Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.     

Characterization of two distinct positive cis-acting elements in the mouse alpha 1 (III) collagen promoter

We have identified two distinct sequence elements in the mouse alpha 1(III) collagen promoter which are protected from DNase I digestion by the binding of factors present in crude nuclear extracts of NIH 3T3 fibroblasts. Small substitution mutations were introduced into these promoter elements and shown by the gel retardation (gel mobility shift) and DNase I protection assays to decrease or eliminate factor binding to the mutated element but not to the remaining wild-type element, indicating that two distinct factors recognize these separate promoter regions. Region A appears to bind a factor related to the Jun/AP-1 protein, whereas the factor binding to region B remains as yet unidentified. Mutagenesis of either region decreased the activity of the alpha 1(III) collagen promoter in DNA transfection assays by about 3-fold for the A region (located between - 122 and - 106) and about 5-fold for the B region (located between -83 and -61). These results indicate that regions A and B in the mouse alpha 1(III) collagen promoter are positive cis-regulatory elements, independently binding two distinct trans-activating factors.     

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.     

Concerted modulation of alpha 1(XI) and alpha 2(V) collagen mRNAs in bovine vascular smooth muscle cells.

We have isolated a partial cDNA for alpha 1(XI) collagen from a bovine smooth muscle cell (SMC) library. Previously, this collagen was not known to be expressed in SMCs. Comparison of the nucleotide and deduced amino acid sequence of the 2.7-kilobase bovine clone and the human alpha 1(XI) sequence indicates 92 and 98% homology, respectively. Bovine SMCs in culture were found to produce alpha 1(XI) mRNA. However, alpha 2(XI) and alpha 1(II) collagen RNA were not detectable; therefore, SMCs cannot synthesize the same type XI collagen as found in cartilage. Since type XI collagen is structurally related to type V collagen, the expression of alpha 1(XI) and alpha 2(V) collagen mRNA in SMCs was characterized. Levels of alpha 1(XI) and alpha 2(V) collagen mRNAs were low in exponentially growing SMCs and increased 3-4-fold as cells became confluent. Increased mRNA levels were also observed when exponentially growing subconfluent SMCs were incubated in medium containing 0.5% fetal bovine serum for 24 h, similar to the effects of serum deprivation on the expression of types I and III collagen genes (Kindy, M. S., Chang, C.-J., and Sonenshein, G. E. (1988) J. Biol. Chem. 263, 11426-11430). However, as cell density increased, serum deprivation resulted in very different responses for these collagen genes. Serum deprivation caused a decrease in expression of alpha 1(XI) and alpha 2(V) collagen mRNAs in cultures as they approached confluence. In contrast, at confluence alpha 1(I) and alpha 2(I) mRNA levels no longer responded to serum concentration whereas expression of alpha 1(III) mRNA remained inducible by serum deprivation. These results suggest concerted regulation of alpha 1(XI) and alpha 2(V) collagen gene expression, which is distinct from that for the chains of type I and type III collagen with respect to cell density and serum.     

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).     

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.