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PubMed1989

Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein.

The enzyme procollagen C-proteinase removes the carboxy-terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl-Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy-terminal propeptide of type I procollagen (PP-Sepharose). This procedure yielded a practically homogeneous, 18,500-fold-purified enzyme preparation and the molecular mass of the purified C-proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl-Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55-kDa protein which was further purified by PP-Sepharose chromatography and identified as an additional form of the 36-kDa and 34-kDa procollagen C-proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6,267-277]. It enhanced the C-proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross-reacted immunologically with the 36-kDa as well as the 34-kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP-Sepharose chromatography, a large proportion of the 55-kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55-kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl-Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C-proteinase from other sources as well.

Animals↗
PubMed1975

Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin.

Extraction of fetal bovine skin at neutral pH and in the presence of protease inhibitors solubilized substantial amounts of type I and type type III collagen and Type III procollagen. Type I and Type III collagen were separated from each other by salt precipitation, and DEAE-cellulose chromatography was used to separate collagen from procollagen. The main chain constituents in type III collagen and procollagen were disulfide-bonded gamma and pgamma components, respectively. Amino acid composition, cross striation banding as observed using electron microscopy, cyanogen bromide peptide patterns in disc electrophoresis gels and resistance of the disulfide regions to pepsin digestion indicated a close similarity to previously described insoluble type III collagen, which was solubilized by limited pepsin digestion. Electron microscopy of long-spacing-segment crystallites and evidence for an extended form of the disulfide-bonded cyanogen bromide peptide suggested that neutral-salt-soluble type III collagon is longer at its C-terminal end by about 10 to 20 amino acid residues than pepsin-treated material. A small elongation was also indicated in the N-terminal portion of the molecule. Procollagen has an additional N-terminal extension with a length of about 160 A, but no difference was observed between collagen and procollagen at their C-terminal ends.

Amino Acids↗
PubMed1976

Radioimmunoassay of procollagen in serum of patients with Paget's disease of bone.

A new radioimmunoassay for human procollagen showed that the sera of 46 of 50 untreated patients with Paget's disease of bone contained increased concentrations of procollagen protein as compared to normal adults. After therapy with disodium etidronate, all the elevated serum procollagen concentrations decreased significantly, falling to normal levels in 33 of 40 patients. The procollagen levels before and after treatment were coordinate with the values for urinary total hydroxyproline and serum alkaline phosphatase activity. The data show that the radioimmunoassay for procollagen is a dependable and useful adjunct to the study of Paget's disease of bone.

Adult↗
PubMed2000

In situ localization of procollagen gene expression on cryosections of undecalcified fracture callus.

OBJECTIVE: To analyze the expression of procollagen gene in fracture callus, and to search for the technique of in situ hybridization for undecalcified skeletal tissue. METHODS: In situ hybridization of procollagen gene expression was performed on the undecalcified cryosections of rat fracture callus at 7, 14, and 28 d. RESULTS: The hybridization signals achieved were clear and easy to be localized with high specificity. On the 7th day, the expressions of pro alpha1 (III) in fibroblasts and some chondrocyte-like cells were dominant; and at the end of second week high expression of type-II procollagen mRNA was observed in chondrocytes. At the end of fourth week, the cartilaginous callus was almost all replaced by woven bone tissue, and some type-I procollagen mRNA positive osteoblasts and hypertrophic chondrocytes were found scattering in the woven bone and remnants of cartilaginous callus. CONCLUSIONS: The modified method employed in this study is easier, quicker, and more sensitive with high specificity than the conventional tec hnique for in situ hybridization of procollagen gene expression of decalcified rat fracture callus. The phenomenon of shared phenotype expression, which was demonstrated among cells engaged in fracture healing, indicates an important approach to reveal the mechanism of the origin, differentiation, and orientation of cells.

Journal Article↗
PubMed2004

[Effect of nandrolone phenylpropionate on hepatic albumin mRNA and granulational alpha 1(I) procollagen mRNA in burned rats].

