Analysis of the role of SHIP2 in the regulation of preadipocyte proliferation and differentiation by PDGF

Abstract

Obesity, coupled with inadequate proliferation and/or differentiation of preadipocytes, favours development of adipose tissue dysfunction, characterized by hypertrophied, inflamed, and insulin-resistant adipocytes that predispose to type 2 diabetes and cardiovascular disease. Platelet-derived growth factor (PDGF) promotes proliferation and inhibits differentiation of preadipocytes, and thus might be relevant to preadipocyte fate and adipose tissue function. I hypothesized that SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) modulates both proliferation and differentiation of preadipocytes by mediating PDGF signalling in these cells. Two principal pathways regulating cell proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SHIP2 is important for proliferation likely due to its ability to dephosphorylate PI(3,4,5)P3 and associate with Shc in response to PDGF. Indeed, overexpression of wild-type SHIP2 inhibits 3T3-L1 preadipocyte proliferation; however, the underlying mechanisms are unclear. My first objective was to determine the role of regulatory regions of SHIP2 for its anti-proliferative action. PKC inhibition attenuated PDGF-stimulated SHIP2 tyrosine phosphorylation and Shc association. In addition, disruption of the SH2 domain, the NPAYY motif, or a novel PDGF-responsive phosphorylation site, Thr958, reduced SHIP2 tyrosine phosphorylation and/or Shc association, but neither altered the anti-proliferative effect of SHIP2. In contrast, inactivation of the 5-phosphatase domain potentiated the ability of SHIP2 to inhibit preadipocyte proliferation. This effect was explained by attenuated PDGF signalling caused by a decrease in receptor levels. My second objective was to characterize the negative effect of PDGF on adipocyte differentiation, and to determine the role of SHIP2 in this process. In 3T3-L1 preadipocytes, pro-adipogenic insulin stimulates production of PI(3,4,5)P3 only, whereas PDGF generates PI(3,4,5)P3 and PI(3,4)P2, suggesting activation of a 5-phosphatase. PDGF, but not insulin, leads to SHIP2 tyrosine phosphorylation and Shc association, suggesting that SHIP2 might mediate the anti-adipogenic action of PDGF. I established that PDGF impaired late markers of adipogenesis, and did not inhibit the required exit from the mitotic clonal expansion phase. Overexpression of catalytically inactive, dominant-negative SHIP2 accelerated differentiation and attenuated the anti-adipogenic effect of PDGF. Overall, this study suggests that SHIP2 mediates PDGF signalling in preadipocytes, thereby affecting both proliferation and differentiation, two distinct processes modulated by PDGF in opposite directions.