Autocrine Regulation of Rat Chondrocyte Proliferation by Natriuretic ...

Vol. 269, No. 14,Issue of April 8,pp. 10729-10733, 1994 Printed in U.S.A.

THEJOURNALOF BIOLWICAL CHEMLSTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Autocrine Regulation of Rat Chondrocyte Proliferationby Natriuretic Peptide C and Its Receptor, Natriuretic Peptide Receptor-B* (Received forpublication, August 17, 1993, and in revised form, January 4, 1994)

Hiromi HagiwaraS, Hajime Sakaguchi, Makoto Itakura, Takanobu Yoshimoto, Mayumi Furuya9, Shoji Tanaka9, and Shigehisa Hirose From the Department of Biological Sciences, Tokyo Znstitute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227 and the §Suntory Znstitute for Biomedical Research, Shimamoto, Mishima, Osaka 618, Japan

Natriuretic peptide receptor-B (NPR-B) was identified in rat chondrocytes, and its physiological functions were investigated. Rat tissues, including the xiphoid cartilage, brain, lung, liver, adrenal gland, and kidney, were screened for NPR-B activity, whichwe assayed by receptor guanylate cyclase activity specifically stimulated by C-type natriuretic peptide (CNP), a known selective activator of NPR-B. Cartilage showed distinctly higher NPR-B activity. Furthermore, exposure of cultured rat chondrocytes to CNP M) resulted in a large increase in intracellular cGMP production (376 38 pmoywell), with threshold responses occurring between M CNP. Atrial natriuretic peptide and brain and lo-@ natriuretic peptide also stimulated cGMP production in rat chondrocytes but with a potency that was at least 10 times less than that of CNP. Polymerase chain reaction analysis also demonstrated NPR-B gene expression in adult rat xiphisternum and cultured chondrocytes. These findings indicate that NPR-B is present in rat chondrocytes. In rat chondrocytes exposed to CNP, [SH]thymidineincorporation was inhibited in a dose-dependent manner (half-maximal response,lo-" M). However, much higher concentrations of atrial natriuretic peptide were required to induce the inhibition of thymidine incorporation. Interestingly, CNP-like immunoreactivity was detected in the conditioned medium from chondrocyte cultures. In addition, TGF-P1, a multifunctional cytokine, induced a marked increase in CNP secretion and CNPmRNA levels in chondrocytes. These results indicate that autocrine CNP inhibits mitogenesis in chondrocytes via NPR-B under the control of TGF-P1.

Atrial natriuretic peptide(ANP)' is the firstdescribed member of a family of hormones known to play important roles in the regulation of body fluid and electrolyte balance (1).Subse-

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan, and by grants from Nissan Science Foundationand Kat0 Memorial Bioscience Foundation. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby 1734 marked "advertisement"in accordancewith18U.S.C.Section solely to indicate this fact. To whom correspondence should be addressed. "el.: 81-45-922-1111 (ext. 2281); Fax: 81-45-921-7790. The abbreviations used are: ANP, atrial natriuretic peptide or natriuretic peptide A; BNP, brain natriuretic peptide or B-type natriuretic peptide; C-ANF, des[Gln'8,Ser's,Gly20,Leu21,Gly22]ANP,,3-NH2; CNP, C-type natriuretic peptide; NPR-A or GC-A, typeA natriuretic peptide receptor; NPR-B or GC-B, type B natriuretic peptide receptor; NPR-C, type C natriuretic peptide receptor; DMEM, Dulbecco's modified Eagle's medium;TGF-Dl,transforminggrowthfactor-Dl;PCR,polymerase chain reaction.

