Oct 25, 2017 - Rajendra Raghow$, David Gossageg, Jerome M. Seyer, and Andrew H. Kang. From the Research Seruice, Veterans Administration Medical ...
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
Vo. 259, No. 20. Issue of October 25, pp. 12718-12723 1984 Printed in C.S.A.
Transcriptional Regulation of Type I Collagen Genes in Cultured Fibroblasts by a Factor Isolated from Thioacetamide-induced Fibrotic Rat Liver* (Received for publication, May 17, 1984)
Rajendra Raghow$, David Gossageg, Jerome M. Seyer, andAndrew H. Kang From the Research Seruice, Veterans Administration Medical Center and Departments of Pharmacology, Biochemistryand Medicine, University of Tennessee Center for the Health Sciences, Memphis, Tennessee38104
Recently Hatahara and Seyer (Hatahara, T., and liver has been investigated in considerable detail. Studies by Seyer, J. M. Biochim. Biophys. Acta (1982) 716,377- Seyer et al. (4) have demonstrated an increase in the ratio of 382) isolated a factor from fibrotic rat liver which type 1:type 111 collagen in human cirrhotic liver. It has also stimulates collagen synthesis in cultured fibroblasts been shown that hepatic fibrosis in humans is accompanied without affecting their rateof proliferation. To inves- by increased content of collagens (4-7). A similar increase in tigate the mechanism of fibrogenic factor-mediated collagen content hasbeen observed in experimentally induced enhancement of type I collagen synthesis, we quanti- hepatic fibrosis in animal models (1-3, 8-10). The molecular tated the levels of mRNAs coding for pro-al(1) and basis for altered collagen metabolism in fibrotic liver is incompro-aZ(1) chains in rat dermal fibroblasts. Cell-free pletely understood. Recently, Hatahara andSeyer (1)isolated translation experiments revealed that the fibrogenic factor caused greater than5-fold increase in the trans- afactor from fibrotic rat liver which stimulated collagen latable levels of type I mRNAs.We also quantitated synthesis in cultured fibroblasts without affecting their rate collagen mRNAs bytechniques of Northern blotting of of proliferation. Although the precise molecular structure of glyoxylated poly(A+)RNA followed by hybridization the fibrogenic factor is unknown, it appears to be a complex to nick-translated human cDNA clones containing the phospholipid-containing polypeptide; smaller amounts of ficoding sequence of pro-al(1) and pro-a2(1) chains. Fur- brogenic factor have also been found in the normal liver (1). of collagen Finally, isolation of. a similar factor from human cirrhotic thermore, we investigated the relative rates mRNA transcription in the isolated nuclei of treated liver has been accomplished recently.’ This study was undertaken to investigate the molecular and control fibroblasts. Similar quantitationof &actin mRNA transcription, which remains unaffected by the mechanisms of the fibrogenic factor-mediated enhancement treatment withfibrogenic factor, was used as an inter- of type I collagen biosynthesis. We quantitated type I collagen nal control. We demonstrate that thefibrogenic factor specific mRNAs in cells treated with fibrogenic factor by in causes a 4-6-fold increase in the rate of transcription vitro translational assays and also by hybridization to nickof pro-al(I) and pro-aZ(I)genes. Finally, we also show translated recombinant plasmids, HF677 and HF32, containthat the rateof intracellular degradationof collagen is ing human pro-d(I)and pro-a2(1) cDNAs, respectively. not significantly altered incells treated withfibrogenic These data indicate that fibrogenic factor selectively enhances factor. These results combined with data on cell-free the transcription of the pro-al(1) and and pro-aB(1) collagen translation strongly suggest that the increased accu- genes coordinately. mulation of type I collagen mRNA in fibrogenic factortreated fibroblasts is a consequence of enhanced rates EXPERIMENTALPROCEDURES of collagen mRNA transcription.
Hepatic fibrosis is a common and important condition in which major amounts of liver parenchyma are replaced by fibrous connective tissue. Experimental hepatic fibrosis has been induced in rats andbaboons with chronic administration of hepatotoxins such as CCL, ethanol, or thioacetamide (13). These animal models mimic to various degrees the pathological processes observed in human hepatic fibrosis. Since interstitial collagens are themajor protein constituents of the fibrous connective tissue, metabolism of collagen in fibrotic * This study was supported by the Veterans Administration and in part by National Institutes of Health Grant 5 R01-AA03732. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom all correspondence should be addressed. §Supported by Medical Student Research Training Grant AM 07405 from the National Institutes of Health.
