Abstract:
We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit
gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase
activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq
or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G
protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13
minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI
hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor
(Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the
association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylationdeficient
RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the
blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-
AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or
NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or
RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced
phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient
RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a
novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF