Multiple Roles of Angiopoietin-Like 4 in Osteolytic Disease (original) (raw)
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The Journal of pathology, 2017
Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature os...
Journal of Orthopaedic Research, 2009
The objective of this article was to investigate the role and expression of a novel adipocytokine, angiopoietin-like-4 (ANGPTL4), in arthropathy. Human chondrocytes were obtained from articular cartilage of patients with rheumatoid arthritis (RA) and osteoarthritis (OA), who underwent total knee or hip arthroplasty. Isolated chondrocytes were cultured under hypoxic (95% N 2 , 5% CO 2 ) or normoxic conditions. The effects of hypoxia on ANGPTL4 expression were determined by real-time reverse transcription polymerase chain reaction and Western blot analysis. We examined the role of ANGPTL4 using small interference RNA or by stimulating chondrocytes with recombinant ANGPTL4 protein. ANGPTL4 expression in the articular cartilage specimens was examined by immunohistochemistry. Hypoxia induced a significant increase in ANGPTL4 production (p < 0.05). Incubation of chondrocytes in vitro with recombinant ANGPTL4 enhanced the expression of matrix metalloproteinase (MMP)-1 and MMP-3. Downregulation of ANGPTL4 mRNA expression by siRNA diminished the expression of MMP-1, but not that of MMP-3, suggesting that each proteinase has a distinct response to ANGPTL4. Although the in vitro responses of chondrocytes to hypoxia were similar between RA and OA samples, the in vivo expression of ANGPTL4 had unique disease-specific patterns, suggesting differences in oxygen tension in vivo. Human chondrocytes expressed ANGPTL4 and the expression was enhanced by hypoxia. ANGPTL4 might modulate cartilage metabolism by regulating MMPs. ß
Angiopoietin-like protein 2 is a positive regulator of osteoblast differentiation
Metabolism: clinical and experimental, 2017
Several studies have reported that angiopoietin-like protein 2 (Angptl2) is expressed abundantly in adipocytes and is associated with adipose tissue inflammation. In the present study, we found that osteoblasts and mesenchymal stem cells also expressed Angptl2 at high levels. The aim of this study was to understand the role of Angptl2 in osteoblastic cell differentiation. Angptl2 expression was examined during osteoblast and adipocyte differentiation. The role of Angptl2 on cell differentiation and associated signaling was analyzed by gene knockdown using Angptl2 small interfering ribonucleic acid (siRNA). Angptl2 was highly expressed in MC3T3-E1 cells, ST2 cells and primary osteoblasts, but not in RAW264 cells. Inhibition of Angptl2 expression using siRNA markedly inhibited alkaline phosphatase (ALP) expression and osteoblastic differentiation in MC3T3-E1, ST2 cells and primary osteoblasts. Angptl2 siRNA also inhibited adipocyte differentiation in ST2 cells. Treatment of MC3T3-E1 c...
2015
The growth, maintenance and repair of bone are regulated by homeostatic interactions between osteoclasts, which resorb bone, and osteoblasts, which produce bone. Disruption of this balance in favor of osteoclast over-activation, in the absence of a balancing amount of bone formation, results in pathological bone loss such as that which occurs in osteoporosis, primary bone cancer, cancer metastasis to bone and rheumatoid arthritis. Hypoxia is a major micro-environmental feature of these conditions which is predictive of disease progression and poor prognosis. There is currently considerable interest in the mechanisms whereby hypoxia, the hypoxia-inducible transcription factors HIF-1α and HIF-2α, and the HIF-regulating prolyl hydroxylase (PHD) enzymes affect bone re-modelling and bone pathologies. This review summarises the evidence for HIF-mediated stimulation of osteogenicangiogenic coupling and the use of PHD inhibitors to stimulate new bone formation and prevent osteolytic disease...
