Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac, ADAM17/TACE, and RhoA in tubular epithelial cells

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α-induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration

Mechanism of Claudin Regulation in Kidney Tubular Epithelial Cells

Abstract The overall objective of my studies was to gain insight into the role and regulation of claudin family tight junction (TJ) proteins in kidney tubular cells. Members of the claudin family generate specific paracellular pathways and thus control epithelial permeability. Each claudin has different properties. However, their regulation is incompletely understood. The inflammatory cytokine Tumor Necrosis Factor-α (TNF- α) is a key pathogenic factor in kidney disease and is also known to alter TJ permeability. Therefore, my first objective was to explore how TNF-α alters claudin expression and to correlate the findings with permeability changes. TNF-α altered the expression of several claudins in cultured tubular cells. We described a novel biphasic effect of TNF-α on claudin-2, a leaky channel TJ protein. TNF-α elevated claudin-2 expression after short-term (1–6 h) and reduced it after long-term treatment through distinct mechanisms. The early increase was due to reduced degradation of claudin-2 mediated by theEGF receptor, ERK, RhoA and Rho kinase. In contrast, prolonged TNF-α treatment reduced claudin-2 synthesis. Moreover, JNK was involved in both effects. Second, ERK and JNK also mediated an increase in claudin-1, 4 and 7 induced by prolonged TNF-α treatment. To explore the role of altered claudin expression we measured TNF-α-induced transepithelial resistance (TER) changes. TNF-α caused an initial decrease and a late increase in TER. Inhibition of ERK and JNK as well as silencing of claudin-2 reduced the second phase (TER increase). In summary these studies suggest a strong causal connection between TNF-α-induced changes in claudin expression and permeability. My second objective was to study how claudin-2 expression is controlled in unstimulated epithelial cells. I found that Cldn-2 expression increased with cell confluency, and was also controlled by the small GTPases RhoA and Rac, key regulators of junction assembly and cytoskeleton. Rho silencing increased Cldn-2 expression, while Rac was required for maintaining Cldn-2 levels. Further, claudin-2 expression also required NFκB and PI3 kinase. These studies point to exciting novel modes of claudin-2 expression regulation. Since claudin-2 also affects proliferation and migration, these findings could have relevance to acute and chronic kidney disease as well as cancer.Ph.D