Abstract
Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that is expressed specifically in striated muscle. Although the contribution of atrogin-1 to cardiac and muscle hypertrophy/atrophy has been examined extensively, it remains unclear whether atrogin-1 plays an essential role in the simulated ischemia/reperfusion-induced apoptosis of primary cardiomyocytes. Here we showed that atrogin-1 markedly enhanced ischemia/reperfusion-induced apoptosis in cardiomyocytes via activation of JNK signaling. Overexpression of atrogin-1 increased phosphorylation of JNK and c-Jun and decreased phosphorylation of Foxo3a. In addition, atrogin-1 decreased Bcl-2, increased Bax, and enhanced the activation of caspases. Furthermore, JNK inhibitor SP600125 markedly blocked the effect of atrogin-1 on cell apoptosis and the expression of apoptotic-related proteins and caspases. Importantly, atrogin-1 induced sustained activation of JNK through a mechanism that involved degradation of MAPK phosphatase-1 (MKP-1) protein. Atrogin-1 interacted with and triggered MKP-1 for ubiquitin-mediated degradation. In contrast, proteasome inhibitors markedly blocked the degradation of MKP-1. Taken together, these results demonstrate that atrogin-1 promotes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby leading to persistent activation of JNK signaling and further cardiomyocyte apoptosis following ischemia/reperfusion injury.
Highlights
mitogen-activated protein kinase (MAPK) become activated by dual phosphorylation on Ser/ Thr and Tyr residues of TEY sites within the activation loop, whereas dephosphorylation of these residues by MAPK phosphatases (MKPs) terminates such activation (4 – 8)
Cleaved Caspases through JNK Signaling—To investigate Because the expression pattern of MAPK phosphatase-1 (MKP-1) protein is inversely whether atrogin-1 could regulate the expression of apoptosis- correlated with atrogin-1 in cardiomyocytes during reperfusion related proteins and caspases, cardiomyocytes were infected (Fig. 6), we sought to determine whether atrogin-1 is involved
We demonstrate that atrogin-1 enhances I/R-induced apoptosis in cardiomyocytes
Summary
Plasmids and Reagents—The plasmids Myc-atrogin-1, Hisubiquitin, and GST-atrogin-1 have been described as previously [31]. Anti-Akt, anti-phospho-Akt (Ser-473), anti-ERK1/2, anti-phospho-ERK1/2, anti-p38, antiphospho-p38, anti-JNK1/2, anti-phospho-JNK1/2, anti-Bcl-2, anti-Bax, anti-cleaved caspase-3, anti-cleaved caspase-9, antiFoxo3a, anti-phospho-Foxo3a, anti-GAPDH and anti-mouseor anti-rabbit-conjugated antibodies were from Cell Signaling Technology. Cell Culture and Adenovirus Infection—H9c2 cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium. Neonatal rat cardiomyocytes were isolated by enzymatic disassociation of 1- to 2-day-old neonatal rat hearts as described previously [30]. Erated as described previously [30]. Twenty-four hours after plating, cells were infected with adenovirus vectors containing Adatrogin-1 or siRNA-atrogin-1 for 24 h before I/R treatment [30]. Simulated I/R Protocol—After 24 h of adenovirus infection, the cells were subjected to simulated ischemia for 1 h by replacing the cell medium with an “ischemia buffer” that contained
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