Procollagen mRNA Levels Research Articles

Surface adhesion-mediated regulation of chondrocyte-specific gene expression in the nontransformed RCJ 3.1C5.18 rat chondrocyte cell line.

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.

Alcohol and iron: a radical combination?

HepatologyVolume 23, Issue 6 p. 1700-1703 Hepatology ElsewhereFree Access Alcohol and iron: A radical combination? K Houglum, K HouglumSearch for more papers by this author K Houglum, K HouglumSearch for more papers by this author First published: June 1996 https://doi.org/10.1002/hep.510230656Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Houglum K, Filip M, Witztum JL, Chojkier M. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest 1990; 86: 1991– 1998. 2 Pierce RA, Glaug MR, Greco RS, Mackenzie JW, Boyd CD, Deak SB. Increased procollagen mRNA levels in CCl4-induced liver fibrosis in rats. J Biol Chem 1987; 262: 1652– 1658. 3 Chojkier M, Houglum K, Solis-Herruzo J, Brenner DA. Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation? J Biol Chem 1989; 264: 16957– 16962. 4 Parola M, Pinzani M, Casini A, Albano E, Poll G, Gentilini A, Gentilini P, et al. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Com-mun 1993; 194: 1044– 1050. 5 Lee KS, Buck M, Houglum K, Chojkier M. Activation of hepatic stellate cells by TGFα and collagen type I is mediated by oxida-tive stress through c-myb expression. J Clin Invest 1995; 96: 2461– 2468. 6 Casini A, Ceni E, Salzano R, Schuppan D, Milani S, Pellegrini G, Surrenti C. Regulation of undulin synthesis and gene expression in human fat-storing cells by acetaldehyde and transforming growth factor-β 1: comparison with fibronectin. Biochem Biophys Res Commun 1994; 199: 1019– 1026. 7 Tuma DJ, Thiele GM, Xu DS, Klassen LW, Sorrell MF. Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during chronic ethanol administration [Abstract]. Hepatology 1995; 22: 226A. 8 Irving MG, Halliday JW, Powell LW. Association between alcoholism and increased hepatic iron stores. Alcohol Clin Exp Res 1988; 12: 7– 13. 9 Houglum K, Bedossa P, Chojkier M. TGF β and collagen α(I) gene expression are increased in hepatic acinar zone 1 of rats with iron overload. Am J Physiol 1994; 267: G908– G913. 10 Pietrangelo A, Gualdi R, Giovanna C, Montosi G, Ventura E. Molecular and cellular aspects of iron-induced hepatic cirrhosis in rodents. J Clin Invest 1995; 95: 1824– 1831. 11 Nakatsukasa H, Evarts RP, Hsia C, Thorgeirsson SS. Transforming growth factor-β1 and type I procollagen transcripts during regeneration and early fibrosis of rat liver. Lab Invest 1990; 63: 171– 180. 12 Sadrzadeh SM, Nanji AA, Price PL. The oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one reduces hepatic-free iron, lipid peroxidation and fat accumulation in chronically ethanol-fed rats. J Pharmacol Exp Ther 1994; 269: 232– 236. 13 Kadiiska MB, MJ Burkitt, Q-H Ziang, Mason RP. Iron supplementation generates hydroxy radicals in vivo, and ESR spin-trapping investigation. J Clin Invest 1995; 96: 1824– 1831. Citing Literature Volume23, Issue6June 1996Pages 1700-1703 ReferencesRelatedInformation

Regulation of Collagen Gene Expression by Prostaglandins and Interleukin-1beta in Cultured Chondrocytes and Fibroblasts.

To compare the modulatory effects of different prostaglandins on collagen gene expression in human chondrocytes, PGE(2), PGE(1), misoprostol (PGE(1) analog), and PGF(2alpha) (10, 50 and 100 ng ml(minus sign1)) were added to human chondrocytes with or without interleukin-1beta (IL-1beta) in the presence of indomethacin to inhibit endogenous prostaglandin synthesis and the effects evaluated on chondrocyte morphology, collagen synthesis, and procollagen mRNA levels. The effects of prostaglandins on the expression of collagen gene regulatory sequences were examined using transient transfection assays of reporter gene constructs in human chondrocytes and BALB/c3T3 fibroblasts, PGE(1), misoprostol, and PGF(2alpha), similar to PGE(2), inhibited type I collagen gene expression in fibroblasts and promoted type II collagen gene expression in chondrocytes. PGE(2), the major inflammatory prostaglandin produced by IL-1-activated chondrocytes and fibroblasts, and PGF(2alpha) were somewhat more potent than the anti-inflammatory prostaglandins PGE(1) and misoprostol in counteracting the IL-1-induced suppression of type II collagen gene expression by chondrocytes and stimulation of type I collagen gene expression by fibroblasts. Rather than promoting degradation of the cartilage matrix in joint diseases, prostaglandins may be somewhat protective, suppressing fibrosis, and maintaining or promoting appropriate cartilage repair.

Enhanced collagen synthesis and transcription by peak E, a contaminant of L-tryptophan preparations associated with the eosinophilia myalgia syndrome epidemic.

The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.

Collagen Biosynthesis in Human Oral Submucous Fibrosis Fibroblast Cultures

To investigate the mechanism of collagen accumulation in oral submucous fibrosis (OSF) tissues, we examined the biosynthesis of collagen in fibroblast cultures established from OSF lesions. Fibroblasts obtained from four of ten OSF specimens showed more than a 1.5-fold increase in the production of collagens compared with fibroblasts from age-, sex-, and passage-matched normal controls (p < 0.05). When the relative amounts of collagen synthesis were estimated by SDS polyacrylamide gel electrophoresis, it was found that both OSF and control cells produced about 85% type I collagen and 15% type III collagen. The ratio of alpha 1(I) to alpha 2(I) chains was about 3:1 in OSF cells instead of the 2:1 expected for type I collagen. The excess alpha 1(I) chains could mean that collagen type I trimer was synthesized by the fibroblasts. These findings suggest that collagen overproduction and a reduced degradation of the structure-stable collagen type I trimer synthesized by OSF fibroblasts might contribute to the accumulation of collagen in OSF lesions in vivo. The mechanism(s) of increased procollagen production were analyzed by Northern blot, slot blot, and Southern blot. The OSF fibroblast strains with elevated collagen production also contained higher-than-normal levels of procollagen mRNA, and the ratios of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs were compatible with the results of corresponding procollagen alpha chains. The gene copy number of pro alpha 2(I) collagen gene in OSF fibroblasts was about 1.05. No gene amplification was found. These results indicate that expression of these procollagen genes in cultured fibroblasts is regulated at the transcriptional level.

Misoprostol Partially Inhibits the Renal Scarring of Chronic Cyclosporine Nephrotoxicity.

Chronic cyclosporine (CY) nephrotoxicity is a well-known complication of this immunosuppressive agent, which may, in part, be attributed to abnormalities in renal prostaglandin content. We utilized misoprostol (M), a prostaglandin E(1) analog, in a rat model of chronic CY toxicity at 7 and 28 days to determine effectiveness in prevention of the renal damage. After 7 days, there were no differences in weight change, creatinine clearance, or renal scarring in rats treated with vehicle (V), CY, or CY + M; however, renal procollagen alpha 1(I) mRNA levels were increased in CY versus V rats (p < 0.05). In contrast, after 28 days CY rats had significant reductions in weight gain, glomerular filtration rate, and renal blood flow with increases in renal scarring and procollagen alpha 1 (IV) mRNA levels (all p < 0.05 versus V). Addition of M resulted in partial but significant improvement in GFR, RBF, and procollagen alpha 1(IV) mRNA levels, with lessening of the renal scarring. Skin fibroblasts also were incubated with CY and M to assess impact on procollagen MRNA levels. CY augmented fibroblast procollagen mRNA levels which enhanced by a large dose of M, but inhibited with a moderate dose. These data suggest that M improves renal scarring induced by CY by hemodynamic and/or direct effects.

Human Dermal Fibroblast Clones Derived from Single Cells Are Heterogeneous in the Production of mRNAs for α1(I) Procollagen and Transforming Growth Factor-β 1

