Journal of Neuroscience Research 3251-59 (1992)
Decorin, A Chondroitin/Dermatan Sulfate Proteoglycan Is Under Neural Control in Rat Skeletal Muscle E. Brandan, M.E. Fuentes, and W. Andrade Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Santiago, Chile been identified and selectively associated with synaptic basal lamina and is recognized by motoneurons (Hunter et al., 1989). An analogous protein, laminin, is a major axon outgrowth-promoting component of ECM in the peripheral nervous system (Sanes, 1989). Proteoglycans (PGs) are widely distributed in the ECM of all mammalian tissues, as well as being associated with the plasma membrane of eukaryotic cells (Fransson, 1987; Ruoslahti, 1988; Brandan and Hirschberg, 1989). Several PGs have been isolated from skeletal muscle ECM. Thus, three major high-buoyantdensity chrondroitin sulfate PGs have been isolated from embryonic chick skeletal muscle (Carrino and Caplan, 1984); a low molecular weight chondroitin sulfate PG has been isolated from rabbit skeletal muscle (Parthasarathy and Tanzer, 1987); and two heparan sulfate PGs (Brandan and Inestrosa, 1987a) and a dermatan sulfate PG, displaced by the glycosaminoglycan (GAG) heparin, have been also isolated from rat skeletal muscle (Brandan and Inestrosa, 1987b). Several observations suggest that PGs present at the muscle basal lamina might play important functional roles. Thus, a heparan sulfate PG is concentrated at the neuromuscular junction (Bayne et al., 1984), and a related PG is responsible for the anchoring of the asymmetric form of acetylcholinesKey words: denervation, muscle activity, adhesive terase to the synaptic basal lamina (Brandan and Inemolecules, decorin strosa, 1984; Brandan et al., 1985). Also acetylcholine receptor clusters are associated with patches of ECM heparan sulfate PGs (Anderson and Fambrough, 1984). INTRODUCTION Some evidence suggests that PGs seem to be important Skeletal muscle fibers are surrounded by a special- for neuronal cell adhesion and growth in the peripheral ized form of extracellular matrix (ECM), the basal lamina. This structure is highly specialized at the neuromuscular junction region (Rubin and McMahan, 1982), Received April 23, 1991; revised November 15, 1991; accepted where it has been implicated in both nerve-muscle inter- November 27, 1991. action and muscle regeneration (Sanes, 1989). Special Address reprint requests to E. Brandan, Molecular Neurobiology Unit, interest has been focused on understanding the signifi- Department of Cell and Molecular Biology, Faculty of Biological cance of the components of the basal lamina. The isola- Sciences, Catholic University of Chile, P.O. Box 114-D, Santiago, tion and characterization of specific ECM components Chile. is, therefore, important in order to ascertain their func- M.E. Fuentes’ present address is Department of Pharmacology, tional roles. For instance, a glycoprotein, s-laminin, has School of Medicine, University of California, La Jolla, CA 92093.
Proteoglycans (PGs) are abundant components of the extracellular matrices (ECM) of skeletal muscle. We have previously found that the synthesis of skeletal muscle PGs present at the ECM increase after denervation. The experiments reported here were undertaken to identify which PG(s) increase after denervation of rat leg muscles. Incorporation of radioactive sulfate demonstrated the presence of a chondroitiddermatan sulfate PG of 70-90 kDa in the skeletal muscle ECM, which increased after denervation. The PG has a core protein of 3 9 4 5 kDa after treatment with chondroitinase ABC. Antibodies against rat decorin, a chondroitin/dermatan sulfate PG synthesized by various cell types, specifically immunoprecipitated this PG from a mixture of PGs. Immunocytolocalization of this PG indicated that the chondroitin/dermatan sulfate PG accumulates at the perimysium of skeletal muscle after denervation. Finally, Northern blot analysis indicated an increase of muscle transcripts for decorin after denervation. The data reported here suggest that a chondroitiddermatan sulfate PG present at the skeletal muscle ECM, very similar if not identical to decorin, increases after denervation. 0 1992 Wiley-Liss, Inc.
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nervous system. Thus it has been suggested that a heparan sulfate PG is involved in axonal regeneration along laminin-rich pathways (Chiu et al., 1986) and in the guidance of axons to original synaptic sites of denervated neuromuscular junctions (Sanes et al., 1986). Little is known regarding the control of the expression of muscle PGs. We have recently shown that denervation of adult rat leg muscles mainly caused an increase in the synthesis of a small PG present at the ECM and that the expression of the PGs in the skeletal muscle appears to be regulated by motor nerve activity. This effect is observed after 2-4 days of denervation (Fadic et al., 1990). In this report, we have identified the PG that mainly increases after denervation of rat skeletal muscle as a chondroitin/dermatan sulfate PG, called decorin. The increase after denervation is due to an increase in the synthesis of this molecule, since their mRNA increase roughly to the same extent.
MATERIALS AND METHODS Materials The following material was purchased from suppliers: Naz3'S0, carrier free was obtained from New England Nuclear, Boston, MA; dCT"P (6,000 Ci/mmol) from Amersham, Aylesbury, Buckinghamshire, England; Na'*'I from the Chilean Nuclear Commission, Santiago, Chile; chondroitinase ABC lyase (EC 4,2,2,4), chondroitinase AC lyase (EC 4,2,2,5), protein A-sepharose, agarose, benzamidine hydrochloride, DEAE-Sephacel, and sepharose CL-6B from Sigma Chemical Co. St. Louis, MO; and heparitinase (EC 4,2,2,8) from Miles Laboratories, Elkhart, IN. Policlonal rabbit antiserum against proteodermatan sulfate purified from rat skin fibroblast (Witsch et al., 1989) was a generous gift of Professor Hans Kresse (University of Munster, Germany). Human decorin cDNA was a kind gift of Dr. Tom Krusius (University of Helsinki, Finland). Other reagents were obtained from commercial sources. Methods Male Sprague-Dawley rats (200-250 g) were denervated by cutting the sciatic nerve as previously described (Fadic et al., 1990). Control or four-day denervated rats were injected intraperitoneally with 1.5-2.0 mCi of 35~-sulfatein saline. Isolation of Muscle Proteoglycans Legs muscles were removed, 18 hr after sulfate injection, and homogenized in 10 mM Tris-HC1 buffer, pH 7.4, containing 5 mM benzamidine, 50 mM 6-aminohexanoic acid, 10 mM N-ethylmaleimide, 10 mM EDTA, and 0.5% (v/v) Triton X-100 and centrifuged in
a Sorvall SS-34 rotor at 12,OOOg for 15 min at 4°C. The pellet was resuspended in the same homogenation buffer and centrifuged again. This last step was repeated twice. Supernatants were pooled and the labeled-PGs, present in the final pellet, were extracted as previously described (Brandan and Inestrosa, 1987a). The detergent insoluble pellet was solubilized with 6 M guanidine-HC1 and 50 mM sodium acetate, pH 5.8, containing protease inhibitors, for 12 hr at 4°C with continuous agitation. The extract was centrifuged as above and unincorporated radioactive precursors, guanidine-HC1, and salts were removed by dialysis with 100 volumes of 4 M urea, 0.1 M NaCI, 0.1% Triton X-100, protease inhibitors, and 50 mM sodium acetate, pH 5.8, for 6 hr and then with 100 volumes of the same solution, but containing 8 M urea, for another 6 hr at 4°C. After dialysis the radioactive material was applied to a DEAE-Sephacel column preequilibrated in the same 8 M urea buffer as previously described (Yanagishita and Hascall, 1984). The column was washed with 8 ml of the same buffer and than eluted with a continuous NaCl gradient, 0.2 to 1.1 M NaCl in the same buffer (60 ml total vol.). Elution was performed at a flow rate of 5.0 ml/hr and fractions of 1.O ml were collected. The NaCl gradient was monitored by measuring conductivity of the fractions. The labeled eluted material was pooled, dialyzed for 12 hr with 10 mM TrisHC1 buffer pH 7.4,0.5% Triton X-100, and 0.1 M NaCl containing protease inhibitors and precipitated by the addition of 2.5 vol. of cold ethanol. The labeled-PGs were collected by centrifugation and resuspended in 0.5 ml of 10 mM Tris-HC1 buffer pH 7.4 and 0.1 M NaCl.
