Session IV. Regulation of Gene Expression (Part I)

Enhanced Collagen Gene Expression in Fibroblast Cultures Treated with AlI-Trans-Retinoic Acid: Evidence for Up-Regulation of the a2(I) Promoter Activity.

Anne Marie Angeles, Veli-Matti Kiihiiri, Yue Qiu Chen, Francesco Ramirez and Jouni Uitto

Jefferson Medical College, Philadelphia, PA and Mt. Sinai School of Med., New York, NY, USA.

Previous studies have suggested that topical application of all-trans-retinoic acid (RA) can alter the metabolism of collagen in the skin. In this study, the effect of RA on type I collagen promoter activity was tested in transient transfec­tions of cultured fibroblasts. Mouse NIH-3T3 cells were grown under serum-free conditions and transfected with a human a2(I) collagen promoter/CAT reporter gene con­struct. After a 24-h incubation following the transfection, RA was added in concentrations varying from 10-5 to 10- 9

M. The cells were harvested 24 h later, and CAT activity, an index of the a2(1) collagen promoter activity, was deter­mined by incubation with [14C]-chloramphenicol as sub­strate. Incubation of cells with increasing concentrations of RA resulted in enhanced collagen promoter activity; the highest enhancement, ~ 14-fold, was noted at 10-5 M con­centration. Parallel transfections with pEP5CAT, an elastin promoter/CAT construct, or with pBSOCAT, a promoter­less CAT construct, revealed that the expression of these plasmids was not affected by RA. Assay of mRNA steady­state levels in 3T3 and human fibroblast cells incubated with RA under similar conditions also suggested activation of type I collagen gene expression. Thus, our results suggest that RA, under culture conditions utilized, upregulates col­lagen promoter activity. These observations may relate to clinical and histopathologic observations made in patients treated with RA for cutaneous aging.

Transcriptional Activation of Type I Collagen Gene Expression by TGF-~ 1 is Inhibited by TNF-a and IFN-y at Different Levels.

Yue Qiu Chen, Yeti-Matti Kiihiiri, Ming Wan Su, Francesco Ramirez and Jouni Uitto

Departments of Dermatology, Jefferson Medical College, Philadelphia, P A and Mt. Sinai School of Medicine, New York, NY, USA.

Activation of type I collagen gene expression by fibro­blasts is an essential component of pathogenesis in fibrotic diseases. Recently, the role of transforming growth factor-~ (TGF-~) in this phenomenon has been emphasized. To elucidate the mechanisms involved in the activation of type I collagen gene expression, we studied the combined effects of TGF-~l, tumor necrosis factor-a (TNF-a) and interfe­ron-y (IFN-y) on the activity of human proa2(1) collagen promoter/CAT construct in transient transfections of human skin fibroblasts and mouse NIH-3T3 cells. Expo­sure of transfected cells to TGF-~l (0.1-10 nglml) resulted in a dose-dependent increase in the proa2(I) promoter activity, and the maximal stimulation (>20-fold) was obtained with 5 nglml. Cells incubated with TGF-~l (5 ngl ml) also showed elevated type I collagen mRNA steady­state levels. Incubation of fibroblasts with TGF-~l (5 ng/ ml) and IFN-y (1000 U/ml) resulted in a marked decrease in type I collagen mRNA levels compared to the cells treated with TGF-~l alone. IFN-y, however, had only a minimal effect on proa2(1) collagen promoter activity either alone or in combination with TGF-~l. Exposure of skin fibroblasts to TNF-a in combination with TGF-~l also resulted in marked decrease in type I collagen mRNA levels compared to cells treated with TGF-~l alone. A similar decrease in proa2(1) collagen promoter activity was also detected in cells exposed to TNF-a alone or in combination with TGF­~l. These results demonstrate that both IFN-y and TNF-a are potent inhibitors ofTGF-~l induced activation of type I collagen gene expression in fibroblasts. Since IFN-y had no effect on the transcriptional activity of type I collagen genes, the inhibiting effect takes place at the post-transcriptional level; whereas TNF-a exerts its inhibitory effect at tran­scriptionallevel.

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Transforming Growth F actor-Beta (TGF-~ 1) Inhibits Growth and Differentially Affects Collagen Expression in Cultured Rat Kidney Epithelial Cells.

Joseph]. Creely, Samuel]. DiMari, Anita M. Howe and Michael A. Haralson

Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN, USA.

The presence of increased amounts and possibly different types of extracellular matrix (ECM) components charac­terizes the end-stage of many renal diseases. Although the pathogenesis of these changes is likely to be multiple and complex, peptide growth factors released during inflamma­tion may be mediators of this altered matrix expression. One approach to obtain insights into this issue is to assess the effects of growth factors on clonal populations of kid­ney epithelial cells. This laboratory has previously reported that NRK52E cells, and epithelioid line derived from nor­mal rat kidney tubular epithelial cells, synthesize types I, III, IV and V collagen, and we have documented that epidermal growth factor (EGF) stimulates the growth of but inhibits collagen production by NRK52E cells. In order to extend our previous studies, we have examined the effects of TGF­~1 on NRK52E cells under conditions of acute (24 h) and chronic exposure (120 h). The secreted and cell-associated collagens produced by NRK cells in both situations were isolated after limited pepsin digestion and differential salt fractionation. Acute exposure to TGF-~l was found to inhibit growth by ~ 15%, to increase type I collagen synthe­sis ~ 70%, but to minimally alter the production of types III, IV and V molecules. Consequently, total collagen pro­duction was increased ~30% and the ratio of type I to type III molecules increased from 0.9 to 1.5 upon acute exposure to this cytokine. Chronic exposure to TGF-~l decreased growth ~50% and stimulated type I collagen production 8-fold. In contrast to effects of acute exposure, the synthesis of types III and V collagen was increased but type IV production remained unaffected by chronic expo­sure to this peptide. Thus conditions of chronic exposure to TGF-~l resulted in total collagen production being enhanced -5-fold and the ratio of type I to type III collagen being increased to 2.9. These findings establish that TGF­~1 and EGF exert directly opposite effects on NRK52E cells. Furthermore, as total collagen production was increased under both conditions of exposure to TGF-~l, these observations suggest that this inflammatory cytokine may be a positive mediator leading to matrix accumulation in kidney disease. (Supported by NIH Grant DK-39 261)

Collagenase Expression in the Lungs of Transgenic Mice.

]. D'Armiento\ H. Tsujinoue\ Y. Okada2, S. Dalal\

R. Berg l and K. Chada I

1 Graduate Program in Biochemistry and Molecular Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway NJ, USA and 2 Kanazawa University, Kanazawa, Japan.

Tissue remodeling is often accompanied by increased synthesis of collagen and increased collagenase activity. In order to study the contribution of interstitial collagenase to tissue injury, repair and remodeling, the human collagenase gene, under the control of an adult specific promoter, was microinjected into fertilized mouse eggs. Initially, 79 founder mice were generated, of which 14 have been shown to be positive for the transgene. Of these 14, 6 died within 3 weeks of age. Histological analysis of the organs demonstrated emphysematous changes in the lungs of these mice. The remaining 8 transgenic mice lived and 3 showed no visible abnormalities. Although they harbor the trans­gene, mice in the latter three lines have been shown not to ex~ress the. collagenase transgene. Two of the remaining 5ltnes of mice have been analyzed in detail, lines Col 50 and Col 52. In these lines mice are generated whereby the prog­eny suffer from emphysema and die. All mice which suffer from emphysema express the trans gene in the lung. In another line Col34 there are various integrations of the transgene into the mouse chromosome. Each integration is being analyzed separately but mice which obtain a combi­nation of integrations have expressed the transgene solely in the lung and develop emphysema between 1-3 weeks of age. More detailed histological analysis is continuing in order to identify the damage to the lung and also other organs of these mice. These studies are expected to lead to a better understanding of the role of metalloproteinases in tissue injury and repair.

Basic Fibroblast Growth Factor and Transforming Growth Factor-a Antagonize the Stimulation of Elastin Production by Transforming Growth F actor-~ 1 in Fibroblasts and Smooth Muscle Cells.

].M. Davidson and O. Zoia

Dept. of Pathology, Vanderbilt Univ. and VA Medical Center Nashville, TN 37212, USA. '

Transforming growth factor-~l (TGF~) binds to one or more cell surface receptors on virtually all cells, a major effect being the induction of matrix accumulation. Basic fi-