quently, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) have been isolatedfrom porcine brain extracts (2,3). Like ANP, both of these hormones can elicit vasorelaxing, natriuretic, and diuretic responses. ANP and BNP have been found in a variety of tissues where they may serve diverse functions. CNP, however, has been detected only in central nervous tissues (4,51, endothelium (6), and humanmonocytic cell (7). On the other hand, threedifferent natriuretic peptide receptors have been identified by molecular cloning studies (8-12): natriuretic peptide receptor-A (NPR-A or GC-A), -B (NPR-B or GC-B), and -C (NPR-C). Two of these, NPR-Aand-B, are membersof the receptor guanylatecyclase family and are capable of synthesizing their own second messenger, cGMP. NPR-C does notcontainguanylate cyclase activity, and its physiological significance other than the clearance of ligands (13)is not clear. The diverse biological activities of the natriuretic peptides are thought to be mediated by intracellular accumulation of cGMP through the activationof receptor guanylate cyclase. Although, as mentioned above, NPR-A and NPR-B are structurally and functionallyvery similar,they have quite different ligandspecificities; NPR-A is sensitive to ANP andBNP, whereas NPR-B is highly specific for CNP (14). To date, Northern blot analysis (12, 15), PCR analysis (16, 171, and in situ hybridization (18)have demonstrated the existence of NPR-B mRNA in many tissuesof the body including brain, lung, kidney, adrenal gland, intestine, uterus, and oviduct. Autoradiographic investigation using '251-[Tyro]CNPhas also demonstrated the localization of NPR-B in ratkidney, adrenal gland, and brain tissues (19). In addition to this tissue level localization, NPR-B has been demonstrated in cultured cells such as rat vascular smooth muscle cells (20), brain endothelial cells (211, glioma cells (221, pheochromocytoma (PC121 cells (231, and human mesangial cells (24). However, the physiological roles of NPR-B on the target tissues have not yet been fully elucidated. A search for an abundantsource of NPR-B will, therefore, be useful for studying its physiological function and for analyzingthesignaltransductionmechanism.Inthe present study, we demonstrated the existence of NPR-B activated selectively by CNP in rat chondrocytes. Furthermore, we showed that the CNPNPR-B system may modulate chondrocyte proliferation i n a nautocrine manner. EXPERIMENTALPROCEDURES Materials-Synthetic and biologically active rat ANP, BNP,and CNP, and human parathyroid hormone were purchased from Peptide Institute, Osaka, Japan. C-ANF, a specific competitor of NPR-C, was obtained from Peninsula Laboratories, Inc.;lZ5I-ANP (74 TBq/mmol) and L3H1thymidine (2.59-3.33 TBq/mmol) were from DuPont NEN; the cyclic GMP assay system was from Yamasa, Chiba, Japan; 3-isobutyl-lmethylxanthine was from Sigma; Dulbecco's modified Eagle's medium (DMEM)and penicillinhtreptomycin antibiotic mixture were from Life