Fibroblast clonal heterogeneity has been reported for growth and protein synthesis, but quantitative studies of synthetic phenotype at the pretranslational level have been limited because of difficulty in reliably growing large numbers of clonal cells. We have recently shown a unique stimulatory activity of low oxygen tension in the early phases of clonal growth, which can be used to establish clonal fibroblast cultures suitable for Northern analysis. Using this methodology, we have measured mRNA levels of alpha 1(I) procollagen and transforming growth factor-beta 1 (TGF-beta) both at baseline and after TGF-beta stimulation in a total of 43 clones derived from single cells and from seven different cell strains. We report a remarkable baseline heterogeneity, commonly four- to six-fold, in procollagen mRNA levels among clones and between clones and their parent cultures. Conversely, differences in baseline TGF-beta mRNA levels among clones were either not present or less than onefold. The clonal phenotypic expression of alpha 1(I) procollagen mRNA remained stable after eight additional cell passages. TGF-beta stimulation of itself (autoinduction) was highly variable among clones (range of increases 30% to 150%), and up-regulation of procollagen mRNA levels after TGF-beta stimulation was detected in only 15 (54%) of 28 clonal cultures (range of increases 30% to 353%). A notable lack of correlation was found between baseline mRNA levels of TGF-beta and alpha 1(I) procollagen in clonal cultures. In conclusion, fibroblast clonal populations are remarkably heterogeneous in their baseline procollagen mRNA levels and in their response to TGF-beta.

Autogenous intrasynovial and extrasynovial tendon grafts: an experimental study of pro alpha 1(I) collagen mRNA expression in dogs.

On the basis of recent evidence that the healing processes of tendon grafts are donor-tissue specific, in situ hybridization, using a 372 bp cDNA fragment complementary to a portion of pro alpha 1(I) collagen mRNA, was utilized to compare the cellular responses to transplantation exhibited by autogenous intrasynovial and extrasynovial flexor tendon grafts. Intrasynovial and extrasynovial tendons from the hindpaw were transferred to synovial sheaths in the forepaw of 12 mongrel dogs (24 tendons) and treated with immediate controlled passive motion. The tendon grafts were harvested at 2, 4, and 6 weeks, and each was divided into a proximal, central (8 mm), and distal portion. Sections from the central portion were embedded in paraffin and subjected to in situ hybridization, autoradiography, and staining; levels of procollagen mRNA then were assessed by microscopic examination. The two types of tendon grafts exhibited different levels of pro alpha 1(I) collagen mRNA expression at all three time points. Intrasynovial tendon grafts displayed no areas of increased type-I procollagen mRNA at 2, 4, and 6 weeks. The extrasynovial tendon grafts displayed increased surface levels of type-I procollagen mRNA at 2 and 4 weeks; the levels decreased to background levels by 6 weeks. The high levels of procollagen mRNA exhibited by the extrasynovial grafts suggest increased collagen synthetic activity, indicative of a cellular response to injury, whereas the preservation of low levels of expression in the intrasynovial grafts may signify a less inflammatory cellular response.

Regulation of Collagen Synthesis and mRNA Expression in Normal and Hypertrophic Scar Fibroblasts in Vitro by Interferon-γ

Development of hypertrophic scarring (HTS) is a major problem for patients who survive extensive thermal injuries. HTS and other fibroproliferative disorders are associated with excessive accumulation of collagen and other extracellular matrix proteins. Recent in vitro and in vivo studies have demonstrated that proteins of the interferon family have an inhibitory effect on collagen production by fibroblasts in some fibroproliferative disorders. This study investigated the effects of interferon-γ (IFN-γ) on cell proliferation, collagen production, and expression of types I and III procollagen mRNA in human postburn HTS fibroblasts. Paired fibroblast cultures were established from explants of biopsies obtained from HTS and normal skin (matched for location and skin tension) in five patients recovering from thermal injuries. Thus, normal dermis from each patient was used as a paired control. Administration of IFN-γ (1000 U/ml) to proliferating fibroblast cultures for 5 days resulted in 51% reduction ( P < 0.05) in HTS cell proliferation. Using hydroxyproline as an index for collagen production, a 34% reduction ( P < 0.05) in collagen synthesis was observed in HTS fibroblast culture media after treatment with IFN-γ (1000 u/ml) for 48 hr. Northern blot analysis demonstrated 55 and 36% reductions ( P < 0.05 for each) in type I and type III procollagen mRNA levels, respectively, after treatment for 12 hr with IFN-γ (1000 u/ml). The effect of IFN-γ on each of these parameters was at least as pronounced in HTS fibroblasts as their normal controls. These results suggest that IFN-γ has a down-regulatory effect on cell proliferation and collagen production in fibroblasts from patients with HTS, a condition resulting from excess collagen synthesis and deposition within the extracellular matrix. Improved understanding of such regulatory mechanisms may eventually be of therapeutic significance in the control of HTS.

