Desialylation of insulin receptors and IGF-1 receptors by neuraminidase-1 controls the net proliferative response of L6 myoblasts to insulin.

We recently established that the subunit of cell surface-residing elastin receptor, neuraminidase-1 (Neu1), can desialylate adjacent insulin-like growth factor 1 receptors (IGF-1R) of arterial smooth muscle cells, thereby quenching their proliferative response to insulin-like growth factor II. In this study, we explored whether Neu1 would also desialylate the insulin receptors (IR), as well as the IGF-1R on rat skeletal L6 myoblasts, and whether desialylation of IR and IGF-1R would affect a net proliferative effect of insulin. First, we found that physiological (0.5-1 nM) and high therapeutic (10 nM) insulin concentrations induced a modest increase in proliferation rate of cultured L6 myoblasts. While IR kinase inhibitor could abolish the mitogenic effect of these insulin concentrations, the observed more pronounced proliferative response to supraphysiological concentration (100 nM) of insulin could be eliminated only by specific inhibition of IGF-1R. Then, we found that treatment of L6 cells with mouse-derived Neu1 or with Clostridium perfringens neuraminidase caused desialylation of IR, which coincided with a significant increase of their proliferative response to lower (0.5-10 nM) concentrations of insulin. In contrast, experimental desialylation of IGF-1R coincided with elimination of the heightened proliferative response of L6 myoblasts to 100 nM insulin. Importantly, we also found that inhibition of endogenous Neu1 abolished the increase in proliferation of L6 cells induced by 1 and 10 nM of insulin, but amplified the proliferative effect of 100 nM insulin. We therefore conclude that desialylation of both IR and IGF-1R by Neu1 controls the net proliferative response of skeletal myoblasts to insulin.

Aldosterone stimulates elastogenesis in cardiac fibroblasts via mineralocorticoid receptor-independent action involving the consecutive activation of Galpha13, c-Src, the insulin-like growth factor-I receptor, and phosphatidylinositol 3-kinase/Akt.

We previously demonstrated that aldosterone, which stimulates collagen production through the mineralocorticoid receptor (MR)-dependent pathway, also induces elastogenesis via a parallel MR-independent mechanism involving insulin-like growth factor-I receptor (IGF-IR) signaling. The present study provides a more detailed explanation of this signaling pathway. Our data demonstrate that small interfering RNA-driven elimination of MR in cardiac fibroblasts does not inhibit aldosterone-induced IGF-IR phosphorylation and subsequent increase in elastin production. These results exclude the involvement of the MR in aldosterone-induced increases in elastin production. Results of further experiments aimed at identifying the upstream signaling component(s) that might be activated by aldosterone also eliminate the putative involvement of pertussis toxin-sensitive Galphai proteins, which have previously been shown to be responsible for some MR-independent effects of aldosterone. Instead, we found that small interfering RNA-dependent elimination of another heterotrimeric G protein, Galpha13, eliminates aldosterone-induced elastogenesis. We further demonstrate that aldosterone first engages Galpha13 and then promotes its transient interaction with c-Src, which constitutes a prerequisite step for aldosterone-dependent activation of the IGF-IR and propagation of consecutive downstream elastogenic signaling involving phosphatidylinositol 3-kinase/Akt. In summary, the data we present reveal new details of an MR-independent cellular signaling pathway through which aldosterone stimulates elastogenesis in human cardiac fibroblasts.