Enzymatic Treatments and Chemical Analyses Chondroitinase ABC and AC treatments of labeledPGs were done exactly as previously described (Brandan and Inestrosa, 1987a). Heparitinase treatment was done as described by Yanagishita and McQuillan (1989). Immunoprecipitation of Decorin Decorin was specifically immunoprecipitated from a mixture of PGs isolated from leg muscles after DEAESephacel chromatography. The PGs were dialyzed with 10 mM Tris HCl, pH 7.4, 0.1 M NaCl and immunoprecipitated with protein A-sepharose coated with IgG from antiserum against rat decorin (Witsch et al., 1989), exactly as previously described by Gloss1 et al. (1984). The immunoprecipitates were subjected to electrophoresis in SDS-polyacrylamide slab gels (SDS-PAGE) as described below. Iodination and SDS-PAGE Analysis of Proteoglycans Purified PGs from the sepharose CL-6B column, eluting with a K,, of about 0.5 were iodinated using the
Motor Nerve Regulates Muscle Proteoglycans
chloramine T method (Carpenter and Cohen, 1976). Sulfated samples eluted from DEAE-Sephacel column and iodinated PGs were analyzed by electrophoresis on 10% and 3-10% SDS-PAGE and fluorographied as described previously (Carlson and Wight, 1987).
RNA Isolation and Northern Blot Analysis Total RNA from human fibroblasts was isolated using the procedure described by Chomczynski and Sacchi (1987). Muscle RNA was isolated from control and denervated groups (4-6 rats) by homogenizing the frozen tissue in guanidine thiocyanate solution followed by centrifugation over a cushion of CsCl as previously described (Goldman et al., 1988). Poly(A)+ RNA was selected by poly U-sepharose chromatography. Total RNA and poly(A)+ RNA were fractionated in 1 % agarose/2.2 formaldehyde gel as described by Krusius and Ruoslahti (1986) and transferred to Hybond-N membranes. A human decorin cDNA probe was radiolabeled by oligonucleotide random primer extension according to the manufacturer’s instructions (Amersham). Prehybridization was done for 3 hr at 44°C in the presence of 30% formamide, 5 X SSPE (0.18 M NaCl, 0.01 M sodium phosphate, 1 mM EDTA, pH 7.7), 1% SDS, 10% dextran sulfate, 5 x Denhardts solution, and 0.1 mg/ml of single stranded salmon DNA. Hybridization was carried out overnight at 44°C in the above solution containing 1 x lo6 c.p.m./ml of the 32P-labeled probe. Blots were washed at 42°C in 100 ml of 2 X SSPE, and four washes of 50 ml of 0.5 X SSPE, 0.1% SDS. Blots were exposed at -30°C overnight for human fibroblast RNA or for four days for rat skeletal muscle RNA. Histology Leg muscles from both control and denervated rats were cross-sectioned at 4 p m in a cryostat. Sections were fixed in 3% paraformaldehyde in phosphate saline, pH 7.4, and incubated sequentially with an antiserum against rat decorin (1/50 dilution in Blotto (Carey et al., 1987), for 1 hr at room temperature and then with a fluorescein-conjugated anti-rabbit antibody for 30 min at room temperature. After rinsing, the sections were mounted on glass slides and viewed with a Nikon Diaphot inverted microscope equipped for epifluorescence. RESULTS Denervation Increases the Synthesis of a Chondroitin/DermatanSulfate-PG Rat leg muscles denervated for 4 days increased in the synthesis of a sulfated PG with a molecular weight ranging from 70-90 kDa as determined by SDS-PAGE, after loading equivalent amounts of material (by weight) in each gel lane (Fig. 1A). The increase was about three-
53
fold, as determined by densotimetric analysis of the autoradiograph. This PG was totally degraded by chondroitinase ABC treatment, while a minor effect was seen after incubation with chondroitinase AC. No degradation was observed when the sample was incubated with heparitinase (Fig. 1B). These results indicate that this denervation sensitive PG corresponds to a chondroitid dermatan sulfate PG of low molecular weight. To further characterize this PG, we studied the size of the core protein. For this purpose, an ECM rich fraction was chromatographied first in a DEAE-Sephacel column (Fig. 2A, top) followed by chromatography on Sepharose CL-6B (Fig. 2A, bottom). The second peak (Kavof 0.50) of the latter column was pooled, concentrated and iodinated. Figure 2B indicates that the material present at this step of purification possesses a molecular weight of 70-90 kDa and a contaminant of 53 kDa. Treatment with chondroitinase ABC completely degraded the PG, with the concomitant appearance of a band with a molecular weight of 39-45 kDa, where only a partially degradation was observed after chondroitinase AC treatment, suggesting that a small proportion of this PG population was sensitive to this treatment. These results strongly indicate that the chondroitin/dermatan sulfate-PG, which increases after denervation, has a core protein of 39-45 kDa.
The ChondroitinlDermatan Sulfate-PG Which Increases After Denervation Corresponds to Decorin A small chondroitin/dermatan sulfate-PG, named DS-PGII or decorin (Krusius and Ruoslahti, 1986) with a core protein of 38-45 kDa has been described in several ECM (Brennan et al., 1984; Gloss1 et al., 1984; Rosenberg et al., 1985; Vogel and Clark, 1989). In order to evaluate if our denervation sensitive PG corresponds to DS-PGII or decorin, a crude rat muscle ECM fraction labeled with sulfate was incubated with an antibody against rat decorin. Figure 3, lane 2, shows that the antiserum precipitated a single component which migrated as a more sharp band with a molecular weight of 70-80 kDa. Lane 1 of Figure 3 indicates that no 35Sradioactivity was precipitated when a normal rabbit serum was used. This result indicates that the PG which increases after denervation correspond to decorin. Decorin Accumulates Around the Muscle Fibers After Denervation To determine where decorin is located in rat skeletal muscle, we used light microscopic immunohistochemistry. Figure 4A shows that decorin accumulates in the interstitial spaces, either around packages of muscle fibers, the perimysium, or around the muscle fiber, the endomysium. Figure 4B shows that after four days of
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Fig. 1 . Increase of a sulfated chondroitinidermatan sulfate proteoglycan after denervation. Rats were denervated and after four days were injected with radioactive sulfate. A: An ECMlike fraction was prepared from control (C) and denervated (D) animals and applied to a DEAE-Sephacel column, as explained in Materials and Methods. Equivalent amounts of material, by weight, were separated on a 10% SDS-PAGE followed by fluorography . Denervation induced an 2.9-fold increase in the amount of a PG of 70-90 kDa, as determined by densitometric quantification of the autoradiograph. B: Sulfated PGs obtained
after DEAE-Sephacel chromatography were incubated with buffer alone (control), heparitinase (Hase), chondroitinase ABC (C. ABC) or chondroitinase AC (C. AC), as explained in Materials and Methods. After the incubation the PGs were fractionated on a 8% SDS-PAGE. The 70-90 kDa was totally degraded by chondroitinase ABC, partially degraded by chondroitinase AC, and totally resistant to heparitinase, suggesting that the 70-90 kDa PG corresponds to a chondroitinidermatan sulfate PG.
denervation, the space occupied by the perimysium increased and a higher reaction for decorin is visualized (arrows). However, at the endomysium, no significant changes in the amount of decorin was detected (arrowheads). These observations suggest that after skeletal muscle denervation there is an increase in the amount of decorin in the muscle interstitial space, the perimysium.
present in control versus denervated rat leg muscles. Poly(A)+ RNA was isolated from both control and 4-day denervated rat leg muscles and equal amounts of poly(A) RNA used for blot hybridization analysis using decorin cDNA. Figure 5 indicates that a substantial higher amount of transcript for decorin is present in denervated muscle compared with the control. Densitometric analysis of the autoradiography indicates a 3.4-fold increase in decorin mRNA. These results strongly suggest that the expression of the muscle mRNA for decorin increases after denervation.
Increase in Transcript for Decorin After Skeletal Muscle Denervation To further evaluate if decorin was expressed in muscle tissue, poly(A)+ RNA was isolated from rat leg muscles and submitted to blot hybridization analysis, using a cDNA against decorin. The results indicated that muscle tissue express an mRNA of 1.9 kb, similar in size to the transcript expressed by fibroblast (Krusius and Ruoslahti, 1986; Heino et al., 1988; data not shown). Then, we evaluated the amount of transcript for decorin
+
DISCUSSION We have recently shown that denervation of rat leg muscles increase the synthesis of PGs present at the ECM (Fadic et al., 1990), however, the PG(s) specie(s) that increase idwere unknown. The results in the present
Motor Nerve Regulates Muscle Proteoglycans
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Fig. 2. The chondroitiddermatan sulfate PG contains a core cold ethanol precipitation. Two peaks are observed; one with a protein of 38-45 kDa. A: Guanidine-solubilizedPGs from rat K,, of 0.25, which corresponds to chondroitin, and heparan leg muscle were fractionated on a DEAE-Sephacel column sulfate PGs, where the peak with a K,, of about 0.5 corre(top). Most of the material was bound to the column and eluted sponds to the chondroitiddermatan sulfate PG. B: The chonat about 0.6 M NaCl. Fractions 12-28 were pooled, concen- droitiddermatan sulfate PG containing fractions were ioditrated, and fractionated on an analytical Sepharose CLdB pre- nated and fractionated on SDS-PAGE after glycosidases pared in 1% sodium dodecyl sulfate (SDS), 0.1 M NaC1, and treatment. The 70-90 kDa PG was partially degraded by chon50 mM Tris-HC1 buffer, pH 8.0 (bottom). The latter column droitinase AC (C. AC), while chondroitinase ABC (C. ABC) was run at a flow rate of 5 .O ml/hr and effluent fractions of 1.O completely degraded the iodinated-PG with the concomitant ml were collected, counted for radioactivity, and pooled before appearance of the 39-45 kDa core protein. study indicate that denervation of skeletal muscles induce the synthesis and expression of a chondroitiddermatan sulfate PG very similar or identical to decorin, based on the following criteria: the size of the PG and its core protein, their specific immunoprecipitation using an anti-rat decorin serum, and the detection of RNA transcript which hybridizes with a specific cDNA probe for human decorin. We found that muscle decorin has a molecular weight of 70-90 kDa; this size is a slightly lower than the one reported for decorin synthesized by other tissues (Fisher et al., 1983; Brennan et al., 1984; Gloss1 et al., 1984; Vogel and Clark, 1989). This muscle PG represent about the 50% of the total PGs present in the skeletal muscle ECM (Brandan and Inestrosa, 1987b). Most of our studies were done after 4 days of denervation; we have recently shown that the synthesis of PGs increase after 2 days of denervation, reaching a steady level after 6 days (Fadic et al., 1990).
