Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

Abstract

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.

Effects of short-term dexamethasone treatment on collagen synthesis and degradation markers in preterm infants with developing lung disease

“…The mechanisms of action are not exactly established. It has been proved that glucocorticoids suppress procollagen gene expression and they also inhibit collagenases belonging to the family of matrix metalloproteinases, which are involved in the degradation of collagen (24,(27)(28). Concerning the possible induction of the hepatic clearance pathway for the propeptides studied here, it has been shown earlier that glucocorticoids do not affect the hepatic clearance of the aminoterminal propeptide of type III collagen (24).…”

Species differences in cis-elements of the Pro?1(I) procollagen promoter and their binding proteins

“…Several studies suggest that pro␣1(I) collagen mRNA stability is decreased by glucocorticoid treatment [Mahonen et al, 1998;Raghow et al, 1986], while others indicate that regulation is at the transcriptional level [Cockayne et al, 1988;Meisler et al, 1997]. Glucocorticoids regulate transcription positively by binding of GR to a palindromic sequence of two hexamers separated by three nucleotides, or negatively by two different mechanisms [Karin, 1998].…”

Regulation of expression of the type I collagen genes

“…Hamalainen et al [391 reported a similar decrease in the stability of both type I procollagen mRNAs in the presence of cortisol. In contrast, Weiner et al [114] reported transcriptional inhibition of the a2 collagen gene by dexamethasone in an in vivo model of hepatic fibrosis, as well as in cultured hepatocytes [1151, and both Cockayne et al [18,191 and Walsh et al [1121 have similarly reported transcriptional downregulation of type I collagen by dexamethasone. Finally, in transient transfection analyses in mouse fibroblasts, dexamethasone decreased expression of chimeric a2-CAT constructs (containing mouse a2 promoter sequences from -2000 to +54) to 3 4 4 1 % of control levels [115].…”