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Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts.

ABSTRACT: Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.

SUBMITTER: Massicotte F

PROVIDER: S-EPMC1794522 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts.

Massicotte Frédéric F Aubry Isabelle I Martel-Pelletier Johanne J Pelletier Jean-Pierre JP Fernandes Julio J Lajeunesse Daniel D

Arthritis research & therapy 20060101 6

Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline ph ...[more]

PMID: 17129375

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Project description:Osteoarthritis (OA) is the most common human joint disease. Recent studies suggest that an abnormal subchondral bone metabolism is intimately involved in the genesis of this disease. Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. RANKL exists as 3 isoforms: RANKL1, 2, and 3. RANKL1 and 2 enhance osteoclastogenesis whereas RANKL3 inhibits this phenomenon. We previously reported that human OA subchondral bone osteoblasts can be discriminated into two subgroups according to their level of PGE2 [low (L) or high (H)]. Moreover, we also showed that L-OA osteoblasts express higher levels of total RANKL compared to H-OA osteoblasts. In this study, we investigated the level of membranous RANKL, comparing L- and H-OA subchondral bone osteoblasts, as well as its modulation by osteotropic factors. The impact of the modulation of RANKL1 and 3 on the membranous RANKL level was also studied.Gene expression was determined using real-time PCR for RANKL1 and semi-quantitative PCR for RANKL3. Membranous RANKL was measured by flow cytometry. The modulation of membranous RANKL and RANKL isoforms was monitored on the L- and H-OA osteoblasts and also following treatment with osteotropic factors, including vitamin D3 (50 nM), IL-1beta (100 pg/ml), TNF-alpha (5 ng/ml), PGE2 (500 nM), PTH (100 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml).Membranous RANKL levels were significantly increased in L-OA osteoblasts compared to normal (p<0.01) and H-OA (p<0.05). The gene expression level of the RANKL1 profile was reminiscent of the membranous RANKL level. Although RANKL3 gene expression was lower on the H-OA osteoblasts than on normal and L-OA osteoblasts (p<0.03), the overall outcome favoured RANKL1. Treatment with the tested factors showed a significant increase in membranous RANKL on the L-OA osteoblasts, with the exception of PTH and IL-17. Interestingly in this subpopulation, the RANKL3 gene expression level was significantly increased upon PTH and IL-17 treatment. No effect of the tested osteotropic factors was found on the H-OA.Our findings showed that the normal, L- and H-OA subchondral bone osteoblasts differentially express membranous RANKL and RANKL isoforms, and that treatment with osteotropic factors generally favours increased membranous localization of RANKL on L-OA compared to H-OA osteoblasts. This phenomenon appears to take place through differential modulation of each RANKL isoform.

Project description:INTRODUCTION: Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularisation parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates since they are up-regulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs. METHODS: Obs from the sclerotic portion of OA tibial plateaus were cultured either under 20% or 2% oxygen tension in the presence or not of 50 nM of 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factors (Hif)-1? and -2? was measured by real-time polymerase chain reaction and leptin production by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1?, Hif-2?, leptin and DKK2 was reduced using silencing (si) RNA technique. Signalling pathway of hypoxia-induced leptin was investigated by western blotting and mitogen-activated protein kinase (MAPK) inhibitors. RESULTS: As expected, hypoxia stimulated the expression of Hif-1 and Hif-2. The expression of leptin and DKK2 in Obs was also stimulated 7-fold and 1.8-fold respectively (p<0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold under normoxia, it further stimulated it to 28- and 6.2-fold under hypoxia (p<0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in presence of VitD3 (p<0.02). Compared to Obs incubated in the presence of siScramble RNAs, siHif-2? inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (p=0.004) and 60% (p<0.02), respectively while it failed to significantly alter the expression of DKK2. SiHif-1? has no effect on these genes. Immunoblotting showed that VitD3 greatly stabilized Hif-2? under hypoxic condition. The increase in leptin expression under hypoxia was also regulated, in addition to the role of Hif-2?, by p38 MAPK (p<0.03) and PI 3-kinase (p<0.05). Finally, we demonstrated that the expression of leptin and DKK2 were not related to each other under hypoxia. CONCLUSIONS: Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin particularly under VitD3 stimulation whereas DKK2 is mainly regulated by VitD3 rather than hypoxia.

Dataset Information

Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts.

Publications

Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts.

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