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broblast growth factor (bFGF) acts through its receptor to stimulate mitogenesis, angiogenesis, and protease expres­sion; while transforming growth factor-a (TGFa) predom­inantly causes cell proliferation. TGFp and bFGF bind to matrix components, and all of these cytokines are likely to be present in varying proportions at sites of injury, for example. We have examined the ability of bFGF or TGFa to modulate the dose-dependent stimulation of elastin syn­thesis elicited by TGFp in two sets of cultured cells at confluency, the porcine aortic vascular smooth muscle cell and the porcine skin fibroblast. Although low concentra­tions (1 ng/ml) of bFGF or TGFa could moderately aug­ment TGFp-induced elastin production by these target cells, the dominant effect of 2:: 1 0 ng/ml was dose-dependent inhibition ofTGF-p stimulated elastin production. Some of the reduction may have been to reprogramming of cells for proliferation, and at least part of the effect was due to reduced steady-state levels of elastin mRNA. These mito­gens may directly interfere with TGFp signaling pathways leading to matrix accumulation, or they may augment the degradation of newly-synthesized elastin. These results emphasize the complex effects of cytokine combinations likely to be influencing cells during development, growth, and tissue repair. (Supported in part by NIH grants AG06528, GM37387, the VA, Genentech, Inc., and Synergen, Inc.)

Changes of Collagen Patterns in Chondrocytes During Retinoic Acid Induced Dedifferentiation.

U. Dietz, K. von der Mark and W. Bertling

Clin. Res. Units Rheumatology, Max-Planck Society Schwabachanlage 10, 8520 Erlangen, FRG.

Modulation of collagen phenotype of chondrocytes by retonic acid as an in vitro system helps to elucidate the mechanism of transcriptional and posttranscriptional con­trol of collagen synthesis.

Freshly isolated chondrocytes express the cartilage specific collagens type II, IX, XI. They change to collagen type I, III and V synthesis after dedifferentiation caused by repeated passaging, treatment with BUdR, phorbolesters, or retinoic acid. Untranslated a1(I) mRNA of freshly iso­lated chondrocytes becomes translatable after dedifferenti­ation. The aim of this study is to elucidate the translational control of a1(I) mRNA isolated from functional and from retinoic acid-modulated chondrocytes.

Modulation or dedifferentiation of sternal chondrocytes from 17d old chicken embryos was induced in monolayer cultures by treatment with 2 f.tg/ml of retinoic acid. We compared the change in expression of the respective colla-

gens at various dedifferentiation states at mRNA level, and at protein level.

As described in other systems type I collagen was not synthesized by freshly isolated chondrocytes, however, sig­nificant levels of mRNA for both a-chains of type I collagen were detectable by northern hybridization.

In the course of dedifferentiation a rapid increase in a1(I) and a2(I) collagen mRNA levels were found coordinately with an increase in a1(I) and a2(I) collagen synthesis; while a1(II) mRNA levels and its translation declined within 2-3 days of retinoic acid treatment. This indicates tran­scriptional and posttranscriptional regulation of a1 (I) and a2(I) collagen genes.

Type IX collagen is presumably involved in the assembly of type II collagen fibers, suggesting coordinate regulation of type II and type IX collagen synthesis. By northern hy­bridization with a a1(IX) specific probe, a rapid decrease in a1 (IX) levels was observed coordinately with the decrease in a1 (II) mRNA levels.

Messenger RNA from type X collagen, a collagen of hypertrophic cartilage also disappeared under the influence of retinoic acid. After one week in culture its expression however increased again, suggesting hypertrophic differ­entiation of a fraction of the cells.

Ascorbate Modulates the Activity of FGF but not Transforming Growth F actor-B on Collagen Synthesis in Human Dermal Fibroblasts.

Jeffrey c. Geesin2, 3, LauraJ. Hendricks3, Joel S. Gordon3

and Richard A. Berg!

1 University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2 Department of Biochemistry, Rutgers University, Piscataway, NJ and 3 Johnson and Johnson Consumer Products, Inc., Skillman, NJ, USA.

Ascorbate has been shown to stimulate collagen synthesis through induction of lipid peroxidation which leads to increased transcription of the collagen genes. The mecha­nism by which lipid peroxidation stimulates collagen syn­thesis is unknown, however, disruption of cell membranes may alter the effects of serum growth factors which may cause a change in gene expression. To test this hypothesis, we treated dermal fibroblasts with transforming growth factor-p (4-2500pM) (TGF-P), epidermal growth factor (0.8-500ng/ml) (EGF), interleukin-1 (0.016-10U/ml) (IL-1), platelet-derived growth factor (0.004-1.43 U/ml) (PDGF), or fibroblast growth factor (0.4-143 ng/ml) (FGF) in the presence of lipid peroxidation stimulating (200 f.tM) and nonstimulating (lIlM) concentrations of

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ascorbate. EGF and IL-1 had no effect on collagen synthesis while PDGF stimulated collagen synthesis two-fold at either concentration of ascorbate. FGF affected collagen synthesis only in the presence of 200 11M ascorbate, produc­ing both a stimulation (0.4-2 ng/ml) and an inhibition (250 ng/ml). TGF-~ stimulated collagen synthesis, produc­ing a four-fold increase at 1 11M ascorbate and an eleven­fold increase at 200 11M ascorbate. These results indicate that regulation of collagen synthesis by ascorbate and TGF­~ occur via different pathways and that ascorbate can mod­ulate the activity of FGF on collagen synthesis.