OBJECTIVE: To assess the mechanism for the effect of nandrolone phenypropionate (NP) on hepatic albumin mRNA and granulational alpha 1(I) procollagen mRNA in burned rats with the aim to underpin the clinical application of anabolic steroids. METHODS: Thirty-two Wistar rats with a deep second-degree cutaneous burn of 20% total body surface area were randomly divided into two groups to receive either 5 mg/kg NP (NP group) or normal saline as placebo(control group) every other day. The expression copy quantities of albumin-mRNA in liver tissue and alpha 1 (I) procollagen mRNA in granulation wound were measured by quantitative fluorescent RT-PCR respectively on the post-burned days 4, 7, 14 and 21. RESULTS: The expression levels of albumin-mRNA and alpha 1 (I) procollagen mRNA in NP group were much higher than those in control group. The 7th and 14th days were the periods in which the albumin-mRNA and alpha 1 (I) procollagen mRNA expression had been increasing obviously (P < 0.01). CONCLUSION: Nandrolone phenylpropionate could effectively up-regulate the expression of albumin-mRNA in liver tissue and the alpha 1 (I) procollagen mRNA in granulation wound.

Albumins↗
PubMed2004

[Effect of cytokine and sandostatin on collagen synthesis and expression of procollagen mRNA by lens epithelial cells].

OBJECTIVE: To study the effects of epidermal growth factor (EGF), interleukin-1 (IL-1), interferon-gamma (IFN-gamma) and sandostatin on collagen synthesis and expression of types I and III procollagen mRNA by bovine lens epithelial cells in vitro. METHODS: Cells were plated in 96-well tissue culture plates. After attached, serum-free medium containing EGF 10(-2) - 10(2) ng/ml, IL-1 10 - 10(5) ng/ml, sandostatin 10(-11) - 10(-7) mol/L and IFN-gamma 10 - 10(5) U/ml were added into the plates. Collagen synthesis was measured with (3)H-proline incorperation, and expression of procollagen mRNA was detected with Northern blot. RESULTS: EGF 10(-1) - 10(2) ng/ml and IL-1 10(2) - 10(5) ng/ml obviously promoted collagen synthesis. EGF 1 ng/ml and IL-1 10(3) ng/ml increased the expression of types I and III procollagen mRNA. IFN-gamma 10(3) - 10(5) ng/ml and sandostatin 10(-10) - 10(-7) mol/L decreased collagen synthesis. IFN-gamma 10(3) U/ml and sandostatin 10(-9) mol/L decreased the expression of types I and III procollagen mRNA. CONCLUSIONS: EGF and IL-1 may enhance the occurrence of posterior capsular opacification, they affect types I and III procollagen gene expression at transcription level. IFN-gamma and sandostatin might be used to prevent the occurrence of posterior capsular opacification.

Animals↗
PubMed1991

Inhibition of prolyl hydroxylation and procollagen processing in chick-embryo calvaria by a derivative of pyridine-2,4-dicarboxylate. Characterization of the diethyl ester as a proinhibitor.

The biochemical and morphological consequences of procollagen prolyl 4-hydroxylase inhibition by pyridine-2,4-dicarboxylic acid (2,4-PDCA) and its diethyl ester (diethyl-2,4-PDC) were studied in chick-embryo calvaria, which predominantly synthesize type I collagen. Half-maximal inhibition of tissue hydroxyproline formation required 650 microM-2,4-PDCA, whereas the Ki with respect to chicken prolyl 4-hydroxylase in vitro was 2 microM. In contrast, half-maximal inhibition was caused by 10 microM-diethyl-2,4-PDC in the intact calvaria, although chicken prolyl 4-hydroxylase in vitro was not inhibited even at 1 mM. The collagenous material produced in the presence of diethyl-2,4-PDC showed an altered 'melting' profile and a lowering of the transition temperature by 10 degrees C, indicating misalignment and thermal instability of its triple-helical structure. Amount and electrophoretic mobility of procollagen type I chains were increased in a dose-dependent manner. The amounts of partially processed species and alpha-chains were decreased, without change in mobility. This marked effect on procollagen-collagen conversion in the intact calvaria suggests that the underhydroxylated collagenous material generated in the presence of diethyl-2,4-PDC is resistant to or acts as endogenous secondary inhibitor of type I procollagen N-proteinase. Electron microscopy of treated calvaria cells showed dilated rough endoplasmic reticulum and numerous phagolysosomes, indicating intracellular retention and lysosomal degradation of the newly synthesized underhydroxylated collagenous material. In summary, these results identify 2,4-PDCA and diethyl-2,4-PDC as the first prolyl 4-hydroxylase-directed inhibitor/proinhibitor pair that affects intra- and extra-cellular events during collagen formation.

