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Biomedical subjects

R Raghow

Publications and source records attributed to R Raghow.

At least 55 records · Page 3Linked to original sources
PubMed1992

Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4.

Interleukin 4 (also known as "B cell stimulatory factor-1"), a cytokine product of T lymphocytes and mast cells, stimulates synthesis of the extracellular matrix proteins, types I and III collagen and fibronectin, by human dermal fibroblasts in vitro. Stimulation of collagen by human recombinant (hr)IL-4 was also demonstrated in several fibroblastic synovial cell lines obtained from patients with rheumatoid arthritis and osteoarthritis. The stimulatory effect of hrIL-4 on fibroblast collagen synthesis was specifically neutralized by rabbit anti-hrIL-4 Ig. IL-4 specifically increased the steady-state levels of types I and III procollagen and fibronectin mRNAs, with no effect on cytoplasmic beta-actin mRNA. Quantitative analysis of the levels of Pro alpha 1(I) collagen transcripts in IL-4-treated fibroblast cultures was also corroborated by antisense RNA-mRNA hybridization and RNAse resistant hybrids which showed that IL-4-treated fibroblasts expressed higher levels of Pro alpha 1(I) collagen transcripts. Nuclear run-off transcription experiments indicated that IL-4 stimulated the rates of mRNA biogenesis. Based on these observations we conclude that IL-4 exerts its effect on collagen and fibronectin synthesis at the pretranslational level, resulting in synthesis of these extracellular matrix proteins. These and other data suggest that IL-4 may be a "fibrogenic cytokine" that could be important in promoting biogenesis of extracellular matrix proteins in normal wound healing and in pathological fibrosis in which mast cells and T lymphocytes play a central role.

Cells, Cultured↗
PubMed1991

Regulation of extracellular matrix production by chemically synthesized subfragments of type I collagen carboxy propeptide.

The complete COOH-propeptide of human alpha 1(I) procollagen was chemically synthesized as a series of overlapping subfragments which were then tested for their effect on extracellular matrix protein production by subconfluent human lung fibroblasts (HFL-1). One peptide (R11; residues 197-241) stimulated production of both collagen and fibronectin by 6-8-fold while a second peptide with a partial overlap with R11 (R9; residues 182-216) enhanced collagen accumulation. The peptide R12 (residues 197-216), which has a sequence common to both R9 and R11, also stimulated collagen production, suggesting that this 20-residues peptide alone contains the required structure for activity. The other synthetic peptides, R1-R13, were inactive in their ability to alter collagen or fibronectin production. Consistent with previously published data, the COOH-terminal peptide, R14, inhibited extracellular matrix production [Aycock, R.A., Raghow, R., Stricklin, G.P., Seyer, J.M., & Kang, A.H. (1986) J. Biol. Chem. 261, 14355-14360]. Both R9 and R11 preferentially stimulated production of collagen types I and III and fibronectin in dose-dependent manner. Elevated collagen and fibronectin production was evident at 4-h posttreatment, and maximal enhancement was seen at 8 h after exposure to peptides. Interestingly, subconfluent cultures of HFL-1 fibroblasts responded vigorously to the stimulatory action of R9 and R11 while confluent cells failed to show any response. Steady-state levels of messenger RNAs encoding type I procollagen and fibronectin were not measurably altered by treatment with R9 or R11, suggesting that the regulation of procollagens and fibronectin by these peptides involves posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Generation of monoclonal antibodies to the zinc finger domain of the eukaryotic transcription factor Sp1.

Using a synthetic peptide that encompasses the zinc finger domain of the eukaryotic transcription factor Sp1, we produced a number of monoclonal antibodies that specifically reacted with the target antigen. Analysis by competitive inhibition assay of five of the monoclonal antibodies revealed that they all recognized a dominant epitope in the synthetic peptide and reacted strongly to recombinantly synthesized beta-galactosidase-Sp1 fusion polypeptide. To determine cellular distribution of Sp1-like molecules, cytoplasmic and nuclear proteins from human lung fibroblasts (HFL-1) and a human rhabdomyosarcoma cell line (A204) were immunoblotted and reacted with our antibodies. In addition to the well characterized 95 Kd and 105 Kd proteins, considered to be the authentic Sp1 polypeptide, a number of other cellular proteins reacted with these antibodies. Immunofluorescence staining of the cells with mAb to the zinc finger of Sp1 also revealed cell-specific differences in intracellular distribution of Sp1-like molecules. Both cytoplasmic and nuclear staining was readily observed in the rhabdomyosarcoma cells. In contrast, while some HFL-1 cells exhibited staining of only cytoplasm, both cytoplasmic and nuclear immunofluorescence was seen in others.