Establishment of Putative TSK/TSK Homozygous cell Lines that Display Marked Elevation in Type I Collagen Gene Expression.

Sergio A. Jimenez, Maureen Bocchieri, Constantinos D. Constantinou, Paul Henriksen, Charles Hanning and Ronald Yankowski

Division of Rheumatology, Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107, USA.

The TSK (tight skin) is an autosomal dominant mutation of the BIO.D2 (5SN)/Sn mouse strain characterized by the excessive production of extracellular matrix. The homozy­gous TSK mutation is lethal and causes death and resorp­tion of affected embryos in utero. Identification of the TSK gene and of the molecular alterations responsible for the TSK phenotype has not been possible because of the lack of TSKITSK homozygous cell lines. The purpose of this work was to attempt the establishment of cell lines that may express the homozygous phenotype. Pregnant TSK females were sacrificed S days post-coitus and viable embryos were submitted to cell isolation by sequential enzymatic diges­tions. We successfully established and expanded S cell lines. Protein and collagen biosynthesis was examined in con­fluent cultures labeled with e4 C]-proline. The steady-state mRNA levels for Type I collagen were determined by Northern and dotblot hybridizations with a murine se­quence-specific eDNA. We found that three cell lines exhi­bited a step-wise progressive increase in collagen produc­tion. One cell line was comparable to normal cells; whereas the second, showed a 3- to 4-fold increase in collagen and protein biosynthesis. The third cell line displayed an inter­mediate level. The elevated phenotype was maintained for up to 16 passages in culture. The steady-state mRNA levels for Type I collagen showed parallel changes. These results suggest that the cell line with the highest levels of Type I col­lagen gene expression is a homozygous TSKlTSK; whereas the cell line with intermediate levels is a heterozygous TSK/ +. The availability of homozygous TSKITSK cell lines will

greatly facilitate the identification of the TSK gene and of the molecular mechanisms that result in the exaggerated collagen gene expression characteristic of this mutant.

Analysis of the Human a 1 (I) Procollagen Gene Promoter.

Sergio A. Jimenez, Anne Olsen, Janet Herhal and Julie Koch

Rheumatology Division, Department of Medicine and JIMM, Jef­ferson Medical College, Philadelphia, PA 19107, USA.

To gain further understanding of the mechanisms responsible for the regulation of expression of human type I collagen genes under normal and pathologic conditions, we cloned and characterized 5 kb of the human a1(I) procolla­gen gene promoter. Reporter gene constructs were prepared in which the a1(I) promoter with varying lengths of up­stream sequence was fused to the bacterial CAT gene. A series of constructs with a1(I) promoter endpoints from -5 kb to -S4 bp were analzyed. The 3' endpoint of all a1(I) promoter fragments was +42 to assure that transla­tion initiates only at the CAT ATG codon. The a1 (I) CAT plasmids were transfected into NIH 3T3 fibroblasts, and cell extracts were analyzed for CAT activity. To control for differences in transfection efficiency all results were nor­malized to a co-transfected reference plasmid containing the SV40 promoter and enhancer fused to a rat alkaline phosphatase eDNA. The results showed maximal expres­sion with 5' endpoints from -S04 bp to -174 bp. Further deletion to -S4 bp caused a significant reduction in activ­ity, although activity was over 10-fold higher than with the promoterless CAT plasmid. Plasmids containing much longer a1(I) gene fragments (-2.3kb or -5kb) showed much lower activity, suggesting the presence of inhibitory sequences. To assess the activity of the a1(I) promoter in a cell line which does not normally express type I collagen, the al(I) CAT plasmids with endpoints at -5, -2.3, and -O.S kb were transfected into HeLa cells. The a1(I) pro­moter was much less active in HeLa cells than in 3T3 fibro­blasts. In 3T3 cells, the -O.S kb construct expressed about 26 times as much CAT activity as the promoterless con­struct, while in HeLa cells the relative activity was tenfold lower. Extending the upstream sequences to - 2.3 and - 5 kb increased the activity about two-fold in He La cells, whereas these sequences were quite inhibitory in 3T3 cells. Thus, there are both positive and negative regulatory regions in the a1(I) promoter, which appear to function differently in collagen-producing and non-producing cells.