Materials-Five- to seven-week-oldSprague-Dawley or Lewis rats were obtained from the National Cancer Institute, Bethesda, MD. Enhance, micrococcal nuclease-treated reticulocyte lysates, [2,3-3H] proline (115 Ci/mmol), [w3’P]dATP, [cx-~’P]~CTP (about 3,000 Ci/ mmol), [a-32P]UTP(about 3,000 Ci/mmol) were all purchased from New England Nuclear, Boston, MA. DNA polymerase I of Escherichia coli, DNase I, and restriction endonucleases were all purchased from Bethesda Research Labs, Gaithersburg, MD and New England BioLabs, Boston, MA. Nitrocellulose filters (BA 85) were bought from Schleicher & Schuell, Keen, NH. Isolation of the Fibrogenic Factor-Rats were injected intraperitoneally daily with 0.5 ml of freshly prepared saline solution of thioacetamide (2.5mg/100 g body weight) for 6-8 weeks. Animals were killed under an atmosphere ofCOZ, livers were resected, and the fibrogenic factor was purified from liver homogenates. Detailed procedures for isolation and assay of fibrogenic factor activity have been described earlier (1). Cell Culture Studies-A continuous cell line of rat dermal fibroblasts was grown in Dulbecco’s minimal essential medium with 10% fetal calf serum to 90-95% confluency. To begin treatment, old media was replaced with 2.0mlof fresh media, and various amounts of R. Raghow,D. Gossage, J. M. Seyer, and A. H. Kang, unpublished results.
12718
12719
Regulation of Collagen Synthesis by Fibrogenic Factor
fibrogenic factor extract isolated from either normal livers or fibrotic isolation of nuclei, in uitro transcription, and subsequent determinalivers were added to duplicate cultures. Pilot experiments revealed tion of the rates of transcription by hybridization. Nuclei were either that the time for maximum stimulation of collagen synthesis was used immediately after isolation or stored a t -70 "C in 25% glycerol, approximately 6-8 h following treatment. Except where otherwise 60 mM KC1, 15 mM Tris-HCI, pH 7.5. To start transcription, nuclei noted, all subsequent determinations were done 6 h following treat- (100-150 pg of DNA) were incubated in a 100-pl reaction mix that contained 10% glycerol, 50mM Tris-HC1, pH 8.0, 5 mMMgC12, 1 mM ment with the fibrogenic factor. Measurement of Intracellular Degradation of Collagen-We deter- MnC12, 150 mM KC], 2.5 mM dithiothreitol, 1 mM each ATP, GTP, at 25 "C for 30 min. Radiolamined the rate of intracellular degradation ofnewly synthesized and CTP, and 250 pCi of [CY-~*P]LJTP collagen according to thetechnique described in detail previously (11, beled RNA was extracted (26) and hybridized to DNA immobilized linearized alkali12). Rat fibroblasts were treated with fibrogenic factor for 4 h. We on nitrocellulose filters. In each case, 2 pgof denaturedplasmid DNA was immobilized on nitrocellulose filter disks then added [2,3-3H]proIine (10 pCi/lOO-mm diameter culture dish) and incubated cultures for further 4 h. After radiolabeling, cells were (0.5-cm diameter). For determining nonspecific background, 1 pg scraped, and combined cell and media were heated for 20 min at each of pBR322 and bacteriophage X charon 4A DNA wereseparately 100 "C and sonicated for 2 min. For determination of the total hybridized with radiolabeled run-off transcripts. Nitrocellulose filters collagen synthesis, an aliquot was hydrolyzed with 6 N HCl, 110 "C were baked as described above, prehybridized, and hybridized a t 42 "C for 24 h and the amount of [3H]hydroxyprolinewas calculated from for 24 and 48 h, respectively (28). These hybridizations were done amino acid analysis on an automated amino acid analyzer equipped under conditions of DNA excess; hybridization for longer than 24 h with a stream splitting device, and appropriate fractionswere counted did not result in increased signal intensity, and therefore we believe in a scintillation spectrometer. Another aliquot was filtered through that maximum hybridization was achieved under these conditions. Amicon (CF-25) membranes and the quantity of [3H]hydroxyproline To measure the efficiency of hybridization, [3H]cRNA wag synthe(representing intracellular degradation) was determined in the fil- sized from HF677 as described (26) and hybridized