Role of nuclear factor-κB in interleukin-1-induced collagen degradation by corneal fibroblasts

Abstract

The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration. The role of nuclear factor (NF)-κB in the IL-1-induced degradation of collagen by corneal fibroblasts that underlies corneal ulceration was investigated. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen with or without IL-1 and sulfasalazine, an inhibitor of NF-κB activation. Collagen degradation was assessed from the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was examined by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP and TIMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation and degradation of the NF-κB-inhibitory protein IκB-α were examined by immunoblot analysis. The subcellular localization and DNA binding activity of the p65 subunit of NF-κB were evaluated by immunofluorescence analysis and with a colorimetric assay, respectively. The transactivation activity of NF-κB was assessed with a reporter gene assay. Sulfasalazine inhibited IL-1-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also inhibited the stimulatory effects of IL-1 on the synthesis or activation of various MMPs in a concentration-dependent manner. IL-1 induced the phosphorylation and degradation of IκB-α, the nuclear translocation and up-regulation of the DNA binding activity of the p65 subunit of NF-κB, and the activation of NF-κB in a manner sensitive to sulfasalazine. These results suggest that NF-κB contributes to the IL-1-induced degradation of collagen by corneal fibroblasts and is therefore a potential therapeutic target for treatment of corneal ulcers.