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Endocrinology Vol. 140, No. 3 1075-1081
Copyright © 1999 by The Endocrine Society

ARTICLES

Retinol-Binding Protein Is Produced by Rabbit Chondrocytes and Responds to Parathyroid Hormone (PTH)/PTH-Related Peptide-Cyclic Adenosine Monophosphate Pathway

Yoshie Kawashima-Ohya1,2, Yoshihiro Kuruta2, Weiqun Yan, Takeshi Kawamoto, Mitsuhide Noshiro and Yukio Kato

Department of Biochemistry, Hiroshima University School of Dentistry, Hiroshima 734, Japan

Address all correspondence and requests for reprints to: Yukio Kato, Department of Biochemistry, Hiroshima University School of Dentistry, 1–2-3, Kasumi, Minami-ku, Hiroshima, 734, Japan. E-mail: ykato{at}ipc.hiroshima-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH and dibutyryl cAMP [(Bu)2cAMP] induced the expression of a 19-kDa protein in the conditioned media of rabbit growth plate chondrocyte cultures. The 19-kDa protein was identified as plasma retinol-binding protein (RBP) by aminoterminal sequence analysis and immunoblot analysis with an anti-RBP monoclonal antibody. Northern blot analysis showed that PTH, PTH-related peptide (PTHrP), and (Bu)2cAMP increased the RBP messenger RNA (mRNA) level in chondrocyte cultures. Further, both PTH and (Bu)2cAMP markedly induced the expression of RBP mRNA by about 10-fold at 3 h and by about 40-fold at 24 h, indicating a pretranslational regulation. The level of the mRNA expression induced by PTH, PTHrP, and (Bu)2cAMP was as high as that by retinoic acid (RA), known as a potent inducer of RBP in hepatoma cells. RBP mRNA was also detected in cartilage tissues at higher levels than in the other tissues examined except liver. Both RBP and PTH/PTHrP inhibited the dedifferentiative activity of RA on growth plate chondrocytes when added to the culture medium. These results demonstrate that chondrocytes synthesize and secrete RBP in vivo and in vitro and suggest that PTH/PTHrP modulates the effect of RA by means of RBP production in chondrocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE GROWTH plate of developing bone, chondrocytes proliferate, synthesize the cartilage matrix, and become hypertrophic. Several hormones and growth factors have been reported to be involved in controlling chondrocyte differentiation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In particular, PTH and PTH-related peptide (PTHrP) are thought to play important roles during endochondral ossification. Using a rabbit chondrocyte culture system, we have shown that PTH/PTHrP increases the synthesis of DNA and aggrecan and suppresses the alkaline phosphatase induction, type X collagen synthesis, and matrix calcification (13, 14, 15). Karaplis et al. (16) and Amizuka et al. (17) reported that the null mutation of the PTHrP gene in mice resulted in imperfect maturation of prehypertrophic chondrocytes and promotion of chondrocytes to hypertrophy in the growth plate. Weir et al. (18) showed that overexpression of PTHrP in mice caused prolonged maturation of prehypertrophic chondrocytes and delayed endochondral ossification. Recently, Vortkamp et al. (19) reported that PTHrP modulated the rate of chondrocyte differentiation in the growth plate. These in vitro and in vivo findings suggest that PTH/PTHrP enhances cartilage matrix synthesis and inhibits hypertrophy of chondrocytes during endochondral bone formation.

PTH and PTHrP bind to a common PTH/PTHrP receptor that mediates their stimulation to intracellular signals such as cAMP, inositol phosphate, and calcium (20, 21, 22). In rabbit chondrocytes, we have shown that PTH and PTHrP bind to the receptor expressing on chondrocytes, increase the intracellular cAMP level within a few minutes, and that dibutyryl cAMP [(Bu)2cAMP] (a permeable analog of cAMP) mimics all of the examined PTH/PTHrP actions (13, 23). Thus, the main effects of PTH/PTHrP in chondrocytes are thought to be mediated by the cAMP pathway. However, little is known about the molecular mechanisms by which the PTH/PTHrP-cAMP pathway controls growth and differentiation of chondrocytes. It is necessary to clarify the events occurring between the increase of intracellular cAMP and the eventual growth and differentiation of the cells.

