The role of osteocalcin in the endocrine cross-talk between bone remodelling and energy metabolism

Abstract Bone remodelling, which maintains bone mass constant during adulthood, is an energy-demanding process. This, together with the observation that the adipocyte-derived hormone leptin is a major inhibitor of bone remodelling, led to the hypothesis that bone cells regulate energy metabolism through an endocrine mechanism. Studies to test this hypothesis identified osteocalcin, a hormone secreted by osteoblasts, as a positive regulator of insulin secretion, insulin resistance and energy expenditure. Remarkably, insulin signalling in osteoblasts is a positive regulator of osteocalcin production and activation via its ability to indirectly enhance bone resorption by osteoclasts. In contrast, leptin is a potent inhibitor of osteocalcin function through its effect on the sympathetic tone. Hence, osteocalcin is part of a complex signalling network between bone and the organs more classically associated with the regulation of energy homeostasis, such as the pancreas and adipose tissue. This review summarises the molecular and cellular bases of the present knowledge on osteocalcin biology and discusses the potential relevance of osteocalcin to human metabolism and pathology

GSK-3β Controls Osteogenesis through Regulating Runx2 Activity

Despite accumulated knowledge of various signalings regulating bone formation, the molecular network has not been clarified sufficiently to lead to clinical application. Here we show that heterozygous glycogen synthase kinase-3β (GSK-3β)-deficient mice displayed an increased bone formation due to an enhanced transcriptional activity of Runx2 by suppressing the inhibitory phosphorylation at a specific site. The cleidocranial dysplasia in heterozygous Runx2-deficient mice was significantly rescued by the genetic insufficiency of GSK-3β or the oral administration of lithium chloride, a selective inhibitor of GSK-3β. These results establish GSK-3β as a key attenuator of Runx2 activity in bone formation and as a potential molecular target for clinical treatment of bone catabolic disorders like cleidocranial dysplasia

New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism, but not in suckling

The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism

cAMP/PKA Regulates Osteogenesis, Adipogenesis and Ratio of RANKL/OPG mRNA Expression in Mesenchymal Stem Cells by Suppressing Leptin

BACKGROUND: Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. METHODS AND FINDINGS: We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX) and forskolin enhances adipogenesis, the gene expression of PPARgamma2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-kappaB Ligand to Osteoprotegerin (RANKL/OPG) gene expression - the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. CONCLUSIONS: Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression

Cross-talk between 1,25-dihydroxyvitamin D-3 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 effecters 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., Rawabata, 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, brit 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 acid vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter

Adventage of mesenchymal stem cells (MSC) expansion directly from purified bone marrow CD105^+ and CD271^+ cells

Mesenchymal Stem Cells (MSC) are employed in gene and cellular therapies. Routinely MSC are isolated from bone marrow mononuclear cells (MNC) by plastic adherence. Here we compared new isolation strategies of bone marrow MSC including immunodepletion of hematopoietic cells and immunomagnetic isolation of CD105+ and CD271+ populations. Four fractions were obtained: MNC MSC, RosetteSep-isolated MSC, CD105+ and CD271+ sorted MSC. We evaluated i) number of CFU-F colonies, ii) cell phenotype, iii) in vitro differentiation of expanded cells and iv) expression of osteo/adipogenesis related genes. Results: Average number of day 9 CFU-F colonies was the highest for CD271 positive fraction. Real-Time PCR analysis revealed expression of RUNX2, PPARgamma and N-cadherin in isolated cells, particularly high in CD271+ cells. Expression of CD105, CD166, CD44, CD73 antigens was comparable for all expanded populations (over 90%). We observed various levels of hematopoietic contamination with the highest numbers of CD45+ cells in MNC-MSC fraction and the lowest in CD105+ and CD271+ fractions. Cells of all the fractions were CD34 antigen negative. Expanded CD105 and CD271 populations showed higher level of RUNX2, osteocalcin, PTHR, leptin, PPARgamma2 and aggrecan1 genes except for alpha1 collagen. After osteogenic differentiation CD105+ and CD271+ populations showed lower expression of RUNX, PPARgamma2 and also lower expression of osteocalcin and PTHR than MNC, with comparable alpha1-collagen expression. Chondrogenic and adipogenic gene expression was higher in MNC. More clonogenic CD105+ and particularly CD271+ cells, which seem to be the most homogenous fractions based on Real-Time PCR and immunostaining data, are better suited for MSC expansion

RUNX2 mutations in Taiwanese patients with cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucleotide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine copy numbers of the promoter, all exons and the 3’UTR region of the RUNX2 gene detected the deletion of a single allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation detected by direct sequencing

Identification of a new pebp2 alpha A2 isoform from zebrafish runx2 capable of inducing osteocalcin gene expression in vitro

Introduction: RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2 alpha A) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone- and cartilage-related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2. Materials and Methods: Zebrafish runx2b gene and cDNA were isolated by RT-PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2 alpha A2 on osteocalcin expression was analyzed by transient co-transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE-PUZZLE 5.2. and MrBayes. Results and Conclusions: We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2, and showed its high degree of sequence similarity with the mammalian pebp2 alpha A. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2-binding elements, and one of them, located at -221 from the ATG, was capable of mediating pebp2 alpha A2 transactivation. In addition, cross-species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2 alpha A2 activated the murine osteocalcin promoter. These results are consistent with the high degree of evolutionary conservation of these proteins. The 3D structure of the runx2b runt domain was modeled based on the runt domain of mouse Runx1. Results show a high degree of similarity in the 3D configuration of the DNA binding regions from both domains, with significant differences only observed in non-DNA binding regions or in DNA-binding regions known to accommodate considerable structure flexibility. Phylogenetic analysis was used to clarify the relationship between the isoforms of each of the two zebrafish Runx2 orthologs and other Runx proteins. Both zebrafish runx2 genes clustered with other Runx2 sequences. The duplication event seemed, however, to be so old that, whereas Runx2b clearly clusters with the other fish sequences, it is unclear whether Runx2a clusters with Runx2 from higher vertebrates or from other fish.info:eu-repo/semantics/publishedVersio

Transcriptional regulation of bone formation by the osteoblast-specific transcription factor Osx

Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblast differentiation occurs through a multi-step molecular pathway regulated by different transcription factors and signaling proteins. Osx (also known as Sp7) is the only osteoblast-specific transcriptional factor identified so far which is required for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely and cartilage is normal. This opens a new window to the whole research field of bone formation. Osx inhibits Wnt pathway signaling, a possible mechanism for Osx to inhibit osteoblast proliferation. These reports demonstrate that Osx is the master gene that controls osteoblast lineage commitment and the subsequent osteoblast proliferation and differentiation. This review is to highlight recent progress in understanding the molecular mechanisms of transcriptional regulation of bone formation by Osx