Sp family of transcription factors is involved in iron-induced collagen alpha1(I) gene expression

The purpose of this study was to identify the cis-acting elements and the trans-acting factors involved in the iron-induced expression of the collagen alpha1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 microM ferric chloride, 50 microM ascorbic acid, and 250 microM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between -220 and -110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the COL1aI promoter.     

Sp1 and Sp3 transcription factors mediate malondialdehyde-induced collagen alpha 1(I) gene expression in cultured hepatic stellate cells

Malondialdehyde, the end product of lipid peroxidation, has been shown to stimulate collagen alpha1(I) (Col1a1) gene expression. However, mechanisms of this effect are unclear. The purpose of this study was to clarify these mechanisms. Rat hepatic stellate cells were cultured in the presence of 200 microm malondialdehyde, and the effects on collagen gene expression and the binding of nuclear proteins to the col1a1 promoter were analyzed. Malondialdehyde treatment induced an increase in the cellular levels of col1a1 mRNA that was abrogated by pretreating cells with cycloheximide, p-hydroxymercuribenzoate, pyridoxal 5'-phosphate, and mithramycin. Transient transfections showed that malondialdehyde exerted its effect through regulatory elements located between -220 and -110 bp of the col1a1 promoter. Gel retardation assays demonstrated that malondialdehyde increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the col1a1 promoter. These bindings were supershifted with Sp1 and Sp3 antibodies. Finally, malondialdehyde increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Our data indicated that treatment of hepatic stellate cells with malondialdehyde stimulated col1a1 gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the col1a1 promoter.     

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.     

Multiple 3'' ends of the chicken pro alpha 2(I) collagen gene

The precise location of the 3' ends of the chicken pro alpha 2(I) collagen gene have been identified by S1 nuclease protection of overlapping genomic fragments by calvaria poly A containing RNA and size determination of the protected fragments on DNA sequencing gels. The gene ends 300 and 306 bp and 754 and 777 bp from the translation stop codon. The two sets of ends explain the major and minor pro alpha 2(I) collagen mRNAs previously observed, which may result from either RNA polymerase readthrough of the first termination site and/or different processing sites.     

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.     

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.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.