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Positive and Negative Control Elements in the Promoter of Two Coordinately Expressed Type I Collagen Genes.

Gerard Karsenty, Roberto Ravazzolo and Benoit de Crombrugghe

Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

Type I collagen, a major biosynthetic product of fibro­blastic cells, is a heterotrimeric protein composed of two a1 and one a2 chain. This same 2:1 stoichiometry is found for the rates of synthesis of the two chains, for the level of the corresponding mRNAs and for their rates of synthesis, suggesting that these two genes are coordinately regulated. Several cis-acting elements and their cognate binding fac­tors have been identified in each gene. In the promoter of the a1(I) gene between -220 and -70 four different cis­acting elements were identified. Three different factors present in nuclear extracts of NIH 3T3 fibroblasts bind to these elements. Competition analysis and site specific mutagenesis indicate that the binding of these factors is specific. DNA transfection experiments using wild-type and mutant a1 (I) promoter -CAT chimeric genes show that two of these factors called Inhibitory Factor 1 and 2 (IF1 and IF2) act as transcriptional inhibitors. IF2 is a metallo­protein requiring zinc for optimum binding to DNA and has been purified to near homogeneity as shown by SDS gel. The third factor, a heterodimeric CCAAT binding protein, acts as a transcriptional activator. These three factors interact with similar sequences approximately in the same location in the a2(I) gene. DNA transfection experiments using mutant a2(1) promoter-CAT chimeric genes suggest that all three factors have similar functions in the a2(I) and a 1 (I) ( promoters. We propose that these factors participate in the coordinate control of these genes in response to different physiological stimuli.

Complex Regulation of Collagen Gene Expression in Normal and Modulated Corneal Endothelial Cells.

EunDuckP. Kay

Doheny Eye Institute and the Dept. of Ophthalmology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA.

The mechanism by which corneal endothelial modula­tion takes place has been shown to involve a phenotypic switch in collagen gene expression. We have previously shown that collagen synthesis does not correlate with the

steady-state collagen RNA levels; substantial amounts of type I collagen RNAs in endothelial cells are not translated into the respective protein. The present studies seek to determine the level of control mechanism in collagen gene expression in normal and modulated endothelial cells. Northern-blot analysis using cloned rabbit types I and IV cDNA probes (same species as RNA sources) demonstrated the presence of specific mRNA transcripts for collagen types I and IV in the endothelial cells, although the steady­state levels of these mRNAs in the modulated endothelial cells are low. The turnover rate of collagen RNAs was determined: normal cells contain very stable a2(1) and a2(IV) mRNAs whose half-lives exceed 24 hours; while the same messages decay with an apparent half-life of approxi­mately 4-8 hours in the modulated cells, suggesting that corneal endothelial modulation exerts a profound effect on the stability of a2(I) and a2(IV) collagen RNAs. Using nuclear run-off transcription, the rate of transcription in normal cells was found to be slightly higher than that of modulated cells. When the relative rate of collagen gene transcription was compared, a2(1) transcription is the lower and a2(IV) is transcribed at a higher rate in both cells. During early stage of modulation, there is a differential effect on both transcription rate and the steady-state levels of these collagen RNAs: preferential enhancement of a2(I) collagen RNA is observed within two passages in the pres­ence of modulation factors released from polymorphonuc­lear leukocytes. The present findings suggest that synthesis of these collagens is primarily translation ally regulated in corneal endothelial cells. However, transcriptional and translational regulation are involved during modulation of endothelial cells.

The Role of Homeobox Genes and Transgenes in Mice.

M. Kessel, R. Balling and P. Gruss

Department of Molecular Cell Biology, Max-Planck-Institute of Biophysical Chemistry, Gottingen, FRG.

The murine genome contains four clusters of genes with homology to a sequence motif, the homeobox, originally found in the homeotic Drosophila gene antennapedia. One example of these around 33 genes is the Hox-1.1 gene located in the Hox-1 cluster on chromosome 6. It is ex­pressed in the developing neuroectoderm and mesoderm of postgastrulation embryos. Hox-1.1 RNA is not present in teratocarcinoma stem cells, but can be induced by retinoic acid, which also initiates differentiation of these cells.

