Cross-talk between TGF-β1 and IL-6 in human trabecular meshwork cells

PURPOSE: To investigate the relationship between transforming growth factor beta-1 (TGF-β1) and interleukin-6 (IL-6) in human trabecular meshwork (HTM) cells as well as to identify the signaling pathway/s involved in the increased IL-6 expression that occurs in response to mechanical stress and TGF-β1. METHODS: All experiments were performed in confluent monolayers of HTM cells at passage 3. Secreted IL–6 was quantified by ELISA. Levels of IL-6 mRNA were evaluated by polymerase chain reaction (PCR) analysis. Activation of the IL-6 and TGF-β1 promoters was monitored by measuring secreted alkaline phosphatase protein (SEAP) released into the culture medium by HTM cells infected with an adenovirus expressing the SEAP reporter gene that was controlled by either the IL-6 promoter (AdIL6–SEAP) or the TGF-β1 promoter (AdTGFβ1-SEAP). Cyclic mechanical stress (5% elongation, one cycle per second) was applied using the Flexcell System. Reagents used in this study included human TGF–β1, human IL-6, and the inhibitors for the p38 mitogen-activated protein kinase (MAPK; SB202190), c-jun NH(2)-terminal kinase (JNK; SP600125), extracellular-regulating kinase (ERK; PD98059), and TGF type I activin receptor (SB431542). RESULTS: Incubation of HTM cells with TGF–β1 (5 ng/ml) resulted in a significant increase in the protein and mRNA levels of IL-6, which was significantly diminished in the presence of the inhibitors for p38 MAPK or JNK. No transcriptional activation of the exogenous IL-6 promoter was observed following TGF-β1 treatment. In addition, the presence of inhibitors for the p38 MAPK, ERK, and TGF-β1 pathways significantly decreased the increased expression of IL-6 by cyclic mechanical stress. Furthermore, exposure of HTM cells to IL-6 (100 ng/ml) demonstrated the transcriptional activation of TGF-β1 promoter, which was severely impaired by blocking the p38 MAPK pathway. CONCLUSIONS: Our results indicate that TGF-β1 participates in the regulation of basal expression and the stretch-induced expression of IL-6 and suggest the possible existence in cultured HTM cells of an autocrine loop between IL-6 and TGF-β1. We also found that p38 MAPK might play a contributing role in the maintenance of such a loop.