hh signaling pathway.
Results
Discussion Beyond the role of vitamin D in calcium homeostasis, severe vitamin D deficiency in humans is associated with a variety of hematopoietic disorders, including anemia and extramedullary hematopoiesis (Yetgin et al., 1989), due to alterations in the bone marrow microenvironment. VDR-deficient mice likewise display extramedullary hematopoiesis (Jeanson and Scadden, 2010). Vitamin D is also important in the immune system, specifically affecting T cell development and function (Kongsbak et al., 2013). Despite these findings, there is minimal investigation into the role of vitamin D regulation in the earliest stages of hematopoiesis. In this study, we demonstrate that vitamin D precursor D3 affects hemogenic vascular niche specification, affecting HSPC numbers during embryogenesis. Excess D3 derived from exogenous exposure or insufficient Cyp2r1 function diminished Hh signaling and disrupted Hh-regulated mesodermal cell fate. Significantly, these effects were not phenocopied by 1,25(OH)D3 or dependent on VDR activity. Rather, our data indicate that D3 acts directly on Smoothened at the recently defined extracellular sterol-binding domain (Figure 3A). The strong synergy observed between D3 and cyclopamine toward Hh inhibition in vivo is consistent with prior mechanistic studies in which 20(S)-OHC and SAG mediated allosteric interactions between the CRD and the heptahelical binding domains of Smoothened to result in maximum pathway activation (Nachtergaele et al., 2012). Finally, the cyp2r1 loss-of-function studies demonstrate a physiological role for D3 in Hh pathway inhibition. These findings are intriguing in the context of human disease, because CYP2R1 mutations result in vitamin D-deficient rickets marked by skeletal abnormalities, with low levels of 25(OH)D3 but relatively normal levels of 1,25(OH)D3 (Al Mutair et al., 2012). While it has been postulated that maintenance of 1,25(OH)D3 sufficiency is due to increased parathyroid hormone activity and alterations in CYP27B1 and CYP24A1 function, it will be important to re-evaluate whether developmental abnormalities in these patients are partially due to reductions in Hh signaling caused by D3 accumulation. It will also be interesting to examine interplay between abnormal vitamin D biosynthesis and Hh signaling in cancer, specifically in tissues where both pathways intersect. In summary, we elucidated the physiological consequences of abnormal vitamin D biosynthesis at the level of D3 hydroxylation on hematovascular specification and characterized the mechanism of action in vivo as an antagonistic interaction of D3 with Smoothened and the Hh signaling pathway.