Effect of the 3'-untranslated region on the expression levels and mRNA stability of alpha 1(I) collagen gene.

Changes in the synthesis of type I collagen, a major extracellular matrix component in skin and bones, are associated with both normal growth or repair processes and with several pathological conditions such as lung fibrosis and liver cirrhosis. The expression of the alpha 1(I) collagen gene is regulated by transcriptional and post-transcriptional mechanisms. Regulation at both these levels are usually utilised when extensive changes occur in collagen synthesis. We constructed plasmids carrying the whole or partially deleted 3'-UTR sequences of the alpha 1(I) collagen gene, fused to two hGH exons and to the promoter of the alpha 1(I) collagen gene. A control plasmid contained the 3'-UTR of the hGH gene. In transient transfections into Rat-1 fibroblasts, no significant differences between plasmids were found, which suggests that although 3'-end of the gene has been shown in previous studies to contain DNaseI hypersensitive sites and to bind sequence-specific nuclear proteins it does not seem to function as a transcriptional regulator. This was further supported by the finding that TGF-beta treatment induced a 2.5-fold expression of hGH mRNA from plasmids containing collagen promoter and either hGH or alpha 1(I) collagen 3'-UTR. In stable transfections, mRNAs using the first polyadenylation site were not as stable as those transcribed from the endogenous alpha 1(I) collagen gene. We suggest that the 3'-UTR alone may not be sufficient to determine the stability of the shorter alpha 1(I) collagen mRNA species.

Fibroblast expression of collagens and proteoglycans is altered in aspartylglucosaminuria, a lysosomal storage disease.

Aspartylglucosaminuria (AGU), a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (E.C. 3.5.1.26), is characterised by progressive mental retardation and variable connective tissue signs. The ultrastructure of collagen fibrils in skin of AGU patients is abnormal and their fibroblasts synthesise reduced amounts of collagens [Näntö-Salonen et al. (1984) Lab invest. 51: 464-468]. In this work we measured the steady-state messenger RNA levels of several extracellular matrix components in skin fibroblast cultures of two patients homozygous for the most prevalent mutation (AGUFin) causing the disease in Finland. In confluent cultures the steady-state mRNA concentrations of type I and III collagens were reduced to 0.5-20% of control values. Almost as marked reduction was observed in the mRNA level of biglycan, a small interstitial proteoglycan whereas that of decorin, a closely related, collagen fibril-associated proteoglycan, was increased several-fold. Elevated decorin and decreased biglycan mRNA levels reflected the amounts of the produced corresponding proteoglycans. The differences in the mRNA levels become more pronounced with the time the cells were in culture. Fibronectin mRNA concentrations were similar in AGU and control fibroblasts. Changes in the expression and synthesis of extracellular matrix components might be related to the connective tissue symptoms of the patients.

Nuclear and cytoplasmic alpha 1 (I) collagen mRNA-binding proteins.

We have recently identified a cytoplasmic protein, alpha 1-RBF67, that specifically interacts with the conserved 3'-untranslated region of the alpha 1 (I) collagen gene. The binding activity was decreased in extracts from dexamethasone treated cells, which correlates with the known accelerated turnover of the COL1A1 RNA [Määttä, A. and Penttinen, R.P.K. (1993) Biochem. J. 295, 691-698]. Now we report that a very similar protein is present in nuclear extracts of NIH 3T3, human fibroblast and HeLa cells, which suggests that determination of cytoplasmic mRNA stability is not the sole function of the alpha 1-RBF67 activity. The binding to the RNA probe can be inhibited by annealing a DNA oligonucleotide or using excess of cold specific competitors. In UV-cross linking assays the nuclear protein has the same molecular weight (67 kDa) as the cytoplasmic one and the RNA-bound peptides generated by CNBr or V8 protease cleavage from both the cytoplasmic and the nuclear protein were identical. This protein was the only one of several nuclear collagen mRNA 3'-UTR binding proteins that was present in both nuclear and cytoplasmic extracts. In fibroblasts heparin-resistant nuclear RNA binding proteins had molecular weights of 45, 67 (alpha 1-RBF67), and 71 kDa. HeLa-cells contained an additional protein of 51 kDa and several non-specific RNA-binding proteins. The binding activity is modified by changes in the redox state, which implicates that in the nucleus the binding affinities of alpha 1(I) collagen RNA-binding protein and AP-1, a redox sensitive nuclear factor, that is important in the transcription of alpha 1(I) collagen gene, can be regulated simultaneously to the same direction.

Highly conserved sequences in the 3'-untranslated region of the COL1A1 gene bind cell-specific nuclear proteins.

Sequencing of the 3' untranslated region (3'-UTR) of the human COL1A1 gene revealed numerous putative regulatory motifs and two highly conserved regions flanking the two polyadenylation sites. The conserved regions were separated by about 700 bp of less conserved sequences. The first region consists of almost all the 3'-UTR of the shorter (4.8 kbp) COL1A1 transcript. The second conserved domain includes a motif shared with several collagen genes. Both conserved domains bind cell-specific nuclear proteins suggesting that the 3'-UTR is important for cell specific expression of the COL1A1 gene.

