In contrast to the TGF-beta1 and beta2 isoforms, TGF-beta3 has shown the ability to downregulate scarring and fibrosis in vivo under certain experimental conditions. In this study, we determined the direct effects of TGF-beta3 on cultures of human dermal fibroblasts. TGF-beta3 (0.1 to 100 pg per ml) increased DNA synthesis up to 50% (p < 0.01, r = 0.970), collagen protein synthesis up to 200% (dose range of 0.1 to 5 ng per ml, p < 0.001, r = 0.990), and increased alpha1(I) procollagen mRNA levels (r = 0.999), with maximal effects (200% of control) observed by 24 h. Collagen lattice contraction was increased by more than 50% in response to TGF-beta3 (p < 0.001), and to a similar extent as the TGF-beta1 isoform. Stimulation of collagen synthesis and of alpha1(I) procollagen mRNA levels in response to TGF-beta3 was partially blocked by a TGF-beta1-specific anti-sense oligonucleotide but was still detectable (35% greater than baseline) when TGF-beta3 was added to dermal fibroblasts from TGF-beta1 knock-out mice. In contrast with these stimulatory effects, however, downregulation of alpha1(I) procollagen, alpha1(III) procollagen, and TGF-beta1 mRNA levels toward baseline occurred when TGF-beta3 (0.1 to 5 ng per ml) was added simultaneously and in combination with TGF-beta1. We conclude that stimulation of collagen synthesis by TGF-beta3 occurs through TGF-beta1-dependent and independent pathways. By downregulating the response to TGF-beta1 and by shifting from one pathway to the other, TGF-beta3 can dampen and provide fine-tuning to the overall TGF-beta's induced program of collagen deposition.
There is evidence that anabolic steroids, which are derived from testosterone and have markedly less androgenic activity, promote tissue growth and enhance tissue repair; however, the mechanisms involved in their anabolic activities remain unclear. In this report, we measured the effect of the anabolic steroid stanozolol on cell replication and collagen synthesis in cultures of adult human dermal fibroblasts. Stanozolol (0.625-5 microg per ml) had no effect on fibroblast replication and cell viability (p = 0.764) but enhanced collagen synthesis (p < 0.01) in a dose-dependent manner (r = 0.907). Stanozolol also increased (by 2-fold) the mRNA levels of alpha1 (I) and alpha1 (III) procollagen and, to a similar extent, upregulated transforming growth factor-beta1 (TGF-beta1) mRNA and peptide levels (p < 0.001). There was no stimulation of collagen synthesis by testosterone. The stimulatory effects of stanozolol on collagen synthesis were blocked by a TGF-beta1 anti-sense oligonucleotide, by antibodies to TGF-beta, and in dermal fibroblast cultures derived from TGF-beta1 knockout mice. We conclude that collagen synthesis is increased by the anabolic steroid stanozolol and that, for the most part, this effect is due to TGF-beta1. These findings point to a novel mechanism of action of anabolic steroids.
Fibroblast clonal heterogeneity has been reported for growth and protein synthesis, but quantitative studies of synthetic phenotype at the pretranslational level have been limited because of difficulty in reliably growing large numbers of clonal cells. We have recently shown a unique stimulatory activity of low oxygen tension in the early phases of clonal growth, which can be used to establish clonal fibroblast cultures suitable for Northern analysis. Using this methodology, we have measured mRNA levels of alpha 1(I) procollagen and transforming growth factor-beta 1 (TGF-beta) both at baseline and after TGF-beta stimulation in a total of 43 clones derived from single cells and from seven different cell strains. We report a remarkable baseline heterogeneity, commonly four- to six-fold, in procollagen mRNA levels among clones and between clones and their parent cultures. Conversely, differences in baseline TGF-beta mRNA levels among clones were either not present or less than onefold. The clonal phenotypic expression of alpha 1(I) procollagen mRNA remained stable after eight additional cell passages. TGF-beta stimulation of itself (autoinduction) was highly variable among clones (range of increases 30% to 150%), and up-regulation of procollagen mRNA levels after TGF-beta stimulation was detected in only 15 (54%) of 28 clonal cultures (range of increases 30% to 353%). A notable lack of correlation was found between baseline mRNA levels of TGF-beta and alpha 1(I) procollagen in clonal cultures. In conclusion, fibroblast clonal populations are remarkably heterogeneous in their baseline procollagen mRNA levels and in their response to TGF-beta.
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