Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I) C DNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley-Liss, Inc.     

DNA and chromatin structure of the human alpha 1 (I) collagen gene

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.     

A combination of cis-acting elements is required to activate the pro-alpha 1(I) collagen promoter in tendon fibroblasts of transgenic mice

The genes encoding the two type I collagen chains are selectively activated in few cell types, including fibroblasts and osteoblasts. By generating transgenic mice, we have previously shown that the activity of the mouse pro-alpha1(I) promoter was controlled by separate cell-specific cis-acting elements. In particular, a sequence located between -3.2 and -2.3 kb was needed to induce expression of the reporter gene at high levels in tendon fibroblasts. In the present work, by using the same transgenic approach, we have identified two short elements in this sequence, named tendon-specific element (TSE) 1 and TSE2, that were necessary to direct reporter gene expression selectively in tendon fibroblasts. Gel shift assays showed that TSE1 and TSE2 bound proteins specifically present in nuclear extracts from tendon fibroblasts and that the sequence of TSE2 binding a tendon-specific protein corresponded to an E-box. Analysis of transgenic mice further indicated that TSE1 and TSE2 needed to cooperate not only with each other but also with other cis-acting elements of the proximal promoter to activate reporter gene expression in tendon fibroblasts. Similarly, it pointed out that the so-called osteoblast-specific element had to interact with downstream sequences to drive reporter gene expression in osteoblasts of transgenic mice. Thus, expression of the mouse pro-alpha1(I) collagen gene in tendon fibroblasts appears to be the result of a unique combination of different cis-acting elements, including TSE1 and TSE2.     

Biosynthesis of α2(IV) and α1(IV) chains of collagen IV and interactions with matrix metalloproteinase-9

In vitro binding studies with latent matrix metalloproteinase-9 (pro-MMP-9) have revealed the existence of nondisulfide-bonded α2(IV) chains on the cell surface capable of forming a high-affinity complex with the enzyme. Here we investigated the biosynthesis and cellular distribution of α2(IV) and α1(IV) chains in breast epithelial (MCF10A and MDA-MB-231) and fibrosarcoma (HT1080) cells by pulse-chase analysis followed by immunoprecipitation with chain-specific monoclonal antibodies (mAb). These studies showed that whereas the α1(IV) chain remained in the intracellular compartment, nondisulfide-bonded α2(IV) chains were secreted into the media in a stable form. Consistently, only α2(IV) was detected on the cell surface by surface biotinylation or indirect immunofluorescence. In agreement with the pulse-chase analysis, media subjected to coprecipitation experiments with pro-MMP-9 or pro-MMP-9-affinity chromatography followed by immunoblotting with chain-specific mAbs resulted in the detection of α2(IV). A preferential secretion of nondisulfide-bonded α2(IV) chains was also observed in CHO-K1 cells transiently transfected with full-length mouse α2(IV) or α1(IV) cDNAs. However, a complex of mouse α1(IV) with pro-MMP-9 was coprecipitated with exogenous enzyme from lysates of CHO-K1 cells transfected with mouse α1(IV), suggesting that under overexpression conditions the enzyme can also interact with the α1(IV) chain. Collectively, these studies further demonstrate the interactions of pro-MMP-9 with collagen IV chains and a unique processing and targeting of nondisulfide-bonded α2(IV) chains that may play a role in the surface/matrix association of pro-MMP-9. J. Cell. Physiol. 180:131–139, 1999. © 1999 Wiley-Liss, Inc.     

Regional chromosome mapping of human collagen genes alpha 2(I) and alpha 1(I) (COLIA2 and COLIA1)

Summary For the assignment of the genes for the pro-2(I) (COLIA2) and the pro-1(I) (COLIA1) collagens, cDNA and genomic DNA probes were used in in situ hybridization experiments on human prometaphase chromosomes. An improved staining method is reported for the simultaneous identification of chromosomes and the autoradiographic grains after the hybridization procedures. With this procedure more cells with higher resolution could be used for the assignment of genes by in situ hybridization. Statistical analysis of the grains located on respectively 660 and 302 metaphases using pro-2(I) and pro1(I) DNA probes, confirmed the assignment of these genes to human chromosomes 7 and 17. Analysis of the grain distribution on prometaphase chromosomes showed that the location of the pro2(I) collagen gene is in the region 7q21.3–22.1. The location of the pro-1(I) collagen gene was found to be in band 17q21.31–2005.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of control elements for position‐independent expression of human α‐lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999. © 1999 Wiley‐Liss, Inc.     

