COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene.

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line Py1a: structure-activity relations and signal transduction mechanisms.

We previously showed that prostaglandin E2 (PGE2) can selectively inhibit collagen synthesis and gene transcription in the immortalized rat osteoblastic clonal cell line Py1a, particularly in the presence of insulin-like growth factor I (IGF-I). In the present study, we examined the structure-activity relations for this effect. PGF2 alpha was approximately 100 times more potent than PGE2. The prostaglandin F receptor (FP) selective agonist, fluprostenol, was the most potent agonist tested, significantly inhibiting incorporation of [3H]proline into both collagen and noncollagen protein at 10(-11) M, with more than 90% inhibition of collagen synthesis at 10(-8) M. The PGE2 analog, sulprostone, and PGD2 showed activity similar to that of PGE2. PGI2 and its stable analog, carbacyclin, were the least effective. Parathyroid hormone (PTH), forskolin, and isobutylmethylxanthine (IBMX) were ineffective. Phorbol myristate acetate (PMA) inhibited collagen synthesis in a manner similar to that of the prostanoids. The inhibitory effects of PGF2 alpha, fluprostenol, and PMA show a similar time course on alpha 1(I) procollagen mRNA levels. The inhibition appeared to be caused by a decrease in collagen gene transcription as measured by nuclear run-on analysis. Further evidence for a transcriptional effect was obtained with COL1A1 promoter-CAT reporter constructs, although these showed somewhat smaller effects of prostanoids on CAT activity than on mRNA levels or labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells.

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.