ABSTRACT
BACKGROUND: Protein kinase C (PKC)s consist of three groups of isoenzyme; conventional, novel and atypical PKCs. Diacylglycerol (DAG) activates both conventional and novel PKCs, but not atypical PKCs. High glucose-induced fibronection production was shown to be mediated by activation of DAG-sensitive PKCs. In this study, we investigated whether PKC mediates IL-1beta-induced fibronectin mRNA expression, and the subtypes of PKC involved in the process. METHODS: Fibronectin mRNA level and phosphorylated PKC zeta/iota in total cell lysate were measured by Northern blot and Western blot, respectively. RESULTS: Pretreatment of HPMCs with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1, 000 nM caused dose-dependent inhibition of IL- 1beta (1 ng/mL)-induced fibronectin mRNA level. GF109203X, another pan-PKC inhibitor, at doses of 1, 5 and 10 microM also downregulated IL-1beta (1 ng/ mL)-induced fibronectin mRNA level in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated fibronectin mRNA level at doses of 1, 10 and 100 nM. After prolonged treatment of the cells for 72 hr with PMA, another dose of PMA did not increase fibronectin mRNA level, while IL-1beta (1 ng/mL) still stimulated it. Pretreatment of the cells with 5, 10, 15 and 20 microM of myristoylated PKC zeta/iota pseudosubstrate inhibited IL-1beta (1 ng/mL)-induced fibronectin mRNA level in a dose-dependent manner, while 20 microM of myristoylated PKC [19-27] pseudosubstrate, given as a control, had no effect. Stimulation of fibronectin mRNA level by IL-1beta (1 ng/mL) was completely prevented by 20 microM of my ristoylated PKC zeta/iotapseudosubstrate. IL-1beta (1 ng/ mL) increased phosphorylated PKC zeta/iota, an active form of the enzyme. CONCLUSION: IL-1beta-induced fibronectin production in HPMCs occurs by way of activation of atypical PKCs (PKC zeta/iota).
Subject(s)
Humans , Blotting, Northern , Blotting, Western , Fibronectins , Interleukin-1beta , Protein Kinase C , Protein Kinases , RNA, MessengerABSTRACT
Protein Kinase C-zeta (PKC-ζ) is a new member of protein kinase C family and has some specific characteristics as comparing with the classical PKC. It lacks the C2 domain making its kinase activity Ca2+ -independent, and it possesses only one zinc-finger region in its regulatory domain. Therefore, PKC-ζ does not bind Ca2+ and can not be activated by diacylglycerol or phorbol esters. In addition, many researches showed that PKC-ζ could induce differentiation, mediate insulin-stimulation protein synthesis, activate immunity of human neutrophils, inhibit proliferation of cancer cells and regulate the function of actin cytoskeleton. What is more, PKC-ζ plays an important role in signal transduction of cells, such as mediating MAPK and NF-κB activation.
ABSTRACT
Protein Kinase C-zeta (PKC-?) is a new member of protein kinase C family and has some specific characteristics as comparing with the classical PKC. It lacks the C 2 domain making its kinase activity Ca 2+ -independent, and it possesses only one zinc-finger region in its regulatory domain. Therefore, PKC-? does not bind Ca 2+ and can not be activated by diacylglycerol or phorbol esters. In addition, many researches showed that PKC-? could induce differentiation, mediate insulin-stimulation protein synthesis, activate immunity of human neutrophils, inhibit proliferation of cancer cells and regulate the function of actin cytoskeleton. What is more, PKC-? plays an important role in signal transduction of cells, such as mediating MAPK and NF-?B activation.