Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

INTRODUCTION: Fibronectin (FN) is commonly used in the development of serum-free media for the expansion of mesenchymal stem cells (MSCs). This study was aimed to observe if thrombin could stimulate FN secretion by human bone marrow MSCs and investigate the potential underlying mechanisms. METHODS: PCR was performed to detect the expression of the protease-activated receptors (PARs) in MSCs. After thrombin treatment, the expression level and secretion of FN were observed by RT-PCR, immunofluorescence staining and ELISA, respectively, and the activation of ERK1/2 and NF kappa B pathways was revealed by Western blotting, with or without pre-treatment of small-molecule blockers specific for PAR-1 and –2. The phenotypic and functional activities of thrombin-treated MSCs were also observed. RESULTS: PCR analysis showed that human bone marrow MSCs expressed two subtypes of PARs, PAR-1 and PAR-2. Thrombin treatment enhanced MSCs to express FN at mRNA and protein levels and promoted FN secretion by MSCs, accompanied by potent adherence to the culture plastic. Thrombin induced prompt phosphorylation of ERK 1/2 and NF kappa B p65 and the stimulatory effects of thrombin on FN secretion were blunted by specific inhibitors of these signaling molecules. Blockage to PAR-1 and PAR-2 partially abrogated thrombin-elicited FN secretion by MSCs and ERK 1/2 phosphorylation, whereas that of NF kappa B p65 was unaffected. Moreover, thrombin-treated MSCs maintained the phenotypic features, in vitro osteogenesis and adipogenesis capacities, and inhibitory activity on Phytohemagglutinin-induced allogeneic lymphocyte proliferation. CONCLUSIONS: Thrombin could promote FN secretion by MSCs via PAR-mediated ERK 1/2 activation, while NF kappa B might be also involved in an undefined manner.