The proinflammatory
cytokine interleukin (IL)-1 is implicated in corneal ulceration. The role of nuclear factor (
NF)-kappaB 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-kappaB 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-kappaB-inhibitory
protein IkappaB-alpha were examined by immunoblot analysis. The subcellular localization and
DNA binding activity of the p65 subunit of
NF-kappaB were evaluated by immunofluorescence analysis and with a colorimetric assay, respectively. The transactivation activity of
NF-kappaB 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
IkappaB-alpha, the nuclear translocation and up-regulation of the
DNA binding activity of the p65 subunit of
NF-kappaB, and the activation of
NF-kappaB in a manner sensitive to
sulfasalazine. These results suggest that
NF-kappaB contributes to the IL-1-induced degradation of
collagen by corneal fibroblasts and is therefore a potential therapeutic target for treatment of corneal
ulcers.