Protein Kinase C-α Signals Rho-Guanine Nucleotide Dissociation Inhibitor Phosphorylation and Rho Activation and Regulates the Endothelial Cell Barrier Function

Abstract

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-␣. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-␣ with Rho. Activated PKC-␣ but not PKC-⑀ induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-␣ is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rhodependent endothelial barrier dysfunction.

ROCK Mediates Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via p21Cip1 Up-regulation and JNK

“…PKC isozymes have been implicated in Rho activation in several models, including cardiomyocytes (36), myoblasts (37), and endothelial cells (38). Although the mechanistic basis of PKC␦ modulation of Rho/ROCK function in LNCaP still remains to be elucidated, it has been shown that phorbol esters can activate Rho GTPases through different mechanisms that involve either Rho-GEF activation or Rho-GDI inactivation (22,23). Rho-GEFs are a very diverse family, with Ͼ70 members being identified in humans (39).…”

“…For example, the decreased invasiveness of PKC⑀-depleted head and neck squamous carcinoma cells is associated with a corresponding inactivation of RhoA and RhoC GTPases (21). PKCs can modulate Rho function through either Rho-GEF activation or Rho-GDI inactivation (22,23) or by direct association to Rho (24). However, it is not known whether Rho or its downstream effectors are implicated in PMA-induced apoptosis in prostate cancer cells.…”

RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry

“…HPAE cells (200,000 cells) grown to confluence on gelatin-coated small gold electrode (4.9 ϫ 10 Ϫ4 cm 2 ) were left untreated or treated with C3, as reported previously (45). Cells were then stimulated with thrombin to measure changes in electrical resistance of endothelial monolayer in real time.…”

“…These proteins can traffic from cytosol to plasma membrane on activation and they also regulate vesicle trafficking (40 -42). We and others have shown that thrombin rapidly induces activation of RhoA (but not Rac or Cdc42) in endothelial cells (43)(44)(45). 2 In the present study, we addressed the possibility that RhoA induces the interaction of IP 3 R and TRPC1 required for activation of store depletion-induced Ca 2ϩ entry.…”

Effects of systemic inhibition of Rho kinase on blood pressure and renal haemodynamics in diabetic rats

“…The RhoA/ROCK pathway seems to have complex interactions with PKC isoforms in the control of a variety of cell functions (Strassheim et al, 1999;Mehta et al, 2001;Holinstat et al, 2003;Rikitake and Liao, 2005) including vasoconstrictor signals (Strassheim et al, 1999;Somlyo and Somlyo, 2003). PKCs, in particular its b isoform, are activated in the diabetic kidney, and play a role in the pathophysiology of nephropathy (Ishii et al, 1996;Koya et al, 2000;Kelly et al, 2003) by mediating signals leading to local alterations in vascular tone, cell growth, extracellular matrix production and oxidative stress (Das Evcimen and King, 2007).…”