It is well known that the level of expression of several muscle macromolecules is strongly influenced by the presence of the nerve. Thus, the level of cell surface acetylcholinesterase (Lomo et al., 1985) and extrajunctional acetylcholine receptor (Reiness and Hall, 1977; Merlie et al., 1984) is regulated by contractile activity. We have previously shown that the level of expression of sulfated PGs is influenced by contractile activity and a new steady state level of expression is observed after reinnervation of the muscle (Fadic et al., 1990). Here, we demonstrated that the level of expression and synthesis of a specific PG decorin is influenced by the presence of the motor nerve; further studies are necessary to establish the mechanism involved in such neural control. Why there is an increase of decorin after denervation is not clear, but it is well known that ECM provides a unique physical and functional boundary which specifies the properties of the normal assembly of the neuromus-
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2
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3727Fig. 3. The chondroitiddermatan sulfate PG is specifically precipitated by an antibody against rat decorin. An aliquot of sulfated PGs obtained after DEAE-Sephacel chromatography was incubated with a non-immune serum (lane 1) or with an antibody against rat decorin (lane 2), as explained in Materials and Methods. The immunoprecipitates were then separated on a 10% SDS-PAGE, followed by fluorography. The antiserum specifically precipitated a sulfated PG of 70-90 kDa.
cular junction. Axons preferentially form new junctions at original synaptic sites (Sanes, 1989). Thus, a muscle fiber might respond to denervation by changing its surface properties, thereby increasing its attractiveness for regenerating axons, where ECM molecules might play an essential role. With respect to decorin expression, it is known that the expression of recombinant decorin in Chinese hamster ovary cells converts these morphologically normal cells and greatly reduces their saturation density; these results indicate that the expression of decorin might be mediating the growth properties of the cell(s) (Yanaguchi and Ruoslahti, 1988). Decorin can bind collagen and fibronectin through its core protein (Vogel et al., 1984; Lewandowska et al., 1987; Schmidt et al., 1987), regulating the structure and thickness of collagen fibrils at the ECM. According with this, we detected that after muscle denervation, decorin accumulated in the interstitial spaces, specifically at the
Fig. 4. Increase of decorin in the perimysium after denervation of rat leg muscles. Rat leg muscles from control (a) and from 4-day denervated animals (b) were fixed and stained with an antibody against decorin, followed by a second fluorescentconjugated antibody as explained in Materials and Methods. An increase in the amount of decorin is observed in the perimysium, around packages of muscle fibers, after denervation (arrows). No significant changes were seen at the endomysium (arrowheads) ( x 660).
perimysium, where collagen fibrils and fibronectin are present. However, we did not see a significant increase in decorin at the endomysium. These observations raise several questions: Is the expression of ECM components coordinately regulated? How does the mechanism by which the cell(s) responsible for the synthesis of ECM molecules respond to muscle activity? There are some lines of evidence that might help to answer some of these questions: It has been shown that the transforming growth factor-beta (TGF-P) stimulates the expression of ECM molecules such as fibronectin, type I collagen, cell adhesion receptor, and decorin in different cell types (Ignotz and MassaguC, 1987; Cheifetz et al., 1987; Bassols and MassaguC, 1988), suggesting a regulated and coordinated expression of several ECM molecules. With
Motor Nerve Regulates Muscle Proteoglycans
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In regard to the cell(s) type responsible for the synthesis of the ECM components, it has been shown that several adhesive molecules, such as tenascin, N-CAM, fibronectin, and a heparan sulfate PG which accumulates at synaptic sites in the muscle after denervation (Covault and Sanes, 1985; Sanes et al., 1986), are synthesized by muscle fibroblasts which proliferate after denervation of skeletal muscle in the interstitial space (Zak et al., 1969; McGeatchie and Allbrook, 1978; Murray and Robbins, 1982; Connor and McMahan, 1987; Gatchalian et al., 2 8 S 1989). Decorin is synthesized and secreted by fibroblasts from several sources (Miyamoto and Nagase, 1980; Coster and Fransson, 1981; Damle et al., 1982; Nakamura et al., 1983; Gloss1 et al., 1984; Vogel and Clark, 18% 1989), however, we have obtained evidence from a skeletal muscle rodent cell line, which differentiates in vitro to form contractile multinucleated myotubes, indicating that decorin is synthesized by them (Brandan et al., 1991). Therefore, in muscle tissue there is the possibility Fig. 5. Decorin muscle transcripts increased after denervation. that both cell types, fibroblasts and myotubes, synthesize Left: 30 pg of poly(A)+ isolated from control (c) and denerdecorin. Which of those cell types respond to denervavated (d) rat leg muscles was separated electrophoretically , tion by increasing the synthesis of decorin is a matter that blotted on nylon membranes, and hybridized with 32-P-labelled we are investigating now. cDNA probe for human decorin. An increase in the amount of
c
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RNA transcript specifically for decorin is observed after four days of denervation. The film was exposed for 4 days. Right: Ethidium bromide staining of the gel indicates no difference between isolated RNA from control and denervated rat leg muscles. Ribosomal RNAs are still present in the fractions and the position is indicated.
ACKNOWLEDGMENTS We thank Dr. N.C. Inestrosa for helpful suggestions and careful reading of the manuscript and Dr. C. Koenig for the sections for histology. This work was supported by a grant from FONDECYT 569-89 to E.B.
respect to the mechanism by which the cell(s) responsible for the synthesis of the PGs respond to muscle activity, one can postulate that the absence of muscle ac- REFERENCES tivity as an effect of denervation might increase the Anderson MJ, Fambrough DM (1984): Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan synthesis of diffusible factor (growth factor-like) which sulfate proteoglycan on the surface of skeletal muscle fibers. J activates the expression of ECM components in a conCell Biol 97:1396-1411. certed fashion. For instance, it has been shown that in- Bassols A, Massague, J (1988): Transforming growth factor-B regusulin (Stevens et al., 1981), insulin-like growth factors lates the expression and structure of extracellular matrix chondroitiddermatan sulfate proteoglycans. J Bio! Chem 263: (Salmon and Daughaday, 1957; Froesch et al., 1976), 3039-3045. fibroblast growth factor (Kato and Gospodarowicz, 1985a), and glucocorticoids (Kato and Gospodarowicz, Bayne EK, Anderson MJ, Fambrough MD (1984): Extracellular matrix organization in developing muscle: Correlation with ace1985b) affect the expression of cartilage specific PGs tylcholine aggregates. J Cell Biol 99: 1486-1501. and that interleukin-1 regulates cellular mRNA level of Brandan E, Hirschberg CB (1989): Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apdecorin (Heino et al., 1988). One can speculate in the paratus and the plasma membrane. J Biol Chem 264:10520case of the muscle tissue that the hypothetical diffusible 10526. factor is not synthesized by the nerve, because either the Brandan E, Inestrosa NC (1984): Binding of the asymmetric forms of level of expression of acetylcholine receptors or the exacetylcholinesterase to heparin. Biochem J 221:415-422. pression of PGs (Lomo and Rosenthal, 1972; Reiness Brandan E, Inestrosa NC (1987a): Isolation of the heparan sulfate proteoglycans from extracellular matrix of rat skeletal muscle. and Hall, 1977; Fadic et al., 1990) is regulated by the J Neurobiol 18:271-282. muscle activity itself. Furthermore, the increase in expression of decorin after denervation might be related to Brandan E, Inestrosa NC (1987b): Co-solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the fact that some growth factors have the ability to bind extracellular matrix of rat skeletal muscles. FEBS Lett 213: PGs, creating a reservoir of growth factors for cells to 159-163. Brandan E, Maldonado M, Garrido J, Inestrosa NC (1985): Anchoruse (Saksela et al., 1988; Ruoslahti, 1989).
