37 research outputs found
Impaired osteoblast differentiation in annexin A2- and -A5-deficient cells.
Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles
Vhl deficiency in osteocytes produces high bone mass and hematopoietic defects
Tissue oxygen (O2) levels vary during development and disease; adaptations to decreased O2 (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes. Because osteocytes orchestrate skeletal development and homeostasis, we evaluated the influence of Vhl or Hif1a disruption in osteocytes. Osteocytic Vhl deletion caused HBM phenotype, but Hif1a was dispensable in osteocytes. Vhl cKO mice revealed enhanced canonical Wnt signaling. B cell development was reduced while myelopoiesis increased in osteocytic Vhl cKO, revealing a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis
Prostaglandin E2 Signals Through PTGER2 to Regulate Sclerostin Expression
The Wnt signaling pathway is a robust regulator of skeletal homeostasis. Gain-of-function mutations promote high bone mass, whereas loss of Lrp5 or Lrp6 co-receptors decrease bone mass. Similarly, mutations in antagonists of Wnt signaling influence skeletal integrity, in an inverse relation to Lrp receptor mutations. Loss of the Wnt antagonist Sclerostin (Sost) produces the generalized skeletal hyperostotic condition of sclerosteosis, which is characterized by increased bone mass and density due to hyperactive osteoblast function. Here we demonstrate that prostaglandin E2 (PGE2), a paracrine factor with pleiotropic effects on osteoblasts and osteoclasts, decreases Sclerostin expression in osteoblastic UMR106.01 cells. Decreased Sost expression correlates with increased expression of Wnt/TCF target genes Axin2 and Tcf3. We also show that the suppressive effect of PGE2 is mediated through a cyclic AMP/PKA pathway. Furthermore, selective agonists for the PGE2 receptor EP2 mimic the effect of PGE2 upon Sost, and siRNA reduction in Ptger2 prevents PGE2-induced Sost repression. These results indicate a functional relationship between prostaglandins and the Wnt/ÎČ-catenin signaling pathway in bone
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Hypoxia Signaling in the Skeleton: Implications for Bone Health
Purpose of reviewWe reviewed recent literature on oxygen sensing in osteogenic cells and its contribution to development of a skeletal phenotype, the coupling of osteogenesis with angiogenesis and integration of hypoxia into canonical Wnt signaling, and opportunities to manipulate oxygen sensing to promote skeletal repair.Recent findingsOxygen sensing in osteocytes can confer a high bone mass phenotype in murine models; common and unique targets of HIF-1α and HIF-2α and lineage-specific deletion of oxygen sensing machinery suggest differentia utilization and requirement of HIF-α proteins in the differentiation from mesenchymal stem cell to osteoblast to osteocyte; oxygen-dependent but HIF-α-independent signaling may contribute to observed skeletal phenotypes. Manipulating oxygen sensing machinery in osteogenic cells influences skeletal phenotype through angiogenesis-dependent and angiogenesis-independent pathways and involves HIF-1α, HIF-2α, or both proteins. Clinically, an FDA-approved iron chelator promotes angiogenesis and osteogenesis, thereby enhancing the rate of fracture repair
HypoxiaâInducible Factorâ2α Signaling in the Skeletal System
ABSTRACT Hypoxiaâinducible factors (HIFs) are oxygenâdependent heterodimeric transcription factors that mediate molecular responses to reductions in cellular oxygen (hypoxia). HIF signaling involves stable HIFâÎČ subunits and labile, oxygenâsensitive HIFâα subunits. Under hypoxic conditions, the HIFâα subunit is stabilized, complexes with nucleusâconfined HIFâÎČ subunit, and transcriptionally regulates hypoxiaâadaptive genes. Transcriptional responses to hypoxia include altered energy metabolism, angiogenesis, erythropoiesis, and cell fate. Three isoforms of HIFâαâHIFâ1α, HIFâ2α, and HIFâ3αâare found in diverse cell types. HIFâ1α and HIFâ2α serve as transcriptional activators, whereas HIFâ3α restricts HIFâ1α and HIFâ2α. The structure and isoformâspecific functions of HIFâ1α in mediating molecular responses to hypoxia are well established across a wide range of cell and tissue types. The contributions of HIFâ2α to hypoxic adaptation are often unconsidered if not outrightly attributed to HIFâ1α. This review establishes what is currently known about the diverse roles of HIFâ2α in mediating the hypoxic response in skeletal tissues, with specific focus on development and maintenance of skeletal fitness. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research
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HypoxiaâInducible Factorâ2α Signaling in the Skeletal System
