A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin‐stimulated proliferation of vascular smooth muscle cells

Abstract

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA 2 ) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA 2 are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA 2 and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA 2 by 1.5-fold ( p < 0.01), which was blocked by the cPLA 2 inhibitor MAFP (10 mM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 mM; 15 min) and ERK1/2 inhibitor PD98059 (20 mM; 15 min) abolished the INS-mediated increase in cPLA 2 phosphorylation by 59% (p < 0.001), and by 75% ( p < 0.001), respectively. Further, inhibition of cPLA 2 with cPLA 2 inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% ( p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% ( p < 0.001). The effect of INS on VSMCs proliferation was significantly ( p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA 2 and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA 2 activity. Ó

Role of PI3K/AKT, cPLA2 and ERK1/2 Signaling Pathways in Insulin Regulation of Vascular Smooth Muscle Cells Proliferation

“…Our observation of an acute INS (10 minutes) stimulation of VSMCs proliferation is consistent with previous observations [56,71] that INS, acting via its INS-linked receptor, also induces rapid tyrosine phosphorylation of IRS-1 [74] and binding of PI3K to IRS-1 in VSMCs [74]. Our recent published data indicate that the stimulation of the VSMCs proliferation by INS involves both Akt and ERK 1/2 [56,71] pathways based on the finding that activation was blocked with pharmacological inhibition of Akt and ERK 1/2. With regard to the Akt pathway, pretreatment with LY (inhibitor of Akt) [75] blocked the stimulation of VSMCs proliferation after acute (10 minutes) exposure to INS.…”

“…Although there is no previous published evidence of the synergistic interaction between the Akt and ERK 1/2 pathways in VSMCs, our published results demonstrating that inhibition of either ERK 1/2 or Akt activity diminished the induction of VSMCs proliferation led us to hypothesize that both pathways contribute to the action(s) of INS. Thus, the ability of INS to induce activation of Akt [56] and ERK 1/2 [71] is consistent with the notion that INS activates the proliferation of VSMCs through both Akt and ERK 1/2 signaling pathways [56,71]. As summarized in Fig.…”

“…Our observation that LY partially but significantly inhibited the INS-induced ERK1/2 phosphorylation in VSMCs [56] suggests that INSinduced ERK1/2 activation could be partially mediated by Akt signaling. In addition, our observation that PD partially but significantly inhibited INS-induced Akt phosphorylation in VSMCs also suggests that Akt activation could be partially mediated by ERK1/2 signaling [56,71]. Whether such an interaction occurs in INS-treated VSMCs remains to be established.…”

“…Activation of MAPK leads to the proliferation and migration of VSMCs, which are somewhat simplistically referred to as proatherogenic cellular processes [61]. There is a body of evidence suggesting that MAPK is a target for INS-mediated proliferation and migration was shown in in vitro studies using PD 98059, an inhibitor of the MAPK pathway [56,60,62]. Treatment of rat SMCs with PD 98059 inhibits the mitogenic response to INS in a concentrationrelated manner [60].…”

“…Our recently published data [56,62] showed that inhibition of ERK1/2 and PI3K/Akt activity abbrogated the INSstimulated VSMC's proliferation. Thus, both pathways are necessary for regulation of proliferation by INS on VSMCs.…”