BMC Cancer
Background: Osteosarcoma is the most common primary bone cancer in children and young adults. It is highly aggressive and patients that present with metastasis have a poor prognosis. Angiopoietin-like 4 (ANGPTL4) drives the progression and metastasis of many solid tumours, but has not been described in osteosarcoma tissue. ANGPTL4 also enhances osteoclast activity, which is required for osteosarcoma growth in bone. We therefore investigated the expression and function of ANGPTL4 in human osteosarcoma tissue and cell lines. Methods: Expression of ANGPTL4 in osteosarcoma tissue microarrays was determined by immunohistochemistry. Hypoxic secretion of ANGPTL4 was tested by ELISA and Western blot. Regulation of ANGPTL4 by hypoxia-inducible factor (HIF) was investigated using isoform specific HIF siRNA (HIF-1α, HIF-2α). Effects of ANGPTL4 on cell proliferation, migration (scratch wound assay), colony formation and osteoblastogenesis were assessed using exogenous ANGPTL4 or cells stably transfected with ANGPTL4. Osteoclastogenic differentiation of CD14+ monocytes was assessed by staining for tartrate-resistant acid phosphatase (TRAP), bone resorption was assessed by lacunar resorption of dentine. Results: ANGPTL4 was immunohistochemically detectable in 76/109 cases. ANGPTL4 was induced by hypoxia in 6 osteosarcoma cell lines, under the control of the HIF-1α transcription factor. MG-63 cells transfected with an ANGPTL4 over-expression plasmid exhibited increased proliferation and migration capacity and promoted osteoclastogenesis and osteoclast-mediated bone resorption. Individually the full-length form of ANGPTL4 could increase MG-63 cell proliferation, whereas N-terminal ANGPTL4 mediated the other pro-tumourigenic phenotypes. Conclusions: This study describes a role(s) for ANGPTL4 in osteosarcoma and identifies ANGPTL4 as a treatment target that could potentially reduce tumour progression, inhibit angiogenesis, reduce bone destruction and prevent metastatic events.
Role of angiogenesis in bone repair
Archives of Biochemistry and Biophysics, 2014
Mechanical loading represents a crucial factor in the regulation of skeletal homeostasis. Its reduction causes loss of bone mass, eventually leading to osteoporosis. In a previous global transcriptome analysis performed in mouse calvarial osteoblasts subjected to simulated microgravity, the most upregulated gene compared to unit gravity condition was Lcn2, encoding the adipokine Lipocalin 2 (LCN2), whose function in bone metabolism is poorly known. To investigate the mechanoresponding properties of LCN2, we evaluated LCN2 levels in sera of healthy volunteers subjected to bed rest, and found a significant time-dependent increase of this adipokine compared to time 0. We then evaluated the in vivo LCN2 regulation in mice subjected to experimentally-induced mechanical unloading by (1) tail suspension, (2) muscle paralysis by botulin toxin A (Botox), or (3) genetically-induced muscular dystrophy (MDX mice), and observed that Lcn2 expression was upregulated in the long bones of all of them, whereas physical exercise counteracted this increase. Mechanistically, in primary osteoblasts transfected with LCN2-expression-vector (OBs-Lcn2) we observed that Runx2 and its downstream genes, Osterix and Alp, were transcriptionally downregulated, and alkaline phosphatase (ALP) activity was less prominent versus empty-vector transduced osteoblasts (OBs-empty). OBs-Lcn2 also exhibited an increase of the Rankl/Opg ratio and IL-6 mRNA, suggesting that LCN2 could link poor differentiation of osteoblasts to enhanced osteoclast stimulation. In fact, incubation of purified mouse bone marrow mononuclear cells with conditioned media from OBs-Lcn2 cultures, or their coculture with OBs-Lcn2, improved osteoclastogenesis compared to OBs-empty, whereas treatment with recombinant LCN2 had no effect. In conclusion, our data indicate that LCN2 is a novel osteoblast mechanoresponding gene and that its regulation could be central to the pathological response of the bone tissue to low mechanical forces.© 2014 American Society for Bone and Mineral Research.
