Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D3 in human osteosarcoma cells

Synthesis of type I and III collagens has been examined in MG-63 human osteosarcoma cells after treatment with the steroid hormone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Analysis of total [3H]proline-labeled proteins and pepsin-derived collagens revealed that 1,25-(OH)2D3 selectively stimulated synthesis of alpha 1I and alpha 2I components of type I collagen after 6-12 h. Consistent with previous reports (Franceschi, R. T., Linson, C. J., Peter, T. C., and Romano, P. R. (1987) J. Biol. Chem. 262, 4165-4171), parallel increases in fibronectin synthesis were also observed. Hormonal effects were maximal (2- to 2.5-fold versus controls) after 24 h and persisted for at least 48 h. In contrast, synthesis of the alpha 1III component of type III collagen was not appreciably affected by hormone treatment. Of several vitamin D metabolites (1,25-(OH)2D3, 25-dihydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) tested for activity in stimulating type I collagen synthesis, 1,25-(OH)2D3 was found to be the most active. Analysis of collagen mRNA abundance by Northern blot hybridization indicated that both types I and III procollagen mRNAs were increased 4-fold after a 24-h exposure to 1,25-(OH)2D3. Pro alpha 1I mRNA remained elevated through the 48-h time point while pro alpha 2I and pro alpha 1III mRNAs returned to control values. These results indicate that the regulation of collagen synthesis by 1,25-(OH)2D3 is complex and may involve changes in translational efficiency as well as mRNA abundance. 1,25-(OH)2D3 also caused at least a 20-fold increase in levels of the bone-specific calcium-binding protein, osteocalcin. These results are consistent with the hypothesis that 1,25-(OH)2D3 is stimulating partial differentiation to the osteoblast phenotype in MG-63 cells.     

Down-regulation of alpha 3(VI) chain expression by gamma-interferon decreases synthesis and deposition of collagen type VI

Treatment of cultured human skin fibroblasts with increasing doses of gamma-interferon produces a distinct reduction of steady-state levels of the alpha 3 chain of collagen VI mRNA by about 60% but not of the alpha 1 and alpha 2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in alpha 3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea-insoluble form in the cell matrix. No other gamma-interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65-80%) of intracellular alpha 3(VI) chains with no decrease found for alpha 1(VI) and alpha 2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of gamma-interferon. It indicates that correctly assembled heterotrimers of the composition [alpha 1(VI) alpha 2(VI) alpha 3(VI)] are formed and secreted also in the absence of an equivalent alpha 3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18,601-18,606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.     

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.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

Activation of type I collagen genes in cultured scleroderma fibroblasts

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.     

Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture.

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.     

Immune interferon inhibits collagen synthesis by rheumatoid synovial cells associated with decreased levels of the procollagen mRNAs

Recombinant immune interferon, (interferon-gamma, IFN-gamma) inhibits types I and III collagen synthesis by rheumatoid synovial fibroblast-like cells in culture. This decrease is associated with a decrease in the levels of types I and III procollagen mRNAs in these cells as measured by dot blot hybridization. In the control synovial cells the level of alpha 2(I) mRNA is disproportionately high compared with that of alpha 1(I) or alpha 1(III) mRNA, and IFN-gamma suppresses the level of alpha 1(I) and alpha 1(III) mRNA to a greater extent than that of alpha 2(I) mRNA. The lymphokine, IFN-gamma, may thus have a role in the regulation of collagen synthesis in inflammatory joint disease and other conditions.     

Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.     

M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: Findings obtained with muscarinic-receptor knockout mouse

Hepatic stellate cells (HSC) differ in their phenotype depending on the initiation and progression of their activation. Our hypothesis was that different mechanisms govern type I collagen synthesis depending on stage of HSC activation. We investigated the role of alpha(5)beta(1)-integrin as a regulator of type I collagen gene COL1A1 expression in primary and passaged HSC cultures using transgenic mouse containing type I collagen gene COL1A1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The alpha(5)beta(1) protein levels increased during the activation and were highest in day 6 primary cultures but decreased in passaged HSC. CAT activity, reflecting COL1A1 expression, was upregulated by alpha(5)beta(1)-integrin. Inhibition of alpha(5)beta(1)-integrin by echistatin and blocking antibody resulted in reduced transgene activity only in early primary cultures (compared with the control, 53.3 +/- 12% echistatin and 58.8 +/- 7% blocking antibody, respectively, P < 0.05). Treatment of passaged HSC with either echistatin or blocking antibody had no effect. Fibronectin, an alpha(5)beta(1)-integrin ligand, increased transgene activity in primary (210 +/- 33%, P < 0.05) but not in passaged HSC cultures (119 +/- 8%). This alpha(5)beta(1)-integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein-beta (C/EBPbeta), because fibronectin increased and alpha(5)-gene silencing by small interfering RNA decreased C/EBPbeta levels. In addition, C/EBPbeta knockout mice showed reduced type I collagen synthesis compared with wild-type littermates. Therefore alpha(5)beta(1)-integrin is an important regulator of type I collagen production in early primary HSC cultures but appears to have no direct role once the HSC are fully activated.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.