Modulation of transglutaminase 2 activity in H9c2 cells by protein kinase A and protein kinase C signalling

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Almami, I., 2014. Modulation of transglutaminase 2 activity in H9c2 cells by protein kinase A and protein kinase C signalling. PhD, Nottingham Trent University.

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Abstract

Transglutaminase 2 (TG2; EC 2.3.2.13) has been shown to protect cardiomyocytes against ischaemia and reperfusion-induced cell death and to mediate cell survival in many cell types. Given the prominent role of PKA and PKC in cardioprotection, this study investigated whether TG2 was involved in the cytoprotection induced by activation of these two kinases in cardiomyocyte-like H9c2 cells.

Cultured H9c2 cells were extracted following stimulation with activators of PKC (phorbol-12-myristate-13-acetate; PMA) and PKA (forskolin; FK). Transglutaminase 2 activity was determined using an amine incorporating (in vitro and in situ) and a protein crosslinking assays. Different protein kinase inhibitors were used to determine the involvement of PKC and PKA in the activation of TG2 in H9c2 cells. To confirm the involvement of TG2 activity via PKC and PKA, TG2 specific (Z-DON and R283) inhibitors were used. Western blot analysis revealed the presence of TG2 and TG1 (TG2 >> TG1) protein, but not TG3. Since the H2O2, a major contributor to reactive oxygen species following damage was used to induce oxidative stress. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. The identification of TG2 substrates in H9c2 cells was investigated using pull down assay coupled with proteomic analysis techniques.

The PMA and FK-induced time and concentration-dependent increases in TG2 catalysed biotin cadaverine incorporation in H9c2 cells. Forskolin but not PMA also increased TG2 catalysed protein crosslinking. The PKC (Ro-31 8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors, blocked PMA and FK-induced TG2 activity. Immunocytochemistry using ExtrAvidin®-FITC revealed in situ TG2-mediated biotin cadaverine incorporation into protein substrates following stimulation of PMA, FK and their receptor agonists. The TG2 inhibitors Z-DON and R283 attenuated the PMA- and FK-induced increases in TG2 activity. Pre-treatment with PMA and FK reversed H2O2-induced cell death as judged by a MTT reduction assay and the release of cellular LDH. The TG2 inhibitors R283 and Z-DON blocked PMA and FK-induced cytoprotection. Proteomic analysis identified more than 25 proteins that serve as intracellular substrates for TG2 following PMA and FK stimulation. Some of these identified proteins have already been reported as TG2 substrates, but not in H9c2 cells e.g. tubulin while others e.g. α-actinin have not been identified before.

In summary, these data have shown TG2 activity to be stimulated via PKA and PKC-dependent signalling pathways in H9c2 cells and suggest a role for TG2 in cytoprotection-induced via these two protein kinases.

Item Type:	Thesis
Creators:	Almami, I.
Date:	June 2014
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	12 Oct 2018 10:22
Last Modified:	12 Oct 2018 10:24
URI:	https://irep.ntu.ac.uk/id/eprint/34659