Investigation of NF-KB / P65 and PKNOX2 protein-protein interactions in mesenchymal stem cell aging

Mesenchymal stem cells undergo replicative senescence by cultivating in vitro and as the passage extends they lose their stem cell properties. Previous study of our group investigating proteomics of the co-immunoprecipitated proteins with PKNOX2 revealed interacting proteins with PKNOX2 in BM-MSCs. Nuclear factor kappa B (NF-κB) subunit p65, a main regulator of senescence-related secretomes, was among the interacting proteins with PKNOX2. Goals of this thesis were 1) to investigate the changes in PKNOX2 and p65/RelA protein levels of BM-MSCs aged by serial passaging in culture, 2) to demonstrate the interaction of PKNOX2 and NF-κB/p65 (RelA) and 3) to examine interaction changes during the cellular aging process if there are any. Replicative senescence was assessed by SA-β-Gal staining and p16, p21 protein levels determined by Western blot analysis. Genomic stress was assessed by γH2AX levels obtained by immunofluorescence imaging. Analysis of western blotting showed that PKNOX2 protein levels tended to decrease with replicative senescence and NF-κB/p65 protein levels overall increased in BM-MSCs exposed to oxidative stress induced by 200 µM H2O2. NF-κB/p65 and PKNOX2 interactions were detected in vitro using co-IP followed by western blot and in situ using proximity ligation assay (PLA) technique. PLA analyzes showed that interactions of NF-κB and PKNOX2 were present both in the cytoplasm and nucleus of BM-MSCs, and the levels of interaction increased at passage 9 and decreased at passage 12.