Pentoxifylline, Pentifylline, and Interferons Decrease Type I and III Procollagen mRNA Levels in Dermal Fibroblasts: Evidence for Mediation by Nuclear Factor 1 Down-Regulation

Pentoxifylline (PTX) is a methylxanthine that exhibits multiple biologic activities, including the inhibition of collagen synthesis by dermal fibroblasts. Because some PTX activities have recently been linked to transcription factor-mediated regulation of gene transcription, we have investigated if PTX acts to inhibit collagen synthesis at a transcriptional locus by measuring procollagen mRNA levels by assaying for the presence of an activator of procollagen gene promoters, nuclear factor (NF)-1. The effects of another methylxanthine, pentifylline (PTF), shown herein to be a tenfold more potent inhibitor of collagen synthesis than PTX, and interferon-alpha, -beta, and -gamma were studied in parallel. Analysis of extracellular protein and RNA from 48-h-treated fibroblasts showed that PTX, PTF, and interferons decreased alpha 1(I), alpha 2(I), and alpha 1(III) procollagens by reducing the steady-state levels of the corresponding procollagen mRNA transcripts. Reduction of procollagen mRNA levels appeared to be dependent on new protein synthesis, as it was prevented by treatment with cycloheximide. Assay for the presence of nuclear NF-1 by gel mobility shift analysis showed that extracts from interferon, PTX, and PTF-treated fibroblasts lacked proteins recognizing the consensus DNA binding sequence for NF-1. Taken together, these observations suggest interferons and methylxanthines may inhibit fibroblast collagen synthesis by a common mechanism requiring new protein synthesis that suppresses procollagen gene transcription through down-regulation of NF-1.

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B.

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin.

Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line pyla: Structure-activity relations and signal transduction mechanisms

We previously showed that prostaglandin E2 (PGE2) can selectively inhibit collagen synthesis and gene transcription in the immortalized rat osteoblastic clonal cell line Py1a, particularly in the presence of insulin-like growth factor I (IGF-I). In the present study, we examined the structure-activity relations for this effect. PGF2 alpha was approximately 100 times more potent than PGE2. The prostaglandin F receptor (FP) selective agonist, fluprostenol, was the most potent agonist tested, significantly inhibiting incorporation of [3H]proline into both collagen and noncollagen protein at 10(-11) M, with more than 90% inhibition of collagen synthesis at 10(-8) M. The PGE2 analog, sulprostone, and PGD2 showed activity similar to that of PGE2. PGI2 and its stable analog, carbacyclin, were the least effective. Parathyroid hormone (PTH), forskolin, and isobutylmethylxanthine (IBMX) were ineffective. Phorbol myristate acetate (PMA) inhibited collagen synthesis in a manner similar to that of the prostanoids. The inhibitory effects of PGF2 alpha, fluprostenol, and PMA show a similar time course on alpha 1(I) procollagen mRNA levels. The inhibition appeared to be caused by a decrease in collagen gene transcription as measured by nuclear run-on analysis. Further evidence for a transcriptional effect was obtained with COL1A1 promoter-CAT reporter constructs, although these showed somewhat smaller effects of prostanoids on CAT activity than on mRNA levels or labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonsynchronous changes in myocardial collagen mRNA and protein during aging: effect of DOCA-salt hypertension

Myocardial fibrosis has been investigated in 3-, 16-, and 24-mo-old normal rats and also in 24-mo-old rats subjected to deoxycorticosterone acetate (DOCA)-salt treatment-induced-hypertension. Collagen content was assessed both histologically and by hydroxyproline assay. Type I and III procollagen mRNA levels were quantitated by Slot Blot analyses. Aging is associated with fibrosis as shown both biochemically (hydroxyproline concentration in 3-, 16-, and 24-mo-old rats was 0.70 +/- 0.05, 0.92 +/- 0.07, and 1.57 +/- 0.13 mg/g of left ventricle, respectively, P < 0.05 and P < 0.0001 vs. 3 mo) and histologically. By contrast, type I procollagen mRNA levels decreased during aging (from -63%, P < 0.001 in 16-mo-old rats and -51%, P < 0.01 in 24-mo-old rats vs. 3-mo-old rats) as well as type III procollagen mRNA levels. DOCA-salt treatment in 24-mo-old rats had no effect on either the degree of fibrosis or the mRNA levels. We conclude that nonsynchronous changes in myocardial collagen mRNA and protein occur during aging, indicating translational and/or posttranslational mechanisms in collagen regulation. Hypertension during senescence did not modify collagen deposition at either the protein or mRNA levels.

