Pellino1 Is Involved Il-1β-induced Cardiac Hypertrophic Response Of Cardiomyocytes

Pathological cardiac hypertrophy is the main risk factor of heart failure and sudden death. The reasons leading to cardiac hypertrophy conclude hypertension, myocardial infarction, valvular heart disease and cardiomyopathy. In the development of cardiac hypertrophy, some biomechnical stress and neurohormonal signal can alter correlated nuclear factors activity through intracelluar signal transduction pathway, leading the abnormal expression of numerous genes and proteins; finally change the morphous, construction and function of heart.Our previous studies have demonstrated that TLR/IL-1R-mideated NF-κB signaling pathway play an important role in the development of cardiac hypertrophy. But the details of intracellular signal transduction mechanism is poorly understood. Pellino superfamily is considered as an important regulating molecular in TLR/IL-1R signaling pathway. In mammals, the Pellino family consists of three members (Pellino-1/-2/-3) that are highly related. Pellinos combine with IRAK1, TRAF6 and TAK1 and act as a E3 ubiquitin ligase in TLR/IL-1R signaling pathway. N-terminal part of Pellino proteins contains a C3HC4 RING-like motif. RING domains mediate protein–protein and protein–DNA interactions in a diverse group of proteins and are best known for their occurrence in RING E3 ubiquitin ligases. C-terminal part of Pellino proteins contains a FHA domain. FHA domains are well-characterized phosphothreonine-binding modules, this cryptic example can drive interaction of Pellino proteins with phosphorylated IRAK1. In vitro studies show that Pellino1 was phosphorylated by IRAK1,and then integrate with some E2 compounds such as Ubc13–Uev1a,UbcH3,UbcH4,UbcH5a and UbcH5b to form polyubiquitin chains. It is reported that phosphorylation of Pellino1 by IRAK1 and IRAK4 enhance Pellino1 E3 ligase activity. But the expression of Pellinos in cardiac hypertrophy and their possible role in regulation of cardiac hypertrophy has not been reported. Therefore, this study was to identify the regulative effect of Pellino in cardiac hypertrophy.We used both in vivo and vitro experiments to investigate the issue. C57BL/6 mice were subjected to transverse aortic constriction (TAC) to duplicate cardiac hypertrophy. Four weeks after operation the hearts were collected to calculate the degree of cardiac hypertrophy and extract the mRNA, plasma protein and nuclear protein. The expression of Pellinos was detected by RT-PCR and weatern blot. The association of IRAK-1 and Pellino1 in the myocardium was detected by co-immunoprecipitation. ShPellino1 plasmids were transfected into IL-1βstimulated primary cultured rat neonatal cardiomyocytes in vitro to determine the role of Pellino1 in cardiomyocytes hypertrophy.We found that TAC could result in cardiac hypertrophy, heart weight-to-body weight ratio (HW/BW) and left ventricular weight to tibial length ratio (LVW/TL) in the aorta banding group were significantly enhanced by 20.89% (4.700±0.05514 Vs 5.682±0.1336,P<0.001, n=6) and 16.11% (4.898±0.05935 Vs 5.687±0.1319, P<0.001, n=6) than sham operation group respectively. The level of Pellino1 mRNA in the TAC group were increased by 194.96% (0.1825±0.0213 Vs 0.5383±0.09187, P<0.01, n=6) compared with sham group, the level of Pellino3 mRNA in the TAC group were decreased by 46.55% (0.3562±0.05083 Vs 0.1904±0.02016, P<0.05, n=6) compared with sham group. The expression level of Pellino2 mRNA in two groups are no significantly changed. We selected Pellino1 for