Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.
OBJECTIVE
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.
Kondo
Yukiko
Y
Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan.
Fukuda
Ken
K
Adachi
Tadafumi
T
Nishida
Teruo
T
D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D003320:Corneal Ulcer / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors*; D015151:Immunoblotting; D007150:Immunohistochemistry; D053583:Interleukin-1beta / Q000494:pharmacology*; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D013876:Thiophenes / Q000494:pharmacology*
D000577:Amides; D024042:Collagen Type I; D053583:Interleukin-1beta; D012333:RNA, Messenger; D013876:Thiophenes; C499326:2-((aminocarbonyl)amino)-5-(4-fluorophenyl)-3-thiophenecarboxamide; D051550:I-kappa B Kinase; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1