The role of mitogen-activated protein kinase and protein kinase C in fibronectin production in human vascular smooth muscle cells.
BACKGROUND
After evaluating various growth factors, cytokines, and extracellular matrix (ECM) proteins, we found that the most potent agonists of smooth muscle cell (SMC) fibronectin (Fn) production were transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF). To determine the possible signaling pathways involved in the production of this matrix protein, we investigated the role of the intracellular proteins, protein kinase C (PKC) and mitogen-activated protein kinase (MAP-K), in TGF-beta- and EGF-induced human vascular SMC Fn production.


METHODS
After stimulation of human SMCs with TGF-beta (10 ng/ml) and EGF (100 ng/ml), Fn in the cell medium was assayed by immunoblotting using a specific antibody. PKC was activated by brief stimulation of SMC with phorbol 12,13-dibutyrate (PDBu) and inhibited by downregulation with PDBu or the inhibitor, GF109203X. MAP-K was inhibited with PD098059.


RESULTS
PKC activation increased basal and synergistically enhanced TGF-beta- and EGF-induced Fn production. However, inhibition of PKC by downregulation and GF109203X did not diminish Fn production by TGF-beta and EGF. Surprisingly, these two methods of inhibition slightly increased basal and agonist-induced Fn production. The MAP-K kinase inhibitor, PD098059, produced an almost complete inhibition of EGF and a partial inhibition of TGF-beta-induced Fn production.


CONCLUSIONS
Activation of PKC stimulates Fn production; however, neither TGF-beta nor EGF produce Fn through a PKC-dependent pathway. EGF and TGF-beta both stimulate Fn production at least in part through the intracellular signaling protein MAP-K. Understanding the signaling pathways involved in extracellular matrix protein production will allow the design of specific inhibitors of intimal hyperplasia.
Kaiura
T L
TL
Division of Vascular Surgery, New York Hospital/Cornell University Medical Center, New York, New York, 10021, USA.
Itoh
H
H
Kent
K C
KC
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005353:Fibronectins / Q000096:biosynthesis*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005353:Fibronectins; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D016212:Transforming Growth Factor beta; D015240:Phorbol 12,13-Dibutyrate; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one