Abstract
Background:
TNFR2 stimulation is known to possess protective effects for the cardiomyocytes, however, the underlying mechanisms remain unknown.
Methods and Results:
Using cultured neonatal cardiomyocytes that were infected with lentivirus containing shRNA targeting TNFR1, we showed that TNFR2 activation by TNFα (5nmol/L) resulted in increased mitochondrial fusion, mitochondrial membrane potential which were associated with both elevated intracellular ATP levels and oxygen consumption rate. Intriguingly, these changes were associated with increased protein levels of OPA1, with no changes in the expression levels of Drp1, Mfn1, Mfn2. We went further and reproduced previously reported data that NF-kB acetylation (Lys310) was increased with TNFR2 activation. Interestingly, however, we also observed dose-dependent effects on increase in Stat3 acetylation. Using shRNA approach, we then demonstrated that either Stat3 or NF-kB knockdown can attenuate TNFR2 induced OPA1 expression. The close interaction between these two signalings was validated by co-IP assay and confocal immunofluorescence staining. Aided by bio-informatics searching, we then performed ChIP assay to show that the binding sites of OPA1 promoter regions for STAT3 (-156 to -167) and NFkB (-192 to -203) were adjacent. We further validated that p300 induced Stat3 acetylation was indispensable for complex formation by the interaction between Stat3-DBD and NF-kB -ΔRHD, which in turn was a key event for OPA1 transcription activation. And silence of p300 can abolish OPA1 upregulation upon TNFR2 activation. Computerized data analysis based on zdock and zrank score followed by molecular dynamic simulation model for the whole Stat3 structure revealed higher value of the exterior dielectric constant (obtained from MM/PBSA calculation) for the two sites, K370 and K383, of Stat3, suggesting the essential roles for these two sites for Stat3-NFkB interaction, which were confirmed by co-IP with Stat3-DBD mutants (K370Q,K370R,K383Q,K383R) approach.
Conclusions:
Our data suggested that p300 mediated Stat3 acetylation cooperates with NF-kB to modulate TNFR2 activation induced OPA1 upregulation, leading to improved mitochondrial morphology and function.