Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR₁ and PAR₄ are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms.

Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H₂DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate.

Thrombin, the PAR₁ selective agonist peptide TFLLRN-NH₂ (PAR₁-AP), and the PAR₄ selective agonist peptide, AYPGKF-NH₂ (PAR₄-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR_1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR₁-AP and the PAR₄-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR₁- and PAR₄-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-l-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR₁-AP and the PAR₄-AP, but had no effect on PAR_1/4-mediated activation of Met and PDGFR in Hep3B cells.

Collectively, our data indicate that PAR₁ and PAR₄ activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR_1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.