Abstract
Reactive oxygen species (ROS) have been implicated in vascular smooth muscle cell (VSMC) apoptosis, a hallmark of advanced atherosclerotic lesions. Transient oxidation and inactivation of protein-tyrosine phosphatases play a critical role in cellular response to ROS production. However, the function of leukocyte antigen-related (LAR) protein-tyrosine phosphatase in ROS signaling is not known. To determine the expression of LAR in ROS-induced apoptosis, we investigated hydrogen peroxide-induced cell death and signaling in aortic VSMCs from wild-type and LAR(-/-) mice. Histone-associated DNA fragmentation and caspase-3/7 activity were significantly enhanced, mitochondrial membrane integrity was compromised, and cell viability was significantly decreased following H(2)O(2) treatment in LAR(-/-) VSMCs compared with wild-type cells. Stronger and sustained increase in autophosphorylation and activity of Fyn, an Src family tyrosine kinase, was observed in LAR(-/-) cells compared with wild-type cells following H(2)O(2) treatment. LAR binds to activated Fyn in H(2)O(2)-treated VSMCs, and recombinant LAR dephosphorylates phosphorylated-Fyn in vitro. In addition, LAR deficiency enhanced H(2)O(2)-induced phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). PP2, a Fyn-specific inhibitor, blocked JAK2, STAT3, and p38 MAPK activation and significantly attenuated apoptosis induced by H(2)O(2). AG490, a JAK2-specific inhibitor, significantly attenuated H(2)O(2)-induced apoptosis, and blocked H(2)O(2)-induced activation of STAT3, but not p38 MAPK in both wild-type and LAR(-/-) VSMCs. Attenuation of Fyn expression by short hairpin RNA significantly decreased H(2)O(2)-induced downstream signaling and apoptosis in VSMCs. Together, these data indicate that LAR regulates Fyn/JAK2/STAT3 and Fyn/p38 MAPK pathways involved in ROS-induced apoptosis.
Highlights
C, leukocyte common antigen-related receptor (LAR) activity was assayed in lysates from growth-arrested Vascular smooth muscle cell (VSMC) that were pretreated with 10 mM
Incubation of recombinant LAR with 1 mM H2O2, in the absence of reducing agents, resulted in complete inactivation of this protein-tyrosine phosphatases (PTPs) (Fig. 1D). These data support the notion that LAR is a redox-sensitive phosphatase, and the residual activity of this PTP in H2O2-treated VSMCs might be due to the inherent reducing capacity in VSMCs
Because the activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in apoptosis of VSMCs [53, 54], we examined the activation of this kinase in wild-type and LAR knockout VSMCs treated with H2O2 (Fig. 5E)
Summary
Materials—AG490, PP2, and H2O2 were purchased from Calbiochem. A MitoCaptureTM mitochondrial apoptosis detection kit was from BioVision. Cell lysates containing 500 g of protein were immunoprecipitated with goat anti-LAR polyclonal antibody overnight at 4 °C, and incubated with Protein A-Sepharose beads for another 2 h. In Vitro Dephosphorylation Assay—Cell lysates obtained from growth-arrested LARϪ/Ϫ VSMCs, treated with H2O2 for 10 min, were immunoprecipitated with either anti-Fyn or antiSTAT3 antibodies and immobilized on protein A-agarose beads. Fyn Kinase Assay—After appropriate treatments, cells were washed with cold phosphate-buffered saline, and lysed on ice for 15 min in lysis buffer containing 20 mM HEPES, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 50 mM -glycerophosphate, 1% Triton X-100, 10 units/ml aprotinin, 2 M leupeptin, 1 mM Na3VO4, and 400 M phenylmethylsulfonyl fluoride. Cell lysates containing 500 g of protein were immunoprecipitated with anti-Fyn antibody for 2 h at 4 °C and incubated with 40 l of 50% (w/v) protein A-Sepharose beads for an additional 1 h.
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