GLUCOCORTICOID AND RETINOID REGULATION OF ALPHA-2 TYPE-I PROCOLLAGEN PROMOTER ACTIVITY

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro-alpha-2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha-2 (I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856. The doublet GRE contains an A/T strand switch of the third base pair as compared to the single GRE and is not necessary for dexamethasone regulation of gene activity. All-trans-retinoic acid increased CAT activity of the same pR40 CAT construct transfected in the mouse fibroblasts. DNA sequencing revealed a RARE and a modified RARE in the stretch of DNA from -981 to -506. Deletion of only the latter DNA region eliminated the elevation of CAT activity elicited by all-trans-retinoic acid. Our results indicate that the singlet GRE and the RARE are required for glucocorticoid and retinoic acid regulation of pro-alpha-2(I) collagen gene activity, respectively.