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Altered phosphorylation of Syp may be responsible for abnormal insulin-like growth factor-1 signaling in human osteoarthritic osteoblasts

Introduction

The subchondral bone plays a prominent role in the pathophysiology of osteoarthritis (OA), possibly related to abnormal osteoblast metabolism. In particular, abnormal responses to insulin-like growth factor-1 (IGF-1) were noted in OA osteoblasts.

Objectives

To investigate whether IGF-1 signaling is abnormal in OA osteoblasts compared with normal osteoblasts.

Methods

We used primary human subchondral osteoblasts from normal and OA individuals. Cells were stimulated, or not, with 100 ng/ml IGF-1 for up to 5 min. Proteins were separated by SDS-PAGE or immunoprecipitated with anti-insulin receptor substrate-1 (anti-IRS-1) antibodies followed by SDS-PAGE, and detected with selective antibodies.

Results

Upon binding to its receptor (IGF-1R), IGF-1 activates the p42/44 mitogen-activated protein kinase (MAPK) pathway using SHC or phosphorylation of IRS-1 via Grb2. The interaction of IRS-1 with IGF-1R is also modulated via the binding of a tyrosine phosphatase, Syp. IGF-1R α-subunits, SHC, IRS-1, Syp, Grb2 and p42/44 MAPK levels, by Western blot analysis, were similar in normal and OA osteoblasts under basal condition and following IGF-1 treatment. IGF-1 stimulated IGF-1R autophosphorylation in normal and OA osteoblasts similarly. However, IRS-1 phosphorylation was reduced whereas p42/44 MAPK phosphorylation was higher in OA osteoblasts than normal osteoblasts in response to IGF-1. In normal osteoblasts, Syp was poorly phosphorylated under basal conditions and rapidly became phosphorylated upon IGF-1 stimulation, while Syp was already highly phosphorylated under basal conditions in OA osteoblasts and was dephosphorylated upon IGF-1 stimulation. Co-immunoprecipitation of Syp using IRS-1 antibodies showed that this interaction is poor under basal conditions in normal cells yet increases following IGF-1 treatment, while in OA osteoblasts this interaction was very strong and rapidly dropped with IGF-1 treatments, indicating that phospho-Syp is the trigger. Co-immunoprecipitation of Grb2 using IRS-1 antibodies showed that Grb2 interaction with IRS-1 was increased in OA osteoblasts compared with normal osteoblasts under basal conditions and following IGF-1 stimulation.

Conclusion

These results suggest that an abnormal interaction of phospho-Syp with IRS-1 in OA osteoblasts leads to a reduced activity of IRS-1-dependent pathways. In addition, the increased interaction of Grb2 with IRS-1 suggests that the SHC7–Grb2 interaction is reduced in OA osteoblasts, and hence could not explain the stimulation of p42/44 MAPK.

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Keywords

  • Normal Osteoblast
  • Altered Phosphorylation
  • MAPK Level
  • Osteoblast Metabolism
  • Subchondral Osteoblast