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We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.

High concentrations of prostaglandin E2 (PGE2) inhibit collagen synthesis and reduce alpha 1(I)procollagen messenger RNA (mRNA) levels in cultured fetal rat calvariae. To examine the mechanism of this effect, we used the immortalized rat osteoblastic clonal cell line, Py1a. PGE2 at 1 microM inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP) and increased incorporation into noncollagen protein, whereas 0.1 microM PGE2 increased both CDP and noncollagen protein labeling. Because insulin-like growth factor-I (IGF-I) is an anabolic hormone in bone and PGE2 can increase its production, we added exogenous IGF-I (10 nM) to Py1a cultures. In the presence of IGF-I, PGE2 from 10 nM to 1 microM had only an inhibitory effect on CDP labeling and alpha 1(I)procollagen mRNA levels. PGE2 at 1 microM decreased the rate of alpha 1(I)procollagen gene transcription in the presence or absence of IGF-I, determined by a nuclear run-on assay. Py1a cells were stably transfected with chimeric genes containing varying lengths of the 5'-upstream region of the rat alpha 1(I)procollagen promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. In cells transfected with ColCAT 3.6, which contains 3520 base pairs of 5'-upstream DNA, CAT activity was inhibited by PGE2, but the inhibition was less than that observed for CDP labeling. With smaller 5'-upstream regions, there was no inhibitory effect of PGE2. These results demonstrate that PGE2 inhibits alpha 1(I)procollagen gene transcription and the activity of a region between -3.5 and -2.3 kilobases of the 5'-upstream collagen gene promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of recombinant human interleukin-1 alpha (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid. Collagen synthesis was assessed as [3H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a 32P-labeled alpha 1(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1-1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75-80%, and a parallel reduction of procollagen alpha 1(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1-100 nM, which inhibited CDP and reduced procollagen alpha 1(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 microM), PTH (10 nM) and prostaglandin E2 (1 microM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 microM) and (Bu)2cAMP (100 microM) failed to alter CDP or procollagen alpha 1(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the alpha 1(I) procollagen gene by 70% and 80%, respectively, while alpha 2(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the beta-actin or beta-tubulin genes.(ABSTRACT TRUNCATED AT 250 WORDS)