The purpose of this study is to find the molecules affected directly by PTH/PTHrP and/or cAMP in cultured chondrocytes. In this study, retinol-binding protein (RBP) was purified from the culture medium of rabbit chondrocytes exposed to (Bu)2cAMP. RBP is known as a carrier protein of retinoids synthesized mainly in liver (24), and its expression is controlled by retinoids (25, 26). This is the first report to demonstrate in chondrocytes that: 1) the cells synthesize and secrete RBP; 2) PTH/PTHrP-cAMP regulates the expression of RBP pretranslationally; and 3) PTH/PTHrP may modulate the effect of retinoic acid (RA) by means of RBP production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant PTH (1–84) and PTHrP (1–34) were supplied by Dr. K. Sato (Chugai Pharmaceutical Co., Tokyo, Japan). (Bu)2cAMP and all-trans RA were purchased from Sigma Chemical Co. (St. Louis, MO). The monoclonal antibody to human RBP was purchased from Biogenesis (London, UK). The rabbit RBP complementary DNA (cDNA) was supplied by Dr. Dianne R. Soprano (Temple University, Philadelphia, PA) (27). Rabbit aggrecan cDNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were generated by the RT-PCR method, from total RNA of rabbit chondrocytes, using a forward (5'-TGCTACTTCATCGACCCCAT-3') and reverse (5'-AAAGACCTCCCCTCCATCT-3') primer for aggrecan and a forward (5'-GCTTCACCACCTTTTGATG-3') and reverse (5'-GTCAAGGCTGAGAACGGGAA-3') primer for GAPDH. These oligonucleotides were synthesized by Kurabo Co. (Osaka, Japan), based on the sequence of mouse aggrecan (EMBL/GenBank/DDBJ database Accession No. L07049) and rabbit GAPDH (No. L23961), respectively. The PCR products were subcloned into the pGEM T-vector (Promega Corp., Madison, WI) and confirmed their nucleotide sequences by the auto sequence analyzer, ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Japan Co., Tokyo, Japan). {alpha}-[32P]deoxycycidine triphosphate (111 terabecquerel/mmol) was obtained from DuPont NEN (Boston, MA). The oligolabeling kit was a product of Pharmacia Biotech (Uppsala, Sweden).

Chondrocyte cultures
Chondrocytes were isolated from growth plates or resting zones of ribs and from the surface zone (0.2 mm) of femur articular cartilage of 4-week-old male Japan white rabbits, as described (28). Our experimental procedures concerning animal care and treatment were performed under the permission, rules, and guidelines of Hiroshima University. Cells were seeded at a density of 3 x 105 cells/35-mm dish, 5 x 105 cells/100-mm dish, or 3 x 104 cells/16-mm dish and grown in MEM-{alpha} medium (Sanko Pharmaceutical, Tokyo, Japan) supplemented with 10% FCS (Mitsubishi Kagaku Co., Tokyo, Japan), 32 U/ml penicillin, 60 µg/ml kanamycin (Meiji Seika Co., Tokyo, Japan), and 250 ng/ml Amphotericin B (Dainippon Pharmaceutical Co., Osaka, Japan) at 37 C, under 5% CO2 in air. RA was dissolved in ethanol and diluted with PBS. Other hormones and metabolic agents were dissolved in saline.

Morphological changes were monitored with an IX70 phase-contrast microscope (Olympus Corp., Tokyo, Japan).

SDS-PAGE
One week after becoming confluent, the cells were exposed to PTH, PTHrP, or (Bu)2cAMP for a maximum of 48 h; and the conditioned media were harvested. The conditioned media of chondrocyte cultures were concentrated using a microconcentrator (Ultrafree C3GC, Millipore Corp. Japan Co., Tokyo, Japan). Proteins in the samples were resolved by SDS-PAGE in the absence of ß-mercaptoethanol (nonreducing condition), then stained using the Daiichi silver staining kit (Daiichi Chemical Co., Tokyo, Japan).

