Recent findings point to low oxygen tension (hypoxia) as an important mechanism for the expression of several eukaryotic genes. We have previously shown that hypoxia (2% O2), when compared to standard oxygen tension (20% O2), upregulates the mRNA levels of the human alpha1(I) (COL1A1) procollagen gene and transforming growth factor-beta1 (TGF-beta1) in human dermal fibroblasts. In this report, we determined the effect of hypoxia on collagen synthesis and transcription. Exposure of human dermal fibroblasts to hypoxia for 24-72 h led to a threefold, dose-dependent increase in collagenous protein (P < 0.0001; r = 0.9794) and to enhanced type I procollagen deposition, as shown by direct immunofluorescence. Transient transfections with a series of luciferase- and CAT-promoter constructs of the human COL1A1 gene (spanning from -2.5 kb to +113 bp) showed that hypoxia increases the transcriptional activity of constructs having 5' endpoints between -804 bp and -107 bp, with loss of stimulation at -84 bp. Maximal increase in promoter activity in hypoxia was observed between -190 and -174 bp of the proximal promoter, once a cKrox repressor site (-199 to -224 bp) was deleted. Upregulation of COL1A1 mRNA levels in hypoxia was blocked by a TGF-beta1 anti-sense oligonucleotide, and failed to occur in fibroblasts from TGF-beta1 knock-out mice. Co-transfection and overexpression with a Smad7 construct abrogated the increase in COL1A1 promoter activity observed in hypoxia. Upregulated transcriptional activity of the TGF-beta1 promoter in hypoxia was found to be maximal between -453 and -175 bp from the transcriptional start site. Since hypoxia is a critical feature of the early phases of wound repair, we conclude that it may act as a potent physiologic stimulus for collagen synthesis. TGF-beta1 appears to be a critical component of this response.

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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.

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.