Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Abstract

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

“…In this aspect, a recent report showed that thrombin induces embryonic fibroblast migration via G␣ 12/13 -coupled GPCRs that target RhoA activation through LARG, PDZ-RhoGEF, and p115 RhoGEF (61). On the other hand, some studies reported that activation of G␣ 12 inhibits thrombin-induced epithelial cell migration (62). However, the present findings indicate that thrombin-induced monocyte/ macrophage migration requires the activation of G␣ 12 but not G␣ q/11 .…”

H 2 O 2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury

“…We analyzed MDCK cells by using ATP depletion/repletion, an established model of epithelial injury (23). Baseline transepithelial resistance (TER) was determined in Tet-off inducible Gα12 (Gα12-MDCK) and Gα12-silenced MDCK cells (shGα12-MDCK) (20,24). As previously reported, Gα12 overexpression in MDCK cells -doxycycline (-dox) led to a decrease in baseline TER (Fig.…”

Constitutive Lymphocyte Transmigration across the Basal Lamina of High Endothelial Venules Is Regulated by the Autotaxin/Lysophosphatidic Acid Axis

“…Our observations that ATX inhibition induced marked lymphocyte accumulation within the EC compartment and that such accumulation could be resolved by local administration of LPA may point to the possibility that such transient lymphocyte retention in the HEV "pocket" structures under physiological conditions is regulated, at least in part, by the ATX/LPA axis. As to the possible mechanism of weakening of cell adhesion between lymphocytes and HEV ECs, Ga 12 activation, which can occur upon the ligation of LPA 4 or LPA 6 , was shown to decrease the phosphorylation of focal adhesion kinase and paxillin, causing integrin displacement from the adhesion substratum (36). Another possibility, not mutually exclusive, is that LPA acts on ECs to increase their motility, which may also interfere with the adhesive interactions between transmigrating lymphocytes and ECs.…”