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 H9c2 cells.
H9c2 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 H2O2-induced oxidative stress in H9c2
showed TG2 >> TG1 protein expression but no detectable TG3. The amine
incorporating activity of TG2 in H9c2 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 (Ro 31-8220) and
PKA (KT 5720 and Rp-8-Cl-cAMPS)
inhibitors respectively. The PMA- and forskolin-induced increases in TG2
activity were attenuated by the TG2 inhibitors Z-DON and R283.
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 H2O2-induced decrease in MTT reduction and
release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and
TG2 activity was stimulated via PKA- and
PKC-dependent signalling pathways in H9c2 cells These results suggest a role
for TG2 in cytoprotection induced by these kinases.