We have generated murine gain of function mutants by introduction of a ubiquitously expressed Hox-1.1 trans­gene into the mouse genome. The transgene interferes with

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cranial neural crest cells and with the somitic mesoderm at the craniocervical transition. The pathology of resulting craniofacial and vertebral abnormalities will be discussed with regard to a proposed developmental control function of Hox-1.1. A developmental model will be presented involving the morphogen retinoic acid and its influence on murine homeobox genes.

Silencer in the Regulatory Region of the Cartilage Matrix Protein Gene.

Ibolya Kiss, Zsuzsa Bosze, Piroska Szabo, Rencendorj Altanchimeg, Endre Barta and Ferenc Deik

Institute of Biochemistry and Genetics, Biological Research Center, Szeged, Hungary 6701.

Cartilage matrix protein (CMP) is a major noncolla­genous protein of hyalin cartilage synthesized by chondro­cytes in a developmentally regulated manner. CMP has a mosaic structure indicating common evolutionary origin with various protein families. The regulation of the chicken CMP gene was investigated in transient expression experi­ments. Selected fragments from the 5-flanking region and the first intron of the gene were coupled to the chloram­phenicol acetyltransferase reporter gene driven from homologous as well as heterologous promoters and the level of gene expression was monitored in various cells. A minimal promoter, which included sequences extending only 15 bp upstream of the TAT A box, was sufficient to drive cell-type specific expression. Apart from the promoter four distinct cis-acting regulatory regions have been iden­tified. 11 A positive regulatory region, located from -1136 to -334 bp relative to the TAT A motif, facilitated tran­scriptional activity. 2/ A tissue-specific enhancer was found to reside in a 388-bp fragment of the first intron. This fragment stimulated gene expression in chondrocytes, but caused severe repression in fibroblasts when inserted upstream of the minimal promoter. 3/ A silencer, located immediately upstream of the TAT A box, inhibited tran­scription in both orientations and over large distances. It exerted negative regulation on the activity of the heterolog­ous thymidine kinase promoter and the RSV enhancer both in chondrocytes and fibroblasts, with a more pronounced effect in the opposite than in the direct orientation. 4/ A chondrocyte specific negative regulatory region was detected 6.8 to 10.7 kb upstream of the CMP gene. The pos­sible involvement of the control regions in the expression of the gene is discussed.

Pre-Translational Regulation of Extracellular Matrix Macromolecules and Collagenase Expression in Fibroblasts by Mechanical Forces.

Ch. M. Lapiere, Ch. A. Lambert and B. V. Nusgens

Lab. Exp. Dermatology, University of Liege, Belgium

In vivo, the extracellular matrix modulates the pheno­type of the mesenchymal cells both through its biochemical composition and its mechanical properties. The biochemi­cal effect can be mimicked by culture in freely retracting collagen type I gels (free lattices). In this study, the mechani­cal effect was investigated in similar gels maintained under tension (stretched lattices). The overall proteins and colla­gen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of 10 and 20 in free lattices as compared to monolayers and increased by a factor of 4 and 6 in stretched versus free lattices. As assayed by the degradation of [3Hl-collagen type I by trypsin-activated medium conditioned by fibro­blasts under the three models of culture, collagenase activ­ity was inversely regulated and increased in lattices when compared to monolayer culture. It was 4 times higher in free lattices than in stretched lattices. The steady-state level of mRNA coding for procollagen types I, III and VI poly­peptides, fibronectin, elastin, ~-actin and procollagenase was determined by cDNA hybridization. The mRNA cod­ing for ~-actin as well as for the various extracellular matrix macromolecules were increased in stretched when com­pared to free lattices while the level of procollagenase mRNA was lower. These data demonstrate the existence of a control of the fibroblasts phenotype performed by the mechanical forces. This regulation operates, at least in part, at a pretranslationallevel.

Regulation of Extracellular Matrix Expression and Mitogenesis by Transforming Growth Factor B 1 in Vascular Smooth Muscle Cells.

G. Liau and M. F. Janat

American Red Cross, Holland Laboratory, Rockville, MD 20855, USA.

