Abstract 159: Fyn Kinase Activation Increases Apoptosis in LAR-Deficient Vascular Smooth Muscle Cells
Apoptosis of vascular smooth muscle cells (VSMC) is a hallmark of advanced atherosclerotic lesions and reactive oxygen species (ROS) have been implicated in VSMC apoptosis. VSMC respond to ROS via activation/deactivation of key intracellular signaling pathways which, in turn, are regulated by phosphorylation status of requisite tyrosine and serine/threonine residues on proteins. Protein tyrosine phosphorylation is regulated by coordinate action of protein tyrosine kinases and protein tyrosine phosphatases (PTP). Previously we reported that leukocyte antigen-related (LAR) PTP regulates IGF-1-induced signaling in VSMC. To determine whether LAR expression is important in apoptosis, we investigated hydrogen peroxide (H2O2; 1 mM)-induced apoptosis and cell signaling in aortic VSMC derived from wild-type (LAR+/+) and LAR knockout (LAR−/ −) mice. Several lines of evidence suggest that Fyn kinase is essential, proximal event signaling event in H2O2-induced apoptosis:
H2O2-induced apoptosis and tyrosine phosphorylation of Fyn kinase were significantly enhanced in LAR−/ − VSMC compared with wild-type cells following H2O2 treatment
LAR directly binds to both Fyn and JAK2 as observed by immunoprecipitation, however, recombinant LAR dephosphorylates only Fyn kinase. PP2, a Fyn-specific inhibitor, blocked H2O2-induced activation of JAK2, STAT3 and p38MAP kinase which suggests that Fyn is upstream of JAK-STAT and p38MAP kinase pathways and significantly attenuated H2O2-induced apoptosis in LAR−/ − cells; and
LAR deficiency enhanced H2O2-induced tyrosine phosphorylation of JAK2, STAT3, activation of p38MAP kinase and phosphorylation of ERK1/2. However, the increase in ERK1/2 phosphor-ylation was observed only at later (20 – 40 min), but not at early (5–15 min) time points.
Pretreatment with AG-490, a JAK2 specific inhibitor, blocked activation of STAT3, but not p38MAP kinase suggesting that JAK-STAT pathway and p38MAP kinase are two divergent signaling cascades in VSMC. AG-490 also inhibited H2O2-induced apoptosis in both LAR−/ − and wild-type VSMC. Together, these data indicate that Fyn-JAK2 pathway activation is necessary for H2O2-induced VSMC apoptosis and LAR negatively regulates Fyn-JAK2-STAT3 and Fyn-p38MAP kinase pathways.