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age of collagen-tailed acetylcholinesterase to the extracellular matrix of rat skeletal muscles. J Cell Biol 101:985-992. Brandan E, Fuentes ME, Andrade W (1991): The proteoglycan decorin is synthesized and secreted by differentiated myotubes and increase during in vitro differentiation. Eur J Cell Biol 551209-216. Brennan MJ, Oldberg A, Pierschbacher MD, Ruoslahti E (1984): Chondroitinidermatan sulfate proteoglycan in human fetal membranes. J Biol Chem 22: 13742-1 3750. Carey DJ, Rafferty CM, Todd S (1987): Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells. J Cell Bid 105:1013-1021. Carlson SS, Wight TN (1987): Nerve terminal anchorage protein I (TAP-1) is a chondroitin sulfate proteoglycan. Biochemical and electron microscopic characterization. J Cell Biol 105:30753086. Carpenter G, Cohen S (1976): ‘Z51-labeledhuman epidermal growth factor. J Cell Biol 71:159-171. Carrino DA, and Caplan A1 (1984): Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart. J Biol Chem 259: 12419-1 2430. Cheifetz S, Weatherbee JA, Tsang M L-S, Anderson JK, Mole JE, Lucas R, Massaguk, J (1987): The transforming growth factorB system, a complex pattern of cross-reactive ligands and receptors. Cell 48:409-415. Chiu AY, Matthew WD, Patterson PH (1986): A monoclonal antibody which blocks the activity of a neurite regeneration promoting factor: Studies on the binding site and its localization in vivo. J Cell Biol 102:1383-1398. Chomczynski P, Sacchi N (1987): Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156-159. Connors EA, McMahan UJ (1987): Cell accumulation in the junctional region of denervated muscle. J Cell Biol 104:109-120. Coster L, Fransson L-A (1981): Isolation and characterization of dermatan sulfate proteoglycans from bovine sclera. Biochem J 1931143-153. Covault J, Sanes JR (1985): Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles. Proc Natl Acad Sci USA 82:4544-4548. Damle SP, Coster L, Gregory JD (1982): Proteodermatan sulfate isolated from pig skin. J Biol Chem 2575523-5527. Fadic R, Brandan E, Inestrosa NC (1990): Motor nerve regulates muscle extracellular matrix proteoglycans expression. J Neurosci 10:3S16-3523, Fisher LW, Termine JD, Dejter SW, Whitson SW, Yanagishita M, Kimura JH, Hascall VC, Kleinman HK, Hassell JR, Nilsson B (1983): Proteoglycans of developing bone. J Biol Chem 258: 6588-6594. Froesch ER, Zapf J, Andhya TK, Ben-Porath E, Segen BJ, Gibson KD (1976): Nonsupressible insulin-like activity and thyroid hormones: Major pituitary-dependent sulfation factors for chick embryo cartilage. Proc Natl Acad Sci USA 73:2904-2908. Fransson L-A (1987): Structure and function of cell-associated proteoglycans. Trends Biochem Sci 12:406-41 I . Gatchalian CL, Schachner M, Sanes JR (1989): Fibroblast that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin ( J l ) , N-CAM, fibronectin, and heparan sulfate proteoglycan. J Cell Biol 108: 1873-1890. Gloss1 J, Beck M, Kresse H (1984): Biosynthesis of proteodermatan sulfate in cultured human fibroblasts. J Biol Chem 259:1414414150.
Goldman D, Brenner HR, Heinemann S (1 988): Acetylcholine receptor alpha, betha, gamma, and deltha-subunit mRNA levels are regulated by muscle activity. Neuron 1 :329-333. Heino J , Kakari V-M, Mauviel A, Krusius T (1988): Human recombinant interleukin-1 regulates cellular mRNA levels of dermatan sulphate proteoglycan core protein. Biochem J 252:309312. Hunter DD, Shah V, Merlie JP, Sanes JR (1989): A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature (London) 338:229-234. Ignotz RA, MassaguC J (1987): Cell adhesion protein receptors as targets for transforming growth factor-B. Cell 51:189-197. Kato Y , Gospodarowicz D (l985a): Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor. J Cell Biol 100:477-485. Kato Y , Gospodarowicz D (1985b): Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro. J Biol Chem 260:23642373. Krusius T, Ruoslahti E (1986): Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 83:7683-7687. Lewandowska K, Choi HU, Rosenberg LC, Zardi LC, Culp LA ( 1987): Fibronectin-mediated adhesion in fibroblast: Inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminiglycan-binding domain. J Cell Biol 105: 1443-1 454. Lomo T, Rosenthal J (1972): Control of ACh sensitivity by muscle activity in the rat. J. Physiol. 221:493-513. Lomo T, MassouliC J , Vigny M (1985): Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms. J Neurosci 5: 1 180-1 187. McGeatchie J , Allbrook D (1978): Cell proliferation in skeletal muscle following denervation or tenotomy. Cell Tissue Res 193:2S9267. Merlie JP, Isenberg IE, Russel SD, Sanes JR (1984): Denervation supersensitivity in skeletal muscle analysis with a cloned cDNA probe. J Cell Biol 99:332-335. Miyamoto I , Nagase S (1980): Isolation and characterization of proteodernatansulfate from rat skin. J Biochem 88: 1793-1 803. Murray MA, Robbins N (1982): Cell proliferation in denervated muscle: Time course, distribution, and relation to disuse. Neuroscience 7: 1817-1822. Nakamura T, Matsunaga E, Shinkai H (1983): Isolation and some structural analysis of a dermatan sulphate from calf skin. Biochem J 213:289-296. Parthasarathy N , Tanzer ML (1987): Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle. Biochemistry 2 6 3 149-3 156. Reiness GC, Hall ZW (1977): Electrical stimulus of denervated muscle reduces incorporation of methionine into the ACh receptor. Nature 268:6SS-657. Rosenberg LC, Choi HU, Tang L-H, Johnson TL, Pal S, Webber C, Reiner A, Poole R (1985): Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages. J Bid Chem 260:6304-63 13. Rubin LL, McMahan UJ (1982): Regeneration of the neuromuscular junctions: Steps toward defining the molecular basis of the interaction between nerve and muscle. In Schotland DL (ed): “Disorders of the Motor Unit.” New York: John Wiley & Sons, pp 187-196. Ruoslahti E (1988): Structure and biology of proteoglycans. Ann Rev Cell Biol 4:229-255.
Motor Nerve Regulates Muscle Proteoglycans Ruoslahti E (1989): Proteoglycans in cell regulation. J Biol Chem 264: 13369-13372. Saksela 0, Rifkin DB (1990): Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activation-mediated proteolytic activity. J Cell Biol 110:767-775. Salmon WD, Daughaday WH (1957): An hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825-836. Sanes JR (1989): Extracellular matrix molecules that influences neural development. Annu Rev Neurosci 12521-546. Sanes JR, Schachner M, Covault J (1986): Expression of several adhesion macromolecules in embryonic, adult, and denervated adult skeletal muscle. J Cell Biol 102:420-43 1. Schmidt G , Robenek H, Harrach B, Gloss1 J , Nolte V, Hormann H, Richter H, Kresse H (1987): Interaction of small dermatan sulfate proteoglycan from fibroblast with fibronectin. J Cell Biol 104:1683-1691. Stevens RL, Nissley SP, Kimura JH, Rechler MM, Caplan AI, Hascall VC (1 981): Effect of insulin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the swarm rat chondrosarcoma. J Biol Chem 256:2045-2052. Vogel K , and Clark P (1989): Small proteoglycan synthesis by skin
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fibroblasts cultured from eldery donors and patients with defined defects in types I and I11 collagen metabolism. Eur J Cell Biol 49:236-243. Vogel KG, Paulsson M, Heinegard D (1984): Specific inhibition of type I and type I1 collagen fibrillogenesis by the small chondroitin sulfate-dermatan sulfate proteoglycan in the human. Biochem J 223587-597. Wjtsch P, Kresse H, Gressner AM (1989): Biosynthesis of small proteoglycans by hepatic lipocytes in primary culture. FEBS Lett 258:233-235. Yanagishita M, Hascall VC (1984): Proteoglycans synthesized by rat ovarian granulosa cells in culture: Isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J Biol Chem 259:10260-10269. Yanagishita M, McQuillan DJ (1989): Two forms of plasma membrane-intercalated heparan sulfate proteoglycan in rat ovary granulosa cells. J Biol Chem 264: 17551-17558. Yamaguchi Y, Ruoslahti E ( I 988): Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation. Nature 336:244-246. Zak R, Grove D, Rabinowitz M (1969): DNA synthesis in the rat diaphragm as an early response to denervation. Am J Physiol 2 16:647-654.