to immobilized trate. These results were expressed as per cent of intracellular deg- HF677 DNA under identical conditions. radation of newly synthesized collagen. For a positive control, cells were treated with various concentrations of dibutyryl cyclic AMP, RESULTS and the rate of intracellular degradation of collagen was similarly Intracellular Breakdown Is Not Affected by Fibrogenic Facdetermined. Isolation of Messenger RNAs-Total cellular RNA was extracted tor-Under a varietyof physiological conditions, intracellular by guanidine thiocyanate solubilization of cells and centrifugation of degradation of newly synthesized collagen has been implicated the extract through a cushion of 5.7 M CsCl as described before (13, as an important regulatory mechanism of collagen biosyn14). Poly(A+) RNA was selected by oligo(dT)-cellulose column chro- thesis (11, 12, 29-32). To determine if the fibrogenic factor matography (15). The quality and quantity of RNA were routinely tested by determining Am/Am, ethidium bromide fluorescence of actedthrough an analogous mechanism, we measured the RNA electrophoresed in agarose gels, and finally its ability to be rates of intracellular degradation of newly synthesized collagen. In fibroblasts either untreated, treated with normal liver translated in cell-free protein synthesizing extracts. Cell-free Translation and Gel Electrophoresis-Nuclease-treated extract, or with fibrogenic factor from the fibrotic liver, the rabbit reticulocyte extract (16) were programmed with 1 pgof rate of intracellular degradation was not significantly differpoly(A+) mRNA and 25pCiof [2,3-3H]proline in a final reaction ent from each other; the rate of intracellular degradation volume of 30 pl. After 1-h incubation at 30 "C, proteins were precipvaried between 5 7 % (Table I). Therefore, the fibrogenic itated with ethanol. Some samples were digested with bacterial collagenase (1 unit/ml) for 5 min a t 37 "cin 50 mM CaC12 and 50 mM factor apparently did not significantly alter the rateof intraN-ethylmaleimide followed by ethanol precipitation. Proteins were cellular degradation of newly synthesized collagen. As a possolubilized in sample buffer and electrophoresed on 6% polyacryl- itive control, similar measurements were done on parallel amide gels as described previously (17); radioactive bands were visu- cultures treated with dibutyryl cyclic AMP, an agent known alized by fluorography (18). to accelerate intracellular breakdown of collagen (30). ConPreparation of cDNA Plasmids-The recombinant plasmids HF677 sistent with earlier observations, dibutyryl cyclic AMP signif(19) and HF32 (20), containing cDNA sequences for human cul(I) and 012(I) collagen chains, respectively, were a gift of Dr. F. Ramirez, TABLEI Rutgers Medical School, Piscataway, NJ. Recombinant clone conIntracellular degradation and proline poob taining nearly complete complement of chicken cytoplasmic @-actin mRNA (21) was generously provided by Dr. Bruce Paterson, National Cultured fibroblasts were treated under various regimens as deCancer Institute, Bethesda, MD. E. coli strains harboring the respec- picted and labeled with [3H]proline for 4 h. The combined cells and tive recombinant plasmids were grownand plasmids were purified by media were heated (100 "C, 20 min) and sonicated for 2 min. For CsCl gradient centrifugation according to published procedures (22). determination of the total hydroxyproline pools, an aliquot was Electrophoretic Fractionation of RNA, Northern Blotting, and Hy- hydrolyzed in 6 N HCI, 110 "Cfor 24 h, and fractions containing [3H] 6ridization"RNA was denatured in 1 M glyoxal, 50% (v/v) Me,SO,, hydroxyproline were collected with the help of an automated amino 10 mM sodium phosphate buffer, pH 7.0, at 50 'C for 1 h. Denatured acid analyzer, equipped with a stream-splitting device. The radioacRNA was electrophoresed on 1%agarose gels (3-mm thick and 20- tivity in the appropriate fractions was determined in a scintillation cm long) submerged in 10 mM phosphate buffer, pH 7.0, according to spectrometer. To determine the rate of intracellular degradation, McMasters and Carmichael (23). RNA was electrophoresed at 100 V