Purification of 19-kDa protein
The conditioned medium of 1 mM (Bu)2cAMP-exposed chondrocytes (1 liter) was concentrated by ultrafiltration, using Microcon (10-kDa cut off membrane, Millipore Corp. Japan Co.). The concentrated medium (60 ml) was applied to a diethylaminoethyl-Sepharose column (1.5 x 20 cm) equilibrated with 10 mM Tris-HCl, pH 8.0. Proteins were eluted with 30 ml of 0.1, 0.5, and 1.0 M NaCl in 10 mM Tris-HCl (pH 8.0), and fractions (1 ml) were collected. Proteins in the fractions were resolved in a 15–25% gel by SDS-PAGE to detect the 19-kDa protein. The 19-kDa protein was eluted in a 0.5 M NaCl fraction. This fraction was dialyzed against 10 mM Tris-HCl (pH 8.0) and applied to a MonoQ-Sepharose column (0.5 x 5 cm) (Pharmacia Biotech), which was eluted with a linear gradient of 0–2 M NaCl in 10 mM Tris-HCl (pH 8.0) at a flow rate of 0.5 ml/min, and fractions (0.5 ml) were collected. The 19-kDa protein was eluted with 0.3 M NaCl (see Fig. 2AGo). Further purification was performed by gel filtration on a Superose-12 column (1 x 30 cm) that was equilibrated with PBS. Proteins were eluted from the column with PBS, and fractions (1 ml) were collected.



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  		Figure 2. Purification of 19-kDa protein from the conditioned media of rabbit chondrocyte cultures. A, MonoQ-Sepharose ion exchange chromatography. Arrowhead indicates the fraction rich in the 19-kDa protein (fraction 8). B, Superose-12 size exclusion chromatography. The fraction containing the 19-kDa protein was no. 19 (arrowhead). C, SDS-PAGE of the purified 19-kDa protein eluted from the Superose-12 column. Proteins of fraction 19 eluted from the Superose-12 column were resolved by SDS-PAGE under nonreducing conditions. Lane 1, Molecular marker; lane 2, purified protein.

 
Determination of amino acid sequence
The purified 19-kDa protein was resolved in a 15–25% gel by SDS-PAGE, then electrophoretically transferred to a ProBlot membrane (PE Applied Biosystems, Foster City, CA). The membrane was stained with Coomassie Brilliant Blue. The spot of 19-kDa protein was cut out and subjected to automatic aminoterminal sequence analysis in a protein sequencer 476A (PE Applied Biosystems).

Immunoblot analysis
Proteins (1 µg) in the conditioned media of rabbit chondrocytes were resolved by SDS-PAGE, then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore Corp. Japan Co.). After treatment for 1 h with 4% skim milk (Yukijirushi Nyugyo Co., Sapporo, Japan), the membrane was incubated with the monoclonal antibody against human RBP for 2 h at room temperature. After washing with PBS containing 0.05% Tween-20, the membrane was incubated with 125I-labeled sheep antimouse IgG (Fab’)2 fragment (Amersham, Buckinghamshire, UK). The membrane was washed with PBS containing 0.05% Tween-20 and exposed to Kodak BMX film (Eastman Kodak Co., Rochester, NY) at -80 C.