Decorin, A Chondroitin/Dermatan Sulfate Proteoglycan Is Under Neural Control in Rat Skeletal Muscle E. Brandan, M.E. Fuentes, and W. Andrade Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Santiago, Chile been identified and selectively associated with synaptic basal lamina and is recognized by motoneurons (Hunter et al., 1989). An analogous protein, laminin, is a major axon outgrowth-promoting component of ECM in the peripheral nervous system (Sanes, 1989). Proteoglycans (PGs) are widely distributed in the ECM of all mammalian tissues, as well as being associated with the plasma membrane of eukaryotic cells (Fransson, 1987; Ruoslahti, 1988; Brandan and Hirschberg, 1989). Several PGs have been isolated from skeletal muscle ECM. Thus, three major high-buoyantdensity chrondroitin sulfate PGs have been isolated from embryonic chick skeletal muscle (Carrino and Caplan, 1984); a low molecular weight chondroitin sulfate PG has been isolated from rabbit skeletal muscle (Parthasarathy and Tanzer, 1987); and two heparan sulfate PGs (Brandan and Inestrosa, 1987a) and a dermatan sulfate PG, displaced by the glycosaminoglycan (GAG) heparin, have been also isolated from rat skeletal muscle (Brandan and Inestrosa, 1987b). Several observations suggest that PGs present at the muscle basal lamina might play important functional roles. Thus, a heparan sulfate PG is concentrated at the neuromuscular junction (Bayne et al., 1984), and a related PG is responsible for the anchoring of the asymmetric form of acetylcholinesKey words: denervation, muscle activity, adhesive terase to the synaptic basal lamina (Brandan and Inemolecules, decorin strosa, 1984; Brandan et al., 1985). Also acetylcholine receptor clusters are associated with patches of ECM heparan sulfate PGs (Anderson and Fambrough, 1984). INTRODUCTION Some evidence suggests that PGs seem to be important Skeletal muscle fibers are surrounded by a special- for neuronal cell adhesion and growth in the peripheral ized form of extracellular matrix (ECM), the basal lamina. This structure is highly specialized at the neuromuscular junction region (Rubin and McMahan, 1982), Received April 23, 1991; revised November 15, 1991; accepted where it has been implicated in both nerve-muscle inter- November 27, 1991. action and muscle regeneration (Sanes, 1989). Special Address reprint requests to E. Brandan, Molecular Neurobiology Unit, interest has been focused on understanding the signifi- Department of Cell and Molecular Biology, Faculty of Biological cance of the components of the basal lamina. The isola- Sciences, Catholic University of Chile, P.O. Box 114-D, Santiago, tion and characterization of specific ECM components Chile. is, therefore, important in order to ascertain their func- M.E. Fuentes’ present address is Department of Pharmacology, tional roles. For instance, a glycoprotein, s-laminin, has School of Medicine, University of California, La Jolla, CA 92093.
Proteoglycans (PGs) are abundant components of the extracellular matrices (ECM) of skeletal muscle. We have previously found that the synthesis of skeletal muscle PGs present at the ECM increase after denervation. The experiments reported here were undertaken to identify which PG(s) increase after denervation of rat leg muscles. Incorporation of radioactive sulfate demonstrated the presence of a chondroitiddermatan sulfate PG of 70-90 kDa in the skeletal muscle ECM, which increased after denervation. The PG has a core protein of 3 9 4 5 kDa after treatment with chondroitinase ABC. Antibodies against rat decorin, a chondroitin/dermatan sulfate PG synthesized by various cell types, specifically immunoprecipitated this PG from a mixture of PGs. Immunocytolocalization of this PG indicated that the chondroitin/dermatan sulfate PG accumulates at the perimysium of skeletal muscle after denervation. Finally, Northern blot analysis indicated an increase of muscle transcripts for decorin after denervation. The data reported here suggest that a chondroitiddermatan sulfate PG present at the skeletal muscle ECM, very similar if not identical to decorin, increases after denervation. 0 1992 Wiley-Liss, Inc.
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nervous system. Thus it has been suggested that a heparan sulfate PG is involved in axonal regeneration along laminin-rich pathways (Chiu et al., 1986) and in the guidance of axons to original synaptic sites of denervated neuromuscular junctions (Sanes et al., 1986). Little is known regarding the control of the expression of muscle PGs. We have recently shown that denervation of adult rat leg muscles mainly caused an increase in the synthesis of a small PG present at the ECM and that the expression of the PGs in the skeletal muscle appears to be regulated by motor nerve activity. This effect is observed after 2-4 days of denervation (Fadic et al., 1990). In this report, we have identified the PG that mainly increases after denervation of rat skeletal muscle as a chondroitin/dermatan sulfate PG, called decorin. The increase after denervation is due to an increase in the synthesis of this molecule, since their mRNA increase roughly to the same extent.
MATERIALS AND METHODS Materials The following material was purchased from suppliers: Naz3'S0, carrier free was obtained from New England Nuclear, Boston, MA; dCT"P (6,000 Ci/mmol) from Amersham, Aylesbury, Buckinghamshire, England; Na'*'I from the Chilean Nuclear Commission, Santiago, Chile; chondroitinase ABC lyase (EC 4,2,2,4), chondroitinase AC lyase (EC 4,2,2,5), protein A-sepharose, agarose, benzamidine hydrochloride, DEAE-Sephacel, and sepharose CL-6B from Sigma Chemical Co. St. Louis, MO; and heparitinase (EC 4,2,2,8) from Miles Laboratories, Elkhart, IN. Policlonal rabbit antiserum against proteodermatan sulfate purified from rat skin fibroblast (Witsch et al., 1989) was a generous gift of Professor Hans Kresse (University of Munster, Germany). Human decorin cDNA was a kind gift of Dr. Tom Krusius (University of Helsinki, Finland). Other reagents were obtained from commercial sources. Methods Male Sprague-Dawley rats (200-250 g) were denervated by cutting the sciatic nerve as previously described (Fadic et al., 1990). Control or four-day denervated rats were injected intraperitoneally with 1.5-2.0 mCi of 35~-sulfatein saline. Isolation of Muscle Proteoglycans Legs muscles were removed, 18 hr after sulfate injection, and homogenized in 10 mM Tris-HC1 buffer, pH 7.4, containing 5 mM benzamidine, 50 mM 6-aminohexanoic acid, 10 mM N-ethylmaleimide, 10 mM EDTA, and 0.5% (v/v) Triton X-100 and centrifuged in
a Sorvall SS-34 rotor at 12,OOOg for 15 min at 4°C. The pellet was resuspended in the same homogenation buffer and centrifuged again. This last step was repeated twice. Supernatants were pooled and the labeled-PGs, present in the final pellet, were extracted as previously described (Brandan and Inestrosa, 1987a). The detergent insoluble pellet was solubilized with 6 M guanidine-HC1 and 50 mM sodium acetate, pH 5.8, containing protease inhibitors, for 12 hr at 4°C with continuous agitation. The extract was centrifuged as above and unincorporated radioactive precursors, guanidine-HC1, and salts were removed by dialysis with 100 volumes of 4 M urea, 0.1 M NaCI, 0.1% Triton X-100, protease inhibitors, and 50 mM sodium acetate, pH 5.8, for 6 hr and then with 100 volumes of the same solution, but containing 8 M urea, for another 6 hr at 4°C. After dialysis the radioactive material was applied to a DEAE-Sephacel column preequilibrated in the same 8 M urea buffer as previously described (Yanagishita and Hascall, 1984). The column was washed with 8 ml of the same buffer and than eluted with a continuous NaCl gradient, 0.2 to 1.1 M NaCl in the same buffer (60 ml total vol.). Elution was performed at a flow rate of 5.0 ml/hr and fractions of 1.O ml were collected. The NaCl gradient was monitored by measuring conductivity of the fractions. The labeled eluted material was pooled, dialyzed for 12 hr with 10 mM TrisHC1 buffer pH 7.4,0.5% Triton X-100, and 0.1 M NaCl containing protease inhibitors and precipitated by the addition of 2.5 vol. of cold ethanol. The labeled-PGs were collected by centrifugation and resuspended in 0.5 ml of 10 mM Tris-HC1 buffer pH 7.4 and 0.1 M NaCl.