Glucocorticoids coordinately regulate type I collagen proα1 promoter activity through both the glucocorticoid and transforming growth factor β response elements: A novel mechanism of glucocorticoid regulation of eukaryotic genes

Glucocorticoids have previously been shown to decrease Type 1 collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type 1 procollagen gene expression. These latter studies have included uridine incorporation into proα1(I) and proα2(1) mRNas and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking regionof the proα1 (1) coullagen gene and the reporter gene, chljoramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibrolblasts that dexamethasone down regulates the promoter activity of the proα1(I) collagen gene. The glucocorticoid-mediated down-regulastionof procolljagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whoulue ColCat 3.6 plasmid did not elimiinatre the effect. The ipossibility existed that another cis-element inthe 5' flanking region of the proα1(I) collagen gene was also required for the glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-β has been shown to stimulate collagen proα1(I) and proα2(I) gene activities. Dexamethasone treatment of non-transfected skin fibroblasts did result in a decrease of transforming growth factor-β. The decrease of CVAT activity by dexamethasone was brought back to control value by the addition of exogenous TGF-β to the culture media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid recptor binding to the GRE and TGF-β activator protein to the TGF-β element which were brought back to control values by coordinate exogenous TGF-β treatment. Thus the interaction of these TGF-β molecules with cellular membrane receptors and subsequent rtransduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expfrssion through both the GRE and TGF-β elements. Depression of procollagen gene expression by glucocorticoids through the TGF-β element is mediated by decreased TGF-β secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the bassis for a novel mechanism of glucocorticoid-mediated regulation of eukaryotic genes containing the TGF-β element. © 1995 Wiley-Liss, Inc.     

Localization of the α7 integrin gene (ITGA7) on human chromosome 12q13: clustering of integrin and hox genes implies parallel evolution of these gene families

Expression of the α7 integrin gene (ITGA7) is developmentally regulated during the formation of skeletal muscle. Increased levels of expression and production of isoforms containing different cytoplasmic and extracellular domains accompany myogenesis. To determine whether a single or multiple α7 genes underlie the structural diversity in this alpha chain that accompanies development, we have examined the rat and human genomes by Southern blotting and in situ hybridization. Our results demonstrate that there is only one α7 gene in both the rat and the human genomes. In the human, ITGA7 is present on chromosome 12q13. Phylogenetic analysis of the integrin alpha chain sequences suggests that the early integrin genes evolved in two pathways to form the I-integrins and the non-I-integrins. The I-integrin alpha chains contain an additional sequence of approximately 180 amino acids and arose as a result of an early insertion into the non-I-gene. The I-chain subfamily further evolved by duplications within the same chromosome. The non-I-integrin alpha chain genes are localized in clusters on chromosomes 2, 12, and 17, and this closely coincides with the localization of the human homeobox gene clusters. Non-I-integrin alpha chain genes appear to have evolved in parallel and in proximity to the Hox clusters. Thus, the Hox genes that underlie the design of body structure and the Integrin genes that underlie informed cell—cell and cell—matrix interactions appear to have evolved in parallel and coordinate fashions.     

Induction of α1-tubulin gene expression during development and regeneration of the fish central nervous system

The α1- and α2-tubulin encoding genes were cloned from a goldfish genomic DNA library. α1- and α2-tubulin RNA expression was examined in developing and adult retinas. These studies demonstrated increased α1-tubulin RNA in presumptive ganglion cells that grow axons early in retinal development and in adult retinal ganglion cells whose optic axons had been damaged. The α2-tubulin RNA was undetectable in developing retina and constitutively expressed in adult retinal ganglion cells regardless of optic nerve crush. To determine if these changes in α1-tubulin RNA reflected changes in α1-tubulin promoter activity, we introduced into zebrafish embryos and adult goldfish retinal explants expression vectors harboring the α1-tubulin gene's promoter. These studies showed that the α1-tubulin promoter confers a developmentally regulated, neuron-restricted pattern of reporter gene expression in vivo and its activity is increased in adult retinal neurons induced to regenerate their axons. Promoter deletions defined regions of α1-tubulin DNA necessary for this pattern of expression. These results suggest that DNA sequences necessary for α1-tubulin gene induction during central nervous system development and regeneration are contained within the α1-tubulin gene's 5′-flanking DNA and that this promoter will be useful for identifying these elements and their DNA binding proteins. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 429–440, 1998