Northern blot analysis
Northern blots were performed using total RNA extracted from cultured rabbit chondrocytes and 4-week-old rabbit tissues with guanidine thiocyanate (29). The RNA samples were denatured by 2.2 M formaldehyde and 50% formamide, electrophoresed on 1% agarose gels containing 2.2 M formaldehyde, as described by Thomas (30), and transferred to Nytran nylon membranes (Schleicher & Schuell, Inc., Dassel, Germany). The membranes were hybridized with the 32P-labeled probes of NcoI-EcoRI 735-bp fragment RBP cDNA or 613-bp GAPDH cDNA in hybridization solution containing 6 x saline-sodium citrate, 5 x Denhardt’s, 10 mM EDTA, 1% SDS, and 0.5 mg/ml sonicated salmon sperm DNA at 68 C. The membranes were washed with 0.5 x saline-sodium citrate containing 0.5% SDS at 50 C and were exposed to Kodak BMX film at -80 C. The radioactivities of the hybridized bands were measured using Bio-imaging Analyzer System BAS2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). After hybridization, the membrane was stained with methylene blue to confirm the equal loading of total RNA on each lane (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH and (Bu)2cAMP induce 19-kDa protein in cultured chondrocytes
One week after becoming confluent, rabbit growth plate chondrocytes were exposed to 0–1.0 µM PTH or 0–1.0 mM (Bu)2cAMP for 48 h. Proteins in the conditioned media were resolved by SDS-PAGE under nonreducing conditions. Figure 1Go shows that some protein bands were altered by the treatment with PTH or (Bu)2cAMP at various concentrations. In particular, 19-kDa protein was obviously induced by both PTH and (Bu)2cAMP, depending on the concentration of the inducers (Fig. 1Go, A and B).



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  		Figure 1. SDS-PAGE of the conditioned media, harvested from rabbit growth plate chondrocyte cultures. Rabbit growth plate chondrocytes were seeded and maintained as described in Materials and Methods. On day 14, the cells were exposed to PTH (A; 0, 0.1, 1, 10, and 100 nM for lanes 1–5, respectively) and (Bu)2cAMP (B; 0, 0.01, 0.1, 0.5, and 1.0 mM for lanes 6–10, respectively) for 48 h, and the conditioned media was harvested. Samples of 1 µg protein were resolved by SDS-PAGE under nonreducing conditions. M, Molecular weight marker.

 
Purification of the 19-kDa protein
The conditioned media of rabbit chondrocytes were concentrated and applied to a diethylaminoethyl-Sepharose column; and subsequently, the fractions rich in 19-kDa protein were applied to a MonoQ-Sepharose column, as described in Materials and Methods. The 19-kDa protein was eluted in fraction 8 from the MonoQ-Sepharose column, as indicated by the arrowhead in Fig. 2AGo. The protein was finally purified to homogeneity by chromatography on a Superose-12 column, and the 19-kDa protein (approximately 30 µg) was eluted at fraction 19 (Fig. 2BGo). The purified 19-kDa protein showed a single band on SDS-PAGE (Fig. 2CGo).

Amino acid sequence of 19-kDa protein was identical to that of RBP
The final preparation of the 19-kDa protein was subjected to SDS-PAGE and blotted to a ProBlot membrane, as described in Materials and Methods. The blotted spot of the 19-kDa protein was cut out and subjected to automatic aminoterminal sequence analysis. The aminoterminal sequence of the protein was determined as Glu-Arg-Asp-X-Arg-Val-Ser-Ser-Phe-Arg-Val-Lys-Glu-Asn-Phe. A homology search, using the SWISS-PROT protein sequence database, revealed that the aminoterminal sequence of the 19-kDa protein was identical to that of rabbit plasma retinol-binding protein (rabbit RBP), except for the 4th unidentified amino acid residue. Thus, the 19-kDa protein was most likely RBP. The size of 19 kDa was also coincident with the reported molecular mass of rabbit RBP (24).

19-kDa protein cross-reacted with anti-RBP antibody
To confirm that the 19-kDa protein is RBP, immunoblot analysis of the purified protein was performed using the antihuman RBP monoclonal antibody. As shown in Fig. 3Go, the purified 19-kDa protein (lane 1) strongly cross-reacted with the antibody. Figure 3Go also shows that the cross-reactive band was hardly detectable in the conditioned medium of control chondrocyte cultures (lane 2), whereas PTH (1 84) (lane 3) and (Bu)2cAMP (lane 5) obviously induced cross-reaction to the antibody in cultured media. In addition, PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (lane 4), the aminoterminal portion of PTHrP, also increased the level of this protein. These findings clearly indicate that the 19-kDa protein induced by PTH, PTHrP, and (Bu)2cAMP is RBP.



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  		Figure 3. Immunoblot analysis of the purified 19-kDa protein and the conditioned media from cultured chondrocytes. The final purified protein (10 ng, lane 1), the conditioned media containing 1 µg of protein prepared from the control culture (lane 2), and the cultures 48 h after the treatment of PTH (100 nM, lane 3), PTHrP (100 nM, lane 4), and (Bu)2cAMP (1.0 mM, lane 5) on day 14 were resolved by SDS-PAGE under nonreducing conditions, then transferred to a polyvinylidene difluoride membrane.

 
PTH, PTHrP, (Bu)2cAMP, and RA induced RBP messenger RNA (mRNA)
Having identified the 19-kDa protein as RBP, we next examined the induction of RBP mRNA by PTH, PTHrP, and (Bu)2cAMP in cultured chondrocytes, by Northern blot analysis. As shown in Fig. 4Go, the basal level of RBP mRNA in the control chondrocytes (lane 1) was hardly detectable, as was the basal protein level (Fig. 3Go, lane 2); whereas PTH (lane 2), PTHrP (lane 3), and (Bu)2cAMP (lane 4) markedly induced 1 kb-RBP mRNA at 24 h after the treatment to the same extent as did RA (lane 5), known as a direct inducer of RBP in cultured hepatocytes (25). This result indicates that the induction of RBP by PTH, PTHrP, (Bu)2cAMP, and RA occurred at the pretranslational level.



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  		Figure 4. Induction of the RBP mRNA in chondrocyte cultures by PTH, PTHrP, (Bu)2cAMP, and RA. Northern blot analysis for RBP, aggrecan, and GAPDH was performed using total RNA (3, 10, and 3 µg, respectively) prepared from the cultured chondrocytes 24 h after the addition of vehicle (lane 1), PTH (100 nM, lane 2), PTHrP (100 nM, lane 3), (Bu)2cAMP (1 mM, lane 4), and RA (1 µM, lane 5). Exposure to x-ray film was performed for 3 h. The equal loading of total RNA sample on each lane was confirmed by the staining with methylene blue.

 
The level of aggrecan mRNA, which is a chondrocyte phenotypic marker, was up-regulated by PTH, PTHrP, and (Bu)2cAMP and down-regulated by RA.

Because the level of GAPDH mRNA examined for comparison showed significant decrease by treatment with RA, we confirmed the equal loading of total RNA sample on each lane by the staining with methylene blue.

To examine the responsiveness to the inducers, a time course study of mRNA induction by PTH or (Bu)2cAMP was carried out. Figure 5Go shows that the RBP mRNA was markedly increased in a time-dependent manner by PTH or (Bu)2cAMP, whereas the GAPDH mRNA level did not show any significant change. Normalization of the radioactivities of the hybridized RBP bands with those of GAPDH bands indicates that about a 10-fold induction of the RBP mRNA occurred as early as 3 h after adding these inducers and that the mRNA level at 24 h was 40-fold that of the respective controls.



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  		Figure 5. Time course of the induction of RBP mRNA by PTH or (Bu)2cAMP. Samples of 3 µg total RNA, prepared from the cultured chondrocytes at 0, 3, 6, and 24 h after the addition of PTH (100 nM) or (Bu)2cAMP (1 mM), were separated on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized to 32P-labeled rabbit RBP cDNA or rabbit GAPDH cDNA. Exposure to x-ray film was performed for 3 h.

 
Expression of the RBP mRNA in cartilages and other tissues
To examine whether cartilage tissue contains RBP mRNA, we performed Northern blot analysis of total RNA prepared from growth plate, resting, and articular cartilages. As shown in Fig. 6AGo, these cartilages expressed a significant level of RBP mRNA. Further, Northern blot analysis was performed to compare the mRNA level of cartilage with those of other tissues. Fig. 6BGo shows that resting cartilage contained a higher level of RBP mRNA than other tissues such as bone, brain, eye, and kidney (except liver, which is a main organ of RBP synthesis). One tenth of the liver RNA still contained 6 times more RBP mRNA than resting cartilage, as shown in the figure. Although it has been reported that kidney and eye (lacrimal gland) synthesize RBP, the mRNA levels in these tissues were much lower than that of cartilage, as shown in Fig. 6BGo.



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  		Figure 6. Tissue distribution of the RBP mRNA. Northern blot analysis for RBP mRNA was performed using total RNA (10 µg) isolated from rabbit growth plate, resting and articular cartilages (A), and other tissues (B). G, R, and A (in panel A) indicate growth plate, resting, and articular cartilage, respectively. In panel B, the following abbreviations are used: Ca, resting cartilage; Bo, bone; Br, brain; Ey, eye; Ki, kidney; and Li, liver. One-tenth of the amount (1 µg) of liver RNA was also analyzed. Exposure to x-ray film was performed for 12 h.

 
RBP and PTH inhibited RA effects on morphological change of the chondrocytes
RA is a potent dedifferentiative factor for chondrogenic cells, and it causes morphological changes of chondrocytes: cells change from polygonal or round shape to fibroblastic spindle shape. To examine the effects of RBP on cultured chondrocytes, we observed the morphological changes of growth plate chondrocytes exposed to RA in the presence or absence of RBP or PTH in preconfluent states. We observed the cells at 6 h of treatment of the factors in subconfluent cultures, because it is easier to detect the morphological changes of chondrocytes in subconfluent than in confluent cultures, and 6 h is long enough for RA to cause morphological changes and for PTH to induce RBP (data not shown).

Incubation of the polygonal chondrocytes with RA for 6 h changed their configuration to the fibroblastic spindle shape, which reflected a dedifferentiated phenotype (Fig. 7BGo). In contrast, RBP made the polygonal cells round, which is characteristic of well-differentiated chondrocytes (Fig. 7CGo). RA plus RBP decreased the number of spindle-shaped (dedifferentiated) cells and increased that of round (matured) cells (Fig. 7DGo). PTH had effects similar to those of RBP, inducing a change from polygonal to round shape, in the absence (Fig. 7EGo) or presence (Fig. 7FGo) of RA, under the culture conditions. Similar results were obtained with RA, RBP, and/or PTH in five independent studies. These results suggest that both RBP and PTH, at least partly, inhibit dedifferentiative effects of RA in cultured chondrocytes.



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  		Figure 7. Effects of RBP, RA, PTH, and their combinations on morphology of the chondrocytes. Rabbit growth plate chondrocytes were seeded and maintained as described in Materials and Methods. Before becoming confluent (70% confluent), the cells were exposed to vehicle (A), RA (1 x 10-7 M) (B), RBP (3 x 10-7 M) (C), a combination of RA plus RBP (D), PTH (1 x 10-7 M) (E), or that of RA plus PTH (F). After 6 h of treatment, photographs were taken, and the cells were counted and classified into three groups under their shapes: round, polygonal, and spindle-like cells. Some cells could not be classified. The control cultures contained polygonal (59%), round (23%), and spindle-like cells (7%). The cultures exposed to RA contained spindle-like (45%), round (27%), and polygonal cells (17%). The cultures exposed to RBP contained round (62%), polygonal (32%), and spindle-like cells (1%). The cultures exposed to RA plus RBP contained round (68%), polygonal (16%), and spindle-like cells (4%). The cultures exposed to PTH contained round (71%), polygonal (16%), and spindle-like cells (7%). The cultures exposed to RA plus PTH contained round (58%), polygonal (23%), and spindle-like cells (15%).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To identify molecules that are affected by PTH, PTHrP, and (Bu)2cAMP directly, we first focused on a 19-kDa protein that was induced in conditioned media of rabbit chondrocyte cultures, depending on the concentrations of PTH and (Bu)2cAMP. Based on its molecular mass, aminoterminal sequence, and cross-reactivity with the anti-RBP antibody, the 19-kDa protein was identified as RBP.