Thrombospondin and type IV collagen are two extracel­lular matrix proteins synthesized by vascular smooth mus­cle cells (SMC) that respectively have important roles in SMC growth and differentiation. Regulation of the expres­sion of these genes by growth factors have important impli­cations for vascular diseases. We have examined the role of transforming growth factor ~1 (TGF-~l) in the regulation

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of these extracellular matrix genes as well as in the regula­tion of SMC mitogenesis. We found that TGF-~ 1 treatment of vascular SMC induced a prolonged increase in steady­state mRNA levels of thrombospondin and at (IV) collagen. The increase began at approximately 2 h, peaked at 24 h and remained considerably elevated 48 h after growth fac­tor addition. The modulation of thrombospondin level by TGF-~1 contrasted sharply with that observe for platelet­derived growth factor (PDGF) which induced a rapid increase in thrombospondin mRNA level, peaking by 2 h and returning to control levels by approximately 6 h after treatment. The TGF-~1 induced increase in thrombospon­din and al (IV) collagen mRNA appeared to occur by a direct mechanism since it was not abolished by cyclohex­amide treatment. Examination of conditioned media revealed that the secretion of at least 4 polypeptides with apparent MW of 240,000, 180,000, 170,000 and 62,000 markedly increased after TGF-~1 addition. The 180,000 MW polypeptide was identified as thrombospon­din using a monoclonal antibody. Addition of both TGF-~1 and PDGF to SMC resulted in an augmentation of the increase in thrombospondin induced by PDGF or by TGF­~1 alone. TGF-~1 alone was unable to enhance [3HJthy­midine incorporation into DNA in 3 smooth muscle cell strains. However, in one smooth muscle cell strain examined, a weak and delayed response was observed. Expression of c-fos or c-myc was detected in this cell strain in their induction by PDGF. In all 4 cell strains TGF-~1 was able to strongly augment the ability of PDGF to induce DNA synthesis. Our results indicate that induction of thrombospondin expression by TGF-~1 and by PDGF occurs by distinct mechanisms and that TGF-~1 alone was a poor mitogen but it was able to strongly synergize with other growth factors to induce DNA synthesis.

Temporal and Spatial Analysis of Collagen Gene Expression During Human Articular Cartilage Development by In Situ Hybridization.

F. Mallein-Gerin, 1. Treilleux-Salles, D. Le Guellec and D.Herbage

Histologie Experimentale, CNRS URA 244, Universite Claude Bernard 69622 Villeurbanne, France.

In situ hybridization with [32PJ-labeled double-stranded eDNA or oligonucleotide probes have been used to visual­ize and localize types I, II and III collagen mRNAs sequences in femoral articular cartilage during fetal human development. The appearance of the corresponding gene products was monitored by immunofluorescence staining. The probes used were (i) a 300-bp double-stranded al(I)

collagen eDNA insert cut out of the Hf 677 clone (Chu et aI., Nucl. Acid Res. 10: 5925 - 5934, 1982) containing the non-translated region. (ii) a 500-bp double-stranded al (III) collagen eDNA insert cut out of the Hf 934 clone (Chu et aI., J. BioI. Chem. 260: 4357 -4363, 1985) containing the non-translated region and (iii) a 33-oligomer probe corre­sponding to a specific al(II) C-propeptide region. Our analysis performed on serial paraffin sections reveals that after 18 weeks of fetal development, types I and III collagen mRNAs are only detected within about 4 superficial cells layers of the articular surface; whereas type II collagen mRNA sequences are only detected deeper in the cartilage. Analysis of the articular surface from a newborn shows that type II as well as types I and III collagen mRNAs are detected within the superficial cell layers up to the surface; whereas only type II mRNAs are detected in the deeper zone. However, in the zone where the perichondrium cov­ers the cartilage and for both stages studied here (18 weeks and newborn) types I, II and III collagen mRNAs are detected within about 2-cell layers immediately adjacent and underneath the perichondrium (which is labeled by types I and III probes); whereas only type II mRNAs are detected deeper. These data suggest that collagen gene expression in the articular surface and in the cartilage covered by the perichondrium is under different regulatory mechanisms.

Chromatin Structure and Transcriptional Regulation of the a2(I) Collagen Gene. Binding of Histone H1.03 Produced in E. Coli to the 5'-TTGGCAnnnTGCCAA-3' Motif on DNA.

Riitta-Maaria Mannermaa and Jouko Oikarinen

Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Qulu, Kajaanintie 52A, SF-90220 Qulu, Finland.