Animals↗
PubMed1997

Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers.

Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.

Animals↗
PubMed1999

Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.

The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.

Ascorbic Acid↗
PubMed1982

Procollagen N-proteinase. Properties of the enzyme purified from chick embryo tendons.

Procollagen N-proteinase was purified about 3700-fold from chick embryo tendons. Electrophoresis of the protein after iodination and denaturation suggested it was homogeneous. However, the native enzyme could not be examined by gel electrophoresis, and therefore homogeneity of the preparation was not conclusively established. Antibodies to the enzyme completely inhibited activity and gave a single precipitant line by double immuno-diffusion. The Km for a native procollagen substrate was 0.3-0.5 microM. The same protein after denaturation inhibited activity. The enzyme did not cleave type III procollagen from human fibroblasts or a type IV procollagen from a mouse sarcoma. Ca2+ was required for maximal enzymic activity. The data suggested a second metal requirement, but this was not identified. Reducing agents and metal chelators inhibited activity, but there was little if any inhibition from several inhibitors of other neutral metalloproteinases.

Animals↗
PubMed1992

Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern.

We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.

Adult↗
PubMed1986

Serum enzymes of collagen synthesis and type III procollagen amino-propeptide in Nigerian patients with sickle cell disease.

Serum immunoreactive prolyl hydroxylase protein, galactosylhydroxylysyl glucosyltransferase activity and the aminoterminal propeptide of type III procollagen were measured in 20 patients with sickle cell disease and the values were compared with those in 20 apparently healthy Nigerians. The means for the two enzymes and serum aminoterminal propeptide of type III procollagen were significantly higher in the sickle cell disease patients. Significant correlations were found between the values for the two enzymes and the protein serum aminoterminal propeptide of type III procollagen within the sickle cell disease patients. The data confirm that collagen formation is found in the liver, bone and other organs of patients with this disease. The measurement of serum immunoreactive prolyl hydroxylase protein, serum galactosylhydroxylysyl glucosyltransferase activity and serum aminoterminal propeptide of type III procollagen in prospective studies might be helpful in predicting hepatic, bone or diffuse fibrogenesis in sickle cell disease.

Adolescent↗
PubMed1983

Synthesis and processing of a type I procollagen containing shortened pro-alpha 1(I) chains by fibroblasts from a patient with osteogenesis imperfecta.

Skin fibroblasts from a patient with a lethal form of osteogenesis imprefecta were found to synthesize equal amounts of normal pro-alpha 1(I) chains and pro-alpha 1(I) chains which are about 10% shorter because of a deletion of about 100 amino acids in the middle of the alpha chain domain. The pro-alpha 1(I) chains were incorporated into three different kinds of trimers: a normal type I trimer with normal length pro-alpha 1(I) chains; a type Is trimer with one shortened pro-alpha 1(I) chain and two normal length chains; and a type Iss trimer containing two shortened pro-alpha 1(I) chains and one normal length pro-alpha 2(I) chain. As judged by resistance to digestion by chymotrypsin and trypsin, the type Is and Iss trimers denatured at a temperature at least 3 degrees C lower than normal type I procollagen. Procollagen containing the shortened pro-alpha 1(I) chains was slowly secreted by the cells but was degraded by extracellular proteinases within 6 h of chase into the medium. The results indicated that the presence of the shortened pro-alpha 1(I) chains in procollagen trimers produces a delay in rate of helix formation, overmodification of the polypeptides by post-translational enzymes, a decrease in the thermal stability of the trimers, and increased susceptibility of the protein to endogenous proteinases. Additionally, the fibroblasts of this patient synthesized and secreted a type III-like species of procollagen with unusual chromatographic properties.

Endopeptidases↗
PubMed1994

Self-assembly into fibrils of collagen II by enzymic cleavage of recombinant procollagen II. Lag period, critical concentration, and morphology of fibrils differ from collagen I.