Animals↗
PubMed1991

In vitro methylation of the promoter and enhancer of Pro alpha 1(I) collagen gene leads to its transcriptional inactivation.

We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Pro alpha 1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Pro alpha 1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Pro alpha 1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Pro alpha 1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Pro alpha 1(I) gene. These findings, along with the extreme methylation sensitivity of the Pro alpha 1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.

Base Sequence↗
PubMed1990

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.

Animals↗
PubMed1990

Expression of calmodulin gene is down-regulated in diabetic BB rats.

In past studies, we have demonstrated that in streptozotocin-induced diabetic or spontaneously diabetic (BB) animal models, low Km cAMP phosphodiesterase and calmodulin are decreased while a low MW inhibitor of calmodulin is increased. To extend these studies, we have determined the rate of [35S]-methionine incorporation into calmodulin in isolated fat cells from these diabetic animals, i.e. streptozotocin-induced diabetic and the BB rats, spontaneous diabetic rat, non-diabetic rat, and control. We found markedly decreased rates of synthesis of calmodulin in the fully diabetic BB rat. In order to investigate the mechanism of the reduced calmodulin biosynthesis, we probed poly A+ mRNA from control and diabetic rat livers with a calmodulin specific anti-sense oligonucleotide probe and found that the fully diabetic animals, streptozotocin-induced diabetic and genetically diabetic BB, contained markedly reduced levels of calmodulin transcripts. Thus, both calmodulin protein and its putative mRNA are decreased in diabetic rat liver. We believe that in uncontrolled diabetes, the observed elevation in the levels of cyclic AMP in plasma and tissue results in part from decreased activity of phosphodiesterase. The insulin-sensitive phosphodiesterase appears to be regulated by calmodulin. We hypothesize that cyclic AMP phosphodiesterase inactivation in diabetes results in part from insulin insufficiency and to a less well-defined genetic lesion leading to calmodulin down-regulation.

Actins↗
PubMed1990

Regulation of TGF beta gene expression in rat liver intoxicated with carbon tetrachloride.

Transforming growth factor beta (TGF beta) has been implicated as having a central role in the postinflammatory tissue regeneration and fibrosis. To test its potential involvement in events that follow hepatotoxin-mediated liver regeneration and fibrosis, we quantitated changes in the steady-state levels of TGF beta mRNA in parenchymal and nonparenchymal cells at various times after an acute treatment with CCl4, and also compared TGF beta gene expression in these two cell types from livers subjected to chronic CCl4 treatment. The parenchymal and nonparenchymal cells from normal liver contained undetectable amounts of TGF beta mRNA. In contrast, we could readily detect TGF beta specific transcripts in both the parenchymal and nonparenchymal cells after acute injury. Nonparenchymal cells from acutely injured liver contained fivefold greater amounts of TGF beta mRNA, which peaked at 48 h and declined thereafter. In chronically treated rat livers (1, 2, 3, and 7 wk after the initiation of CCl4 treatment), increased expression of TGF beta mRNA was found only in nonparenchymal cells obtained after 2-3 wk of treatment. Strikingly large elevations in the steady-state levels of beta-actin mRNA in CCl4-treated liver were also observed, which may be related to the known regenerative processes associated with acute liver toxicity. Changing dynamics of TGF beta gene expression, therefore, appear to be an important attribute of regenerating liver after acute or chronic CCl4 toxicity.

Actins↗
PubMed1990

Extracellular matrix phenotype of rat mesangial cells in culture. Biosynthesis of collagen types I, III, IV, and V and a low molecular weight collagenous component and their regulation by dexamethasone.

Rat glomerular cells were cultured in vitro to evaluate their collagen phenotype. The enriched epithelial cell population that grew out from glomeruli in the initial stages of culture primarily synthesized type IV collagen. By 14 days, mesangial cell proliferation occurred with a coordinate change in collagen phenotype, after which time these cells mainly synthesized type I collagen and lesser amounts of types III, IV, and V collagens. In addition to the standard collagens, mesangial cells synthesized and accumulated in their extracellular matrix a small molecular weight collagenous protein that accounted for 16.6% of the total radioactivity incorporated into extracellular matrix proteins. This short-chain collagen, with an approximate molecular mass of 70,000 (70 kd), appears to be a unique product of mesangial cells. Collagen biosynthesis by the mesangial cells was down-regulated by dexamethasone in a time- and dose-dependent manner. Treatment with 10(-6) mol/L dexamethasone reduced synthesis of type I and type IV collagens by about 37% and 24%, respectively. Steady-state levels of the corresponding procollagen messenger RNAs were diminished in dexamethasone-treated mesangial cells. This effect of glucocorticoid was found to be specific because dexamethasone did not affect biosynthesis and secretion of noncollagenous proteins.

