Interferon-gamma reverses the stimulation of collagen but not fibronectin gene expression by transforming growth factor-beta in normal human fibroblasts.

It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effects of the simultaneous addition of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) on normal human dermal fibroblast collagen and fibronectin production. In addition, in vitro transcription rates and steady-state mRNA levels for these molecules were determined by nuclear run-off assays and Northern and dot-blot hybridization using specific human cDNA probes. Treatment of cultures with TGF-beta caused stimulation of collagen and fibronectin production. Addition of IFN-gamma to the TGF-beta-treated cultures abrogated the stimulatory effects of TGF-beta on collagen production in a dose-dependent manner and resulted in a net inhibition of collagen production. In contrast, the increase in fibronectin synthesis induced by TGF-beta was augmented further by IFN-gamma. These changes in collagen and fibronectin production were accompanied by parallel changes in the steady-state mRNA levels for these proteins. The effects of TGF-beta plus IFN-gamma on fibronectin gene expression appeared to be mediated entirely by transcriptional mechanisms, whereas the effects on collagen gene expression resulted from a combination of transcriptional and post-transcriptional events.

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.