RBP is mainly synthesized in liver and secreted in the form of retinol-RBP complex. The liver regulates circulating concentrations of retinol and RBP over a wide range of dietary vitamin intakes. In hepatoma cells, it has been shown that RA and retinol regulate RBP expression (25). Panariello et al. (26) reported that RA response elements (RARE) were present in the promoter region of the human RBP gene. In the present study, we showed that RBP mRNA was rapidly and strongly induced by PTH/PTHrP, as well as RA, in cultured chondrocytes. RBP expression, in response to PTH/PTHrP or (Bu)2cAMP, clearly demonstrates that synthesis and secretion of RBP are controlled by a PTH/PTHrP-cAMP pathway in chondrocytes. The induction of RBP by the PTH/PTHrP-cAMP pathway occurred, at least at the pretranslational level. Concerning the regulation of RBP mRNA expression by cAMP, it is noteworthy that a computer search of the 5'-flanking sequence of the human RBP gene revealed a cAMP-responsive element-like sequence (TTACGAAA) (32). Thus, in chondrocytes, there may be at least two mechanisms for regulation of RBP expression, the RA pathway and the PTH/PTHrP-cAMP pathway; the latter is demonstrated in this study as a novel mechanism. In other words, RBP is a common molecule, functioning in both PTH/PTHrP-cAMP and RA pathways.

Most of the retinol-RBP complex in plasma is found in complex with the second protein, transthyretin (TTR). Retinol is transported from liver into plasma in the form of retinol-RBP-TTR complex and delivered to retinoid-requiring target tissues (24). In target cells, the retinol molecules are thought to be taken up from the retinol-RBP-TTR complex through a specific cell surface receptor for RBP (33, 34, 35). Moreover, the RBP mRNA is also expressed in several extrahepatic organs, including kidney, adipose tissue (36, 37), visceral yolk sac (38, 39), uterine (40), and lacrimal gland (27). The physiological functions of RBP produced by these nonhepatic tissues are unknown, but it has been suggested that RBP controls the transportation of retinol into these tissues, most of which have a relatively high sensitivity to the surrounding retinol concentration (24).

The function of RBP in cartilage is also unknown. Previous in vivo studies have clarified that newborn animals show abnormal skeletal tissues, including growth plates, both in hypovitaminosis A and hypervitaminosis A (41, 42). An excessive dosage of retinol in calves resulted in a narrow epiphyseal growth plate because of the reduction of the number of columns and columnar-zone cells and led to immature ossification (43). In in vitro studies, Benya et al. (44) showed that RA suppresses the phenotype of matured chondrocytes and the production of type II collagen and aggrecan, and changes chondrocytes from a rounded shape to a flattened spindle shape. These studies indicate that chondrocytes are also sensitive to retinoids and that appropriate control of retinol- or RA concentration is important for endochondral bone formation. It is plausible that RBP promotes the transportation of retinol into cartilages, as well as in other nonhepatic RBP-producing tissues. On the other hand, Dingle et al. (45) reported that the addition of apo-RBP prevented retinol- induced cartilage matrix degradation in the organ culture of chick limb rudiments. Our results showed that both RBP and PTH/PTHrP, which is a potent inducer of RBP, suppress the dedifferentiative action of RA. We believe that RBP serves as a modulator of the retinoid bioactivities and regulates the availability of retinoids in cartilages.

In conclusion, this study demonstrated that chondrocytes synthesize and secrete RBP, PTH/PTHrP-cAMP regulates the expression of RBP pretranslationally, and cartilage tissues contain RBP mRNA at significant levels. Clarification of the mechanisms of function and expression of RBP may help us to understand the molecular mechanisms of cartilage formation and endochondral bone formation.


   Acknowledgments
 
We thank Dr. D. R. Soprano (Temple University) for the gift of rabbit RBP cDNA, Dr. K. Sato and T. Mori (Chugai Pharmaceutical Co.) for the human recombinant PTH (1 84), and Dr. H. Pan (Hoechst Marion Roussel, Inc., Tokyo, Japan) for performing protein sequence analysis. We also thank the Research Center for Molecular Medicine of Hiroshima University School of Medicine and the Research Center of Hiroshima University School of Dentistry for the use of their facilities.


   Footnotes
 
1 Present address: AIST-NIBHT CREST Centre of Structural Biology, Tsukuba. Back

2 These authors contributed equally to this work. Back

Received March 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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