Abstract
We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.
Highlights
Thromboxane A2 Stimulates VascularSmooth Muscle Hypertrophy by Up-regulating the Synthesis and ReleaosfeEndogenous Basic Fibroblast Growth Factor*
Had increased the phosphotyrosine content of vascular smooth muscle cellproteins, and since staurosporine, a protein kinase inhibitor which exhibits some selectivity for protein kinase C, appeared to inhibit U46619-stimulated tyrosinephosphorylation of only the 58.5-kDa protein, we examined the effects of staurosporine treatment on thromboxane-stimulated hypertrophy
The most significant finding is that endogenous vascular smooth muscle synthesis and release of bFGF is necessary for thromboxanemediated hypertrophy
Summary
Muscle cells were grown to confluence on 75-cm2flasks and growth- Measurement of Thromboxane-stimulated Protein andDNA arrested by culturing for 48 h in serum-free Dulbecco's modifiedEagle Synthesis-In ourpriorstudy,thromboxane exposure inmedium containing 1%antibiotic/antimycotic solution and 10 pg/ml each of insulin and transferrin (added to maintain cell viability in serum-free medium). The thromboxane A, agonist U46619 (1PM) or ethanolic vehicle was added directly to the culture medium, and the culture vessels were returned to the incubator for various periods of creased vascular smooth muscle cell proteinsynthesisas measured by [3H]leucine incorporation, but did not increase DNA synthesis or stimulate cell proliferation [10]. As these results were at variance with thoseof Hanasaki etal. This figurealsoshows that tyrosine phosphorylationwas stimulated by addition of PMA a
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