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Transforming growth factor-beta 1 induces alpha-smooth muscle-actin expression in cultured human and monkey
Abstract:
Transforming growth factor-beta 1 (TGF-beta 1) induces alpha-smooth muscle (sm)-actin expression and a contractile myofibroblast-like phenotype in a considerable number of different cell types. Since alpha-sm-actin is expressed in some of the resident human trabecular meshwork (TM) cells in situ, and TGF-beta 1 is synthesized by TM in vitro, alpha-sm-actin expression in TM might also be under the influence of TGF-beta 1. To assess this question, TM cultures were initiated from the eyes of three human donors and three cynomolgus monkeys. Various doses of TGF-beta 1 (0.5-5 ng ml-1) were added to confluent cultures from third to fourth passages. Experiments were performed in the presence of fetal calf serum or under chemically defined serum-free conditions. Four days after treatment, cells were stained immunocytochemically for alpha-sm-actin, and the number of positively labelled cells was quantitatively evaluated. In addition, reverse transcription polymerase chain reaction (PCR) was performed using oligonucleotide primers specific for alpha-sm-actin. In control cultures supplemented with serum, 19.0 +/- 9.4% of human meshwork cells, and 10.2 +/- 4.5% of monkey meshwork cells expressed immunoreactivity for alpha-sm-actin. In human cultures, this number was significantly higher in serum-free conditions (34.1 +/- 7.7%). Treatment with TGF-beta 1 induced alpha-sm-actin expression in a dose-dependent manner. At 5 ng ml-1 TGF-beta 1, 75.5 +/- 7.1% of human meshwork cells expressed distinct stress fibers that stained positively for alpha-sm-actin (P < or = 0.01). A similar albeit smaller increase in alpha-sm-actin positive cells was observed in monkey cultures following treatment with TGF-beta 1. These effects were seen with and without serum, but not when TGF-beta 1 was supplemented in the presence of neutralizing antibodies. In PCR experiments, a distinct product was amplified with cDNA derived from cells treated with 0.1 ng and 1 ng ml-1 TGF-beta 1, but not in control cultures. We conclude that TGF-beta 1 may play a role in differentiating TM cells towards a myofibroblast-like cell type by modulating the expression of alpha-sm-actin.