Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels.
OBJECTIVE
To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated.


METHODS
Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis.


RESULTS
The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts.


CONCLUSIONS
These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.
Department of Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Takashi
T
Nagano
Ji-Long
JL
Hao
Masatsugu
M
Nakamura
Teruo
T
Nishida
D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D006909:Hydroxyproline / Q000096:biosynthesis; D008297:Male; D008666:Metalloendopeptidases / Q000096:biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D024042:Collagen Type I; D004791:Enzyme Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D008666:Metalloendopeptidases; D006909:Hydroxyproline