Abstract
The effects of human recombinant interleukin (IL)-1 beta on elastin gene expression were studied in human skin fibroblast cultures by Northern hybridization and transient transfection experiments. Incubation of the cells with IL-1 beta elevated the elastin mRNA steady-state levels by approximately 3- to 4-fold. A similar increase was noted at the protein level, when estimated by indirect immunofluorescence of cultured cells. This effect was independent of the on-going protein synthesis, as tested by incubation with cycloheximide. Transient transfections of the dermal fibroblasts with a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct suggested transcriptional regulation, since the CAT activity in cells incubated with IL-1 beta was similarly increased approximately 3-fold. Enhancement of the human elastin promoter activity by IL-1 beta was also noted in fibroblast cultures established from the skin and lungs of transgenic mice which have integrated the human promoter/CAT construct into their genome and express it in a tissue-specific manner. Furthermore, subcutaneous injection of IL-1 beta to the mice resulted in a approximately 4-fold elevation of the CAT activity in the skin after a 30-h incubation, as compared to the CAT activity in the skin of control animals. Collectively, these data indicate that IL-1 beta up-regulates elastin gene expression in fibroblast cultures as well as in the skin in vivo, and the activation occurs at the transcriptional level.
Highlights
From the Departments of Dermatology,and Biochemistry and Molecular Biology, Jefferson Medical College, and Section of Molecular Dermatology,Jefferson Institute of Molecular Medicine, Thomas Jefferson Uniuersity, Philadelphia, Pennsylvania 19107
16 on elastin gene expression were studied in human tially synthesized as 70-kDa polypeptides which polymerize skin fibroblast culturebsy Northern hybridization and into an insoluble fiber network through the formation of transient transfection experiments
Transient transfections of the dermal fibroblasts with a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct suggested transcriptional regulation, since the CAT activity incells incubated with to be synthesized by a variety of cell types both i n vivo and in uitro
Summary
Transient transfections of the dermal fibroblasts with a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct suggested transcriptional regulation, since the CAT activity incells incubated with to be synthesized by a variety of cell types both i n vivo and in uitro. Treatment of Transgenic Mice-To test the effects of IL-1 on the elastin promoter activity in viuo, 100 units of recombinant human IL-10 in 200 p1 of 0.15 M NaCl was injected subcutaneously into 5day-old transgenic mice which we have recently developed and express the human elastinpromoter linked to the CAT gene (see above).The control animals were injected with 200 pl of 0.15 M NaCl alone. Transient Transfections of Cultured Cells-Human neonatal foreskinfibroblasts in late logarithmic growth phase were transfected with 20 pg of plasmid DNA, pEPl/CAT, which contains 5.2 kb of 5'flanking DNA of the human elastingene cloned into thepromoterless plasmid pBSOCAT (Fazio et al, 1990; Kahariet al., 1990). Mined with a commercial assay kit (Bio-Rad), and identical amounts of protein (50-100 pg) were used for each CAT assay, using ["C] chloramphenicol as substrate (Gorman et al, 1982)
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