A recently developed recombinant system for synthesis of human procollagen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibrils. The procollagen II was cleaved to pCcollagen II by procollagen N-proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically purified, and the pCcollagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase. The kinetics for assembly of collagen II fibrils were similar to those observed previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. However, under the same experimental conditions, the lag time for assembly of collagen II fibrils was 5-6-fold longer, and the propagation rate for collagen II fibrils was about 30-fold lower than for collagen I fibrils. The relatively long lag time for the assembly of collagen II into fibrils made it possible to demonstrate that most of the conversion of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37 degrees C for collagen II was about 50-fold greater than the critical concentration for collagen I. The Gibbs free energy change for the assembly of collagen II into fibrils was -40 kJ/mol, a value that was about 14 kJ/mol less than the free energy change for collagen I and about the same as the free energy change for the homotrimer of collagen I. Dark-field light microscopy and negative-staining electron microscopy demonstrated that the collagen II fibrils were thin and formed network-like structures. The results demonstrated, therefore, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.

Cell Line↗
PubMed1999

The early molecular natural history of experimental osteoarthritis. I. Progressive discoordinate expression of aggrecan and type II procollagen messenger RNA in the articular cartilage of adult animals.

OBJECTIVE: To quantify changes in the chondrocyte metabolism of aggrecan core protein and type II procollagen messenger RNA (mRNA) during the early and middle phases of experimental osteoarthritis (OA) in animals. METHODS: Experimental OA was induced by transecting the cranial cruciate ligament of the stifle joint in adult animals; articular cartilage was harvested and analyzed after 4, 10, and 32 weeks. RESULTS: Northern blot analysis revealed no change in aggrecan mRNA 4 weeks after surgery compared with aggrecan mRNA in the unoperated contralateral control joints; aggrecan mRNA levels became significantly elevated by 10 and 32 weeks after surgery. In OA cartilage, type II procollagen mRNA was dramatically and progressively elevated at all times after surgery. The relative increases in type II procollagen mRNA exceeded the relative increases in aggrecan mRNA at all times after surgery, and these differences increased progressively over time. Articular chondrocytes became activated globally (total RNA increases) and specifically (mRNA increase) early after joint injury and remained activated throughout the early and middle phases of this experimental OA. CONCLUSION: The early natural history of experimental OA is characterized by a progressive imbalance in the mRNA expression of aggrecan and type II procollagen in articular chondrocytes. These results suggest that the stimuli for the transcription of these 2 genes are fundamentally different in this animal model.

Aggrecans↗
PubMed1992

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing.

The effect of transforming growth factor-beta 1 (TGF-beta 1) on collagen biosynthesis was investigated in confluent primary monolayer cultures of rabbit articular chondrocytes (RAC). Exposure to TGF-beta (0.1, 1, and 10 ng/ml) in serum-free medium caused a dose- and time-dependent stimulation of collagen biosynthesis associated with an increase of steady-state levels of procollagen type II mRNA. Elevation of the mRNA steady-state did not result from a stabilization of the transcript, as shown by measure of the mRNA half-life. Electrophoresis (SDS-PAGE) showed that TGF-beta stimulates the synthesis of most collagen isotypes, including type II, without qualitative change in their distribution. Moreover, pulse-chase experiments revealed that TGF-beta did not affect the processing rate of type II procollagen. TGF-beta slightly stimulated the production of prostaglandin E2 (PGE2), which could in turn exert an inhibition on collagen synthesis. However, addition of indomethacin to block prostaglandin synthesis did not further enhance the TGF-beta-induced stimulation of collagen production, suggesting that this mediator was not implicated in the effect. Moreover, TGF-beta increased steady-state levels of procollagen type II, I, and III mRNAs even in the presence of indomethacin. Despite these increased mRNA levels, only the production of type II collagen was significantly augmented, suggesting that type I procollagen mRNA was not fully translated. In addition, the TGF-beta-induced stimulation of collagen synthesis was observed whenever ascorbic acid is added or not in the culture medium. In conclusion, TGF-beta, which is present in great amount in bone and cartilage, can increase the collagen production of cultured RAC and might therefore play a role in the early events of cartilage repair, such as those observed in osteoarthritis.

Animals↗
PubMed1995

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.

Blotting, Northern↗