Pressure activates epidermal growth factor receptor leading to the induction of iNOS via NFkappaB and STAT3 in human proximal tubule cells.

Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-kappaB (NFkappaB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0-36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NFkappaB, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NFkappaB p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 min. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NFkappaB, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation.

Effects of transforming growth factor-beta1 on human vocal fold fibroblasts.

OBJECTIVES: We studied the effect of transforming growth factor (TGF)-beta on immortalized human vocal fold fibroblasts. METHODS: Normal human vocal fold fibroblasts were subjected to sequential lentiviral transduction with genes for human telomerase (hTERT) and SV40 large T antigen in order to produce an "immortalized" cell line of normal phenotype. After confirmation of vocal fold fibroblast transfection, these cells, referred to as HVOX, were treated with various concentrations of exogenous TGF-beta1 and assayed for collagen secretion, migration, and proliferation. In addition, components of the TGF-beta signaling pathway were examined in this cell line. RESULTS: TGF-beta stimulated collagen secretion and migration without altering proliferation of HVOX. HVOX constitutively expressed type I and II TGF-beta receptors, as well as messenger RNA for the Smad signaling proteins and for all TGF-beta isoforms. Exogenous TGF-beta1 induced temporally dependent alterations in Smad2 and Smad3 gene expression. TGF-beta increased Smad7 expression at both 4 and 24 hours. Prolonged exposure to TGF-beta decreased TGF-beta1 gene expression. CONCLUSIONS: Insight into the underlying pathophysiology of vocal fold fibrosis is likely to yield improved therapeutic strategies to mitigate vocal fold scarring. Our data suggest that TGF-beta signaling may be both paracrine and autocrine in this vocal fold fibroblast cell line, and we therefore propose that TGF-beta may be a reasonable target for therapies to prevent and/or treat vocal fold fibrosis, given its putative role in both acute and chronic vocal fold injury, as well as its effects on vocal fold fibroblasts.

Early upregulation of iNOS mRNA expression and increase in NO metabolites in pressurized renal epithelial cells.

Pressure is an important physiological regulator, but under abnormal conditions it may be a critical factor in the onset and progression of disease in many organs. In vivo, proximal tubular epithelial cells are subjected to pressure as a result of ureteral obstruction, which may influence the production of nitric oxide (NO), a ubiquitous multifunctional cytokine. To directly explore the effect of pressure on the expression and activity of NO synthase (NOS) in cultured proximal tubular epithelial cells, a novel pressure apparatus was developed. Cells were subjected to pressures of 20-120 mmHg over time (5 min-72 h). RT-PCR demonstrated an increase in inducible NOS (iNOS) and sGC, while endothelial NOS remained unchanged. Real-time PCR (qPCR) confirmed an earlier induction of iNOS transcript subjected to 60 mmHg compared with cytokine mix. iNOS protein expression was significantly increased following 60 mmHg of pressure for 24 h. Use of nuclear factor-kappaB inhibitors was shown to prevent the increase in iNOS expression following 60 mmHg for 2 h. NO and cGMP were increased with the application of pressure. The addition of the irreversible iNOS inhibitor (1400W) was shown to prevent this increase. We demonstrate that with the use of a simply designed apparatus, pressure led to an extremely early induction of iNOS and a rapid activation of NOS activity to increase NO and cGMP in proximal tubule epithelial cells. The rapid effects of pressure on iNOS may have important implications in the obstructed kidney.