Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

PURPOSE: Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts is involved in the progression of osteoarthritis (OA). Human osteoblasts isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin pathway, and a reduced mineralization in vitro as well as in vivo. These alterations were linked with an abnormal response to BMP-2. OA osteoblasts release factors such as the hepatocyte growth factor (HGF) that contribute to cartilage loss whereas chondrocytes do not express HGF. HGF can stimulate BMP-2 expression in human osteoblasts, however, the role of HGF and its effect in OA osteoblasts remains unknown. Here we investigated whether elevated endogenous HGF levels in OA osteoblasts are responsible for their altered response to BMP-2. METHODS: We prepared primary human subchondral osteoblasts using the sclerotic medial portion of the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals obtained at autopsy. The expression of HGF was evaluated by qRT-PCR and the protein production by western blot analysis. HGF expression was reduced with siRNA technique whereas its activity was inhibited using the selective inhibitor PHA665752. Alkaline phosphatase activity (ALPase) and osteocalcin release were measured by substrate hydrolysis and EIA respectively. Canonical Wnt/β-catenin signaling (cWnt) was evaluated both by target gene expression using the TOPflash TCF/lef luciferase reporter assay and western blot analysis of β-catenin levels in response to Wnt3a stimulation. Mineralization in response to BMP-2 was evaluated by alizarin red staining. RESULTS: The expression of HGF was increased in OA osteoblasts compared to normal osteoblasts and was maintained during their in vitro differentiation. OA osteoblasts released more HGF than normal osteoblasts as assessed by western blot analysis. HGF stimulated the expression of TGF-β1. BMP-2 dose-dependently (1 to 100ng/ml) stimulated both ALPase and osteocalcin in normal osteoblasts whereas, it inhibited them in OA osteoblasts. HGF-siRNA treatments reversed this response in OA osteoblasts and restored the BMP-2 response. cWnt is reduced in OA osteoblasts compared to normal, and HGF-siRNA treatments increased cWnt in OA osteoblasts almost to normal. Smad1/5/8 phosphorylation in response to BMP-2, which is reduced in OA osteoblasts, was corrected when these cells were treated with PHA665752. The BMP-2-dependent mineralization of OA osteoblasts, which is also reduced compared to normal, was only partially restored by PHA665752 treatment whereas 28days treatment with HGF reduced the mineralization of normal osteoblasts. CONCLUSION: OA osteoblasts expressed more HGF than normal osteoblasts. Increased endogenous HGF production in OA osteoblasts stimulated the expression of TGF-β1 and reduced their response to BMP-2. Inhibiting HGF expression or HGF signaling restored the response to BMP-2 and Smad1/5/8 signaling. In addition, decreased HGF signaling partly corrects the abnormal mineralization of OA osteoblasts while increased HGF prevents the normal mineralization of normal osteoblasts. In summary, we hypothesize that sustained elevated HGF levels in OA osteoblasts drive their abnormal phenotype and is implicated in OA pathophysiology

Lack of Adiponectin Drives Hyperosteoclastogenesis in Lipoatrophic Mice.

Long bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlighted the dual bone phenotype of PPARγ null mice: one the one hand, the increased bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explored the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibited mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis

Agonists of peroxisome proliferators-activated receptors (PPAR) α, β/δ or γ reduce transforming growth factor (TGF)-β-induced proteoglycans' production in chondrocytes

SummaryObjectiveTo investigate the potency of selective agonists of peroxisome proliferators-activated receptors' (PPAR) isotypes (α, β/δ or γ) to modulate the stimulating effect of transforming growth factor-β1 (TGF-β1) on proteoglycans' (PGs) synthesis in chondrocytes.MethodRat chondrocytes embedded in alginate beads and cultured under low serum conditions were exposed to TGF-β1 (10ng/ml), alone or in combination with the following agonists: Wy14643 for PPARα, GW501516 for PPARβ/δ, rosiglitazone (ROSI) for PPARγ, in the presence or absence of PPAR antagonists (GW6471 for PPARα, GW9662 for PPARγ). PGs' synthesis was evaluated by radiolabelled sulphate incorporation and glycosaminoglycans' (GAGs) content by Alcian blue staining of beads and colorimetric 1.9 dimethyl-methylene blue assay after beads' solubilization. Phosphorylation of Extracellular Signal-related Kinase1/2 (ERK1/2), Smad2/3 and p38-MAPK was assessed by Western Blot and production of prostaglandin E2 (PGE2) by Enzyme immuno-assay (EIA). Levels of mRNA for PPAR target genes [acyl-CoA oxidase (ACO) for PPARα; mitochondrial carnitin palmitoyl transferase-1 (CPT-1) for PPARβ/δ and adiponectin for PPARγ], aggrecan, TGF-β1 and genes controlling GAGs' side chains' synthesis were quantified by real time polymerase chain reaction and normalized over RP29 housekeeping gene.ResultsACO was selectively up-regulated by 100μM of Wy14643, CPT-1 by 100nM of GW501516 and adiponectin by 10μM of ROSI without cell toxicity. TGF-β1 increased PGs' synthesis by four-fold, GAGs' content and deposition by 3.5-fold and six-fold, respectively, while inducing aggrecan expression around 10-fold without modifying mRNA levels of GAGs' controlling enzymes. PPAR agonists inhibited the stimulating effect of TGF-β1 by 24–44% on PGs' synthesis and over 75% on aggrecan, GAGs' content and deposition with the following rank order of potency: ROSI>GW501516≥Wy14643. TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 was reduced by ROSI over GW501516 but not by Wy14643 whereas stimulated PGE2 production was inhibited by Wy14643 over GW501516 but not by ROSI. The effect of PPAR agonists on PPAR target genes and TGF-β1-induced aggrecan expression was reversed selectively by PPAR antagonists.ConclusionIn chondrocytes' beads, PPAR agonists reduced the stimulating effect of TGF-β1 on PGs by inhibiting TGF-β1-induced aggrecan expression in an isotype-selective manner. Thus, PPAR agonists could be deleterious in situation of cartilage repair although being protective in situation of cartilage degradation