ATF4 promotes bone angiogenesis by increasing vegf expression and release in the bone environment
Journal of Bone and Mineral Research, 2013
Activating transcription factor 4 (ATF4) is a critical transcription factor for bone remodeling; however, its role in bone angiogenesis has not been established. Here we show that ablation of the Atf4 gene expression in mice severely impaired skeletal vasculature and reduced microvascular density of the bone associated with dramatically decreased expression of hypoxia-inducible factor 1a (HIF-1a) and vascular endothelial growth factor (VEGF) in osteoblasts located on bone surfaces. Results from in vivo studies revealed that hypoxia/ reoxygenation induction of HIF-1a and VEGF expression leading to bone angiogenesis, a key adaptive response to hypoxic conditions, was severely compromised in mice lacking the Atf4 gene. Loss of ATF4 completely prevented endothelial sprouting from embryonic metatarsals, which was restored by addition of recombinant human VEGF protein. In vitro studies revealed that ATF4 promotion of HIF-1a and VEGF expression in osteoblasts was highly dependent upon the presence of hypoxia. ATF4 interacted with HIF-1a in hypoxic osteoblasts, and loss of ATF4 increased HIF-1a ubiquitination and reduced its protein stability without affecting HIF-1a mRNA stability and protein translation. Loss of ATF4 increased the binding of HIF-1a to prolyl hydroxylases, the enzymes that hydroxylate HIF-1a protein and promote its proteasomal degradation via the pVHL pathway. Furthermore, parathyroid hormone-related protein (PTHrP) and receptor activator of NF-kB ligand (RANKL), both well-known activators of osteoclasts, increased release of VEGF from the bone matrix and promoted angiogenesis through the protein kinase C-and ATF4-dependent activation of osteoclast differentiation and bone resorption. Thus, ATF4 is a new key regulator of the HIF/VEGF axis in osteoblasts in response to hypoxia and of VEGF release from bone matrix, two critical steps for bone angiogenesis.
Osteopontin inhibits HIF-2α mRNA expression in osteoarthritic chondrocytes
Experimental and therapeutic medicine, 2015
The aim of the present study was to investigate the in vitro effect of osteopontin (OPN) on the expression of hypoxia-inducible factor-2α (HIF-2α) in chondrocytes and the role of OPN in osteoarthritis (OA). Cartilage was purified from the tibial surfaces of patients with OA of the knee and cultured in vitro to obtain chondrocytes. Recombinant human OPN (rhOPN) and OPN small interfering RNA (siRNA) were used to treat the chondrocytes, and the changes in the expression levels of the HIF-2α gene were measured. An anti-CD44 blocking monoclonal antibody (mAb) was used to determine the probable ligand-receptor interactions. Reverse transcription-quantitative polymerase chain reaction assays were designed and validated with SYBR® Green dyes for the simultaneous quantification of the mRNA expression levels of OPN and HIF-2α. The mRNA expression level of HIF-2α was markedly decreased in the rhOPN-treated group compared with that in the control group; by contrast, OPN siRNA increased HIF-2α ge...
Scientific Reports
Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Hypoxia modulates this relationship partially via direct and indirect effects of the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor on osteoclast formation and bone resorption. Little data is available on the role(s) of the HIF-2α isoform of HIF in osteoclast biology. Here we describe induction of HIF-1α and HIF-2α during the differentiation of human CD14+ monocytes into osteoclasts. Knockdown of HIF-1α did not affect osteoclast differentiation but prevented the increase in bone resorption that occurs under hypoxic conditions. HIF-2α knockdown did not affect bone resorption but moderately inhibited osteoclast formation. Growth of osteoclasts in 3D gels reversed the effect of HIF-2α knockdown; HIF-2α siRNA increasing osteoclast formation in 3D. Glycolysis is the main HIF-regulated pathway that drives bone resorption. HIF knockdown only affected...