The data demonstrate that UV irradiation causes reduction of type I and type III procollagen mRNA and protein expression in fibroblasts in human skin in vivo. UV reduction of procollagen mRNA and protein expression was most prominent in the upper one-third of skin connective tissue. This localization likely reflects the depth of UV penetration into the skin, which increases with increasing wavelength. We used a UVB/UVA2 source that emits wavelengths primarily between 290–340 nm, which penetrates into the upper region of the connective tissue (33
In nonirradiated human skin, type I and III procollagen proteins are present within fibroblasts and extracellularly. In the upper dermis, this procollagen pool is substantially reduced within 8 hours and essentially absent within 24 hours after UV irradiation. This depletion of existing procollagen protein presumably reflects ongoing conversion into mature collagen coupled with decreased new procollagen synthesis due to reduced procollagen mRNA levels. However, our data do not allow us to directly ascertain the fate of the existing procollagen protein pool. It is possible that UV irradiation stimulates degradation and/or processing of procollagen.
We have reported previously that UV irradiation induces collagenase and other matrix metalloproteinases in human skin in vivo (19
). Collagenase is induced 12–16 hours after UV irradiation, trailing the initial loss of procollagen protein, which is observed 8 hours after UV irradiation. This difference in the time of onset of collagenase induction and reduction of procollagen protein makes it unlikely that initial loss of procollagen results from UV-induced, collagenase-mediated degradation. In general, however, the time course for UV induction of matrix-metalloproteinase mRNA and protein expression inversely mirrors that for UV reduction of procollagen mRNA and protein expression. During the first 24 hours after UV exposure, matrix-metalloproteinase mRNAs and proteins are induced, whereas procollagen mRNAs and proteins are reduced. During the second and third days after UV exposure, matrix-metalloproteinase expression declines, whereas procollagen expression increases. Multiple UV exposures cause sustained elevations of matrix-metalloproteinase mRNAs and proteins (19
) and sustained repression of procollagen mRNAs and proteins. Simultaneous reduction of procollagen expression and induction of mature collagen degradation maximizes mature collagen loss during the initial 24 hours after a single UV exposure. During the succeeding 48 hours (i.e., 2–3 days after UV exposure) matrix-metalloproteinase expression wanes as procollagen expression rises. This temporal coordination of matrix metalloproteinase–mediated collagen breakdown and procollagen mRNA and protein expression protects newly synthesized mature collagen (and procollagen) from rapid degradation.
Evidence suggests that transcriptional control is the major mechanism of regulation of type I procollagen expression (26
). Transcription factor AP-1, which is composed of Jun and Fos proteins, negatively regulates both type I(α1) and type I(α2) procollagen gene transcription (31
). UV irradiation rapidly upregulates AP-1 in human skin in vivo. This induction of AP-1 results primarily from increased c-Jun expression, since c-Fos is constitutively expressed in human skin (20
). c-Jun remains maximally elevated for at least 24 hours after UV irradiation in human skin in vivo. This sustained induction of c-Jun is consistent with the observed time course for repression of procollagen gene expression. These observations led us to investigate the role of c-Jun in UV inhibition of type I procollagen gene expression in primary adult human skin fibroblasts. UV irradiation of skin fibroblasts rapidly induced c-Jun and inhibited endogenous type I and type III procollagen gene expression. We found that human type I(α1) procollagen-promoter CAT-reporter constructs, which were previously characterized in mouse 3T3 fibroblasts (29
), were not transcriptionally active in adult human skin fibroblasts and therefore could not be studied. In contrast, the human type I(α2) procollagen promoter CAT reporter (containing 714 bp upstream from the transcription start site) was active in human skin fibroblasts. Promoter activity was reduced by UV irradiation and further reduced by overexpression of wild-type c-Jun. Overexpression of dominant-negative mutant c-Jun completely abrogated UV inhibition of promoter activity. In cultured fibroblasts, c-Jun remained elevated for at least 8 hours after UV irradiation (as opposed to 24 hours in human skin in vivo). In cultured fibroblasts, UV inhibition of endogenous type I(α2) procollagen gene expression and type I(α2) procollagen promoter reporter was maximal 8 hours after UV exposure. Therefore, the kinetics of induction of c-Jun and repression of type I(α2) procollagen promoter activity coincided. These data indicate that UV inhibition of type I(α2) procollagen gene expression in adult human fibroblasts is mediated, at least in part, by UV-induced c-Jun. However, these data do not rule out the possibility that there may be other factors, including reduced mRNA stability, that contribute to UV inhibition of procollagen gene expression.
Treatment of human skin in vivo with all-trans
retinoic acid before UV irradiation protected against UV-induced loss of type I and type III procollagen transcripts and proteins. Protection by all-trans
retinoic acid was observed after 24 hours of pretreatment, but not after 8 hours of pretreatment (data not shown). We have shown previously that pretreatment of human skin with all-trans
retinoic acid for 24 hours, but not 8 hours, inhibits induction of c-Jun and AP-1 (19
). These data support the involvement of c-Jun in UV inhibition of type I and type III procollagen expression in human skin in vivo. The requirement for prolonged pretreatment with all-trans
retinoic acid presumably reflects a mechanism that involves new gene expression, however, this mechanism remains to be clarified.
Type I procollagen and type III procollagen are reduced in chronically photodamaged human skin, not recently exposed to UV (5
). Treatment of such photodamaged skin with all-trans
retinoic acid increases type I procollagen expression (11
). The mechanism of this increase is not known, however, it may involve increased expression or activation of TGF-β (38
). In addition, treatment of skin with all-trans
retinoic acid before UV exposure inhibits collagen breakdown by matrix metalloproteinases (19
) and protects against UV-induced reduction of procollagen expression, as demonstrated in this study. Therefore, all-trans
retinoic acid and its metabolic precursor all-trans
retinol (vitamin A) should have the capacity to both repair existing photodamage and retard accumulation of new photodamage.