Differential regulation of mitogenic activated protein kinases by thymosin β4 sulphoxide (Tb4SO)

Background

Thymosin β4 (Tb4) is a 4.9 kDa ubiquitously expressed intracellular peptide known to sequester G-actin, with a growing number of extracellular biological functions. We have shown that oxidation of its single methionine residue at position 6 (Tb4 sulphoxide [Tb4SO]) gives the peptide potent anti-inflammatory properties. In addition, Tb4 promotes wound healing in various in vitro and in vivo models. The mechanism by which Tb4SO exerts its extracellular biological functions is unknown. Its cell receptor remains unidentified and intracellular signalling consequences of Tb4SO require elucidation.

Objective

The study was conducted to investigate the intracellular signalling consequences of the addition of Tb4SO to various cell types, specifically MAP kinase activation.

Methods

Cell types used were treated with Tb4SO and lysed using RIPA buffer. Lysates were run on SDS PAGE, transferred to PVDF membrane and probed with antiphospho ERK1/2, p38, JNK1/2 or AKT antibodies. Membranes were then stripped and reprobed with pan-antibodies to allow normalization of samples.

Results

Tb4SO induced the phosphorylation of ERK1/2 in peripheral blood-derived monocytes in dose-dependent manner. This was inhibited by addition of U0126, an inhibitor of the upstream activator of ERK1/2, implicating ERK1/2 in Tb4SO-mediated functions in monocytes. Neither p38 MAPK nor JNK1/2 were activated in peripheral blood monocytes. In peripheral blood-derived macrophages, Tb4SO induced rapid and marked activation p38 MAPK, pAkt and ERK1/2, but not of JNK (p-JNK1/2). Interestingly, only JNK1 was activated by Tb4SO in HeLa cells, and this occurred in a delayed (after 15 min) and transient manner. Short-term activation of JNKs has previously been shown to be involved in the upregulation of apoptosis. Preliminary studies have shown an acceleration in neutrophil but not monocyte/ macrophage apoptosis in response to Tb4SO.

Conclusions

These observations suggest that Tb4SO activates MAPKs and that activation is dependent on cell type, which may in turn regulate differential functions in these different cell types.