Age-related increase in collagen production in cultured human osteoblast-like periosteal cells

Since collagen synthesis is a primary function of differentiated osteoblasts, we investigated the age-related changes in hydroxyproline content, procollagen mRNA level, collagenase activity and collagenase inhibitory activity in human osteoblast-like periosteal cell (OP cell) cultures established from donors of various ages. After achieving confluent growth, the OP cells were treated with 10 −8 M 1,25(OH) 2 vitamin D 3 in the presence of 2 mM α-glycerophosphate for 10 days to facilitate osteoblast differentiation. Hydroxyproline content of the treated cells increased with donor age. But pro α 1(I) collagen mRNA level increased until a donor age of 65, and decreased thereafter with increasing donor age. Collagenase activity was not detected in cell extracts or the conditioned medium concentrated more than 10 times. However, OP cells had collagenase inhibitory activity which was five times higher in cells from aged donors than in cells from young donors. It is possible that cells from elderly donors accumulated much more collagen than cells from younger donors by inhibiting collagen degradation.

Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts

Dermal fibroblasts exposed to low oxygen tension show upregulated synthesis of transforming growth factor-beta 1 (TGF-beta 1), an established stimulatory peptide in the formation of extracellular matrix proteins. In this report, procollagen synthesis was measured in cultures of confluent adult human dermal fibroblasts exposed to either standard (20%) or low (2%) oxygen tension. By Northern blot analysis the steady state levels of alpha 1 (I) procollagen mRNA were increased by 75 to 150% of control (standard oxygen) as early as 12 hours and more than 200% 96 hours after exposure of cells to low oxygen. Similar increases in procollagen mRNA levels were obtained in hypoxic fibroblast cultures in a collagen lattice. The stimulatory effect of hypoxia on procollagen mRNA levels in fibroblast monolayers was diminished by antibodies to TGF-beta, and could not be augmented further by the addition of TGF-beta 1, evidence that hypoxic fibroblasts may already be maximally stimulated by TGF-beta 1. We conclude that low oxygen tension enhances steady state mRNA levels of alpha 1 (I) procollagen, and that this effect is mediated at least in part by TGF-beta 1.

Regulation of Collagen Gene Expression in Keloids and Hypertrophic Scars

The synthesis of type I and III collagens in cultured skin fibroblasts from normal skin, normal scar, hypertrophic scar, and keloids was examined. The ratio of type I/III collagen was significantly elevated in keloids compared to that in the other groups. When mRNA steady-state levels coding for α1(I) procollagen were determined, it was apparent that this increase in the type I/III collagen ratio in keloids was paralleled by a specific increase in α1(I) procollagen mRNA. This specific increase in α1(I) procollagen mRNA in keloids was the result of increased gene expression because the transcription rate of the α1(I) procollagen gene was significantly elevated in keloids, as determined by nuclear runoff transcription. The rate of transcription of the α(I) procollagen gene was also elevated in hypertrophic scars, although no concomitant increase in α1(I) procollagen mRNA levels or alteration in the type I/III collagen ratio was observed. These data indicate that the rate of gene transcription of α1(I) procollagen is increased in both hypertrophic scars and keloids, but only keloids exhibit increased steady-state levels of α1(I) procollagen mRNA and concurrent increases in type I collagen. These results suggest that at least two distinct mechanisms, one pretranscriptional and one post-transcriptional, regulate type I collagen synthesis. It is possible, therefore, that in keloids, neither mechanism functions efficiently to down-regulate type I collagen. In hypertrophic scars, however, the post-transcriptional mechanisms are able to decrease elevated levels of mRNA coding for α1 (I) procollagen that result from increased transcriptional of the α1(I) procollagen gene.

Regulation of osteoblastic gene expression by lead.

Although it is well recognized that lead accumulates in bone, skeletal tissue is considered primarily a sequestering compartment and not a site of toxic action for lead. However, exposure to lead is associated with impaired skeletal growth in children and reductions in indices of bone formation in laboratory animals. Osteoblastic ROS 17/2.8 cells were used in an effort to better understand the consequences of lead exposure on skeletal homeostasis. Studies on confluent cultures of ROS 17/2.8 cells revealed that lead (2-200 microM) had no effect on cell number or DNA and protein synthesis. However, alkaline phosphatase activity was reduced by lead in a dose- and time-dependent manner. Reductions in steady state alkaline phosphatase mRNA levels paralleled the lead-induced inhibition of enzyme activity. Moreover, lead exposure resulted in similar dose-dependent reductions in steady state type 1 procollagen and bone Gla protein mRNA levels. The effect of lead on osteoblastic gene expression in ROS 17/2.8 cultures, however, was selective in nature, as similar lead exposures resulted in no alterations in beta-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA levels. These data demonstrate that lead, in the absence of over toxicity, specifically restricts the expression of certain aspects of the differentiated osteoblast phenotype. Such alterations in osteoblast function may contribute to the skeletal abnormalities observed in settings of lead intoxication.

