Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

Abstract

BACKGROUND AND PURPOSETissue 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. EXPERIMENTAL APPROACHH9c2 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 cells. KEY RESULTSWestern blotting 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 forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONSTG2 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. AbbreviationsMTT, (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); PMA, phorbol-12-myristate-13-acetate; TG2, transglutaminase type 2

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…Our previous studies have shown that TG2 is involved in cytoprotection triggered by PKA and PKC-dependent signalling pathways in H9c2 cells [27]. In the current study the role …”

“…Protein was determined using the bicinchoninic acid (BCA) protein assay [24], using a commercially available kit (Sigma-Aldrich Co. Ltd), with bovine serum albumin (BSA) as the standard Biotin-labeled cadaverine-incorporation assays were performed according to Slaughter et al. [25] with modifications [26], as described previously [27]. The biotin-labeled peptide cross-linking assay was performed according to the method of Trigwell et al [28] with minor modifications [27].…”

“…[25] with modifications [26], as described previously [27]. The biotin-labeled peptide cross-linking assay was performed according to the method of Trigwell et al [28] with minor modifications [27]. The reaction was started by the addition of 50 μl of samples, positive control (50 ng/well of guinea-pig liver TG2) or negative control (100 mM TrisHCl, pH 8.0) and allowed to proceed for 1 h at 37°C.…”

“…Proteins were transferred to nitrocellulose membranes in a Bio-Rad Trans-Blot system, using electro-transfer buffer comprising 25 mM Tris, 192 mM glycine pH 8.3 and 20% (v/v) MeOH. Following transfer, the membranes were blocked and probed with antibodies as described by Alamami et al [27]. The primary antibodies (1:500 dilutions unless otherwise indicated) used were phospho-specific ERK1/2 (1:1000), phospho-specific PKB, phospho-specific p38 MAPK, phospho-specific JNK.…”

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…Possible explanations for this include reversal of amine incorporation by TG (Stamnaes et al 2008), targeting of modified proteins for degradation or their rapid removal from the cell. Our previous studies have reported the rapid expulsion of biotinylated proteins from H9c2 cells following treatment with PMA or forskolin (Almami et al 2014). It could be that the rapid expulsion of biotinylated proteins from N2a cells occurs via exosomes (Kalani et al 2014).…”