During the fiscal year we accomplished the following: 1. Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. We showed that HGF both induced co-localization of IQGAP1 with Asef (a Rac/Cdc42-specific guanine nucleotide exchange factor) at the cell cortex and stimulated formation of an Asef-IQGAP1 functional protein complex. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Silencing IQGAP1 attenuated HGF-induced enhancement of EC barrier. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1- Arp3/cortactin interactions. 2. Tight junction (TJ) formation, which contributes to cell-cell adhesion of polarized epithelia, is crucial for tissue homeostasis. The junctional complex is compromised in several pathological states, including carcinoma, inflammatory bowel disease and diabetic retinopathy. We observed that silencing IQGAP1 in kidney cells enhanced a transient increase in transepithelial electrical resistance. Analysis by quantitative microscopy and biochemical assays suggested that the effect of IQGAP1 is mediated by reduced expression and recruitment of claudin 2, and increased TJ recruitment of claudin 4. In addition, IQGAP1 knockdown increases the activity of the Cdc42 effector JNK (c-Jun N-terminal kinase). These data reveal that IQGAP1 modulates TJ formation by both controlling the recruitment of claudin 2 and claudin 4 to the TJ and by transient inhibition of the Cdc42-JNK pathway.
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