Animals↗
PubMed1989

Molecular mechanisms of collagen gene expression.

Collagens are a structurally and functionally heterogenous group of proteins encoded by a family of genes that share evolutionary history. Collagen gene expression is regulated both in developmental, tissue-specific manners as well as in response to a variety of biologic and pharmacologic inducers. In the present review we have attempted to synthesize a conceptual overview of the available information from studies aimed at deciphering the molecular mechanisms of collagen gene expression. We have chosen to focus our discussion mainly, although not exclusively, to observations relating to type I collagen gene for a number of practical reasons. The underlying theme that emerges from this survey of the literature is that the regulation of collagen gene expression is complex, utilizing transcriptional, posttranscriptional and translational mechanisms. Although the transcriptional control mechanisms that involve activation and modulation of collagen gene transcription by RNA polymerase II appear to predominate, preferential stabilization of collagen mRNAs and modulation of translational discrimination appear to play significant roles in the regulation of collagen biosynthesis under some physiological situations. Molecular organization of the regulatory regions of collagen genes reveal a mosaic of subdomains with overlapping sequence motifs, involved in positive and negative transcriptional regulation. The precise identity of the cis-acting subdomains of the promoter/enhancer-proximal DNA of collagen gene and how they interact with the trans-acting nuclear protein(s) have yet to be elucidated and will remain the focus of future studies.

Chromatin↗
PubMed1988

Regulation of mRNAs for type-1 plasminogen activator inhibitor, fibronectin, and type I procollagen by transforming growth factor-beta. Divergent responses in lung fibroblasts and carcinoma cells.

Transforming growth factor-beta (TGF beta) is a growth modulator which stimulates the growth of fibroblasts but acts as a strong growth inhibitor for cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins and of proteases and protease inhibitors by cultured cells. One important protease inhibitor whose production is affected rapidly by TGF beta is the type-1 plasminogen activator inhibitor (PAI-1). To investigate the relationships between PAI-1 and the extracellular matrix, we analyzed the regulation by TGF beta of PAI-1, fibronectin, and type I procollagen in malignant human lung carcinoma cells (A549) and in human lung fibroblasts (WI-38). The expression of the respective genes was examined by polypeptide analyses and by measurements of the steady-state levels of the corresponding mRNAs by Northern hybridization. The mRNA levels for PAI-1 were elevated rapidly by TGF beta in both cell lines. This induction occurred in the presence of cycloheximide and thus was not dependent on protein synthesis. In fact, the effects of TGF beta and cycloheximide on PAI-1 mRNA were additive. In contrast, the induction of fibronectin, beta-actin, and type I procollagen (synthesized only in WI-38 cells) was abrogated by cycloheximide. In general, the effects of TGF beta on the steady-state levels of mRNAs for fibronectin, actin, and type I procollagen mRNA were similar in the two cell types. However, the production of PAI-1 mRNA in response to TGF beta differed in the two cell types, being transient (peak within 5 h) in the carcinoma cells and more persistent in fibroblasts. Thus, the major difference in TGF beta regulation of PAI-1 mRNA between lung fibroblasts and carcinoma cells was the duration of the effect. These results, together with previous data, suggest that PAI-1 and plasminogen activators may be regulated oppositely by TGF beta. We therefore propose a model for TGF beta in the regulation of extracellular proteolytic activity.

Actins↗
PubMed1988

Interaction of fibronectin with DNA/anti-DNA complexes from systemic lupus erythematosus: role of activated complement Cl in modulation of the interactions.

