COL1A1 Transgene Expression in Stably Transfected Osteoblastic Cells

Abstract

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.Type I collagen is the most abundant protein in vertebrates with levels reaching 5-65% of total protein synthesis in bone cells (1, 2). Two coordinately regulated genes, COL1A1 and COL1A2, give rise to the mRNA transcripts, each containing 50 -51 exons, which are processed, translated, and post-transcriptionally modified. The resulting procollagen chains are assembled into a heterotrimeric structure and modified further before entering into the collagen fibril, which in turn is modified by its interaction with other extracellular molecules. The primary site of regulation for this complex series of events is thought to be at the level of transcription initiation. Most studies aimed at understanding the transcriptional regulation of COL1A1 have focused on cell-specific regulatory domains within the proximal promoter, the first intron, and more distal 5Ј-upstream domains without attention to sequences accounting for the extremely high level of gene transcription (3-8).We have been studying the regulation of the rat COL1A1 gene utilizing a family of transgenes, designated ColCAT, which are derived from the expression vector pSV 2 CAT. These transgenes are expressed in a tissue-specific manner in bone, tendon, and skin of transgenic mice and have been used to elucidate a bone-specific regulatory ele...

The Y-box Binding Protein YB-1 Suppresses Collagen α1(I) Gene Transcription via an Evolutionarily Conserved Regulatory Element in the Proximal Promoter

“…To create the promoter deletion constructs pColCAT Ϫ1.3/ϩ1.6 and Ϫ0.4/ϩ1.6, B16 was digested with NheI (Ϫ3382) together with Tth111I (Ϫ1282) or MunI (Ϫ390), respectively, and religated. The intron-less pColCAT Ϫ2.3/0 is identical to B47 (17) except that it lacks the 5Ј HindIII fragment between Ϫ3544 and Ϫ2369 present in B16.…”

Activation of Fibroblast Procollagen α1(I) Transcription by Mechanical Strain Is Transforming Growth Factor-β-dependent and Involves Increased Binding of CCAAT-binding Factor (CBF/NF-Y) at the Proximal Promoter

“…This plasmid construct is identical to B15, described by Breault et al (29), except an EcoRV site in the polylinker has been converted to a ClaI site. The construct contains sequences between Ϫ3518 and ϩ1594 (relative to the transcription start site) of the rat COL1A1 gene, which includes the upstream promoter region, the first exon (the translational start site, ATG, has been converted to a NotI site), and most of the first intron (3.6/1.6 refers to 3.6 kb of upstream promoter sequence and 1.6 kb of downstream sequence).…”

“…The full-length construct, pColCAT-3.6/1.6, contains 3.6 kb of the 5Ј upstream promoter sequence and 1.6 kb of sequence downstream of the transcription start site, including the first exon and most of the first intron. This construct was chosen because these regions of the COL1A1 gene, including the first intron, are known to contain potentially important positive and negative regulatory sequences (29,(35)(36)(37), and it therefore provides a suitable starting point for modeling responses of the endogenous gene. Cells transfected with the full-length pColCAT-3.6/1.6 construct displayed an ϳ2-fold increase in CAT protein levels in response to continuous cyclic strain for 48 h compared with rigid control cells.…”

“…The intronless pColCAT3.6/0 has the first intron and the distal third of the first exon replaced with the 16 S splice donor site from the SV40 virus (compare B47 in Ref. 29). The set of proximal promoter deletion constructs were generated using an ExoIII/Mung Bean deletion kit (Stratagene) following the manufacturer's recommendations.…”

Mutation of the 5′-Untranslated Region Stem-Loop Structure Inhibits α1(I) Collagen Expression in Vivo

“…Previous studies have reported that interfering with the first intron of ␣1(I) collagen mRNA can have deleterious effects on collagen expression (25)(26)(27). Although these studies dealt with deletions in the first intron, evidence suggests that increased intron size can also influence transcript expression (28,29).…”