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.     

Heparan mimetic regulates collagen expression and TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle cells.

Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-beta1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-beta1 distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a carboxymethylated and sulfated dextran (RG-1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After (60)Co irradiation (10 Gy), HISM were labeled with [(3)H] proline (+/-RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-beta1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85-fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF-beta1 was increased (up to twofold) in irradiated HISM. RG-1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG-1503 decreased the amount of TGF-beta1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF-beta1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.     

Interferon-alpha and interferon-gamma reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture

The effects of interferon-alpha and interferon-gamma on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-gamma treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-gamma reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-alpha on procollagen mRNA levels was somewhat weaker than that of interferon-gamma. The results suggest potential use of interferon-gamma in treatment and prevention of human fibrotic conditions.     

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.     

Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.     

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.     

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.     

Effect of cardiac lymph flow obstruction on cardiac collagen synthesis and interstitial fibrosis

The effect of chronic cardiac lymphatic obstruction on the myocardial synthesis of collagen type I and III was investigated in a rabbit model. In the lymphatic obstruction group (n=16), plasma C-terminal propeptide type I procollagen (PICP) and N-terminal propeptide type III procollagen (PIIINP) were elevated at 7, 14 and 30 days after the operation (p<0.05). The elevated PICP and PIIINP returned to the pre-operation values 60 days after the operation. The myocardial expression of collagen type I and III mRNA were also enhanced in the lymphatic flow obstruction group. Plasma PICP, PIIINP and myocardial collagen type I and III mRNA remained unchanged in the control group (n=16). We concluded that chronic obstruction of cardiac lymph flow leads to enhanced myocardial collagen synthesis in rabbits. The enhanced collagen synthesis starts within seven days after lymphatic obstruction and subsides after 60 days.     

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.