another aliquot was filtered through an Amicon membrane (CF 25, for 4-6 h while buffer was constantly recirculated. The portion of the cut off molecular weight 20,000)and theamount of [3H]hydroxyprogel containing markers was stained with EtBr and theremaining gel line was similarly determined in the filtrate. was subjected to Northern blotting without presoaking as described Proline Total Intracellularly by Thomas (24). The dried nitrocellulose blots were baked in a Treatment pool degraded Degradation vacuum oven a t 80 'C for 2 h. hvdroxwroline -. The cDNA plasmid probes were nick-translated with [ ( U - ~ ~ P J ~ A T P cpmlnmol cprn/IOO-mrn dish % or [ ~ I - ~ ~ P ] ~toCaTspecific P activity of greater than 5 X 10' cpm/pg Control, no 76.6 1040 72 6.9 of DNA according to published techniques (25). The nitrocellulose treatment filters were incubated in prehybridization buffer for 12-24 h at 42 'C. Extract from 80.1 1089 58 5.3 The nick-translated probes were denatured a t 100 'C for 10 min, normal liver cooled, and added to hybridization buffer (4 parts of prehybridization Fibrogenic factor, 20678.5 2866 7.2 buffer and one part of 50% (w/v) dextran sulfate). Following hybrid6h ization for 18-24 h at 42 "C, the RNA blots were washed extensively, Fibrogenic factor, 74.9 3182 184 5.8 dried, wrapped in Saran Wrap, and exposed to x-ray film a t -60 "C 12 h using Cronex Hi-plus intensifying screen. Radioactivity in the indidbc-AMP, 0.1 mM 82.3 1188 314 26.5 vidual bands was quantitated in a scintillationspectrometer. dbc-AMP, 1.0 mM 375 77.3 1215 30.9 In Vitro Transcription and Hybridization-Previously published dbc-AMP, 10 mM 81.2 1178 565 47.9 techniques (26, 27), with minor modifications, were used for the
-
12720
Regulation of Collagen Synthesis by Fibrogenic Factor
icantly elevated the ratesof intracellular degradation of colNorthern Blot Hybridizations-The results from cell-free lagen; morethan 30% of radiolabeled [3H]hydroxyproline was translation experiments implicatedsignificantly higher levels represented by the intracellularly degraded fraction (Table I). of procollagen mRNAs in the cells treated with fibrogenic Finally, it is important to point out that our simultaneous factor. However, these experiments did not descriminate bemeasurements of intracellular proline pool size also revealed tween an increase in the absolute amounts of mRNAs and no significant changes after fibrogenic factor treatment (Ta- their relative stability and/or translational efficiency. Thus, ble I). using radiolabeled recombinantDNA probes, we directly Effect of Fibrogenic Factor on Translatable Type Z Collagen quantitated the relative amounts of pro-d(1) and pro-a2(1) rnRNAs-Since a n increased accumulation of collagen could mRNAs along with mRNA coding for @-actin (a ubiquitous not be accounted for by altered rates of intracellular degra- non-collagenous eukaryotic protein) asa control in ratfibrodation, we quantitated the levels of translatable procollagen blasts before and after treatment withfibrogenic factor. mRNAs in cells treated with or without fibrogenicfactor. Poly(A+)RNA from control and fibrogenic factor-treated Initiation-competent reticulocyte extracts were programmed cells was size-fractionated, transferred to nitrocellulose, and with poly(A+) mRNA and [3H]proline-labeledpolypeptides hybridized to nick-translated recombinant plasmids containwere synthesized in vitro. The cell-free translation products ing appropriatecDNA sequences.For pro-cul(1) and pro-a2(I) were analyzed by sodium dodecyl sulfate-polyacrylamide gel mRNAs, we used HF 677 and HF 32 plasmids that contain electrophoresis and fluorography. Although the overall pat- cDNA sequences coding for human pro-cul(1) and pro-a2(1) tern of polypeptides synthesized in vitroby mRNAs from chains, respectively (19,20). A nearly full-lengthcDNA clone control and fibrogenic factor-treated cells was similar (Fig. representing a chicken@-actinmRNA sequences (21) was I), the pro-al(1) and pro-a2(1) bands are more intense in the used for quantitation of @-actin mRNA in ratfibroblasts. In fibrogenic factor-treated sample. The quantitation of radio- a preliminary experiment, we