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Technologies, Inc.;fetal bovine serum was from Filtron, Victoria, Australia; TGF-pl was from King Brewing, Hyogo, Japan; mouse tumor necrosis factor-cr and basic fibroblast growth factor were from Genzyme Corp. and Oncogene Science, Inc., respectively; mouse interleukin-lp and -6 were from Upstate Biotechnology,Inc.;Moloney murine leukemia virus reverse transcriptase was from LifeTechnologies,Inc.; GeneAmpm PCR reagent kit with AmpliTaqa DNA polymerase was from Perkin-Elmer. Specific antiserum against CNP was a kind gift from Dr. H. Matsuo and Dr. K. Kangawa, National Cardiovascular Center Research Institute, Osaka, Japan. Zsolation and Cultivation of Chondrocytes-The xiphisterna of two adult Wistar rats (male, 150 g) were aseptically separated and cut into pieces.Chondrocytesweredissociated by treatment with 10 ml of DMEM containing 1 mg/mlcollagenase and 1000 protease unitdml Dispase at 37 "C for 1 h. The isolated cells were plated in three 55-cm2 dishes and grown in DMEM supplemented with 10% fetal bovine serum, 50 unitdml penicillin, and 50 pg/ml streptomycin in a humidified atmosphere of 5% CO, in air at 37 "C. The cells were maintained in culture medium until they reached confluency and then detached by 0.05% trypsin and replated in 12-well plates (3.8 cm2).Early passages (primary to 3) of the cells were used for the experiments. Guanylate Cyclase Activity in Plasma Membranes of Various Rat Tissues-Rat brain, xiphoid cartilage, lung, liver, adrenal gland, and kidney plasma membranes were prepared as described previously(25, 26). Fresh rat tissues were homogenized with a Waring Blendor for 30 s in 3 volumes of 50 m~ Tris-C1 buffer (pH 7.6) containing 1m~ EDTA, 0.2 m~ phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 10pg/ml pepstatin, and 0.2 m~ phosphoramidon (BufferA), and thehomogenate was centrifuged at 100,000 x g for 20 min at 4 "C. The membranes were suspended in 3 volumes of Buffer A and recentrifuged. This washing step was repeated twice. Guanylate cyclase activities were assayed by incubating 50 pg of the membrane preparation for 15 min at 37 "C in a 100-pl reaction mixture containing 50 m~ Tris-C1 buffer (pH 7.6), 0.5 m~ 3-isobutyl-l-methyluanthine, 0.1% bovine serum albumin, 15 m~ creatine phosphate, 20 pg (125 unitdmg) of creatine phosphokinase, 1 m~ GTP, 4 m~ MnCl,, and lo4 M ANP or CNP. The amounts of cGMP were measured by radioimmunoassay with Yamasa radioimmunoassay kits. Intracellular Cyclic GMP Measurement-The rat chondrocytes were seeded onto 12-well cluster plates. The confluent cell monolayer was washed twice with 1ml of serum-free DMEM and then preincubated at 37 "C for 15 min with 0.5 ml of DMEM containing 0.5 m~ 3-isobutyl-lmethylxanthine. After the incubation, various concentrations of ANP, BNP, and CNP were added into the medium, and the cell monolayerwas further incubated at 37 "C for 15 min. The medium was rapidly removed by aspiration, and 200 pl of 1N HCl solution containing 0.5 m~ EDTA were added to lysethe cells and to stop the production of cGMP. The amounts of cGMP were measured as described above. Reverse lkanscription of RNA and Polymerase Chain Reaction-RNA from rat xiphisternum or cultured rat chondrocytes wasprepared by the acid g u a n i d i n i d p h e n o l o r o f o r m extraction (27). Total RNA (1 pg) was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase, Superscript (200 units), using oligo(dT)primers (5 nmol) lof reaction mixture. Amplification or random primers (5 nmol) in 20 p was performed using 4 i o of this cDNA by 35 cyclesof PCR in 100 pl of AmpliTaq polymerase mixture containing 1 p~ sense primer, 5'-CTGGATCCTCGCACAGATGACCGGCC-3'(nucleotides585-610), and antisense primer, 5'-CACTGCAGTCTGAAAGGCCTCTCTGAGGGCCTG3' (nucleotides 956-924), for rat NPR-B, or sense primer, 5"CGCACCATGCACCTCTCCCAGCTGAT-3' (nucleotides -6 to 20), andanti(nucleotides sense primer, 5'-CGCTGCACTAACATCCCAGACCGC-3' 388-365), for rat CNP. The reaction cycle consisted of 94 "C for 1 min, 45 "C for 1min, and 75 "C for 2 min. PCR products were extracted with phenol and chloroform and ethanol-precipitated. Samples were electrophoresedon a 3% agarose gel and visualized by ethidium bromide staining. The PstI fragment of the NPR-B PCR product was sequenced by the dideoxy chain termination method (28) after subcloning into pBluescript I1 (Stratagene). For analysis of CNP, PCR products were blotted onto nylon membranes (MagnaGraph, Micron Separations Inc.) after electrophoresis. Southern blots wereprehybridized at 37 "C for 2 h in 6 x SSC (1x SSC: 0.15 M NaCl and 15 m~ sodium citrate, pH 7.0) containing 5 x Denhardt's (1 x Denhardt's: 0.1% each of bovine serum albumin, polyvinylpyrrolidone,and Ficoll) and 0.1% SDS,and thenhybridized at 37 "C for16 h in the same solution containing 3ZP-end-labeledprobe (5'AATCAGAAAAAGGGTGACAAGACT-3')at lo6cpdml. The blots were washed twice in 1 x SSC and 0.1% SDS at 40 "C for 1 h. Washed blots

Chondrocytes

were analyzed with a Fuji Film bioimage analyzer model Fujix BAS 2000. Cell Proliferation-Primary culture cells were seeded onto 24-well plates at a density of 10,000 celldwell and cultured for 48 h in the presence of 10% fetal bovine serum. Subsequently, cells were washed twice with serum-free DMEM and incubated with serum-free DMEM for 24h. After 18 h of incubation in fresh serum-free DMEM containing ANP or CNP, [3Hlthymidine(1pCi/well) was added for an additional 4 h, and the cells were washed twice with ice-cold phosphate-buffered saline. Acid-soluble radioactivity was removed by treatment with 5% trichloroacetic acid for 20 min at 4 "C. Cells were washed twice with ethanol, solubilized by incubation with 0.1 M NaOH and 2% Na,C03 for 30 min, and the radioactivity was determined by liquid scintillation counting. to confluRadioimmunoassay for CNP-Chondrocytes were grown ence in 55-cm2dishes (-6 x lo6 cellddish). The cells were washed twice with serum-free DMEM and incubated in 8 ml of serum-free DMEM in the presence or absence of TGF-pl(1 PM),tumor necrosis factor-a (200 , fibroblast growth unitdml), humanparathyroid hormone (10 n ~ )basic factor (1ng/ml), interleukin-lp (0.5 ng/ml), and interleukin-6 (1ng/ml) for48h.After the incubation, the conditioned medium (8 ml) was collected and centrifuged a t 700 x g for 5 min. CNP in the 8 mlof medium was extracted using Sep-Pak C,, cartridges (Waters Chromatography Division, Millipore), lyophilized, and reconstituted. Cells were washed twice with serum-free DMEM, scraped from the dish with a rubber policeman, centrifuged at 700 x g for 5 min, and thenboiled for 7 min in 400 pl of 0.1 M acetic acid containing 0.1% Triton X-100 and disrupted by sonication (Microson, Heat Systems Ultrasonic Inc.). The cell homogenates were centrifuged at 10,000 x g for 30min at 4 "C and the supernatants subjected to radioimmunoassay.The amounts of CNP in the conditioned media(8 ml) and cell lysate obtained from the cells in 55-cmzdishes were estimated as fmol/dish/48 h. Radioimmunoassay forCNP was performed using specific antiserum raised against CNP according to the method of Minamino et al. (29). 1261-[TyrolCNPwas prepared by the lactoperoxidase method (30) and purified by reverse phase high pressure liquid chromatography. In this radioimmunoassay system, a-rat ANP and rat BNP-45 showed 0.015 and 0.1% cross-reactivity,respectively, on a molar basis. RESULTS

Identification ofNPR-B in Rat Chondrocytes-CNP activates NPR-B more selectively and potently than ANP, while ANP potently activates NPR-A. Using this selectivity, we screened various rat tissues for NPR-B activity. The tissues examined included cartilage (xiphisternum), brain, lung, liver, adrenal gland, andkidney. The relativelevels of NPR-B activity in each tissue wereexpressed as the ratioof guanylate cyclase activity stimulated by CNP M) to thatstimulated by ANP M) (Fig. 1).Cartilage showed a high NPR-B activity compared with thatof NPR-A (cGMP formation by CNPkGMP formation by ANP = 2.43) and responded to CNP (10" M) with a marked increase incGMP content (53.9 pmoVlOO pg of protein). Brain tissue, in which NPR-B is present (191, showed almost equivalent NPR-A and NPR-B activities. Other tissues suchas lung, liver, adrenalgland,and kidneypredominantlyexpressed NPR-A. To study NPR-B of rat cartilage in detail, we established a cell culture system for chondrocytes from adult rat xiphisternum. Fig. 2 shows the dose-response curves for the intracellular accumulation of cGMP by natriuretic peptides in cultured cGMP chondrocytes. CNP caused a rapid and steep increase in levels, with threshold responses occurring between 10"' and lo-' M CNP. Addition of 1O"j M CNP resulted in an approximately 116-fold increase in intracellular cGMP over the basal level. The rank order of potency of the different natriuretic peptides was CNP>> ANP 2 BNP. To further confirm the presence of NPR-B in chondrocytes, we carried out PCR analysis using rat xiphisternum and cultured chondrocyte cDNAs. Oligonucleotide probes (see "Experimental Procedures" for nucleotidesequences) were prepared based on the publishedsequence of rat NPR-B cDNA (12). As expected, PCR produced a 372-base pair product exclusively, as demonstrated by agarose

CNP and ItsReceptor in Rat Chondrocytes

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8 Ca Br Lu Li Ki Ad FIG.3. Detection of NPR-B mRNA by PCR The productsof PCR FIG.1. Relative abundanceof NPR-Bover NPR-Adetermined by comparing CNP-stimulated and ANP-stimulated particulateamplification of cDNAs from rat cartilage (xiphisternum) and cultured guanylate cyclase activities.Cyclic GMP generation was measured rat chondrocytes were electrophoresedon a 3% agarose gel. The arrow in the presence of ANP or CNP as described under "Experimental Pro- points to a 372-base pair ( b p ) fragment corresponding to rat NPR-B V Boehringer cedures'' using membranes from various tissues. Values are expressed cDNA. Size markers (DNA molecular weight marker from as ratios of cGMP formation with CNP (NPR-B activity) tocGMP for- Mannheim) are shown on the left. mation with ANP (NPR-A activity); Ca,cartilage (cGMP formation with CNP (pmoV100 pg of protein)/cGMP formation with ANP(pmoV100 pg protein) = 53.9122.2 = 2.43); Br, brain (21.0/15.7 = 1.34); Lu,lung (80.9/ 100 178.2 = 0.45);Li, liver(4.4/18.3 = 0.24);Ki, kidney(12.0128.3 = 0.42);Ad. adrenal gland(117.6/406.9 = 0.29). Values represent the means of three determinations. 80 0 ANP

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FIG.4. Dose-responsecurvesshowinginhibitoryeffects of ANP and CNP on [SH1thymidine incorporation into rat chondrocyte DNA. Values are percentages of control (4950 dpdwell). Control represents thymidine incorporation cells in in serum-free medium without ANP or CNP. Cells and samples were preparedas described under "Experimental Procedures."

FIG.2. CNP-induced cGMPaccumulationin cultured rat chondrocytes. Rat chondrocytes wereg r o w n to confluence in 12-well plates (3.8 cm'). Cells wereexposed to increasing concentrationsofANP, BNP, cyte-conditioned medium was parallelto the standard curve of and CNP at 37 "C for 15 min in the presence of 0.5 mM 3-isobutyl-lCNP as shown in Fig. 5 A . The basalconcentrations of CNP-like methylxanthine, and then intracellular cGMP levels were determined immunoreactivity in the conditioned medium (8 ml) and cell as described under "Experimental Procedures." lysate corresponding to -6 x lo6 cells in 55-cm2 dishes were

gel electrophoresis (Fig. 3). DNA sequence analysis showed the 591.4 and 39.2 fmoVdisW48 h, respectively. The level of CNPobtained cDNA fragments to be identical to the rat NPR-B like immunoreactivity in the conditioned medium was 15-fold higher than the cellular content of CNP-like immunoreactivity, cDNA. These results indicate that chondrocytes in cartilage suggesting that CNP synthesized in chondrocytes was immespecifically express NPR-B. diately secreted to the surrounding medium without intracelCell Proliferation-Recently, some studiesusingcultured cells (31-38) have suggested that ANP regulates cell prolifera- lular storage. We next examined the effects of various agents, tion. Thus, we investigated theeffects of natriuretic peptides on including TGF-p1, tumor necrosis factor-a,human parathyroid factor, interleukin-lp, and the incorporation of thymidine into the DNA of cultured rat hormone, basic fibroblast growth chondrocytes. Fig. 4shows a typicalexample of significant interleukin-6, on CNP secretion from cultured chondrocytes. inhibition of chondrocyte mitogenic activity by ANP or CNP. Interestingly, TGF-p1 significantly augmented the accumulaThe inhibitory effect of CNP on chondrocyte mitogenesis was tion of CNP-like immunoreactivity in theconditioned medium, whereas other agents did not affect CNP production by chonM) inhibited DNA-associated dose dependent, and CNP [3Hlthymidine incorporationby about 50%. The potency of CNP drocytes. As shown in Fig. 5B,exposure to TGF-pl(1 PM)for 48 in inhibitingchondrocyte mitogenesis was 102-103 times higher h increased the levels of CNP in chondrocyte-conditioned methan thatofANP. Furthermore, C-ANF, a specific competitor of dium from 0.59 pmol (74 p~)/dish/48h up to 1.85 pmol (231 NPR-C, did not attenuate thymidine incorporation into chon- p~)/dish/48h. To further support the TGF-pl-stimulatedprodrocytes. These results suggest that rat chondrocyte prolifera- duction of CNP in culturedchondrocytes, Southern blotting of PCR products from cells treated with TGF-p1 was carried out tion isregulated by the action of natriuretic peptidesvia NPR-B. with 32P-end-labeledoligonucleotides corresponding to a cDNA Detection of CNP-like Immunoreactivity in Chondrocytes and sequence located between the two PCR primers. Fig. 6 shows Its Conditioned Medium-The serial dilution curve of chondro- the effects of TGF-P1 on CNP mRNA levels in cultured chon-

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Control

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FIG.5. Production ofCNP by cultured chondrocytes andits stimulation by TGF-f31 measured by radioimmunoassay.A, a typical standard curve of CNP (open circles) and a dilution curveof the conditioned medium from rat chondrocyte culture (closed circles). B, CNP-like immunoreactivity in the culture medium and cell lysate of rat chondrocyte culture exposed to TGF-PI. Cells of primary culture were grown to confluence in55-cm2dishes and incubated with 8 ml of serum-free DMEM in thepresence or absence of 1 PMTGF-Pl for48 h. Cell lysates obtained from these cells and the conditioned medium (8 ml) were subjected to radioimmunoassayfor CNP. Three dishes were used per group.*, p < 0.05 uersus control.

1

2

FIG. 6. Demonstration of TGF-Pl stimulation of CNP transcript levels in cultured chondrocytesby Southern blottingof PCR products. Poly(A)' RNA (50 ng) from cultured rat chondrocytes treated or untreated with 1PM TGF-PI (lanes I and 2, respectively) for 48 h were amplifiedby PCR (40 cycles) as described under "Experimen2% agarose gels, and tal Procedures." PCR products were separated on Southern blotting was performed with a 32P-end-labeled oligonucleotide probe (5'-AATCAGAAAAAGGGTGACAAGACT-3') corresponding to coding sequences located between the two PCR primers. The arrow points to a 395-base (pair b p )fragment corresponding rat to CNPcDNA.

drocytes. When cells were treated with TGF-Pl(1 PM)for 48 h, the levels of CNP mRNA increased markedly. DISCUSSION