Fibronectin (Fn) is an integral constituent of the endothelial cell surface and the basement membrane. The mechanism for binding DNA/anti-DNA complexes to Fn was examined in a solid-phase assay. In physiological buffer, a low-affinity binding of DNA was observed with Fn and optimal binding was seen at pH 6.5 and in the absence of Ca2+. Further, the interaction of DNA to Fn was inhibited when DNA was complexed to anti-DNA antibody. However, complement Clq mediated the binding of complexes to Fn at pH 7.4 and it was proportional to the extent of the dissociation of Cl. Cl inactivator (Cl-In) appeared to play a modulating role; whereas at low concentrations (Cl:Cl-In::4: less than 1) it enhanced the binding of complexes to Fn, higher concentrations inhibited the binding. Further, sera from patients with active systemic lupus erythematosus reacted with Fn, which was shown to be dependent on the presence of Clq and was minimally affected by DNase treatment of sera, indicating a relatively minor role of DNA in the direct binding of DNA to Fn. These findings support "circulating immune complex" hypothesis in the pathogenesis of lupus glomerular immune complex deposition disease.

Antibodies, Antinuclear↗
PubMed1988

Modulation of fibroblast functions by interleukin 1: increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta.

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.

Cell Division↗
PubMed1987

Phenotypic plasticity of extracellular matrix gene expression in cultured hamster lung fibroblasts. Regulation of type I procollagen and fibronectin synthesis.

Enhanced synthesis and accumulation of interstitial collagens in the lungs of bleomycin-treated animals have been ascribed to drug-induced alterations in the cellular phenotype as judged by preferentially elevated rates of transcription of collagen and fibronectin genes (Raghow, R., Lurie, S., Seyer, J. M., and Kang, A. H. (1985) J. Clin. Invest. 76, 1733-1739). To explore the biosynthetic contribution of mesenchymal cells to the fibrotic process we cultured hamster lung fibroblasts from animals at 0, 1, 2, and 4 weeks after bleomycin treatment and compared their ability to synthesize type I collagen and fibronectin. Our analyses involved (i) measurements of the rates of procollagen and fibronectin synthesis in relation to the total protein synthesis, (ii) quantitative analyses of the steady-state levels of mRNAs coding for type I procollagen chains and fibronectin by Northern hybridization, and finally (iii) a semiquantitative comparison of the steady-state levels of type I procollagen transcripts by in situ hybridization to recombinant cDNA probes. We show that in the earliest stages of the growth in vitro, the patterns of type I procollagen and fibronectin biosynthesis by cells in primary culture closely corresponded with those found in the intact lung. With increasing duration in culture, the distinctive phenotypes of cells from control as well as fibrotic lungs changed steadily; the biosynthetic profiles of fibroblasts cultured for 10 days from untreated or bleomycin-treated lungs were nearly identical. Based on these data, we conclude that the biosynthetic phenotype of fibroblasts, as judged by the parameters of collagen and fibronectin synthesis, appears to be reversible. The characteristic biosynthetic phenotype of fibroblasts, in the interactive milieu of the intact lung (both control and bleomycin-treated), is maintained by cellular and/or humoral factor(s) which are removed or inactivated by the procedures used for isolation and in vitro culture of fibroblasts.

Animals↗
PubMed1987

A hepatic fibrogenic factor stimulates the synthesis of types I, III, and V procollagens in cultured cells.

A hepatic fibrogenic factor (HFF) isolated from fibrotic rat livers has previously been shown to stimulate the transcription of type I procollagen genes in cultured fibroblasts (Raghow, R., Gossage, D., Seyer, J. M., and Kang, A.H. (1984) J. Biol. Chem. 259, 12718-12723). To test if the expression of other collagen genes was similarly affected by the fibrogenic factor, we measured the rates of types I, III, and V procollagen synthesis in two different cell lines after treatment with HFF. The effect of fibrogenic factor on types I and III procollagens was tested in rat fibroblasts, while a human rhabdomyosarcoma cell line was used to evaluate the effect of HFF on type V procollagen synthesis. Incubation with rat fibroblasts resulted in a 3-4-fold stimulation of the synthesis of both types I and III procollagens in a time-dependent manner. The stimulated rates of types I and III procollagen synthesis accompanied an increase in the steady-state levels of their corresponding mRNAs. When A204 cells, which are derived from a rhabdomyosarcoma and exclusively synthesize type V procollagen, were incubated with the fibrogenic factor, a 3-4-fold stimulation of the synthesis of both pro-alpha 1(V) and pro-alpha 2(V) chains was seen. Using a cDNA probe for pro-alpha 2(V), we also observed that there was a 2-3-fold increase in the steady-state level of pro-alpha 2(V) mRNA in A204 cells after treatment with the fibrogenic factor. In both rat fibroblasts and A204 cells the steady-state levels of beta-actin mRNA were minimally affected by fibrogenic factor, suggesting that the procollagen genes were preferentially affected. Since types I, III, and V collagens are present in the normal liver and accumulate aberrantly in the fibrotic liver, we suggest that fibrogenic factor may play an important role in determining the altered collagen composition of the fibrotic liver. Based on these data, we also speculate that the regulation of the biosynthesis of a variety of procollagens in diverse cell types by HFF possibly occurs by a common mechanism.