further study. The level of Pellino1 protein in the TAC group were increased by 74.85% (0.4092±0.03334 Vs 0.7155±0.03945, P<0.01, n=5) compared with sham group. And association of IRAK-1 with Pellino1 in the TAC group were increased by 48.93% (0.2624±0.03442 Vs 0.3908±0.040205, P<0.05, n=5) compared with sham group. NF-κB binding activity in the TAC group were increased by 128.76% (5508±343.8 Vs 12600±629.8, P<0.001, n=5) compared with sham group. In vivo studies indicated that Pellino1 maybe involved in cardiac hypertrophy induced by pressure overload.We select IL-1βas stimulant to determine role of Pellino1 in IL-1R induced NF-κB signaling pathway in cardiomyocytes hypertrophy. In vitro experiments showed that 10ng/ml IL-1βstimulation could significantly induce cardiomyocytes hypertrophy, the areas of cardiomyocytes were increased by 41.69% (4749±276.3 Vs 6729±327.5, P<0.001, n=3) than those of control,expression Pellino1 mRNA in and protein IL-1βtreated group were increased by 182.6% (0.1121±0.01471 Vs 0.3168±0.03206, P<0.01, n=3) and 43.19% (0.1197±0.01166 Vs 0.1714±0.007792, P<0.05, n=3) than those of control. The areas of cardiomyocytes treated with shPellino1 and IL-1βwas significantly decreased by 47.41% (6729±327.5 Vs 3539±189.8, P<0.001, n=3) compared with IL-1βtreated group. Meanwhile,we oberserved that areas of cardiomyocytes treated with shPellino1 and IL-1βwas decreased by 25.48% (4749±276.3 Vs 3539±189.8, P<0.05, n=3) compared with those of control. IL-1βcould induce increase in protein synthesis, 3H-leucine incorporation were increased by 23.64% (7714±275.0 Vs 9538±386.8, P<0.01, n=3) than those of control. 3H-leucine incorporation of cardiomyocytes treated with shPellino1 and IL-1βwas decreased by 68.89% (9538±386.8 Vs 2967±148.2, P<0.001, n=3) compared with IL-1βtreated group. Similarly, 3H-leucine incorporation of cardiomyocytes treated with shPellino1 and IL-1βwas decreased by 61.54% (7714±275.0 Vs 2967±148.2, P<0.001, n=3) compared with untreated group. The myocardial atrial natriuretic peptide (ANP) mRNA expression level of IL-1βgroup was significantly increased by 238.10% (0.2614±0.03181 Vs 0.8838±0.04803 P<0.001, n=3) than that of control. When transfection of shPellino1 plasmid, the level of ANP mRNA was decreased by 80.76% (0.8838±0.04803 Vs0.1700±0.06283, P<0.001, n=3) than that of IL-1βstimulation group. The myocardial brain natriuretic peptide (BNP) mRNA expression level of IL-1βgroup was significantly higher, by 232.4% (0.4179±0.01199Vs 0.6363±0.02676, P<0.001, n=3) than that of control. When transfection of shPellino1 plasmid, the level of BNP mRNA was decreased by 42.57% (0.6363±0.02676 Vs 0.2297±0.2297, P<0.001, n=3) than that of IL-1βstimulation group. NF-κB binding activity was significantly increased in IL-1βtreated group by 59.26% (8330±404.7 Vs 13266±434.5, P<0.05, n=3) than that of control. Transfection of shPellino1 plasmid lead a decreased of NF-κB binding activity by 49.13% (13266±434.5 Vs 6749±1012, P<0.01, n=3) compared with IL-1βtreated group. The ubiquitination of TRAF6 was increased in IL-1βtreated group,and transfection of shPellino1 plasmid lead a decreased of ubiquitination of TRAF6(need to be determined by more repeats). Vitro experiments suggested that expression of Pellino1 was increased in IL-1βtreated neonatal cardiomyocytes, interference of Pellino1 with shPellino1 decreased IL-1β-induced hypertrophy and NF-κB binding activity.Our results suggested that Pellino1 act as an important regulator in IL-1R-mediated NF-κB signaling pathway within cardiac hypertrophy.