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Modulation of transglutaminase 2 activity in H 9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection
Author(s) -
Almami Ibtesam,
Dickenson John M,
Hargreaves Alan J,
Bonner Philip L R
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12756
Subject(s) - forskolin , cytoprotection , tissue transglutaminase , protein kinase c , phorbol , microbiology and biotechnology , protein kinase a , chemistry , stimulation , biochemistry , biology , signal transduction , endocrinology , enzyme , oxidative stress
Background and Purpose Tissue transglutaminase ( TG2 ) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte‐like H 9c2 cells. Experimental Approach H 9c2 cells were extracted following stimulation with phorbol‐12‐myristate‐13‐acetate ( PMA ) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms ( TG1 , 2, 3) was determined using Western blot analysis. The role of TG2 in PMA ‐ and forskolin‐induced cytoprotection was investigated by monitoring H 2 O 2 ‐induced oxidative stress in H 9c2 cells. Key Results Western blotting showed TG2 >> TG1 protein expression but no detectable TG3 . The amine incorporating activity of TG2 in H 9c2 cells increased in a time and concentration‐dependent manner following stimulation with PMA and forskolin. PMA and forskolin‐induced TG2 activity was blocked by PKC ( R o 31‐8220) and PKA ( KT 5720 and R p‐8‐ C l‐ cAMPS ) inhibitors respectively. The PMA ‐ and forskolin‐induced increases in TG2 activity were attenuated by the TG2 inhibitors Z ‐ DON and R 283. Immunocytochemistry revealed TG2 ‐mediated biotin‐ X ‐cadaverine incorporation into proteins and proteomic analysis identified known (β‐tubulin) and novel (α‐actinin) protein substrates for TG2 . Pretreatment with PMA and forskolin reversed H 2 O 2 ‐induced decrease in MTT reduction and release of LDH . TG2 inhibitors R 283 and Z ‐ DON blocked PMA ‐ and forskolin‐induced cytoprotection. Conclusions and Implications TG2 activity was stimulated via PKA ‐ and PKC ‐dependent signalling pathways in H 9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases.