Gene expression of syndecans and betaglycan in ... - Springer Link

Cell Tissue Res (1996) 285:11–16

© Springer-Verlag 1996

161815.729 070514.125 120922.500 080516.666 192514.919 020520.375 180120*429

Gene expression of syndecans and betaglycan in isolated rat liver cells O.H. Weiner, M. Zoremba, A.M. Gressner Department of Clinical Chemistry and Central Laboratory, Philipps University, Baldingerstrasse, D-35033 Marburg, Germany &misc: Received: 20 July 1995 / Accepted: 6 February 1996

&Abstract. p.1: Membrane-bound heparan sulfate proteoglycans act as coreceptors for cytokines and are involved in cell-matrix or cell-cell adhesion. We have determined the gene expression of all four members of the syndecan-like integral membrane proteoglycans and of betaglycan, the transforming growth factor-β type III receptor, in various types of isolated hepatic cells of the rat. Fat-storing cells express syndecan-1, -2, -3, -4, and betaglycan. During the transformation of fat-storing cells into myofibroblasts (the key process in the development of liver cirrhosis), the levels of mRNA for syndecan-1, -3, and -4 remain constant, whereas the amount of syndecan-2 mRNA increases and that for betaglycan decreases. Liver macrophages express syndecan-3 and -4, but only small amounts of syndecan-1. Freshly isolated hepatocytes express only syndecan-1, -2, and -4, but fail to express betaglycan. During cultivation, hepatocytes start to express betaglycan. Syndecan-3, -4, and betaglycan are transcribed into one mRNA population, whereas syndecan-1 and -2 are expressed in different-sized mRNA populations. The data show that the genes of all tested membrane heparan sulfate proteoglycans are expressed by hepatic cells, but that each cell type is characterized by its specific heparan sulfate proteoglycan mRNA profile. &Key k w d : words: Membrane proteoglycans – Syndecans – Betaglycan – Gene expression – Liver cells – Rat (Sprague Dawley).

Introduction Proteoglycans (PG) are complex glycoconjugates harboring different types of glycosaminoglycan (GAG) chains, such as heparan sulfate (HS), chondroitin sulfate This study was supported by grants Gr 463 (10–1 and 9–2) from Deutsche Forschungsgemeinschaft. Correspondence to: A.M. Gressner&kl / f o n - b:c

isomers, or dermatan sulfate on a core protein. Cell-surface proteoglycans, which contain mainly HS GAG, participate in cell-matrix and cell-cell recognition processes and act as receptors for various pathogenic agents, such as the Herpes simplex virus (Elenius and Jalkanen 1994). After the finding that transforming growth factor-β (TGF-β) isoforms bind to the protein core of the membrane heparan sulfate proteoglycan (HSPG) betaglycan, an increasing number of cytokines, including hepatocyte growth factor (HGF), acidic and basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor, and the heparin-binding epidermal growth factor, have been identified to bind to the HS GAG moiety (Cheifetz et al. 1987; Segarini et al. 1987; Elenius and Jalkanen 1994). Although the precise function of this cytokine-HS binding is unclear, it is generally accepted that several cell surface HSPG take part in the regulation of cytokine stability, activation and conformation, and that they act as coreceptors facilitating the transfer of cytokines to their signaling receptors (Gallagher 1994). With regard to their domain structure, membrane-anchored HSPG are divided into two major groups. The first group includes the four syndecan-like integral membrane proteoglycans, termed syndecan-1, -2, -3, -4, and betaglycan, the TGF-β type III receptor (David 1993).They are transmembrane proteins with an amino-terminal extracellular domain containing clustered sites for the potential attachment of GAG chains, a potential proteasecleavage site near the single transmembrane domain, and a short cytoplasmic tail. The second group is made up of the glypican-related integral membrane proteoglycans that are anchored to the membrane via glycosyl phosphatidylinositol. The liver is composed of various cell types but mainly consists of hepatocytes, fat-storing cells, liver macrophages (Kupffer cells), and endothelial cells. Fat-storing cells, also called hepatic stellate or Ito cells, play a central pathogenetic role in liver fibrogenesis, because they are activated in necroinflammatory tissue areas and are transformed into myofibroblasts that are the main source of extracellular matrix (ECM) in fibrosis (Friedman

