help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palmer, G.
Right arrow Articles by Caverzasio, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palmer, G.
Right arrow Articles by Caverzasio, J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 141, No. 6 2236-2243
Copyright © 2000 by The Endocrine Society

ARTICLES

Transforming Growth Factor-ß Stimulates Inorganic Phosphate Transport and Expression of the Type III Phosphate Transporter Glvr-1 in Chondrogenic ATDC5 Cells1

Gaby Palmer, Jerôme Guicheux, Jean-Philippe Bonjour and Joseph Caverzasio

Division of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis and Bone Diseases, Department of Internal Medicine, University Hospital, CH-1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Joseph Caverzasio, Division of Bone Diseases, Department of Internal Medicine, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland. E-mail: caverzas{at}cmu.unige.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Members of the transforming growth factor (TGF)-ß family are important regulators of skeletal development. In this study, we investigated the effect of TGF-ß1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-ß1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-ß1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-ß1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-ß1. Ram-1 messenger RNA expression was not affected by TGF-ß1. TGF-ß1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-ß1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-ß1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-ß family members in bone-forming cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEMBERS of the transforming growth factor (TGF)-ß family are important regulators of skeletal development, which influence the proliferation, differentiation, and activity of bone-forming cells. TGF-ßs initiate their cellular action by binding to the types I and II TGF receptor serine/threonine kinases (1). Receptor activation then leads to the phosphorylation of Smad2 and Smad3, which are essential components of TGF-ß signaling in many cell types. These phosphorylated Smads form complexes with Smad4 and are translocated to the nucleus, where they regulate gene transcription. In addition to the Smad pathway, TGF-ßs have been reported to activate several other signaling molecules, including the TGF-ß-activated kinase TAK-1, as well as the three major types of mitogen-activated protein kinases (MAPK), namely: extracellular signal-regulated kinase (ERK), p38 MAPK, and Jun-N-terminal kinase (JNK) (1, 2, 3).

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells, in relation to extracellular matrix mineralization (4, 5). Several observations indicate that type III sodium-dependent Pi transporters are involved in regulated Pi handling in bone-forming cells in vitro (6, 7). Moreover, in situ hybridization recently revealed expression of the type III transporter Glvr-1 in a subpopulation of early hypertrophic chondrocytes during endochondral bone formation in vivo, inferring a potential role for this Pi carrier in extracellular matrix calcification (8).

Types I and II TGF receptors are both expressed in proliferating and early hypertrophic chondrocytes in the growth plate, suggesting that TGF receptors and Glvr-1 are coexpressed to some extent during chondrocyte differentiation in vivo (9, 10). The mouse embryonal carcinoma-derived ATDC5 chondroprogenitor cells reproduce in vitro the various stages of chondrocyte differentiation, including sequential production of type II and type X collagen and mineralization of the deposited collagenous matrix (11, 12). The functional characteristics of Pi transport in ATDC5 cells are representative of a type III Pi transporter, and we recently observed Glvr-1 messenger RNA (mRNA) expression in these cells (13). The aims of the present study were to examine the effect of TGF-ß1 on Pi transport and on the expression of type III Pi transporters in chondrocytes, using the ATDC5 cell line as an in vitro model, as well as to investigate initial signaling events triggered by TGF-ß1 in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
FCS, antibiotics, and trypsin/EDTA were obtained from Gibco Life Technologies Ltd. (Paisley, UK). A 1:1 mixture of DMEM and Ham’s F12 medium (DMEM/F12) was provided by ICN Biochemicals, Inc. (Munich, Germany). Human TGF-ß1, actinomycin D, and cycloheximide were purchased from Sigma (St. Louis, MO). TGF-ß1 was dissolved as a concentrated solution in 4 mM HCl containing 1 mg/ml BSA. Actinomycin D and cycloheximide were dissolved as concentrated solutions in dimethylsulfoxide (DMSO). PD98059 (2'-amino-3'-methoxyflavone), calphostin C, Rö320432, Gö6983, and Gö6976 were purchased from Calbiochem-Novabiochem (La Jolla, CA). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) was a generous gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). All inhibitors were dissolved as concentrated solutions in DMSO.

