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/β-catenin signaling pathway in bone

Hypoxia‐Inducible Factor‐2α Signaling in the Skeletal System

ABSTRACT 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‐β subunits and labile, oxygen‐sensitive HIF‐α subunits. Under hypoxic conditions, the HIF‐α subunit is stabilized, complexes with nucleus‐confined HIF‐β 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

Hypoxia‐Inducible Factor‐2α 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-β subunits and labile, oxygen-sensitive HIF-α subunits. Under hypoxic conditions, the HIF-α subunit is stabilized, complexes with nucleus-confined HIF-β 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

PGE₂ 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), PGE₂, EP2 agonist butaprost, or EP4 agonist CAY10580 (each 500 nM). <i>Sost</i> expression was analyzed by qPCR and normalized to <i>Rpl32</i>. n = 5 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. n = 4 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. n = 4 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 µM PGE₂ for 3 hours, after which time total RNA was collected and analyzed for <i>Sost</i> and <i>Rpl32</i>. n = 5 samples. Compared to vehicle control, <b>b</b> indicates <i>p</i><0.01.</p

PGE₂ decreases <i>Sost</i> expression.

<p>(<b>A</b>) Human PTH(1–34) (100 nM) or PGE₂ (5 nM–5 µM) 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. n = 6–10 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 PGE₂. (<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 µM PGE₂ or vehicle control. n = 4 samples per treatment. For PGE₂, 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

PTH decreases <i>Sost</i> expression independent of prostaglandins.

<p>Cells were exposed to 1 µM indomethacin for 24 hours, then treated with 100 nM hPTH(1–34) or vehicle control for 24 further hours. <i>Sost</i> expression was analyzed by qPCR and normalized to <i>Rpl32</i>. Compared to vehicle or indomethacin control, <b>a</b> indicates <i>p</i><0.05.</p