Reduction in Gsalpha induces osteogenic differentiation in human mesenchymal stem cells

We hypothesized that a decrease in Gsalpha expression occurs with osteogenic differentiation and that when Gsalpha expression was decreased by antisense oligonucleotides or direct inhibition of protein kinase A there was a concomitant increase in Runx2/Cbfa1. We also investigated the mechanism involved in the change in Runx2/Cbfa1 levels and whether the expression of other genes known to be involved in bone formation was altered. There was a decrease in Gsalpha expression with osteogenic differentiation and antisense oligonucleotides, and protein kinase A inhibition led to increased expression and DNA binding of the osteoblast-specific Runx2/Cbfa1. Additionally, with decreased Gsalpha expression or protein kinase A inhibition, Runx2/Cbfa1 protein was serine phosphorylated and ubiquitinated less. Microarray analysis, after the addition of antisense Gsalpha, showed a more than 10-fold increase in collagen Type I Alpha 2 mRNA (a target of Runx2/Cbfa1). These data show that reduced expression of Gsalpha can induce an osteoblast-like phenotype. The results also indicate a potential pathophysiologic role in patients with heterozygous inactivating mutations in GNAS1, the gene for the alpha chain (Gsalpha) of the heterotrimeric G protein, present in three disorders with ectopic intramembranous bone: Albright's hereditary osteodystrophy, progressive osseous heteroplasia, and osteoma cutis.     

Evaluation of 9.4-T MR microimaging in assessing normal and defective fetal bone development: comparison of MR imaging and histological findings

We evaluated 9.4-T magnetic resonance (MR) microimaging in assessing normal and defective bone development in mouse embryos. For this purpose, we performed 9.4-T MR microimaging on developing bones in normal embryos, and also in Runx2/Cbfa1-/- embryos with severely defective bone development. MR images were compared with the histological and histochemical features of these fetal bones. MR microimaging delineate successfully the normal long bone development in embryos. The T1- and T2-weighted MR microimaging demonstrated chondrocyte maturation in different regions of growing cartilage, such as epiphysis, physis, hypertrophic cartilage, and zone of provisional calcification. These developmental changes were detectable in as early as E14.5 embryos. The MR microimaging clearly demonstrated defective bone development in Runx2/Cbfa1-/- embryos. The femur from E18.5 homozygous Runx2/Cbfa1-/- embryos lacked MR signal intensity patterns including the hypertrophic cartilage, which are characteristic of the bone from the age-matched Runx2/Cbfa1+/+ embryos. Interestingly, however, the tibia from the same mutants was associated with MR signal patterns indicative of hypertrophic cartilage but not of the primary spongiosa and ossifying perichondrium, suggesting that bone development is differently regulated in these two long bones. On the other hand, the bones from heterozygous Runx2/Cbfa1+/- embryos exhibited an MR phenotype intermediate between the Runx2/Cbfa1+/+ and Runx2/Cbfa1-/- embryos; the primary spongiosa and ossifying perichondrium formation occurred normally even in the absence of preceding organized maturation of chondrocytes, a phenotype that was not detected by histological examinations. We concluded that MR microimaging is useful in assessing the bone development.     

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.     

Dentin matrix protein 1, a target molecule for Cbfa1 in bone, is a unique bone marker gene.

Dentin matrix protein 1 (Dmp1), a phosphoprotein highly linked to dentin formation, has also been reported to be expressed in the skeleton. However, the role of Dmp1 in skeletal tissues remains unclear. To clarify the role of Dmp1 in bone formation, we characterized the expression profile of Dmp1 in bone and cartilage and examined whether Dmp1 expression was regulated by core-binding factor a1 (Cbfa1). Studies of fetal rat calvarial (FRC) cell cultures showed that the expression of Dmp1 was associated closely with "bone nodule" formation and mineralization in vitro. In situ hybridization studies were performed to examine the spatial and temporal expression patterns of Dmp1 during development in mouse embryos from 12.5 day postcoitus (dpc) to 8 weeks postnatal; these studies showed that Dmp1 first appeared in hypertrophic cartilage cells, followed by osteoblasts, and later was expressed strongly in osteocytes. The expression profiles of Cbfa1 and Dmp1 overlapped in both cartilage and bone during development, with Cbfa1 preceding Dmp1. Examination of Dmp1 expression in Cbfa1-/- mice revealed that Dmp1 was absent in the developing bones of Cbfa1-null mice, whereas there was essentially no change in Dmp1 expression in the arrested tooth bud. Transient transfection studies showed forced expression of Dmp1 under the control of Cbfa1 and gel shift data indicated the presence of a functional osteocalcin-specific element (OSE)-2 response element in the Dmp1 proximal promoter region. However, in vitro promoter studies suggested that regulation of Dmp1 by Cbfa1 was not mediated by direct binding of Cbfa1 to this site and may be through indirect mechanisms. These studies highlight Dmp1 as a unique marker gene for osteoblastic differentiation. The close association of Dmp1 and Cbfa1 in the developing skeleton suggests that Dmp1 may play an important role in bone formation.     

Involvement of parathyroid hormone-related protein in vascular calcification of chronic haemodialysis patients

Aims: To investigate the role of parathyroid hormone‐related protein (PTHrP) in vascular calcification of patients with chronic hemodialysis. Methods: The inferior epigastric arteries were obtained from 23 patients on chronic haemodialysis and 16 patients with renal carcinoma as control. Haematoxylin‐eosin staining, elastic fibre staining, Alizarin Red calcium staining and immunohistochemical staining of PTHrP, bone morphogenetic protein‐2 (BMP‐2), Cbfa1/Runx2 were performed. Real‐time polymerase chain reaction (PCR) was used to examine mRNA expressions of PTHrP, BMP‐2 and Cbfa1/Runx2. Western blot and real‐time PCR were used to detect the effects of PTHrP‐siRNA and rh‐PTHrP‐1–34 on the expressions of PTHrP, BMP‐2 and Cbfa1/Runx2 in human aortic smooth muscle cells (HASMC). Alkaline phosphatase (ALP) activities and intracellular calcium content in HASMCs were assessed after treatment with 10 mmol/L β‐glycerol phosphoric acid for 48 h. Results: Vascular calcification was confirmed in 78.2% of patients on chronic haemodialysis, and the expressions of PTHrP, BMP‐2 and Cbfa1 in the arteries were significantly upregulated. PTHrP‐siRNA could downregulate the expression of PTHrP by 60%, BMP‐2 by 25% and Cbfa1 by 25% at 24 h (P < 0.05). Exogenous rh‐PTHrP‐1–34 could upregulate the expressions of BMP‐2 and Cbfa1 by 1.37‐fold and 1.46‐fold, respectively, at 24 h in a time‐independent manner (P < 0.05), which were attenuated by PTHrP‐siRNA. Moreover, it could promote intracellular calcium deposition and increase ALP activities, which were partially blocked by PTHrP‐siRNA (P < 0.05). Conclusions: Vascular calcification was common in patients with chronic haemodialysis, to which PTHrP might contribute by activating BMP‐2/ Cbfa1 signalling pathway.     

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.

Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. CONCLUSION: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.