Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.     

Palmitoyl ascorbate: selective augmentation of procollagen mRNA expression compared with L-ascorbate in human intestinal smooth muscle cells.

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.     

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.     

Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.     

Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells

Hsp47 is a heat stress protein that interacts with procollagen in the lumen of the endoplasmic reticulum, which is vital for collagen elaboration and embryonic viability. The precise actions of Hsp47 remain unclear, however. To evaluate the effects of Hsp47 on collagen production we infected human vascular smooth muscle cells (SMCs) with a retrovirus containing Hsp47 cDNA. SMCs overexpressing Hsp47 secreted type I procollagen faster than SMCs transduced with empty vector, yielding a greater accumulation of pro alpha1(I) collagen in the extracellular milieu. Interestingly, the amount of intracellular pro alpha1(I) collagen was also increased. This was associated with an unexpected increase in the rate of pro alpha1(I) collagen chain synthesis and 2.5-fold increase in pro alpha1(I) collagen mRNA expression, without a change in fibronectin expression. This amplification of procollagen expression, synthesis, and secretion by Hsp47 imparted SMCs with an enhanced capacity to elaborate a fibrillar collagen network. The effects of Hsp47 were qualitatively distinct from, and independent of, those of ascorbate and the combination of both factors yielded an even more intricate fibril network. Given the in vitro impact of altered Hsp47 expression on procollagen production, we sought evidence for interindividual variability in Hsp47 expression and identified a common, single nucleotide polymorphism in the Hsp47 gene promoter among African Americans that significantly reduced promoter activity. Together, these findings indicate a novel means by which type I collagen production is regulated by the endoplasmic reticulum constituent, Hsp47, and suggest a potential basis for inherent differences in collagen production within the population.     

3,4-methylenedioxymethamphetamine ("Ecstasy") stimulates the expression of alpha1(I) procollagen mRNA in hepatic stellate cells.

3,4-Methylenedioxymethamphetamine, MDMA ("Ecstasy"), has been previously shown to produce cell necrosis and fibrosis in the liver. Our aim was to study the effect of MDMA on the type I collagen production by a cell line of hepatic stellate cells (HSC), the cell type mainly responsible for collagen synthesis in the liver. We demonstrated that MDMA increases alpha1(I) procollagen mRNA levels and that this increase correlates with glutathione depletion and enhanced hydrogen peroxide production by HSC. Pre-treatment with either glutathione monoethyl ester or deferoxamine prevents the MDMA-induced alpha1(I) procollagen mRNA expression, indicating oxidative stress to be a mediator of this effect. Lipid peroxidation was not detected in MDMA-treated cells and therefore does not seem to be involved in the pro-fibrogenic action of MDMA on HSC.     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

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.     

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.     

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.     

Matrix protein synthesis by glomerular mesangial cells in culture: Effects of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA

The pathogenesis of glomerular scarring is multifactional; recent evidence suggests that transforming growth factor β (TGFβ), a pleiotropic cicatricial mediator, may promote mesangial sclerosis by enhancing the production of extracellular matrix proteins. We studied the effect of TGFβ1 and TFGβ2 on collagen type IV and fibronectin (FN) synthesis in human glomerular mesangial cells in culture (GMC). Two hours after addition of TGFβ, an up to twofold increase in abundance of collagen type IV mRNA was found, which further increased up to fivefold within 24 h. Addition of cycloheximide did not inhibit the TGFβ effect, but caused by itself an up to twofold increase in the abundance of collagen type IV mRNA after 2 h. Together with collagen mRNA, the mRNA for FN and for platelet-derived growth factor (PDGF) was also enhanced. PDGF was found to enhance abundance of the collagen type IV and fibronectin mRNA in GMC. A neutralizing antibody to PDGF or a PDGF-antisense oligonucleotide partly inhibited the TGFβ-induced increase of collagen type IV mRNA, suggesting that TGFβ can affect the collagen type IV synthesis not only directly but also indirectly via the synthesis of PDGF. © 1995 Wiley-Liss, Inc.     

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.     

Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation

Four clonal cell lines subcloned from a clonal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of alpha 1(I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases MspI and HpaII. It was found that the loss in collagen type I expression correlated positively with the degree of methylation of alpha 1(I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen gene regulation.