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1990; Gressner and Bachem 1990). Although normal liver tissue contains mainly HS GAG, as analyzed in detail by Gressner (1991) and Lyon et al. (1994), surprisingly little is known about the expression of individual membrane HSPG and their cellular source. Because of their central role in various aspects of cell regulation, liver membrane HSPG are suspected to play important roles in the maintainance of liver homeostasis, liver regeneration, neoplastic transformation, and liver fibrogenesis. We have therefore studied the gene expression of all four syndecans and of betaglycan in rat hepatocytes, fat-storing cells, myofibroblasts, and Kupffer cells.

Materials and methods Materials α-[32P]-dCTP (≈8000 Ci/mmol) and α-[33P]-dATP (≈2000 Ci/mmol) were purchased from NEN-Du Pont (Dreieich, Germany); the random primed DNA labeling kit, MMLV-reverse transcriptase, RNase inhibitor, and Taq-polymerase were from Boehringer Mannheim (Mannheim, Germany); dithioerythritol, and the oligo-(dT)12–18 primer were from Gibco BRL (Eggenstein, Germany); 2′-deoxynucleoside 5′-triphosphates (dNTPs) were from Pharmacia Biotech (Freiburg, Germany), and the Hybond N membrane was from Amersham (Braunschweig, Germany).

Isolation and culture of cells Fat-storing cells, Kupffer cells, and hepatocytes were prepared from male Sprague-Dawley rats (Lippische Versuchstieranstalt, Extertal, Germany) using the pronase or the pronase-collagenase method as described previously (Bachem et al. 1992). Accordingly, the purity of Kupffer cells was found to be greater than 85%, that of hepatocytes to be 98%, and that of fat-storing cells to be greater than 90%. For cultivation, freshly isolated hepatocytes or fat-storing cells were seeded in plastic wells at a density of 4.5×106 cells/75 cm2, or 3.8×106 cells/75 cm2, respectively, in Dulbecco’s modified Eagle medium containing 4 mmol/l L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), 10% (v/v) fetal calf serum, and in the case of hepatocytes, insulin (0.02 U/ml); they were cultured in a humidified atmosphere of 7.5% CO2 and 92.5% air. Fat-storing cells that had been passaged twice (≈16 d after isolation) were considered to be fully transformed into myofibroblasts according to several criteria, viz., αactin and desmin immunofluorescence staining, and the reduction in the number and size of their fat droplets.

RNA extraction and RNA analysis Total cellular RNA was extracted following established procedures (Chomczynski and Sacchi 1987). To increase sensitivity, approximately 30 µg (hepatocytes, fat-storing cells, and myofibroblasts) and 90 µg (Kupffer cells) total RNA were separated using a 1.2% agarose, 6% formaldehyde gel according to Noegel et al. (1985). cDNA fragments specific for rat syndecan-1, -2, -3, -4, and betaglycan, and for the ribosomal protein S6 were labeled using the random primed DNA labeling kit according to the instructions manual. In order to prepare ultrasensitive probes, high concentrations of α-[32P]-dCTP (≈8000 Ci/mmol) were used. Exposure was performed using two intensifying screens at –70°C and analyzed densitometrically.

To detect mRNAs for betaglycan, S6, decorin, and biglycan by the polymerase chain reaction (PCR), total RNA was reversely transcribed using the MMLV-reverse transcriptase, and the resultant cDNA was amplified by PCR. Briefly, 1 µg RNA was denatured by heating for 10 min at 70°C and added to a solution containing 500 nM oligo (dT)12–18 primer and 500 nM of each dNTP. Each cDNA synthesis was performed in a total volume of 22.5 µl for 60 min at 37°C and terminated by incubating at 95°C for 10 min. PCR was carried out with 150 pmol primer pairs and 1 µl cDNA in 50 µl 10 mM TRIS-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.4 mM dNTPs, and 0.5 U Taq DNA polymerase, for 39 cycles at 94°C for 35 s, and then at 55°C for 45 s for betaglycan, 58°C for decorin, 60°C for biglycan, or 59°C for S6, and 72°C for 60 s using a DNA thermocycler (Biometra, Göttingen, Germany). Equal volumes of the PCR mixes were analyzed by agarose gel electrophoresis. Several controls were performed. The quality of the synthesized cDNA and the concentration of cDNA were tested by using primer pairs specific for ribosomal protein S6. To ensure that contaminating plasmid or genomic DNA had not been amplified, RNA samples were amplified that had not been converted into cDNA by reverse transcriptase. The possibility of fat-storing cells contaminating the hepatocyte cultures was controlled by reverse transcriptase/PCR (RT-PCR) using primers for decorin and biglycan, viz., proteoglycans expressed exclusively in fat-storing cells (Meyer et al. 1992). The specificity of amplification was confirmed in each case by Southern-blot analysis. Primers were based on published nucleotide sequences for betaglycan (5′-GGC AGT GAC ATG CCA CCA C-3′; 5′-GTG AAC GAAGTC ACT GCT CC-3′; Wang et al. 1991; López-Casillas et al. 1991), S6 (5′-ATG AAG CTG AAT ATC TCC TTC-3′; 5′-TTA TTT TTG ACT GGA CTC AGA T-3′; Chan and Wool 1988), decorin (5′CGC GAA TTC CAT ATG GAT GAG GCC TCT GGC ATA ATC-3′; 5′-CGC GAA TTC GAG TTA CTT GTA GTT CCC AAG-3′; Abramson and Woessner 1992), and biglycan (5′-GAT GAG GAG GCT TCA GGC-3′, 5′-CCA AGT GAA GTT CCC TCA G-3′; Dreher et al. 1990), and custom-synthesized by Pharmacia Biotec (Freiburg, Germany). All given results were representative of three independent experiments.

Results Gene expression of syndecans in isolated hepatic cells Northern-blot experiments have been carried out in order to identify the expression of syndecan-1, -2, -3, and -4 in freshly isolated rat hepatocytes, fat-storing cells, Kupffer cells, and approximately 16-day-old transformed fatstoring cells (myofibroblasts). Syndecan-1 (formerly called syndecan) mRNA is expressed in fat-storing cells as a major 2.5-kb transcript and a less abundant 3.4-kb mRNA (Fig. 1A). These cells play the central pathogenetic role in liver fibrogenesis because they are activated in necroinflammatory tissue, for example, and are transformed into myofibroblasts that are the main source of the ECM in fibrosis (Friedman 1990; Gressner and Bachem 1990). During transformation into myofibroblasts, the amounts of both transcripts remain almost constant. Both transcripts are also found in hepatocytes, but in comparison with fat-storing cells, at much lower steady state levels. Using comparable amounts of RNA, no syndecan-1 mRNA is detectable in Kupffer cells. Both mRNAs are only found in Kupffer cells by increasing the amount of RNA about threefold. Syndecan-2 (fibroglycan) mRNA is found in fat-storing cells and in higher concentrations in hepatocytes

13

C

FS

B

MF

KC

B

C

PC

MF

FS

KC

PC

A

A

3.4 2.5 B

6

3.5

B

2.2 1.1 0.8 Fig. 2A, B. Gene expression of betaglycan in isolated hepatic cells. Total RNA from isolated hepatic cells was separated (as in Fig. 1) and labeled with ultrasensitive radioactive cDNA probes for (A) betaglycan, and (B) the ribosomal protein S6 used as an internal reference. Exposure was performed in both cases for 5 h. The size of the labeled RNAs is indicated right (in kb)&.i c/ :gf

C

5.6

(Fig. 1B). It increases about fivefold during the transformation of fat-storing cells into myofibroblasts. Interestingly, all cell types contain crosshybridizing 3.5-kb, 2.2kb, 1.1-kb, and 0.8-kb transcripts. Even after extreme overexposure, no detectable amounts of syndecan-2 can be found in Kupffer cells, although it is highly expressed in hepatocytes, fat-storing cells, and myofibroblasts. This underlines the purity of the Kupffer cell preparation and excludes that the observed syndecan-1 gene expression in Kupffer cells is the result of contaminating hepatocytes or fat-storing cells. Syndecan-3 (N-syndecan) mRNA is found as a single 5.6-kb transcript in fat-storing cells and myofibroblasts, whereas Kupffer cells express the major 5.6-kb mRNA and small amounts of the 5.0-kb and 4.6-kb transcripts (Fig. 1C). The syndecan-3 mRNA concentration increases slightly during the transformation of fat-storing cells to myofibroblasts. Syndecan-3 mRNA is not detectable in hepatocytes. Syndecan-4 (ryudocan, amphiglycan) mRNA is expressed as a 2.6-kb transcript in hepatocytes, Kupffer

D

2.6

Fig. 1A–D. Gene expression of syndecans in isolated hepatic cells. Total RNAs from freshly isolated fat-storing cells (FSC), in vitro transformed FSC (myofibroblasts, MFB), Kupffer cells (KC), and rat hepatocytes (PC) were separated on a 1.2% agarose gel, 6% formaldehyde gel, transferred to Hybond-N, and probed with ultrasensitive probes for (A) syndecan-1, (B) syndecan-2, (C) syndecan-3, (D) syndecan-4, and for the ribosomal protein S6 as a control (see Fig. 2B). Exposure was performed in the case of syndecan-1 for 18 h and in the other cases for 3–6 h. The size of the labeled RNAs is indicated right (in kb)&.i c/ :gf

Table 1. Gene expression and mRNA distribution of membrane heparan sulfate proteoglycans (HSPG) in isolated rat liver&l . .c/ : b&t t :b

Syndecan-1 Syndecan-2 Syndecan-3 Syndecan-4 Betaglycan

PC

KC

FSC

MFB

mRNA size (kb)

+ ++ O ++ O

± O + + O

+ + + + +

+ ++ + + ±

2.5; 3.4a 0.8; 1.1; 2.2; 3.5 5.6 2.6 6

+, Expression; ++, strong expression; ±, weak expression; O, no detectable expression; PC, hepatocytes; KC, Kupffer cells; FSC, fat-storing cells; MFB, FSC-derived myofibroblasts a The major size fraction is underlined&l ./ bt :b

14

Fig. 3A, B. Gene expression of betaglycan in cultivated rat hepatocytes. Total RNAs from freshly isolated (0 h) and 24 h, 48 h, 72 h, and 96 h cultivated rat hepatocytes (0, 24, 48, 72, 96, respectively) were reversely transcribed. Equal amounts of the resultant cDNAs were amplified under identical conditions by polymerase chain reaction (PCR) using specific primers for betaglycan (A), and for the housekeeping gene of ribosomal protein S6 (B) as a control. Equal volumes of the PCR mixes were analyzed by agarose gel electrophoresis. N, Negative control; M, 100-bp DNA marker. The size of the DNA fragments is indicated to the left (in bp)&.i c/ :gf

Fig. 4A, B. Purity of the hepatocyte cultures. Equal amounts of the hepatocyte cDNA fractions analyzed in Fig. 3, and cDNA from cultured fat-storing cells for 2 days (P) as a positive control were amplified by PCR using primers for the proteoglycan decorin, which is specific for fat-storing cells (A), and the housekeeping gene S6 (B) as a control. Equal volumes of the PCR mixes were analyzed by agarose gel electrophoresis. N, Negative control; M, 100-bp DNA marker. The size of the DNA fragments is indicated to the left (in bp)&.i c/ :gf

cells, and fat-storing cells. Although the level of syndecan-4 mRNA increases slightly during transformation into myofibroblasts, the overall mRNA concentration in these cells is much lower than that in hepatocytes (Fig. 1D). The expression profile of the various HSPG mRNAs in isolated liver cells is compiled in Table 1. Gene expression of betaglycan in isolated hepatic cells Because of its low abundance, the gene expression of betaglycan was analyzed by Northern-blot analysis and ad-

