Abstract
Plaque disruption with superimposed thrombosis is the main cause of unstable angina, myocardial infarction and sudden death. Instability of a plaque depends on the balance between release of extracellular matrix by human coronary artery smooth muscle cells (hCASMCs) and cytokines involved in inflammation. Insulin-like growth factor (IGF-i) is one of the most potent inducers of proliferation in hCASMCs, mainly through the activation of phosphatidyl inositol3 (Pl3)-Kinase and mitogen induces protein (MAP) Kinase pathways. Cytokines on the other hand use other pathways such as Jak/STAT (Janus activated kinase/ signal transducers and activators of transcription) to induce their effects. Recent studies have shown the role of suppressors of cytokine signaling, (SOCS) not only in inhibition cytokine signaling, but also in inhibiting growth factor signaling, such as insulin and IGF-i. They act in an auto regulatory negative feedback loop. For the first time I observed a dose dependent increase of SOCS3, which is mostly understood as an inhibitor of STAT3 related pathways, in response to both IGF-i and tumor necrosis factor (TNF)-a, in human coronary artery smooth muscle cells. I also found that SOCS3 inhibits cell proliferation mediated by IGF-i. However, its role in the regulation of smooth muscle cell proliferation in presence of growth factors and cytokines is not yet understood. In addition, underlying cellular and molecular mechanisms of SOCS3 action are unknown. Therefore I proposed an overall hypothesis that SOCS3 involves STAT3 pathway to attenuate IGF-i mediated human coronary artery smooth muscle cell proliferation and atheromatous cytokine TNF-a regulates this effect. I used SOCS3 over-expression and SOCS3 siRNA approaches to confirm the role of SOCS3 in hCASMCs proliferation. Following IGF-i stimulation, cells over-expressing SOCS3 were susceptible to a higher degree of apoptosis and became resistant to IGF-i induced proliferation. SOCS3 siRNA on the other hand, increased IGF-i induced hCASMCs proliferation. Western blot analysis revealed a reduction in IGF-i induced phosphorylation of Akt and Insulin Receptor Substrate-i in cells overexpressed SOCS3. For the first time, my studies showed activation of STAT3 in response to IGF-i. AG490, an inhibitor of STAT3, inhibited IGF-i induced proliferation of hCASMCs as detected by BrdU cell proliferation assay. Over-expression of SOCS3 in hCASMCs also resulted in suppression of IGF-i induced STAT3 activation and nuclear localization as detected by fluorescence microscopy. IGF-l and not TNF-a induced STAT3 phosphorylation; this effect was blocked upon over-expression of SOCS3 in hCASMCs. Inununoprecipitation studies revealed that SOCS3 associated with IGF-i receptor (IGF-iR) upon IGF-i stimulation, indicating its inhibitory role on IGF-i pathway upon association with the receptor. Finally to test the effect of SOCS3 on IGF-i treated hCASMCs in presence of atheromatous cytokine TNF-a, the cells were treated with both IGF-i and TNF-a and SOCS3 expression was found to be completely blocked. Nuclear factor-xB (NF- kB) promoter activity was activated with treatment of TNF-a in contrast to STAT3 activation by IGF-i. Since SOCS3 promoter region has response elements to both these transcription factors, a series of immunoprecipitation experiments revealed that antagonistic interaction of transcription factors STAT3 (activated by IGF-i) and NF-kB (activated by TNF-a) blocked induction of SOCS3 upon adding IGF-i together with TNF- a. Pre-treatment of cells with tyrphostin AG490 resulted in appearance of SOCS3 upon treatment of cells with IGF-i together with TNF-a. These novel findings suggest that SOCS3 expression in hCASMCs regulates the activity of IGF-l signaling. This leads to decreased cell proliferation in hCASMCs which might result in instability of atherosclerotic plaque. Thus, blocking SOCS3 in an atherosclerotic plaque lesion may serve as an important target in regulating IGF-i signaling.