Hypoxia-inducible factors (HIFs) are oxygen-dependent heterodimeric transcription factors that mediate molecular responses to reductions in cellular oxygen (hypoxia). HIF signaling involves stable HIF-ÎČ subunits and labile, oxygen-sensitive HIF-α subunits. Under hypoxic conditions, the HIF-α subunit is stabilized, complexes with nucleus-confined HIF-ÎČ subunit, and transcriptionally regulates hypoxia-adaptive genes. Transcriptional responses to hypoxia include altered energy metabolism, angiogenesis, erythropoiesis, and cell fate. Three isoforms of HIF-α-HIF-1α, HIF-2α, and HIF-3α-are found in diverse cell types. HIF-1α and HIF-2α serve as transcriptional activators, whereas HIF-3α restricts HIF-1α and HIF-2α. The structure and isoform-specific functions of HIF-1α in mediating molecular responses to hypoxia are well established across a wide range of cell and tissue types. The contributions of HIF-2α to hypoxic adaptation are often unconsidered if not outrightly attributed to HIF-1α. This review establishes what is currently known about the diverse roles of HIF-2α in mediating the hypoxic response in skeletal tissues, with specific focus on development and maintenance of skeletal fitness. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research
Gap Junctional Intercellular Communication in Bone
Gap junctional intercellular communication has been demonstrated in bone cells and may contribute to the mechanism by which osteoblasts integrate and amplify extracellular signals, both chemical (hormonal) and biophysical (electrical and mechanical). Connexin 43 (Cx43) is the predominant gap junction protein expressed by bone cells. Experiments with osteoblastic cells in which Cx43 expression was diminished by antisense transfection demonstrate that cell-to-cell coupling in osteoblastic ROS 17/2.8 cells is via gap junctions composed of Cx43. Cellular networks of these coupling deficient clones are dramatically less responsive to parathyroid hormone (PTH) suggesting that coupling contributes to hormonal responsiveness. Furthermore, PTH per se can upregulate cell-to-cell communication in these networks. Membrane deformation-induced Ca2+ signals propagate from the deformed cell to neighboring undeformed cells. This phenomenon is blocked by octanol, a gap junction uncoupler. Physiologically relevant electric fields, i.e., induced by mechanical load, stimulate alkaline phosphatase activity in ROS 17/2.8 cells, but this response is greatly reduced in coupling deficient Cx43 antisense transfectants. Furthermore, electric fields per se regulate Cx43 expression in osteoblastic cells. Gap junctional intercellular communication appears critical for the regulation of osteoblastic behavior and thus bone metabolism by extracellular signals
PGE<sub>2</sub> signals through <i>Ptger2</i> to decrease <i>Sost</i> expression.
<p>(<b>A</b>) Cells were cultured for 3 hours in the presence of PTH (100 nM), PGE<sub>2</sub>, EP2 agonist butaprost, or EP4 agonist CAY10580 (each 500 nM). <i>Sost</i> expression was analyzed by qPCR and normalized to <i>Rpl32</i>. nâ=â5 samples. Compared to vehicle control, <b>b</b> indicates <i>p</i><0.01 and <b>c</b> indicates <i>p</i><0.001; <b>a</b> indicates <i>p</i><0.01 versus CAY10580. (<b>B</b>) UMR106.01 cells were cultured with 50 nM of scrambled or <i>Ptger2</i> siRNA for 48 hours, after which <i>Ptger2</i> expression was examined by qPCR. nâ=â4 samples. Compared to vehicle control, <b>a</b> indicates <i>p</i><0.05. (<b>C</b>) UMR106.01 cells were cultured with 50 nM of scrambled or <i>Ptger4</i> siRNA for 48 hours, after which <i>Ptger4</i> expression was examined by qPCR. nâ=â4 samples. Compared to vehicle control, <b>a</b> indicates <i>p</i><0.05. (<b>D</b>) UMR106.01 cells were cultured with 50 nM of scrambled, <i>Ptger2</i>, or <i>Ptger4</i> siRNA for 48 hours, then with 5 ”M PGE<sub>2</sub> for 3 hours, after which time total RNA was collected and analyzed for <i>Sost</i> and <i>Rpl32</i>. nâ=â5 samples. Compared to vehicle control, <b>b</b> indicates <i>p</i><0.01.</p
PGE<sub>2</sub> decreases <i>Sost</i> expression.
<p>(<b>A</b>) Human PTH(1â34) (100 nM) or PGE<sub>2</sub> (5 nMâ5 ”M) or vehicle control (0.05% DMSO) was added to UMR 106.01 cells for 3 hours. Total RNA was collected and analyzed for <i>Sost</i> and <i>Rpl32</i> expression by qPCR. nâ=â6â10 samples per treatment. <b>a</b> indicates <i>p</i><0.05 <i>versus</i> Control; <b>b</b> indicates <i>P</i><0.05 versus 5 nM PGE<sub>2</sub>. (<b>B</b>) <i>Sost</i> mRNA expression was quantified in UMR 106.01 cells after 0, 1, 2, 3, 6, or 24 hours treatment with 5 ”M PGE<sub>2</sub> or vehicle control. nâ=â4 samples per treatment. For PGE<sub>2</sub>, each time point is significantly different (<i>p</i><0.05) from Control, while for PTH, every time point except 1 hr is significantly different (<i>p</i><0.05) from Control.</p