Enzymatic Treatments and Chemical Analyses Chondroitinase ABC and AC treatments of labeledPGs were done exactly as previously described (Brandan and Inestrosa, 1987a). Heparitinase treatment was done as described by Yanagishita and McQuillan (1989). Immunoprecipitation of Decorin Decorin was specifically immunoprecipitated from a mixture of PGs isolated from leg muscles after DEAESephacel chromatography. The PGs were dialyzed with 10 mM Tris HCl, pH 7.4, 0.1 M NaCl and immunoprecipitated with protein A-sepharose coated with IgG from antiserum against rat decorin (Witsch et al., 1989), exactly as previously described by Gloss1 et al. (1984). The immunoprecipitates were subjected to electrophoresis in SDS-polyacrylamide slab gels (SDS-PAGE) as described below. Iodination and SDS-PAGE Analysis of Proteoglycans Purified PGs from the sepharose CL-6B column, eluting with a K,, of about 0.5 were iodinated using the
Motor Nerve Regulates Muscle Proteoglycans
chloramine T method (Carpenter and Cohen, 1976). Sulfated samples eluted from DEAE-Sephacel column and iodinated PGs were analyzed by electrophoresis on 10% and 3-10% SDS-PAGE and fluorographied as described previously (Carlson and Wight, 1987).
RNA Isolation and Northern Blot Analysis Total RNA from human fibroblasts was isolated using the procedure described by Chomczynski and Sacchi (1987). Muscle RNA was isolated from control and denervated groups (4-6 rats) by homogenizing the frozen tissue in guanidine thiocyanate solution followed by centrifugation over a cushion of CsCl as previously described (Goldman et al., 1988). Poly(A)+ RNA was selected by poly U-sepharose chromatography. Total RNA and poly(A)+ RNA were fractionated in 1 % agarose/2.2 formaldehyde gel as described by Krusius and Ruoslahti (1986) and transferred to Hybond-N membranes. A human decorin cDNA probe was radiolabeled by oligonucleotide random primer extension according to the manufacturer’s instructions (Amersham). Prehybridization was done for 3 hr at 44°C in the presence of 30% formamide, 5 X SSPE (0.18 M NaCl, 0.01 M sodium phosphate, 1 mM EDTA, pH 7.7), 1% SDS, 10% dextran sulfate, 5 x Denhardts solution, and 0.1 mg/ml of single stranded salmon DNA. Hybridization was carried out overnight at 44°C in the above solution containing 1 x lo6 c.p.m./ml of the 32P-labeled probe. Blots were washed at 42°C in 100 ml of 2 X SSPE, and four washes of 50 ml of 0.5 X SSPE, 0.1% SDS. Blots were exposed at -30°C overnight for human fibroblast RNA or for four days for rat skeletal muscle RNA. Histology Leg muscles from both control and denervated rats were cross-sectioned at 4 p m in a cryostat. Sections were fixed in 3% paraformaldehyde in phosphate saline, pH 7.4, and incubated sequentially with an antiserum against rat decorin (1/50 dilution in Blotto (Carey et al., 1987), for 1 hr at room temperature and then with a fluorescein-conjugated anti-rabbit antibody for 30 min at room temperature. After rinsing, the sections were mounted on glass slides and viewed with a Nikon Diaphot inverted microscope equipped for epifluorescence. RESULTS Denervation Increases the Synthesis of a Chondroitin/DermatanSulfate-PG Rat leg muscles denervated for 4 days increased in the synthesis of a sulfated PG with a molecular weight ranging from 70-90 kDa as determined by SDS-PAGE, after loading equivalent amounts of material (by weight) in each gel lane (Fig. 1A). The increase was about three-
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fold, as determined by densotimetric analysis of the autoradiograph. This PG was totally degraded by chondroitinase ABC treatment, while a minor effect was seen after incubation with chondroitinase AC. No degradation was observed when the sample was incubated with heparitinase (Fig. 1B). These results indicate that this denervation sensitive PG corresponds to a chondroitid dermatan sulfate PG of low molecular weight. To further characterize this PG, we studied the size of the core protein. For this purpose, an ECM rich fraction was chromatographied first in a DEAE-Sephacel column (Fig. 2A, top) followed by chromatography on Sepharose CL-6B (Fig. 2A, bottom). The second peak (Kavof 0.50) of the latter column was pooled, concentrated and iodinated. Figure 2B indicates that the material present at this step of purification possesses a molecular weight of 70-90 kDa and a contaminant of 53 kDa. Treatment with chondroitinase ABC completely degraded the PG, with the concomitant appearance of a band with a molecular weight of 39-45 kDa, where only a partially degradation was observed after chondroitinase AC treatment, suggesting that a small proportion of this PG population was sensitive to this treatment. These results strongly indicate that the chondroitin/dermatan sulfate-PG, which increases after denervation, has a core protein of 39-45 kDa.
The ChondroitinlDermatan Sulfate-PG Which Increases After Denervation Corresponds to Decorin A small chondroitin/dermatan sulfate-PG, named DS-PGII or decorin (Krusius and Ruoslahti, 1986) with a core protein of 38-45 kDa has been described in several ECM (Brennan et al., 1984; Gloss1 et al., 1984; Rosenberg et al., 1985; Vogel and Clark, 1989). In order to evaluate if our denervation sensitive PG corresponds to DS-PGII or decorin, a crude rat muscle ECM fraction labeled with sulfate was incubated with an antibody against rat decorin. Figure 3, lane 2, shows that the antiserum precipitated a single component which migrated as a more sharp band with a molecular weight of 70-80 kDa. Lane 1 of Figure 3 indicates that no 35Sradioactivity was precipitated when a normal rabbit serum was used. This result indicates that the PG which increases after denervation correspond to decorin. Decorin Accumulates Around the Muscle Fibers After Denervation To determine where decorin is located in rat skeletal muscle, we used light microscopic immunohistochemistry. Figure 4A shows that decorin accumulates in the interstitial spaces, either around packages of muscle fibers, the perimysium, or around the muscle fiber, the endomysium. Figure 4B shows that after four days of
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Fig. 1 . Increase of a sulfated chondroitinidermatan sulfate proteoglycan after denervation. Rats were denervated and after four days were injected with radioactive sulfate. A: An ECMlike fraction was prepared from control (C) and denervated (D) animals and applied to a DEAE-Sephacel column, as explained in Materials and Methods. Equivalent amounts of material, by weight, were separated on a 10% SDS-PAGE followed by fluorography . Denervation induced an 2.9-fold increase in the amount of a PG of 70-90 kDa, as determined by densitometric quantification of the autoradiograph. B: Sulfated PGs obtained
after DEAE-Sephacel chromatography were incubated with buffer alone (control), heparitinase (Hase), chondroitinase ABC (C. ABC) or chondroitinase AC (C. AC), as explained in Materials and Methods. After the incubation the PGs were fractionated on a 8% SDS-PAGE. The 70-90 kDa was totally degraded by chondroitinase ABC, partially degraded by chondroitinase AC, and totally resistant to heparitinase, suggesting that the 70-90 kDa PG corresponds to a chondroitinidermatan sulfate PG.
denervation, the space occupied by the perimysium increased and a higher reaction for decorin is visualized (arrows). However, at the endomysium, no significant changes in the amount of decorin was detected (arrowheads). These observations suggest that after skeletal muscle denervation there is an increase in the amount of decorin in the muscle interstitial space, the perimysium.
present in control versus denervated rat leg muscles. Poly(A)+ RNA was isolated from both control and 4-day denervated rat leg muscles and equal amounts of poly(A) RNA used for blot hybridization analysis using decorin cDNA. Figure 5 indicates that a substantial higher amount of transcript for decorin is present in denervated muscle compared with the control. Densitometric analysis of the autoradiography indicates a 3.4-fold increase in decorin mRNA. These results strongly suggest that the expression of the muscle mRNA for decorin increases after denervation.
Increase in Transcript for Decorin After Skeletal Muscle Denervation To further evaluate if decorin was expressed in muscle tissue, poly(A)+ RNA was isolated from rat leg muscles and submitted to blot hybridization analysis, using a cDNA against decorin. The results indicated that muscle tissue express an mRNA of 1.9 kb, similar in size to the transcript expressed by fibroblast (Krusius and Ruoslahti, 1986; Heino et al., 1988; data not shown). Then, we evaluated the amount of transcript for decorin
+
DISCUSSION We have recently shown that denervation of rat leg muscles increase the synthesis of PGs present at the ECM (Fadic et al., 1990), however, the PG(s) specie(s) that increase idwere unknown. The results in the present
Motor Nerve Regulates Muscle Proteoglycans
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Fig. 2. The chondroitiddermatan sulfate PG contains a core cold ethanol precipitation. Two peaks are observed; one with a protein of 38-45 kDa. A: Guanidine-solubilizedPGs from rat K,, of 0.25, which corresponds to chondroitin, and heparan leg muscle were fractionated on a DEAE-Sephacel column sulfate PGs, where the peak with a K,, of about 0.5 corre(top). Most of the material was bound to the column and eluted sponds to the chondroitiddermatan sulfate PG. B: The chonat about 0.6 M NaCl. Fractions 12-28 were pooled, concen- droitiddermatan sulfate PG containing fractions were ioditrated, and fractionated on an analytical Sepharose CLdB pre- nated and fractionated on SDS-PAGE after glycosidases pared in 1% sodium dodecyl sulfate (SDS), 0.1 M NaC1, and treatment. The 70-90 kDa PG was partially degraded by chon50 mM Tris-HC1 buffer, pH 8.0 (bottom). The latter column droitinase AC (C. AC), while chondroitinase ABC (C. ABC) was run at a flow rate of 5 .O ml/hr and effluent fractions of 1.O completely degraded the iodinated-PG with the concomitant ml were collected, counted for radioactivity, and pooled before appearance of the 39-45 kDa core protein. study indicate that denervation of skeletal muscles induce the synthesis and expression of a chondroitiddermatan sulfate PG very similar or identical to decorin, based on the following criteria: the size of the PG and its core protein, their specific immunoprecipitation using an anti-rat decorin serum, and the detection of RNA transcript which hybridizes with a specific cDNA probe for human decorin. We found that muscle decorin has a molecular weight of 70-90 kDa; this size is a slightly lower than the one reported for decorin synthesized by other tissues (Fisher et al., 1983; Brennan et al., 1984; Gloss1 et al., 1984; Vogel and Clark, 1989). This muscle PG represent about the 50% of the total PGs present in the skeletal muscle ECM (Brandan and Inestrosa, 1987b). Most of our studies were done after 4 days of denervation; we have recently shown that the synthesis of PGs increase after 2 days of denervation, reaching a steady level after 6 days (Fadic et al., 1990).