Expression of extracellular matrix genes: transforming growth factor (TGF)-beta1 and ras in tibial fracture healing of lathyritic rats.

Experimental osteolathyrism, induced by dietary aminoacetonitrile (AAN), was used to study the effect of altered extracellular matrix on the expression of connective tissue components in long bone healing. AAN inhibits lysyl oxidase, which is needed for the formation of collagen cross-link precursors, and is also shown to act as a regulator of Ras. Fractured tibias in lathyritic rats develop excessive amounts of mechanically weak callus tissue with irregular cartilage and reduced glycosaminoglycan accumulation. Cartilage-specific proteins (collagen types II, IX, and X and aggrecan) were expressed temporally much wider in lathyritic calluses than in the controls, and active transcription was observed even during the fibrous and ossifying stages. Soft connective tissue was still present in 2- and 3-week-old lathyritic calluses and could explain the elevated type III collagen, biglycan, and decorin mRNA levels. Both transforming growth factor (TGF)-beta1 and c-Ha-ras, which control cell growth and differentiation, were upregulated during the cartilaginous stage. The maximal expression of TGF-beta1 preceded that of ras in osteolathyrism.

A fibroblast protein binds the 3'-untranslated region of pro-alpha 1(I) collagen mRNA.

Post-transcriptional regulation of the expression of the pro alpha 1(I) chain of type I collagen (COL1A1) was studied by analysing cytoplasmic RNA-binding proteins and by transient transfections with collagen minigene plasmids. In this paper we present evidence for a factor from NIH 3T3 cells and human skin fibroblasts that interacts with the conserved 3'-untranslated region (UTR) of the shorter 4.8 kb mRNA species of the COL1A1 gene. The specificity of the interaction was confirmed by using (i) unlabelled specific and non-specific competitor RNAs and (ii) oligodeoxyribonucleotides annealed to the probe or used as single-stranded competitors. An antisense oligonucleotide annealed to the RNA probe near its 3'-terminus [20-42 nucleotides upstream of the first polyadenylation signal of the alpha 1(I) collagen mRNA] inhibited the binding, whereas other sense or antisense oligonucleotides had no effect on the interaction. The binding was sensitive to alkylation of free SH groups but not to phosphatase treatment of the extracts. In u.v. cross-linking analysis this factor migrated as a single polypeptide chain of about 67 kDa, and was named alpha 1-RBF67 (type I collagen alpha 1 chain RNA-binding factor). Dexamethasone treatment of fibroblasts, which is known to accelerate the turnover of COL1A1 mRNA, decreased the alpha 1-RBF67 activity markedly as evaluated by gel-retardation and u.v. cross-linking assays. Transient transfections with plasmids carrying the alpha 1(I) collagen promoter and 3'-UTR sequences demonstrated that the 3'-UTR participates in the response to dexamethasone. Thus the loss of alpha 1-RBF67 activity might be associated with decreased alpha 1(I) collagen mRNA levels after dexamethasone treatment.

Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate synthesis of extracellular matrix proteins, both in animals and in cell culture. We found that mRNAs for alpha 1(I) collagen, fibronectin, and thrombospondin were markedly increased in TGF-beta-treated 3T3 (mouse) cells. For collagen and fibronectin this increase was 10-to 20-fold, as measured by quantitative blot hybridization analysis. A maximal value was reached at 16-24 hr, with a subsequent gradual decline. Concomitant treatment with cycloheximide prevented the stimulation observed with TGF-beta. Under conditions of confluent growth a clear increase in alpha 1(I) collagen mRNA stability was observed, whereas in subconfluent cells no change in mRNA half-life was found, despite an equally large increase in mRNA levels. We suggest that the mode of action of TGF-beta varies with the target cell and depends on the interplay of a number of complex cellular factors.

Abnormal collagen metabolism in cultured cells in osteogenesis imperfecta.

Cells obtained from normal human skin synthesize predominantly type I collogen in culture. Cells obtained from the skin of an infant with a severe form of osteogenesis imperfecta were found to synthesize as much type III as type I collagen. Decreased synthesis of type I collagen could explain the tissue fragility observed in this case.

Nuclear factor binding to an AP-1 site is associated with the activation of pro-alpha 1(I)-collagen gene in dedifferentiating chondrocytes.

Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.

Extended expression of cartilage components in experimental pseudoarthrosis.

The healing of femoral fractures in an experimental rat pseudoarthrosis model was followed by studying the expression of cartilage specific genes coding for type II and X collagens and aggrecan, soft tissue and bone specific type I collagen, and decorin. Severe impairment of healing was observed with cartilage gene expression continuing until the seventh week and then declining rapidly. The abnormal healing pattern results in an inactive scar-like callus after the ninth week of healing even though house-keeping (e.g., GAPDH) genes are continuously expressed in the tissue. These results could be explained on the basis of continuous chondrogenic stimulus extending much beyond the normal range. If union is not achieved because of mechanical instability, signal of endochondral ossification persists until it becomes exhausted and callus at the fracture gap becomes an inactive fibrous scar. The disturbed matrix gene expression was confirmed by histology.