Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.     

1,25-Dihydroxyvitamin D3 interaction with dexamethasone and retinoic acid: effects on procollagen messenger ribonucleic acid levels in rat osteoblast-like cells

We previously have reported that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], dexamethasone, and retinoic acid inhibit collagen synthesis in rat osteoblast-like cell primary cultures. We also have found that dexamethasone increases 1,25-(OH)2D3 receptor levels in these cells. Furthermore, this increase in 1,25-(OH)2D3 receptor level is paralleled by an enhanced inhibition of collagen synthesis when dexamethasone and 1,25-(OH)2D3 are used in combination. In contrast, retinoic acid at high doses decreases 1,25-(OH)2D3 receptor level in rat osteoblast-like cells and attenuates 1,25-(OH)2D3 inhibition of collagen synthesis. In the present study, we have used a [32P]cDNA probe for rat pro alpha 1 (I) to determine if these osteotropic agents act by modulating steady state procollagen mRNA levels. Hybridization with a [32P]cDNA probe for human actin was used as a control. We find that the steady state levels of procollagen mRNA are decreased in all cases, while there are negligible changes in actin mRNA levels. Dexamethasone, at the low dose of 13 nM, acts synergistically with 1,25-(OH)2D3 in decreasing procollagen mRNA levels. The effects of retinoic acid and 1,25-(OH)2D3 are additive at low doses (13 and 130 nM); however, at a high dose of retinoic acid (1.3 microM), combined treatment with 1,25-(OH)2D3 does not reduce procollagen mRNA levels beyond the decrease due to retinoic acid alone. The reduction in procollagen mRNA level after each of these treatments falls in the same range as inhibition of collagen synthesis measured at the protein level. These data suggest that the synthesis of collagen under these treatments is controlled primarily through modulation of steady state procollagen mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts

Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-alpha decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-alpha diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is down-regulated by TNF-alpha in a concentration- and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-alpha does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts.     

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.     

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-³H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line. © 1995 Wiley-Liss, Inc.     

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.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures

The deposition of insoluble functional collagen occurs following extracellular proteolytic processing of procollagens by procollagen N- and C-proteinases, fibril formation, and lysyl oxidase dependent cross-linking. Procollagen C-proteinases in addition process and activate lysyl oxidase. The present study evaluates a possible role for procollagen C-proteinases in controlling different aspects of collagen deposition in vitro. Studies determine whether inhibition of procollagen C-proteinase activity with a specific BMP-1 inhibitor results in perturbations in lysyl oxidase activation, and in collagen processing, deposition, and cross-linking in phenotypically normal cultured murine MC3T3-E1 cells. Data show that BMP-1 Inhibitor dose dependently inhibits lysyl oxidase activation by up to 50% in undifferentiated proliferating cells. In differentiating cultures, BMP-1 inhibitor decreased collagen processing but did not inhibit the accumulation of mature collagen cross-links. Finally, electron microscopy studies show that collagen fibril diameter increased. Thus, inhibition of procollagen C-proteinases results in perturbed collagen deposition primarily via decreased collagen processing.     

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.     

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.     

Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.     

Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.     

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.