It is well known that the level of expression of several muscle macromolecules is strongly influenced by the presence of the nerve. Thus, the level of cell surface acetylcholinesterase (Lomo et al., 1985) and extrajunctional acetylcholine receptor (Reiness and Hall, 1977; Merlie et al., 1984) is regulated by contractile activity. We have previously shown that the level of expression of sulfated PGs is influenced by contractile activity and a new steady state level of expression is observed after reinnervation of the muscle (Fadic et al., 1990). Here, we demonstrated that the level of expression and synthesis of a specific PG decorin is influenced by the presence of the motor nerve; further studies are necessary to establish the mechanism involved in such neural control. Why there is an increase of decorin after denervation is not clear, but it is well known that ECM provides a unique physical and functional boundary which specifies the properties of the normal assembly of the neuromus-
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2
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3727Fig. 3. The chondroitiddermatan sulfate PG is specifically precipitated by an antibody against rat decorin. An aliquot of sulfated PGs obtained after DEAE-Sephacel chromatography was incubated with a non-immune serum (lane 1) or with an antibody against rat decorin (lane 2), as explained in Materials and Methods. The immunoprecipitates were then separated on a 10% SDS-PAGE, followed by fluorography. The antiserum specifically precipitated a sulfated PG of 70-90 kDa.
cular junction. Axons preferentially form new junctions at original synaptic sites (Sanes, 1989). Thus, a muscle fiber might respond to denervation by changing its surface properties, thereby increasing its attractiveness for regenerating axons, where ECM molecules might play an essential role. With respect to decorin expression, it is known that the expression of recombinant decorin in Chinese hamster ovary cells converts these morphologically normal cells and greatly reduces their saturation density; these results indicate that the expression of decorin might be mediating the growth properties of the cell(s) (Yanaguchi and Ruoslahti, 1988). Decorin can bind collagen and fibronectin through its core protein (Vogel et al., 1984; Lewandowska et al., 1987; Schmidt et al., 1987), regulating the structure and thickness of collagen fibrils at the ECM. According with this, we detected that after muscle denervation, decorin accumulated in the interstitial spaces, specifically at the
Fig. 4. Increase of decorin in the perimysium after denervation of rat leg muscles. Rat leg muscles from control (a) and from 4-day denervated animals (b) were fixed and stained with an antibody against decorin, followed by a second fluorescentconjugated antibody as explained in Materials and Methods. An increase in the amount of decorin is observed in the perimysium, around packages of muscle fibers, after denervation (arrows). No significant changes were seen at the endomysium (arrowheads) ( x 660).
perimysium, where collagen fibrils and fibronectin are present. However, we did not see a significant increase in decorin at the endomysium. These observations raise several questions: Is the expression of ECM components coordinately regulated? How does the mechanism by which the cell(s) responsible for the synthesis of ECM molecules respond to muscle activity? There are some lines of evidence that might help to answer some of these questions: It has been shown that the transforming growth factor-beta (TGF-P) stimulates the expression of ECM molecules such as fibronectin, type I collagen, cell adhesion receptor, and decorin in different cell types (Ignotz and MassaguC, 1987; Cheifetz et al., 1987; Bassols and MassaguC, 1988), suggesting a regulated and coordinated expression of several ECM molecules. With
Motor Nerve Regulates Muscle Proteoglycans
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In regard to the cell(s) type responsible for the synthesis of the ECM components, it has been shown that several adhesive molecules, such as tenascin, N-CAM, fibronectin, and a heparan sulfate PG which accumulates at synaptic sites in the muscle after denervation (Covault and Sanes, 1985; Sanes et al., 1986), are synthesized by muscle fibroblasts which proliferate after denervation of skeletal muscle in the interstitial space (Zak et al., 1969; McGeatchie and Allbrook, 1978; Murray and Robbins, 1982; Connor and McMahan, 1987; Gatchalian et al., 2 8 S 1989). Decorin is synthesized and secreted by fibroblasts from several sources (Miyamoto and Nagase, 1980; Coster and Fransson, 1981; Damle et al., 1982; Nakamura et al., 1983; Gloss1 et al., 1984; Vogel and Clark, 18% 1989), however, we have obtained evidence from a skeletal muscle rodent cell line, which differentiates in vitro to form contractile multinucleated myotubes, indicating that decorin is synthesized by them (Brandan et al., 1991). Therefore, in muscle tissue there is the possibility Fig. 5. Decorin muscle transcripts increased after denervation. that both cell types, fibroblasts and myotubes, synthesize Left: 30 pg of poly(A)+ isolated from control (c) and denerdecorin. Which of those cell types respond to denervavated (d) rat leg muscles was separated electrophoretically , tion by increasing the synthesis of decorin is a matter that blotted on nylon membranes, and hybridized with 32-P-labelled we are investigating now. cDNA probe for human decorin. An increase in the amount of
c
d
c
d
RNA transcript specifically for decorin is observed after four days of denervation. The film was exposed for 4 days. Right: Ethidium bromide staining of the gel indicates no difference between isolated RNA from control and denervated rat leg muscles. Ribosomal RNAs are still present in the fractions and the position is indicated.
ACKNOWLEDGMENTS We thank Dr. N.C. Inestrosa for helpful suggestions and careful reading of the manuscript and Dr. C. Koenig for the sections for histology. This work was supported by a grant from FONDECYT 569-89 to E.B.
respect to the mechanism by which the cell(s) responsible for the synthesis of the PGs respond to muscle activity, one can postulate that the absence of muscle ac- REFERENCES tivity as an effect of denervation might increase the Anderson MJ, Fambrough DM (1984): Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan synthesis of diffusible factor (growth factor-like) which sulfate proteoglycan on the surface of skeletal muscle fibers. J activates the expression of ECM components in a conCell Biol 97:1396-1411. certed fashion. For instance, it has been shown that in- Bassols A, Massague, J (1988): Transforming growth factor-B regusulin (Stevens et al., 1981), insulin-like growth factors lates the expression and structure of extracellular matrix chondroitiddermatan sulfate proteoglycans. J Bio! Chem 263: (Salmon and Daughaday, 1957; Froesch et al., 1976), 3039-3045. fibroblast growth factor (Kato and Gospodarowicz, 1985a), and glucocorticoids (Kato and Gospodarowicz, Bayne EK, Anderson MJ, Fambrough MD (1984): Extracellular matrix organization in developing muscle: Correlation with ace1985b) affect the expression of cartilage specific PGs tylcholine aggregates. J Cell Biol 99: 1486-1501. and that interleukin-1 regulates cellular mRNA level of Brandan E, Hirschberg CB (1989): Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apdecorin (Heino et al., 1988). One can speculate in the paratus and the plasma membrane. J Biol Chem 264:10520case of the muscle tissue that the hypothetical diffusible 10526. factor is not synthesized by the nerve, because either the Brandan E, Inestrosa NC (1984): Binding of the asymmetric forms of level of expression of acetylcholine receptors or the exacetylcholinesterase to heparin. Biochem J 221:415-422. pression of PGs (Lomo and Rosenthal, 1972; Reiness Brandan E, Inestrosa NC (1987a): Isolation of the heparan sulfate proteoglycans from extracellular matrix of rat skeletal muscle. and Hall, 1977; Fadic et al., 1990) is regulated by the J Neurobiol 18:271-282. muscle activity itself. Furthermore, the increase in expression of decorin after denervation might be related to Brandan E, Inestrosa NC (1987b): Co-solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the fact that some growth factors have the ability to bind extracellular matrix of rat skeletal muscles. FEBS Lett 213: PGs, creating a reservoir of growth factors for cells to 159-163. Brandan E, Maldonado M, Garrido J, Inestrosa NC (1985): Anchoruse (Saksela et al., 1988; Ruoslahti, 1989).