Radiolabeled H3[32P]O4 and GeneScreen membranes were purchased from Dupont de Nemours (Brussels, Belgium). The random nonamer-primed DNA labeling kit, [{alpha}-32P] deoxycytidine triphosphate and [3H]-alanine were obtained from Amersham International plc (Little Chalfont, UK). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories, Inc. (Munich, Germany). The antiphospho-ERK monoclonal antibody, as well as anti-ERK2, anti-p38 MAPK, and anti-Smad2/3 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiphospho-p38 MAPK polyclonal antibody was obtained from New England Biolabs, Inc. (Beverly, MA); and the antiphospho-Smad2 polyclonal antibody, from Upstate Biotechnology, Inc. (Lake Placid, NY). All other reagents were of highest purity available from standard laboratory suppliers.

Cell culture
Murine chondrogenic ATDC5 cells were cultured, as previously described, in differentiation medium consisting of DMEM/F12 (1:1) containing 5% FCS, 10 µg/ml insulin, 10 µg/ml transferrin, 3 x 10-8 M sodium selenite, 100 IU/ml penicillin, and 100 µg/ml streptomycin (11). All cultures were maintained at 37 C in a humidified atmosphere of 5% CO2- 95% air. Subcultures were obtained once a week by removing the cells from the dishes using 0.25% trypsin in a Ca2+ and Mg2+ free Earle’s salt solution containing 2 mM EDTA.

The cells were plated in 24-well tissue culture clusters for Pi and alanine uptake measurements, in 25 cm2 cell culture flasks for RNA extraction, and in 6-well tissue-culture clusters for Western blotting analyses. The cells reached confluence within 3 days, and experiments were performed on days 9–10. For all experiments, ATDC5 cells were preincubated for 18 h in differentiation medium without insulin before stimulation with TGF-ß1 or its vehicle for the indicated times.

Uptake of Pi or alanine
Transport studies were performed in Earle’s buffered salt solution (EBSS), as previously described (14). Pi transport was initiated by adding 0.3 ml EBSS containing 0.1 mM H3[32P]O4 (1 µCi/ml), except for kinetic analyses, where H3[32P]O4 concentrations ranged from 0.05–1.5 mM. For alanine transport, the EBSS solution contained 0.1 mM [3H]-alanine (1 µCi/ml) and 1 mM Na2HPO4. After 6 min for Pi or 2 min for alanine, solute uptake was stopped by washing the cell layer with ice-cold, substrate-free EBSS. The cells were then solubilized with 300 µl 0.2 N sodium hydroxide, and the radioactivity contained in a 200-µl aliquot was counted using a standard liquid scintillation technique. The results were normalized for protein content, which was determined in the remaining 100 µl using the Coomassie Plus Protein Assay Reagent from Pierce Chemical Co. (Rockford, IL), with BSA as a standard. The sodium-independent components of Pi and alanine uptake were determined by replacing NaCl in the uptake solution with choline chloride.

RNA extraction and Northern blotting
Total RNA was isolated using TriPure Isolation Reagent (Roche Molecular Biochemicals, Rotkreuz, Switzerland). The cell layer was scraped into 1 ml TriPure Reagent and homogenized with a pipette. The cell lysates were then cleared by centrifugation before addition of 0.2 ml chloroform. After vigorous mixing, the samples were separated into three phases by centrifugation. Finally, the RNA contained in the upper aqueous phase was precipitated with 0.5 ml isopropanol, washed with 75% ethanol, air-dried, and resuspended in water.