tested the suitabilityof human activity in polypeptide bands by scintillation counting conand chickencDNA probes to quantitate mRNAs in rat fibrofirmed the finding. The results from three separate determi- blasts. Hybridization of human a2(I) cDNA clone (HF 32) to nationsindicatedthattherewas 4-5-fold increaseinthe human and rat mRNAs in a dot blothybridizationassay amount of translatable procollagen mRNA in fibroblasts after revealed a high degree of homology; similar homology was treatment with fibrogenic factor for 12 h. The direct quanti- also apparent when human al(1)and chicken @-actincDNA tation of procollagen mRNAs by Northern blotting and hy- clones were hybridized to rat fibroblast mRNAs (data not bridization corroborated these results (see below). shown). ThecDNA clone of chicken cytoplasmic @-actinused The use of [3H]proline incell-free translation assays limited in our experiments haspreviously been shown to share some the background of noncollagenous proteins. However, in ad- homology with the chicken y-actin mRNAs (21). Since we did dition to authentic pro-al(1) and pro-aZ(1) chains, therewere not vigorously test if this clone hybridized to only @-actin several lower-molecular-weight species observed in both con- mRNA, it is possible that we are measuring @ as well as ytrolandtreatedsamples (Fig. 1). Since several of these actin mRNAs. It should be noted, however, that even under additional polypeptide bands were susceptible to cleavage by more stringent conditionsfor hybridization and washing the highly purified bacterial collagenase, these were assumed to overall results were similar to those obtained under standard be products of premature termination. Similar subsized pro- conditions described under “Experimental Procedures” (data collagen polypeptide products have recently been observed by not shown). The efficiency and specificity of these probes other investigators (33, 34). were therefore adequate for these analyses. A comparative Northern blot analysisof poly(A+) mRNAs CELL FREE TRANSLATION from control and fibrogenic factor-treated cells is shown in Fig. 2. The radiolabeled probe for pro-d(1) detected two 1 2 3 4 distinct mRNAspecies of 7.1 and 5.5 kb,2 the latter accounting for 70% of radioactivity (Table 11). Nick-translated H F 32 (pro-a2(1)) on the other handrevealed the presence of three ,-Pro o ( 1 (I) distinct mRNA species; the major mRNA species of4.8 kb accounts for 70% radioactivitywhile the 5.8 and 7.0 kb 7-Pro 2 (I) account for 17 and 13%,respectively (Table 11). The radiolabeled probes for both pro-d(1) and pro-a2(1) chainsclearly revealed greater signal intensities in fibrogenic factor-treated samples (Fig. 2). The effect of fibrogenic factor on collagen mRNA accumulation was studied quantitatively by dot blot hybridizations. The results from three different determinations on the course of type I procollagen mRNA accumulation revealed a consistent 6-7-fold increase in thed ( 1 ) a n d a 2 ( I ) specific mRNA sequences; the maximum effect was achieved as early as 6-h post-treatment and thislevel was maintained for 24 h (Fig. 3). In contrast to the effect on type I collagen FIG. 1. Levels of translatable type I procollagen mRNAs mRNAs, the accumulation of @-actin mRNA remained virare increased in fibrogenic factor-treated rat fibroblasts. Cell- tually unaffected by treatment of fibrogenic factor (Figs. 2 free translation products of poly(A+) RNA from rat fibroblasts un- and 3). It appears, therefore, thatfibrogenic factor selectively treated or treated with fibrogenic factor are depicted. [3H]Proline influences the accumulation of collagen mRNAs; similar selabeled polypeptides translated in reticulocyte extracts were electrolectivity has been shown at the protein synthesislevel (I).’ phoresed in 7.5% polyacrylamide gels and fluorographed as described Thereis a close correspondencebetween the collagen under “Experimental Procedures.” Lune 1, no mRNA; lane 2, globin mRNA; lane 3, mRNA from untreated rat fibroblasts, and lane 4, mRNA levels as determined by dot blot/Northern blot anal-
-
translation products of mRNA from rat fibroblasts treated with fibrogenic factor for 6 h.