ditionally by RT-PCR using the expression of the housekeeping gene of ribosomal protein S6 as an internal standard. A single, approximately 6-kb betaglycan mRNA was detectable only in fat-storing cells and in approximately tenfold lower concentrations in myofibroblasts (Fig. 2A). No significant betaglycan transcripts were found in freshly isolated Kupffer cells or hepatocytes. During the cultivation of hepatocytes, the cells started to express the betaglycan mRNA (Fig. 3). The amount of detectable mRNA increased constantly, reaching a maximum after 72 h, and decreased during the next 24 h. The observed increase in betaglycan gene expression was not the result of contaminating fat-storing cells, because no significant gene expression of the fat-storing cell/myofibroblast-specific proteoglycans decorin and biglycan was detectable in the analyzed hepatocyte RNA (Fig. 4). Discussion Although normal liver mainly contains HS GAG, little is known about the expression, the cellular sources, and the functions of the individual hepatic HSPG. In order to identify and characterize the four structurally related members of the syndecan gene family and of betaglycan, we have determined, as the first step, their gene expression in hepatic cells isolated from normal rat livers. In the second step, we have determined the expression of HSPG genes in transformed fat-storing cells, which play the central pathogenetic role in liver fibrogenesis (Gressner 1991). Rat hepatocytes express small amounts of syndecan-1 mRNA, but high levels of syndecan-2 and -4 mRNA. Kupffer cells express syndecan-1, -3, and -4, whereas fat-storing cells, the main producer of ECM components of the liver, express all four syndecans. The transformation of fat-storing cells into myofibroblasts has been recognized as the key pathogenetic event in the initiation of liver fibrosis and includes the stimulation of cell proliferation, phenotypic transformation, and increased secretion of matrix proteins and cytokines (Gressner 1991). During this transformation, the gene expression pattern of the syndecans remains qualitatively unchanged, but the amount of syndecan-2 gene expression increases. The gene expression of syndecan-1 in hepatic cells of the rat has recently also been shown by Ramadori and coworkers (Kovalszky et al. 1994), who have additionally detected small amounts of syndecan-1 mRNA in endothelial cells. In immunohistochemical studies of human liver biopsies, syndecan-1 is detectable in hepatocytes and in sinusoidal endothelial and bile-duct epithelial cells. Syndecan-2 is found in mesenchymal cells, whereas syndecan-4 has been detected at the bile canalicular pole of hepatocytes. Syndecan-3 staining is present in arterial and portal venous endothelial cells, mesenchymal cells, and in fat-storing cells (Roskams et al. 1995). Syndecan-1 is the best-characterized member of the syndecan family. In vitro, it binds to several matrix proteins including fibrillar collagens and fibronectin, and has been colocalized with actin filaments in polarized

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epithelial cells (Koda et al. 1985; Rapraeger et al. 1986; Saunders and Bernfield 1988). This, and several in vivo observations, indicate that syndecan-1 functions as a cell-matrix receptor, perhaps by modulating integrin-induced cell adhesion (Chun and Bernfield 1993). The determined gene expression pattern of syndecan-1 in hepatic cells, and the reported cellular distribution on human hepatocytes fits well with its proposed function as a matrix receptor. It is expressed by hepatocytes, fat-storing cells, and myofibroblasts that are tightly connected to neighboring cells and matrix components located in the Disse space. Immunhistochemical analysis of human hepatocytes has shown that it is mainly located at the sinusoidal and intercellular plasma membranes (Roskams et al. 1995). As in human, mouse, and other rat tissue, syndecan-1 is expressed as a major 2.5-kb mRNA and a minor 3.4kb mRNA in hepatic cells (Mali et al. 1993; CizmeciSmith et al. 1992; Yeaman and Rapraeger 1993).The functional differences between the two transcripts are unknown but, for the mouse syndecan-1 gene, these mRNAs are not generated by alternative splicing; instead, the gene possesses three transcriptional start sites and two polyadenylation signals (Hinkes et al. 1994). Because 3′-untranslated trailers of mRNAs contain several regulatory elements, this might lead to different lifetimes or intracellular distributions of the two syndecan-1 transcripts. The second alternatively transcribed syndecan is syndecan-2. At the cellular level, it has been identified so far only in rat hepatocytes (Pierce et al. 1992), which express crosshybridizing 3.5-kb, 2.2-kb, 1.1-kb, and 0.8-kb transcripts. The three larger isoforms arise from a differential use of alternative polyadenylation sites, whereas the 0.8-kb message represents a crosshybridizing unknown gene product that is specific to kidney and liver (Pierce et al. 1992). Gallagher and coworkers (Lyon and Gallagher 1991; Pierce et al. 1992) have shown that the syndecan-2 protein represents the major membrane HSPG of the rat liver. This is supported by our finding that, with the exception of Kupffer cells, all tested hepatic cells express syndecan-2 transcripts. Cell surface HSPG act as coreceptors in the signaling process of cytokines. The observation that syndecan-1 simultaneously binds bFGF and fibronectin or type I collagen indicates that it might serve, at the same time, as a cell-matrix and cytokine receptor (Salmivitra et al. 1992). Among these cytokines, HGF, bFGF, and TGF-β are the most relevant. Betaglycan, the type III TGF-β receptor, is present in a variety of tissues and has been detected in isolated fat-storing cells of rat liver (Friedman et al. 1994). The amount of mRNA decreases during transformation to myofibroblasts. We have confirmed the gene expression of betaglycan in fat-storing cells and the reduction in gene expression during transformation. The betaglycan message is not detectable in hepatocytes and Kupffer cells isolated from normal rat livers. Although betaglycan mRNA is absent in freshly isolated hepatocytes, they activate betaglycan gene transcription in culture, reaching a maximum steady state level after 72 h. This might be relevant to the observation that the treatment of cultivated rat hepatocytes with activated

