VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells

Osteoblasts and adipocytes are thought to derive from a common bone marrow stromal cell (BMSC) precursor. Activation of the canonical Wnt signaling pathway plays a pivotal role in the differentiation of BMSCs along either of these two lineages, promoting osteogenesis and inhibiting adipogenesis. Liganded nuclear receptors, including the vitamin D receptor (VDR) and peroxisomal proliferator-activated receptor gamma (PPARgamma), can also affect BMSCs differentiation. To address whether VDR ablation modulates the differentiation of BMSCs into the osteoblast or adipogenic lineages, BMSCs were isolated from VDR null mice and from their wild-type littermates. VDR ablation did not alter osteoblastic differentiation. However, when cultured under adipogenic conditions, BMSCs from the VDR null mice expressed higher mRNA levels of PPARgamma and of markers of adipogenic differentiation. An increase in the size and number of mature adipocyte foci was also observed in cultures isolated from VDR null mice relative to those isolated from wild-type mice. To address whether the increased adipogenesis observed in the VDR null cultures was associated with inhibition of the canonical Wnt signaling pathway, mRNA levels for DKK1 and SFRP2 were examined. Cultures from the VDR null mice expressed higher levels of mRNA encoding DKK1 and SFRP2 than did the wild-type cultures. This difference is, at least in part, due to ligand-dependent actions of the VDR, since 1,25-dihydroxyvitamin D3 suppressed DKK1 and SFRP2 expression in wild-type cultures. Thus, the VDR inhibits adipogenesis of BMSCs at least in part by suppressing the expression of inhibitors of the canonical Wnt signaling pathway.     

Role of the vitamin D receptor in hair follicle biology

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.     

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.     

Functions of 1alpha,25-dihydroxyvitamin D(3) in mammary gland: from normal development to breast cancer

This review examines the role of 1alpha,25(OH)(2)D(3) (1,25D) and the vitamin D(3) receptor in growth regulation of normal and transformed mammary epithelial cells. 1,25D exerts both anti-proliferative and pro-apoptotic functions in transformed mammary cells such as MCF-7. The anti-proliferative effects of 1,25D have been linked to suppression of growth stimulatory signals and potentiation of growth inhibitory signals, which lead to changes in cell cycle regulators such as p21, p27, cyclins and Rb. The pro-apoptotic effects of 1,25D involve alterations in the relative ratios of the bcl-2 family members which regulate mitochondrial integrity. In MCF-7 human breast cancer cells, 1,25D mediated apoptosis is associated with translocation of the pro-apoptotic protein Bax to the mitochondria, generation of reactive oxygen species, dissipation of the mitochondrial membrane potential and release of cytochrome c. These mitochondrial events trigger apoptosis in a caspase-independent manner, since caspase inhibitors do not rescue 1,25D treated cells from death. The potential role of 1,25D in growth and differentiation of normal mammary epithelial cells has been examined in VDR null mice. Initial data indicates a significant decrease in ductal differentiation in VDR null mice compared to age matched wild type mice, reflected as an increased number of undifferentiated terminal end buds in the VDR null mouse. These data suggest that 1,25D promotes differentiation during early mammary gland development. In summary, our studies suggest an expanding role for the vitamin D(3) endocrine system in control of proliferation, differentiation and apoptosis of mammary epithelial cells.     

Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation

Both calcium and 1,25(OH)(2)D promote the differentiation of keratinocytes in vitro. The autocrine or paracrine production of 1,25(OH)(2)D by keratinocytes combined with the critical role of the epidermal calcium gradient in regulating keratinocyte differentiation in vivo suggest the physiologic importance of this interaction. The interactions occur at a number of levels. Calcium and 1,25(OH)(2)D synergistically induce involucrin, a protein critical for cornified envelope formation. The involucrin promoter contains an AP-1 site essential for calcium and 1,25(OH)(2)D induction and an adjacent VDRE essential for 1,25(OH)(2)D but not calcium induction. Calcium regulates coactivator complexes that bind to the Vitamin D receptor (VDR). Nuclear extracts from cells grown in low calcium contain an abundance of DRIP(205), whereas calcium induced differentiation leads to reduced DRIP(205) and increased SRC 3 which replaces DRIP in its binding to the VDR. In vivo models support the importance of 1,25(OH)(2)D-calcium interactions in epidermal differentiation. The epidermis of 1alphaOHase null mice fails to form a normal calcium gradient, has reduced expression of proteins critical for barrier function, and shows little recovery of the permeability barrier when disrupted. Thus in vivo and in vitro, calcium and 1,25(OH)(2)D interact at multiple levels to regulate epidermal differentiation.     

Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development

Wdr5 accelerates osteoblast and chondrocyte differentiation in vitro, and is developmentally expressed in osteoblasts as well as in proliferating and hypertrophic chondrocytes. To investigate the role of Wdr5 during endochondral bone development, transgenic mice overexpressing Wdr5 under the control of the 2.3-kb fragment of the mouse alpha(1) I collagen promoter were generated. The transgene was specifically expressed in the osteoblasts of transgene positive mice and was absent in the growth plate. Histological analyses at embryonic day 14.5 demonstrated that the humeri of transgene positive embryos were longer than those isolated from wild-type littermates largely due to an expansion of the hypertrophic chondrocyte layer. Acceleration of osteoblast differentiation was observed with greater and more extensive expression of type I collagen and more extensive mineral deposition in the bone collar of transgene positive embryos. Acceleration of vascular invasion was also observed in transgene positive mice. Postnatal analyses of transgenic mice confirmed persistent acceleration of osteoblast differentiation. Targeted expression of Wdr5 to osteoblasts resulted in earlier activation of the canonical Wnt signaling pathway in the bone collar as well as in primary calvarial osteoblast cultures. In addition, overexpression of Wdr5 increased the expression of OPG, a target of the canonical Wnt signaling pathway. Overall, our findings suggest that Wdr5 accelerates osteoblast differentiation in association with activation of the canonical Wnt pathway.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

Accelerated mammary gland development during pregnancy and delayed postlactational involution in vitamin D3 receptor null mice

The vitamin D receptor (VDR) is present in mammary gland, and VDR ablation is associated with accelerated glandular development during puberty. VDR is a nuclear receptor whose ligand, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] is generated after metabolic activation of vitamin D by specific vitamin D hydroxylases. In these studies, we demonstrate that both the VDR and the vitamin D 1-alpha hydroxylase (CYP27B1), which produces 1,25-(OH)(2)D are present in mammary gland and dynamically regulated during pregnancy, lactation, and involution. Furthermore, we show that mice lacking VDR exhibit accelerated lobuloalveolar development and premature casein expression during pregnancy and delayed postlactational involution compared with mice with functional VDR. The delay in mammary gland regression after weaning of VDR knockout mice is associated with impaired apoptosis as demonstrated by reductions in terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling staining, caspase-3 activation and Bax induction. Under the conditions used in this study, VDR ablation was not associated with hypocalcemia, suggesting that altered mammary gland development in the absence of the VDR is not related to disturbances in calcium homeostasis. Furthermore, in the setting of normocalcemia, VDR ablation does not affect milk protein or calcium content. These studies suggest that the VDR contributes to mammary cell turnover during the reproductive cycle, and its effects may be mediated via both endocrine and autocrine signaling pathways. Unlike many mammary regulatory factors that exert transient, stage-specific effects, VDR signaling impacts on mammary gland biology during all phases of the reproductive cycle.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.