* The abbreviation used is: kb, kilobases.
Regulation of Collagen Synthesis by Fibrogenic Factor
12721
J-actin
FIG. 2. Accumulation of type I collagen and &actin mRNAs in fibrogenic factor-treated rat fibroblasts. Five micrograms of poly(A+) RNA from control and fibrogenic factor-treated cells were denatured with glyoxal and dimethyl sulfoxide and electrophoresed in 1.0% agarose gels. After blotting to nitrocellulose, filters were hybridized with nick-translated cDNA plasmids. Left, Northern blot hybridization of rat fibroblast mRNA to nick-translated human pro-al(1) plasmid. Lanes I and 2 contain mRNA from control cells while lanes 3 and 4 from cells treated with fibrogenic factor for 6 and 12 h, respectively. Lane M depicts EtBr stained 28 and 18 S ribosomal RNA markers. Center, hybridization of rat fibroblast mRNA to nick-translated human pro-a2(I) cDNA plasmid. All five lanes have identical RNA samples as depicted on the left. Right, the nitrocellulose filter depicted in A was washed in water a t 100 "C for 20 min to remove the first probe and rehybridized with nick-translated chicken cytoplasmic @-actincDNA probe. The unwashed probe from the previous experiment representing 7.1and 5.5-kb al(1) procollagen mRNAs (arrows) can also be seen.
TABLE I1 Effect of fibrogenic factor on multiple mRNA species coding for proal(I) and pro-a2(1) collagen chains Five micrograms of poly(A+) mRNA from untreated or fibrogenic factor-treated rat fibroblasts (12 h following treatment) were electrophoresed, blot-transferred to nitrocellulose, and hybridized with nicktranslated pro-al(1) and pro-a2(I) cDNA plasmids. Areas covering the various molecular weight procollagen mRNA species were excised and radioactivity was determined in a Beckman scintillation spectrometer. The background incorporation of 46 cpm was subtracted from each value. The per cent distributions into different mRNA sizes within each sDecies is shown in Darentheses. Collagen chain
Pro-al(1) Pro-a2(I)
mRNA size kb 7.2 5.5 7.0 5.8 4.8
Incowrated Control Treated
Treated/control ratio
CPm
165 (29.8) (28.5) 759 388 (70.1) (71.4) 1902 112 (12.8) 461 (11.1) 158 (18.0) 736 (17.6) 603 (69.2) 2926 (71.3)
4.6 4.9 4.1 4.6 4.8
ysis and cell-free translation experiments. These experiments suggested that fibrogenic factor most likely acted at the transcriptional level. Thus, we tested the transcriptional activity directly by the run-off transcription assay(27). The isolated nuclei from control or treatedcells were incubated in vitroto permit elongation of nascent transcripts in the presence of ["PIUTP and quantitatedby hybridization to cDNA plasmids immobilized on nitrocellulose, under conditions of DNA excess. Since inclusion of 0.8 pM a-amanitin in the reaction completely abolished the incorporation of ["P]UTP into proal(1) and pro-a2(I) transcripts (Table III), this reflected assay theaction of RNA polymerase I1 on actively transcribed genes. The transcription of al(1) and a2(I)genes was stimulated greater than 5-fold in cells treated withfibrogenic factor. The rateof transcription of @-actingenes was notsignificantly
affected undersimilarconditions(Table 11). The run-off transcripts assays conducted at different times following fibrogenic factor treatmentrevealed that maximum stimulation occurred at 6-8-h post-treatment; the transcriptional activity was maintained at similar levels up to 24-h post-treatment (the longest duration of treatment tested for transcriptional stimulation). The experimentalevidence strongly indicates a selective stimulation of type I collagen gene expression by the fibrogenic factor. DISCUSSION