Regulation of osteoblastic gene expression by lead

Although it is well recognized that lead accumulates in bone, skeletal tissue is considered primarily a sequestering compartment and not a site of toxic action for lead. However, exposure to lead is associated with impaired skeletal growth in children and reductions in indices of bone formation in laboratory animals. Osteoblastic ROS 17/2.8 cells were used in an effort to better understand the consequences of lead exposure on skeletal homeostasis. Studies on confluent cultures of ROS 17/2.8 cells revealed that lead (2-200 microM) had no effect on cell number or DNA and protein synthesis. However, alkaline phosphatase activity was reduced by lead in a dose- and time-dependent manner. Reductions in steady state alkaline phosphatase mRNA levels paralleled the lead-induced inhibition of enzyme activity. Moreover, lead exposure resulted in similar dose-dependent reductions in steady state type 1 procollagen and bone Gla protein mRNA levels. The effect of lead on osteoblastic gene expression in ROS 17/2.8 cultures, however, was selective in nature, as similar lead exposures resulted in no alterations in beta-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA levels. These data demonstrate that lead, in the absence of over toxicity, specifically restricts the expression of certain aspects of the differentiated osteoblast phenotype. Such alterations in osteoblast function may contribute to the skeletal abnormalities observed in settings of lead intoxication.

Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures.

To determine the effects of an intact extracellular matrix on collagen synthesis, arterial smooth muscle cells (SMCs) were plated sparsely on a cell-free, SMC-derived matrix and examined the following day. Collagen synthesis during a 5-hour incubation by cells on the matrix was reduced to 67% of the control values obtained from cultures on plastic. Total protein synthesis was unaffected. Treatment of the matrix with heparitinase to remove basic fibroblast growth factor (bFGF) before seeding the SMCs abolished the inhibitory effect of the matrix on collagen synthesis. The inhibitory effect was also eliminated by treating the matrix with a neutralizing polyclonal antibody directed against bFGF. Collagen synthesis by SMC cultures grown in wells coated with purified bFGF was only 61% that of control cultures, whereas total protein synthesis remained unchanged. Slot-blot analysis revealed that the relative message level for alpha 1(III) procollagen was reduced in cultures grown on the preexisting matrix or on plastic precoated with bFGF, whereas the alpha 1(I) procollagen message was unaffected. These results demonstrate the ability of the extracellular matrix to modulate the synthesis of collagen by arterial SMCs and indicate that bFGF in the matrix is responsible for these effects.

Parallel regulation of procollagen I and colligin, a collagen-binding protein and a member of the serine protease inhibitor family.

A potential regulatory linkage between the biosynthesis of colligin, a collagen-binding protein of the ER, and procollagen I was examined under a variety of experimental conditions. Cell lines which did not produce a significant amount of procollagen I mRNA also lacked the capacity to produce colligin mRNA. Anchorage-dependent cell lines like L6 myoblasts and normal rat kidney fibroblasts produced both colligin and procollagen I mRNA, but the level of both was concurrently reduced considerably in their ras-transformed counterparts. Similarly, during the differentiation of L6 myoblasts, levels of both colligin and procollagen declined together. Treatment of myoblasts by dexamethasone or EGF led to a decrease in the steady-state levels of procollagen I mRNA, and this was, again, accompanied by a decrease in colligin mRNA synthesis. On the other hand, when the rate of procollagen I synthesis was stimulated by treatment of myoblasts with TGF beta, it led to the concurrent augmentation of both the mRNA and protein levels of colligin. A linkage between the regulation of synthesis of procollagen I and colligin thus seems to exist. The only exception to this generalization is provided by the heat induction behavior of the two proteins. Treatment of myoblasts for a very short period leads to an increase in the synthesis of both the mRNA and protein levels of colligin. This, however, is not accompanied by a change in the mRNA levels of procollagen I. These studies establish that colligin and procollagen are generally tightly co-regulated except after heat shock, suggesting an important functional linkage.