Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

Abstract

PURPOSE:

Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-kappaB and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-kappaB activation is mediated by phosphorylation of the inhibitor IkappaB by IkappaB kinase (IKK)-2 and consequent IkappaB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts.

METHODS:

Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IkappaBalpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively.

RESULTS:

IL-1beta-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration- and time-dependent manner. TPCA-1 inhibited the IL-1beta-induced expression of MMP-1, -3, and -9 in these cells at both the mRNA and protein levels and the IL-1beta-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IkappaBalpha and the nuclear translocation of NF-kappaB induced by IL-1beta.

CONCLUSIONS:

An IKK-2 inhibitor blocked IL-1beta-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-kappaB signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.

• Authors: Yukiko Kondo, Ken Fukuda, Tadafumi Adachi, Teruo Nishida
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 49 Issue 11 Pg. 4850-7 (Nov 2008) ISSN: 1552-5783 [Electronic] United States
• PMID: 18676628 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Amides
• Collagen Type I
• Interleukin-1beta
• RNA, Messenger
• Thiophenes
• 2-((aminocarbonyl)amino)-5-(4-fluorophenyl)-3-thiophenecarboxamide
• I-kappa B Kinase
• Matrix Metalloproteinase 3
• Matrix Metalloproteinase 9
• Matrix Metalloproteinase 1

Topics

• Amides (pharmacology)
• Animals
• Cells, Cultured
• Collagen Type I (metabolism)
• Cornea (drug effects, metabolism, pathology)
• Corneal Ulcer (drug therapy, metabolism, pathology)
• Disease Models, Animal
• Fibroblasts (drug effects, metabolism, pathology)
• Gene Expression (drug effects)
• I-kappa B Kinase (antagonists & inhibitors)
• Immunoblotting
• Immunohistochemistry
• Interleukin-1beta (pharmacology)
• Male
• Matrix Metalloproteinase 1 (biosynthesis, genetics)
• Matrix Metalloproteinase 3 (biosynthesis, genetics)
• Matrix Metalloproteinase 9 (biosynthesis, genetics)
• Microscopy, Fluorescence
• RNA, Messenger (genetics)
• Rabbits
• Reverse Transcriptase Polymerase Chain Reaction
• Spectrophotometry
• Thiophenes (pharmacology)