Tumor Necrosis Factor-α Induces Claudin-3 Upregulation in Kidney Tubular Epithelial Cells through NFκB and CREB1.

Claudins are essential for tight junction formation and paracellular transport, and affect key cellular events including proliferation and migration. The properties of tight junctions are dynamically modulated by a variety of inputs. We previously showed that the inflammatory cytokine Tumor Necrosis Factor-α (TNFα), a major pathogenic factor in kidney disease, alters epithelial permeability by affecting the expression of claudin-1, 2 and 4 in kidney tubular cells. Here we explored the effect of TNFα on claudin-3 (Cldn-3), a ubiquitous barrier-forming protein. We found that TNFα elevated Cldn-3 protein expression in tubular epithelial cells (LLC-PK1 and IMCD3) as early as 3h post-treatment. Bafilomycin A and Bortezomib, inhibitors of lysosomal and proteasomes, respectively, reduced Cldn-3 degradation. However, TNFα caused a strong upregulation of Cldn-3 in the presence of bafilomycin, suggesting an effect independent from lysosomes. Blocking protein synthesis using Cycloheximide prevented Cldn-3 upregulation by TNFα, verifying the contribution of de novo Cldn-3 synthesis. Indeed, TNFα elevated Cldn-3 mRNA levels at early time points. Using pharmacological inhibitors and siRNA-mediated silencing we determined that the effect of TNFα on Cldn-3 was mediated by extracellular signal regulated kinase (ERK)-dependent activation of NFκB and protein kinase A (PKA)-induced activation of CREB1. These two pathways were turned on by TNFα in parallel and both were required for the upregulation of Cldn-3. Since Cldn-3 was suggested to modulate cell migration and epithelial-mesenchymal transition, and TNFα were shown to affect these processes, Cldn-3 upregulation may modulate regeneration of the tubules following injury.