58
Brandan et al.
age of collagen-tailed acetylcholinesterase to the extracellular matrix of rat skeletal muscles. J Cell Biol 101:985-992. Brandan E, Fuentes ME, Andrade W (1991): The proteoglycan decorin is synthesized and secreted by differentiated myotubes and increase during in vitro differentiation. Eur J Cell Biol 551209-216. Brennan MJ, Oldberg A, Pierschbacher MD, Ruoslahti E (1984): Chondroitinidermatan sulfate proteoglycan in human fetal membranes. J Biol Chem 22: 13742-1 3750. Carey DJ, Rafferty CM, Todd S (1987): Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells. J Cell Bid 105:1013-1021. Carlson SS, Wight TN (1987): Nerve terminal anchorage protein I (TAP-1) is a chondroitin sulfate proteoglycan. Biochemical and electron microscopic characterization. J Cell Biol 105:30753086. Carpenter G, Cohen S (1976): ‘Z51-labeledhuman epidermal growth factor. J Cell Biol 71:159-171. Carrino DA, and Caplan A1 (1984): Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart. J Biol Chem 259: 12419-1 2430. Cheifetz S, Weatherbee JA, Tsang M L-S, Anderson JK, Mole JE, Lucas R, Massaguk, J (1987): The transforming growth factorB system, a complex pattern of cross-reactive ligands and receptors. Cell 48:409-415. Chiu AY, Matthew WD, Patterson PH (1986): A monoclonal antibody which blocks the activity of a neurite regeneration promoting factor: Studies on the binding site and its localization in vivo. J Cell Biol 102:1383-1398. Chomczynski P, Sacchi N (1987): Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156-159. Connors EA, McMahan UJ (1987): Cell accumulation in the junctional region of denervated muscle. J Cell Biol 104:109-120. Coster L, Fransson L-A (1981): Isolation and characterization of dermatan sulfate proteoglycans from bovine sclera. Biochem J 1931143-153. Covault J, Sanes JR (1985): Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles. Proc Natl Acad Sci USA 82:4544-4548. Damle SP, Coster L, Gregory JD (1982): Proteodermatan sulfate isolated from pig skin. J Biol Chem 2575523-5527. Fadic R, Brandan E, Inestrosa NC (1990): Motor nerve regulates muscle extracellular matrix proteoglycans expression. J Neurosci 10:3S16-3523, Fisher LW, Termine JD, Dejter SW, Whitson SW, Yanagishita M, Kimura JH, Hascall VC, Kleinman HK, Hassell JR, Nilsson B (1983): Proteoglycans of developing bone. J Biol Chem 258: 6588-6594. Froesch ER, Zapf J, Andhya TK, Ben-Porath E, Segen BJ, Gibson KD (1976): Nonsupressible insulin-like activity and thyroid hormones: Major pituitary-dependent sulfation factors for chick embryo cartilage. Proc Natl Acad Sci USA 73:2904-2908. Fransson L-A (1987): Structure and function of cell-associated proteoglycans. Trends Biochem Sci 12:406-41 I . Gatchalian CL, Schachner M, Sanes JR (1989): Fibroblast that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin ( J l ) , N-CAM, fibronectin, and heparan sulfate proteoglycan. J Cell Biol 108: 1873-1890. Gloss1 J, Beck M, Kresse H (1984): Biosynthesis of proteodermatan sulfate in cultured human fibroblasts. J Biol Chem 259:1414414150.
Goldman D, Brenner HR, Heinemann S (1 988): Acetylcholine receptor alpha, betha, gamma, and deltha-subunit mRNA levels are regulated by muscle activity. Neuron 1 :329-333. Heino J , Kakari V-M, Mauviel A, Krusius T (1988): Human recombinant interleukin-1 regulates cellular mRNA levels of dermatan sulphate proteoglycan core protein. Biochem J 252:309312. Hunter DD, Shah V, Merlie JP, Sanes JR (1989): A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature (London) 338:229-234. Ignotz RA, MassaguC J (1987): Cell adhesion protein receptors as targets for transforming growth factor-B. Cell 51:189-197. Kato Y , Gospodarowicz D (l985a): Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor. J Cell Biol 100:477-485. Kato Y , Gospodarowicz D (1985b): Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro. J Biol Chem 260:23642373. Krusius T, Ruoslahti E (1986): Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 83:7683-7687. Lewandowska K, Choi HU, Rosenberg LC, Zardi LC, Culp LA ( 1987): Fibronectin-mediated adhesion in fibroblast: Inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminiglycan-binding domain. J Cell Biol 105: 1443-1 454. Lomo T, Rosenthal J (1972): Control of ACh sensitivity by muscle activity in the rat. J. Physiol. 221:493-513. Lomo T, MassouliC J , Vigny M (1985): Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms. J Neurosci 5: 1 180-1 187. McGeatchie J , Allbrook D (1978): Cell proliferation in skeletal muscle following denervation or tenotomy. Cell Tissue Res 193:2S9267. Merlie JP, Isenberg IE, Russel SD, Sanes JR (1984): Denervation supersensitivity in skeletal muscle analysis with a cloned cDNA probe. J Cell Biol 99:332-335. Miyamoto I , Nagase S (1980): Isolation and characterization of proteodernatansulfate from rat skin. J Biochem 88: 1793-1 803. Murray MA, Robbins N (1982): Cell proliferation in denervated muscle: Time course, distribution, and relation to disuse. Neuroscience 7: 1817-1822. Nakamura T, Matsunaga E, Shinkai H (1983): Isolation and some structural analysis of a dermatan sulphate from calf skin. Biochem J 213:289-296. Parthasarathy N , Tanzer ML (1987): Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle. Biochemistry 2 6 3 149-3 156. Reiness GC, Hall ZW (1977): Electrical stimulus of denervated muscle reduces incorporation of methionine into the ACh receptor. Nature 268:6SS-657. Rosenberg LC, Choi HU, Tang L-H, Johnson TL, Pal S, Webber C, Reiner A, Poole R (1985): Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages. J Bid Chem 260:6304-63 13. Rubin LL, McMahan UJ (1982): Regeneration of the neuromuscular junctions: Steps toward defining the molecular basis of the interaction between nerve and muscle. In Schotland DL (ed): “Disorders of the Motor Unit.” New York: John Wiley & Sons, pp 187-196. Ruoslahti E (1988): Structure and biology of proteoglycans. Ann Rev Cell Biol 4:229-255.
Motor Nerve Regulates Muscle Proteoglycans Ruoslahti E (1989): Proteoglycans in cell regulation. J Biol Chem 264: 13369-13372. Saksela 0, Rifkin DB (1990): Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activation-mediated proteolytic activity. J Cell Biol 110:767-775. Salmon WD, Daughaday WH (1957): An hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825-836. Sanes JR (1989): Extracellular matrix molecules that influences neural development. Annu Rev Neurosci 12521-546. Sanes JR, Schachner M, Covault J (1986): Expression of several adhesion macromolecules in embryonic, adult, and denervated adult skeletal muscle. J Cell Biol 102:420-43 1. Schmidt G , Robenek H, Harrach B, Gloss1 J , Nolte V, Hormann H, Richter H, Kresse H (1987): Interaction of small dermatan sulfate proteoglycan from fibroblast with fibronectin. J Cell Biol 104:1683-1691. Stevens RL, Nissley SP, Kimura JH, Rechler MM, Caplan AI, Hascall VC (1 981): Effect of insulin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the swarm rat chondrosarcoma. J Biol Chem 256:2045-2052. Vogel K , and Clark P (1989): Small proteoglycan synthesis by skin
59
fibroblasts cultured from eldery donors and patients with defined defects in types I and I11 collagen metabolism. Eur J Cell Biol 49:236-243. Vogel KG, Paulsson M, Heinegard D (1984): Specific inhibition of type I and type I1 collagen fibrillogenesis by the small chondroitin sulfate-dermatan sulfate proteoglycan in the human. Biochem J 223587-597. Wjtsch P, Kresse H, Gressner AM (1989): Biosynthesis of small proteoglycans by hepatic lipocytes in primary culture. FEBS Lett 258:233-235. Yanagishita M, Hascall VC (1984): Proteoglycans synthesized by rat ovarian granulosa cells in culture: Isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J Biol Chem 259:10260-10269. Yanagishita M, McQuillan DJ (1989): Two forms of plasma membrane-intercalated heparan sulfate proteoglycan in rat ovary granulosa cells. J Biol Chem 264: 17551-17558. Yamaguchi Y, Ruoslahti E ( I 988): Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation. Nature 336:244-246. Zak R, Grove D, Rabinowitz M (1969): DNA synthesis in the rat diaphragm as an early response to denervation. Am J Physiol 2 16:647-654.