TGF-β induces apoptosis (Oberhammer et al. 1991; Bursch et al. 1993). Hepatocytes start or increase the synthesis of TGF-β isoforms during cultivation (Bissell et al. 1995; Chunfang et al. 1996), and thus an increase of betaglycan gene expression might be a possible mechanism for the autoinduction of hepatocyte apoptosis. In conclusion, the present study provides further insights into the expression profile of membrane HSPG by hepatic cells. Their cell-type-specific expression pattern might be involved in a cell-anchorage mechanism to maintain liver structure. With regard to the cytokinebinding properties of membrane HSPG, this pattern might represent a possible mechanism to render the various hepatic cell types differentially sensitive to cytokines. &Acknowledgements. p.2: We thank Brigitte Heitmann and Stefan Petri for excellent technical assistance, Drs. D.J. Carey, J. Massague, and I.G. Wool for providing the cDNA clones, and Dr. H. Ratz for reading the manuscript.

References Abramson SR, Woessner JF (1992) cDNA sequence for rat dermatan sulfate proteoglycan-II (decorin). Biochim Biophys Acta 1132:225–227 Bachem MG, Meyer DM, Melchior R, Sell KM, Gressner AM (1992) Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblast-like cells – a potential mechanism of self-perpetuation in liver fibrogenesis. J Clin Invest 89:19–27 Bissell DM, Wang SS, Jarnagin WR, Roll FJ (1995) Cell- and isoform-specific expression of TGF-beta in rat liver. Hepatology 20:222A Bursch W, Oberhammer F, Jirtle RL, Askari M, Sedivy R, GraslKraupp B, Purchio AF, Schulte-Hermann R (1993) Transforming growth factor-beta1 as a signal for induction of cell death by apoptosis. Br J Cancer 67:531–536 Chan YL, Wool IG (1988) The primary structure of rat ribosomal protein S6. J Biol Chem 263:2891–2896 Cheifetz S, Weatherbee JA, Tsang MLS (1987) The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors. Cell 48:409–415 Chomczynski P, Sacchi V (1987) Single-step method of RNA isolation by acidic guanidinium thiocyanate-phenol chloroform extraction. Anal Biochem 162:156–159 Chun JS, Bernfield M (1993) Syndecan-1 and integrin alpha5/beta1 appear to associate physically in the absence of their ligand fibronectin. Mol Biol Cell 4:283A Chunfang G, Gressner G, Zoremba M, Gressner AM (1996) Transforming growth factor-beta (TGF-beta) expression in isolated and cultured rat hepatocytes. J Cell Physiol 167:394–405 Cizmeci-Smith G, Asundi V, Stahl RC, Teichman LJ, Chernousov M, Cowan K, Carey DJ (1992) Regulated expression of syndecan in vascular smooth muscle cells and cloning of rat syndecan core protein cDNA. J Biol Chem 267:15729–15736 David G (1993) Integral membrane heparan sulfate proteoglycans. FASEB J 7:1023–1030 Dreher KL, Asundi V, Matzura D, Cowan K (1990) Vascular smooth muscle biglycan represents a highly conserved proteoglycan within the arterial wall. Eur J Cell Biol 53:296–304 Elenius K, Jalkanen M (1994) Function of the syndecans – a family of cell surface proteoglycans. J Cell Sci 107:2975–2982 Friedman SL (1990) Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis 10:20–29 Friedman SL, Yamasaki G, Wong L (1994) Modulation of transforming growth factor beta receptors of rat lipocytes during