We have demonstrated that the fibrogenic factor isolated from thioacetamide-induced fibrotic rat liver stimulates the synthesis of type I collagen in cultured rat fibroblasts. The combined evidence from cell-freeprotein synthesis, Northern blot hybridizations with nick-translated plasmids containing pro-al(I), pro-aZ(I), andcytoplasmic @-actincDNA, and the measurements of the relative rates of collagen and @-actin genes by run-off transcription assays reveal that the fibrogenic factor acts at the transcriptional level. The measurements on the relative accumulation of collagen and @-actin mRNAs in cells treated with fibrogenic factor indicate that the effect is selective. Although the rate of transcription /3actin is elevated slightly (less than 2-fold), the rate of transcription of pro-al(1) and pro-aB(1) genes is affected to a much greater extent (more than&fold). The selective stimulation of collagen mRNA synthesis is consistent with the earlier observations that showed about a 6-fold increase in the collagen synthesis in these cells treated with fibrogenic factor (I).' We did not examine the influence of fibrogenic factor on the mRNAs coding for pro-al(II1) collagen and, therefore, it is impossible to say whether there was significant change in the biosynthesisof type I11 collagen. Although type I and I11 collagen genes are expressed co-ordinately under a variety of
Regulation of Collagen Synthesis by
12722
Fibrogenic Factor
the run-off transcription assay? we believe that thefibrogenic factor needs to be “activated intracellularly. Alternatively, there may be an intermediate messenger molecule between the plasma membrane action and/or entry event and the nuclear transcriptional event. Some of these aspects of fibrogenic factor actionwill beinvestigated as thefibrogenic factor is further purified. It is difficult to extrapolate the in vitro results presented here to their significance in the pathogenetic mechanism of hepatic fibrosis in uiuo. Although our preliminary results suggest that thefibrogenic factor can stimulate type I collagen biosynthesis in the primary hepatocytes, we do not yet know the primary cellular target of fibrogenic factor in uiuo. Finally, it is also uncertain whether the accelerated rate of collagen production by hepatocytes in vivo alone would completely account for the increased collagen deposition during hepatic fibrosis.
I
I
I
Acknowledgments-We are grateful to Dr. F. Ramirez, Rutgers University Medical School, Piscataway, NJ and Dr. Bruce Paterson, National Cancer Institute for supplying us with recombinant cDNA plasmids used inthese studies. We also acknowledge the expert technical assistance of Yolanda Towner and Pam Asa.
I
3
6 9 12 FlBROGENlC FACTOR TREATMENT (HOURS)
FIG. 3. Time-course of accumulation of al(1). a2(I), and 8actin mRNAs in fibrogenic factor-treated rat fibroblasts. Cell cultures were treated for various times as depicted, total RNA was extracted, and poly(A+)RNA was selected. Five 2-fold serial dilutions (starting with 2.0 fig) were spotted onnitrocellulose filter and hybridized with nick-translated cDNA plasmids (see “Experimental Procedures’’). Areas representing different dots were cut out with the help of autoradiographs and radioactivity was quantitated by scintillation spectrometer. The average value of three different determinations is plotted. All values were normalized against the zero-time untreated control cultures.
TABLEI11 Effect of fibrogenic factor on transcriptional rates of collagen and 8actin genes Nuclei were isolated from control or fibrogenic factor-treated (12 h after treatment) ratfibroblasts. Purified, radiolabeled nascent RNA (1 X lo’ cpm) from each sample was hybridized to 2 pgofDNA immobilized on nitrocellulose. The average values from three different determinations, as parts/million, are denoted. The transcription of all three genes was completely inhibited by 0.75 p~ a-amanitin. Fibrogenic factor-treated PPm
al(1) 123.5 a2(1) @-Actin 81.7 pBR322 1.6 X Charon 4A
4.1
26.1 29.2 67.1 2.1 1.8
Transcriptional stimulation -fold
4.7 119.6 1.2
2.1
experimental conditions (35-37), the ratio of type I to type I11 collagen has been shown to be increased in the fibrotic human liver (4). Therefore, a direct measurement of pro(ul(II1) mRNA sequences in fibrogenic factor-treated cells would be very important. Efforts to develop a suitable cDNA probe for type I11 collagen arecurrently underway in our laboratory. Several fundamental aspects of the mechanism of actions of the fibrogenic factor remain to be investigated. For instance, we do not know whether the entry of the fibrogenic factor into the cells is necessary for its action. Since the addition of the factor directly into thenuclear extract fails to stimulate transcriptional activity of type I collagen genes in
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