Animals↗
PubMed1987

Fibroblast stimulation in schistosomiasis. VII. Egg granulomas secrete factors that stimulate collagen and fibronectin synthesis.

Tissue fibrosis in schistosomiasis is largely responsible for the important morbidity that results from infection with the trematode worms, Schistosoma. Neither the migrating larval forms (cercariae) nor the intravascular adult worms appear to incite pathological responses that are important in chronic schistosomiasis. On the other hand, eggs deposited in tissue incite chronic granulomatous inflammatory responses that are the hallmark of infection and precede the onset of adjacent tissue fibrosis. We previously reported that products of the egg granulomas can stimulate a number of relevant responses in fibroblast cultures that in vivo would be expected to promote tissue fibrosis. We report here that the granulomas secrete factors that in vitro can stimulate collagen and fibronectin synthesis in fibroblasts. We determined that activity stimulating collagen synthesis is congruent to 10 Kd (gel filtration) with a pI of congruent to 5.5 (isoelectric focusing); additional activity is also detected in some other fractions (congruent to Kd; pI approximately 7.0). In contrast, the activity stimulating fibronectin synthesis was congruent to 22 Kd with a pI of 5.5. Activity was also present in fractions of 50 Kd with pI of approximately 7.5. Fibroblasts grown in granuloma supernatant-containing medium contained greater steady-state levels of specific mRNA coding for type I procollagen and fibronectin compared with cells cultured in unsupplemented medium. These observations support the hypothesis that biologically active molecules secreted by granuloma cells are instrumental in the initiation of tissue fibrosis in schistosomiasis.

Animals↗
PubMed1987

Transforming growth factor-beta increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts.

Transforming growth factor-beta (TGF beta), when injected subcutaneously into newborn mice, induces a rapid fibrotic response, stimulates chemotaxis, and elevates the rates of biosynthesis of collagen and fibronectin by fibroblasts in vitro. We explored the molecular mechanisms of TGF beta-mediated stimulation of collagen and fibronectin synthesis in cultured human foreskin fibroblasts. TGF beta preferentially stimulated the synthesis of fibronectin and type I procollagen chains 3-5-fold as shown by polypeptide analysis. Concomitant elevation in the steady state levels of messenger RNAs (mRNAs) coding for type I procollagen and fibronectin also occurred but without a net increase in the rate of transcription of either of these genes. The preferential stabilization of mRNAs specifying type I procollagen and fibronectin provides a partial explanation for the mechanisms by which TGF beta enhances the synthesis of type I procollagen and fibronectin in mesenchymal cells.

Collagen↗
PubMed1986

Post-transcriptional inhibition of collagen and fibronectin synthesis by a synthetic homolog of a portion of the carboxyl-terminal propeptide of human type I collagen.

We evaluated the effects of a synthetic copy of a highly conserved portion (residues 225-246) of the COOH-propeptide of human pro-alpha 2(I) procollagen on collagen, fibronectin, and total protein synthesis by human fibroblasts. Incubation of COOH-propeptide 225-246 with fibroblasts resulted in a concentration-dependent inhibition of both type I procollagen and fibronectin when compared with controls; a 50% inhibition of both fibronectin and type I collagen was observed at a concentration of 45 microM. Since the overall cellular protein synthesis was only minimally affected, COOH-propeptide appeared to specifically inhibit collagen and fibronectin synthesis. The peptide was nontoxic to cells and the inhibition was completely reversible upon removal of the peptide. We measured the steady-state levels of mRNAs coding for procollagen, fibronectin, and beta-actin by hybridization to specific recombinant cDNA probes; there was no significant change in the steady-state level of mRNAs of the three proteins. These results strongly suggest that the biosynthesis of procollagen and fibronectin in COOH-propeptide-treated cells is inhibited at a post-transcriptional level. These data establish a link between collagen and fibronectin synthesis and further define the important interaction of these molecules in the formation of the extracellular matrix.

Amino Acid Sequence↗