Localization of CNP in the brain(4, 5) and endothelium (6) and a wider tissue distribution of NPR-B than initially envisaged have been demonstrated. However, the precise functional role of the CNPNF'R-B system remains to be elucidated. Recent studies, however, have suggested the action of the CNPI NPR-B system inspecific tissues other than the cardiovascular system; for example, porcine seminal plasma contains large amounts of CNP, and NPR-B mRNA has been demonstrated in the uterus and oviduct, indicating that the CNPNF'R-B system may play a role in fertilization (15).NPR-B has alsobeen cloned from human retina, and ATP was found to be obligatory for CNP signaling in the retina (39). In the present study, we clearly demonstrated the existence of NPR-B and its specific ligand, CNP, in rat chondrocytes and the augmentation of CNP

by TGF-P1. We further showed that CNP modulateschondrocyte proliferation in an autocrine manner. These findings suggest that the CNPNF'R-B system acts as a local regulator in the cartilage. I t h a sbeen proposed that ANP has growth regulatory properties in a variety of tissues and cultured cells. For example, Appel(31) and Itoh et al. (34) havedescribed that ANP inhibits mitogenesis of rat mesangial and vascularsmooth muscle cells, respectively, through cGMP produced by receptor guanylate cyclase. Furthermore, Pines and Hurwitz(33)showed that the proliferation of avian epiphyseal growth plate chondroprogenitor cells is modulated by ANP, probably mediated by cGMP as a second messenger. Recent studies by Garg and Hassid (40) support theconcept that cGMP is important in inhibition the of mitogenesis in rat mesangial cells and vascularsmooth muscle cells (41). However, their results have not determined which receptor guanylate cyclase, NPR-A or NPR-B, regulates cell proliferation. Our results also indicate that natriuretic peptides inhibit rat chondrocyte proliferation. Furthermore, CNP was muchmore effective than ANP in the inhibition and C-ANF had no effect (Fig. 4), suggestingthat NPR-B is involved in cell proliferation. Growth and proliferation of the epiphyseal cartilage is controlled by various hormones such as growth hormone (42,43) andthyroid hormones(44,45). We avoided the use of serum because growth hormones and many poorly defined componentsin serum make interpretation of results more difficult. The findings indicate that natriuretic peptide ( A N Por CNP) itself participates in the proliferation of chondrocytes. Further investigation is necessary to determine the role of cGMP in thecontrol of chondrocyte proliferation. If CNP can act as a growth inhibitor, the question of where CNP originates remains. CNP is synthesized by endothelium (6) and secreted into the circulation. However, cartilage, in which chondrocytes reside, has no blood vessels of its own and so is seemingly unrelated to the circulatory system. In the present study, we detected CNP in cultured chondrocytes and chondrocyte-conditioned medium (Fig. 51, indicating that CNP may induce the inhibition of chondrocyte proliferation in an autocrine manner. TGF-P1 modulates the phenotype of chick embryo sternal chondrocytes (46) and the proliferation of rabbit growth plate chondrocytes (47). The production of CNP in rat chondrocytes was also modified by TGF-P1. This is consistent with the reportby Suga et al. (6), who showed that endothelial production of CNP is markedly augmented by TGF-P1. As a

CNP and Its Receptor in Rat Chondrocytes consequence of the stimulation by 1 PM TGF-P1, CNP concentration in the conditioned medium reached a concentration of about 2 x 10"' M (Fig. 51, which is sufficient to activate chondrocyte NPR-B (Fig. 2) and inhibit thymidine incorporation (Fig. 4). The mechanisms of signal transduction and transcriptional regulation of TGF-P1 in the chondrocytes remain to be elucidated. Acknowledgments-We are grateful to Dr. N. Takahashi (Showa University, Tokyo, Japan) for helpful discussions concerning the cultivation. We also thank Kazuko Tanaka and Setsuko Satoh for technical and secretarial assistance.

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