For Northern blotting analyses, 10 µg total RNA were denatured in glyoxal and separated by electrophoresis on a 1.2% agarose gel. RNA was transferred to GeneScreen membranes by capillary transfer. Membranes were UV cross-linked and stained with methylene blue to control equal sample loading and RNA integrity. The membranes were then hybridized to a PCR-amplified fragment (bp 1208–1920) of the human Glvr-1 complementary DNA (cDNA) (GenBank accession no. L20859) or to a PCR-amplified fragment (bp 1195–1680) of the rat Ram-1 cDNA (GenBank accession no. L19931) (15, 16). These central regions of Glvr-1 and Ram-1 exhibit low homology to each other. The primers used for the PCR amplifications were: Glvr-1 forward 5'-gaagaccatgaagaaacaaa-3' (bp 1208–1227 of the human Glvr-1 cDNA), Glvr-1 reverse 5'-aactggaagaggagagagac-3' (antisense to bp 1901–1920 of the human Glvr-1 cDNA), Ram-1 forward 5'-gcttcctacggccgagcact-3' (bp 1195–1214 of the rat Ram-1 cDNA), and Ram-1 reverse 5'-atcctcgtgaggctggtcag-3' (antisense to bp 1661–1680 of the rat Ram-1 cDNA). To correct for differences in sample loading, the expression of the housekeeping gene cyclophilin was assessed by simultaneous hybridization with a BamHI fragment of the human cyclophilin cDNA (17). The probes were labeled with 50 µCi [{alpha}-32P] deoxycytidine triphosphate by random priming. Prehybridization (30 min) and hybridization (2 h) were performed in Quickhyb buffer (Stratagene, La Jolla, CA), which was supplemented for the hybridization with 100 µg/ml salmon sperm DNA. Membranes were washed twice for 15 min in 2 x SSC, 0.1% SDS at 25 C and for 10–30 min at 60 C in 0.1 x SSC, 0.1% SDS before exposition to Kodak (Rochester, NY) X-OMAT AR films for 18–72 h at -80 C. Signals were quantitated by densitometry, using the 300A Computing Densitometer from Molecular Dynamics, Inc. (Sunnyvale, CA).

Western blotting
After stimulation of the cells, for the indicated times, with 5 ng/ml TGF-ß1 or its vehicle, the culture medium was removed, and the cells were immediately frozen in liquid nitrogen. Whole-cell lysates were obtained by solubilizing the cells at 4 C in 1 ml lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM Na3VO4, 0.01 µM calyculin A, 0.1 µM mycrocystin LR, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS. The cell lysates were cleared by centrifugation at 6000 rpm at 4 C for 30 min, and the supernatant was diluted with an equal amount of 2x reducing sample buffer consisting of 125 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 200 mM dithiothreitol, and 0.025% bromophenol blue. The samples were boiled for 5 min, fractionated by reducing SDS-PAGE on 6–15% acrylamide gradient gels, and transferred to Immobilon P membranes (Millipore Corp., Bedford, MA). Immunoblotting was performed as described previously, with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies (18). Immunoreactive bands and biotinylated molecular weight standards were visualized by ECL (Amersham International plc).

Statistical analysis
Results are expressed as mean ± SEM. Significance of differences was calculated by the two-tailed unpaired Student’s t test or by ANOVA for multiple comparisons. A difference between experimental groups was considered significant when the P value was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of Pi transport stimulation by TGF-ß1 in ATDC5 cells
The effect of TGF-ß1 on sodium-dependent Pi uptake was investigated in ATDC5 cells cultured for 9–10 days in differentiation medium. At this stage, ATDC5 cells express an early chondrocytic phenotype, as indicated by type II collagen and proteoglycan production (11). A selective increase in sodium-dependent Pi transport was observed in ATDC5 cells stimulated for 24 h with 5 ng/ml TGF-ß1 (Table 1Go). Sodium-dependent alanine transport was not significantly affected in the same conditions, suggesting that the effect of TGF-ß1 on Pi uptake is mediated by a selective cellular mechanism, rather than by a general alteration in the sodium gradient driving force. The sodium-independent component of Pi uptake accounted for less than 20% of total Pi uptake in vehicle-treated cells and was not affected by TGF-ß1. It was thus neglected in further experiments (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Selectivity of sodium dependent Pi transport stimulation by TGF-ß1 in ATDC5 cells

 
The stimulation of Pi uptake by TGF-ß1 was dose dependent (Fig. 1AGo). A significant increase was observed in response to 0.5 ng/ml TGF-ß1, and maximal stimulation was achieved at 5 ng/ml. The response to TGF-ß1 was also time dependent and progressive. A stimulation of Pi uptake was first detectable after 2 h, reached maximal values after 8 h, and was still maintained after 24 h (Fig. 1BGo). Furthermore, the stimulatory effect of TGF-ß1 on Pi uptake seemed to be dependent on RNA and protein synthesis, because it was completely blocked in cells pretreated with either 1 µg/ml actinomycin D (Fig. 2AGo) or 10 µM cycloheximide (Fig. 2BGo). Finally, kinetic analysis indicated that the stimulatory effect of TGF-ß1 on Pi uptake is related to an increase in the maximal rate (Vmax) of the transport system, without a significant change in its apparent affinity constant (KM) for phosphate (Table 2Go).



View larger version (9K):
[in this window]
[in a new window]
  		Figure 1. Dose- and time-dependent effects of TGF-ß1 on Pi uptake in ATDC5 cells. A, ATDC5 cells were stimulated with various concentrations of TGF-ß1, or with its vehicle, for 24 h, before determination of Pi uptake. Each symbol represents the mean ± SEM of five to six determinations in a representative experiment. *, P < 0.001, compared with vehicle-treated cells. B, ATDC5 cells were stimulated with 5 ng/ml TGF-ß1, or with its vehicle, for the indicated times, before determination of Pi uptake. Values for TGF-ß1-treated cells are expressed in percent, compared with vehicle-treated cells at the same time point. Each symbol represents the mean ± SEM of six determinations in a representative experiment. *, P < 0.01; **, P < 0.001, compared with vehicle-treated cells at the same time point.

 


View larger version (10K):
[in this window]
[in a new window]
  		Figure 2. Effect of actinomycin D and cycloheximide on Pi uptake stimulation by TGF-ß1 in ATDC5 cells. A, ATDC5 cells were preincubated with 1 µg/ml actinomycin D or with its solvent (DMSO) for 2 h. The cells were then stimulated with 5 ng/ml TGF-ß1 (black columns) or with its vehicle (white columns), in the presence of the inhibitor or of DMSO, for 8 h, before determination of Pi uptake. Each column represents the mean ± SEM of six determinations in a representative experiment. *, P < 0.001, compared with DMSO and vehicle-treated cells. B, ATDC5 cells were preincubated with 10 µM cycloheximide or its solvent (DMSO) for 2 h. The cells were then stimulated with 5 ng/ml TGF-ß1 (black columns) or with its vehicle (white columns), in the presence of the inhibitor or of DMSO, for 8 h, before determination of Pi uptake. Each column represents the mean ± SEM of six determinations in a representative experiment. *, P < 0.001, compared with DMSO and vehicle-treated cells.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Kinetic analysis of Pi uptake stimulation by TGF-ß1 in ATDC5 cells