16 the hepatic wound healing response – enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo. J Biol Chem 269:10551–10558 Gallagher JT (1994) Heparan sulphates as membrane receptors for the fibroblast growth factors. Eur J Clin Chem Clin Biochem 32:239–247 Gressner AM (1991) Liver fibrosis: perspectives in pathobiochemical research and clinical outlook. Eur J Clin Chem Clin Biochem 29:293–311 Gressner AM, Bachem MG (1990) Cellular sources of noncollagenous matrix proteins: role of fat storing cells in fibrogenesis. Semin Liver Dis 10:30–46 Hinkes MT, Goldberger OA, Neumann PE, Kokenyesi R, Bernfield M (1994) Organization and promoter activity of the mouse syndecan-1 gene. J Biol Chem 268:11440–11448 Koda JE, Rapraeger A, Bernfield M (1985) Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens. J Biol Chem 260:8157–8162 Kovalszky I, Gallai M, Armbrust T, Ramadori G (1994) Syndecan-1 gene expression in isolated rat liver cells (hepatocytes, Kupffer cells, endothelial and Ito cells). Biochem Biophys Res Commun 204:944–949 López-Casillas F, Cheifetz S, Doody J, Andres JL, Lane WS, Massagué J (1991) Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system. Cell 67:785–795 Lyon M, Gallagher JT (1991) Purification and partial characterization of the major cell-associated heparan sulfate proteoglycan of rat liver. Biochem J 273:415–422 Lyon M, Deakin JA, Gallagher JT (1994) Liver heparan sulfate structure – a novel molecular design. J Biol Chem 269: 11208–11215 Mali M, Elenius K, Miettinen HM, Jalkanen M (1993) Inhibition of basic fibroblast growth factor-induced growth promotion by overexpression of syndecan-1. J Biol Chem 268:24215–24222 Meyer DH, Krull N, Dreher KL, Gressner AM (1992) Biglycan and decorin gene expression in normal and fibrotic rat liver:

cellular localization and regulatory factors. Hepatology 16: 204–216 Noegel A, Metz BA, Williams KL (1985) Developmentally regulated transcription of Dictyostelium discoideum plasmid Ddp1. EMBO J 4:3797–3803 Oberhammer F, Bursch W, Parzefall W, Breit P, Erber E, Stadler M, Schulte-Hermann R (1991) Effect of transforming growth factor beta on cell death of cultured rat hepatocytes. Cancer Res 51:2478–2485 Pierce A, Lyon M, Hampson IN, Cowling GJ, Gallagher JT (1992) Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver. J Biol Chem 267: 3894–3900 Rapraeger AC, Jalkanen M, Bernfield M (1986) Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol 103:2683–2696 Roskams T, Moshage H, Vos RD, Guido D, Yap P, Desmet V (1995) Heparan sulfate proteoglycan expression in normal human liver. Hepatology 21:950–958 Salmivitra M, Heino J, Jalkanen M (1992) Basic fibroblast growth factor-syndecan complex at cell surface or immobilized to matrix promotes cell growth. J Biol Chem 267:17606–17610 Saunders S, Bernfield M (1988) Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor on interstitial matrix. J Cell Biol 106:423–430 Segarini PR, Roberts AB, Rosen MD, Seyedin SM (1987) Membrane binding characteristics of two forms of transforming growth factor beta. J Biol Chem 262:14655–14662 Wang XF, Lin HY, Ng-Eaton E, Downward J, Lodish HF, Weinberg RA (1991) Expression, cloning and characterization of the TGF-beta type III receptor. Cell 67:797–805 Yeaman C, Rapraeger A (1993) Membrane-anchored proteoglycans of mouse macrophages-P388D1 cells express a syndecan-4 like heparan sulfate proteoglycan and a distinct chrondroitin sulfate form. J Cell Physiol 157:413–425