Histone HI binds to the nucleosome hinge region where two DNA strands enter and leave the complex. It is thought to trigger nucleosome aggregation to ordered solenoid structures and thereby to act as a eukaryotic repressor. HI binds preferentially to AfT-rich DNA regions such as the nuclear scaffold-associated regions and this interaction is thought to take place through its K-rich C tail. There is in addition a central homeodomain-like globular domain in HI which may be capable interaction with DNA. We have here cloned the chicken HI.03 gene and produced a lacZ­HI fusion protein in E. coli. From functional dissection data it is evident that the fusion protein binds to the CTFI NF-I recognition motif through its globular domain. These data confirm thus the fact that there are several DNA contact domains in HI. The C tail interacts in a non-

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sequence-specific manner while DNA recognition by the globular domain is sequence-dependent. The present results are in agreement with the hypothesis that H1 recog­nizes a specific DNA motif through its globular domain and that the C tail serves to gather the two DNA strands and sealing the nucleosome. The role of H1 binding to the - 300 CTFINF-I site in mouse a2(I) collagen promoter in the chromatin formation and counteraction of this process by other regulatory factors will be studied further.

Chromatin Structure and Transcriptional Regulation of the u2(I) Collagen Gene. Nucleotide Modulation of Binding of Histone H 1 to the Nucleosome Hinge Region.

Jouko Oikarinen and Riitta-Maaria Mannermaa

Collagen Research Unit, Biocenter and Department of Medical Biochemisty, University of Oulu, Kajaanintie 52A, SF-90220 Oulu, Finland.

Several regulatory elements have recently been charac­terized in the promoter region of the mouse a2(I) collagen gene. Some of these have also been shown to be of crucial importance for adequate tissue-specific expression of the gene. There is a CTFINF-I binding site at -300 with respect to the start of transcription, and the protein which binds to this region is different from that one which binds to a CCAAT box at -80. Histone H1 is one of the proteins that recognize the -300 motif and therefore we have initiated studies on the role of this presumptive eukaryotic repressor in the regulation of chromatin formation along the a2(I) promoter. It is shown here that there is a nucleotide binding site in H1, preferentially interacting with ATP and GTP. The nucleotides stimulate binding of H1 to its recognition motif that resembles that of CTFINF-I, and may become hydrolyzed upon binding of H1 to DNA. Acidic domains such as the ones identified here in histones H3 and H4 interact with H1 and stimulate nucleotide hydrolysis. The nucleotide hydrolysis by H1-H3-H4 may thus be of impor­tance in H1-directed nucleosome and chromatin assembly and in subsequent repression of the eukaryotic chromatin activity in general. Interference by transcriptional activators with this process may serve as a mechanism of regulating gene expression. This approach will be applied in the future to studies on negative and positive regulation of the a2(I) collagen gene expression.

Co-Localization of Type I and VI Collagen and Transforming Growth Factor-~l mRNAs in Keloid Tissue.

Juha Peltonen, Li Li Hsiao, SirkkuJaakkola, Stephan Sollberg, Monique Aumailley, Rupert Timpl, Mon-Li Chu and Jouni Uitto

Departments of Dermatology and Biochemistry and Molecular Biology, Jefferson Medical College and Jefferson Institute of Mo­lecular Medicine, Philadelphia, PA, USA and Max-Planck-Institut fiir Biochemie, Martinsried, West Germany.

The expression of collagen types I, III and VI was exam­ined in nine cases with classic keloids. In situ hybridizations localized active expression of type I and VI collagen genes to the areas apparently representative of the expanding border of the lesion. Also, active expression of these colla­gen genes was detectd in the proximity of blood vessels and around mononuclear cell infiltrates within the lesions. Slot­blot hybridizations of total RNA isolated from keloids suggested that, in most cases, the expression of type I and VI collagen genes was selectively enhanced, as compared with type III collagen gene expression. On the average, proa 1 (I) collagen mRNA levels were 2- to 3-fold higher, and a1(VI) collagen mRNA levels were 4- to 6-fold higher in keloids than in control skin samples. The levels of type proal (III) collagen mRNAs were increased in keloids by about 1.4-fold.

Immunohistochemistry indicated the presence of type VI collagen epitopes within the keloids, and the presence of type VI collagen was also quantitatively demonstrated by protein isolation methods. The expression of transforming growth factor-~l (TGF-~l) gene in keloids was examined by peroxidase anti-peroxidase and in situ hybridization techniques. TGF-~l protein and mRNA were detected in areas active in type I and type VI collagen gene expression, indicating that TGF-~l gene is transcribed and the corre­sponding protein is deposited in keloids in areas of elevated collagen gene expression. Thus, TGF-~l may participate in the upregulation of type I and VI collagen gene expression, leading to collagen accumulation and development of clini­cally detectable keloids.