 
Stimulation of Glvr-1 expression by TGF-ß1 in ATDC5 cells
Northern blotting analysis showed a dose-dependent increase in Glvr-1 mRNA expression in response to TGF-ß1 in ATDC5 cells, which was approximately parallel to the dose-dependent increase in Pi transport. An increase in Glvr-1 mRNA was indeed detectable from 0.1 ng/ml and progressive up to 2 ng/ml TGF-ß1 (Fig. 3Go). The induction of Glvr-1 mRNA expression was time dependent, detectable already after 1 h and maximal after 2 h (Fig. 4Go). Thereafter Glvr-1 mRNA levels in TGF-ß1-treated cells remained high and were still elevated after 24 h. The expression of the housekeeping gene cyclophilin was not significantly affected by TGF-ß1 at any time point. Steady-state Glvr-1 mRNA levels in cells exposed to 5 ng/ml TGF-ß1 for 2 h were consistently more than 6-fold higher than those observed in vehicle-treated cells (Figs. 3Go and 4Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Dose-dependent effect of TGF-ß1 on Glvr-1 mRNA expression in ATDC5 cells. A, Ten micrograms of total RNA, obtained from ATDC5 cells stimulated with various concentrations of TGF-ß1 or with its vehicle (veh), for 2 h, were analyzed by Northern blotting, with probes for Glvr-1 and cyclophilin. A representative experiment is shown. The positions of 28S and 18S ribosomal RNAs (rRNAs) are indicated on the right. B, The autoradiography was quantified by densitometry and the integrated optical density of the Glvr-1 band, corrected for cyclophilin, is shown for each concentration.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 4. Time-dependent effect of TGF-ß1 on Glvr-1 mRNA expression in ATDC5 cells. A, Ten micrograms of total RNA, obtained from ATDC5 cells stimulated with 5 ng/ml TGF-ß1 or with its vehicle, for the indicated times, were analyzed by Northern blotting, with probes for Glvr-1 and cyclophilin. A representative experiment is shown. The positions of 28S and 18S rRNAs are indicated on the right. B, The autoradiography was quantified by densitometry, and the integrated optical density of the Glvr-1 band, corrected for cyclophilin, is shown for each sample.

 
Preincubation of the cells with actinomycin D, which markedly reduced the abundance of Glvr-1 mRNA in both TGF-ß1 and vehicle-treated cells, completely blocked the induction of Glvr-1 expression by TGF-ß1, suggesting that this response is dependent on transcription (Fig. 5AGo). Inhibition of protein synthesis by preincubation with cycloheximide slightly reduced the induction of Glvr-1 mRNA by TGF-ß1, without, however, completely blocking the response, suggesting that the effect of TGF-ß1 is, at least in part, independent of new protein synthesis (Fig. 5BGo).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. Effect of actinomycin D and cycloheximide on the stimulation of Glvr-1 mRNA expression by TGF-ß1 in ATDC5 cells. A, ATDC5 cells were preincubated with 1 µg/ml actinomycin D or with its solvent, for 1 h, before stimulation with 5 ng/ml TGF-ß1 or with its vehicle for 2 h. Total RNA was extracted and analyzed by Northern blotting, with probes for Glvr-1 and cyclophilin. A representative experiment is shown. The positions of the 28S and 18S rRNAs are indicated on the right. B, ATDC5 cells were preincubated with 10 µM cycloheximide or with its solvent, for 90 min, before stimulation with 5 ng/ml TGF-ß1 or with its vehicle for 2 h. Total RNA was extracted and analyzed by Northern blotting, with probes for Glvr-1 and cyclophilin. A representative experiment is shown. The positions of 28S and 18S rRNAs are indicated on the right.

 
Interestingly, Northern blotting analysis also showed expression of the related type III Pi transporter Ram-1 in ATDC5 cells (Fig. 6Go) (16). However, in contrast to Glvr-1 expression, the level of Ram-1 mRNA was not notably affected by TGF-ß1.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 6. Expression of Ram-1 mRNA in ATDC5 cells. Ten micrograms of total RNA obtained from ATDC5 cells stimulated with 5 ng/ml TGF-ß1 or with its vehicle, for the indicated times, were analyzed by Northern blotting, with probes for Ram-1 and cyclophilin. A representative experiment is shown. The positions of 28S and 18S rRNAs are indicated on the right.

 
Signaling pathways activated by TGF-ß1 in ATDC5 cells
Western blotting analysis revealed sustained phosphorylation of Smad2 in response to TGF-ß1 in ATDC5 cells, which express both Smad2 and Smad3 (Fig. 7Go). Smad2 phosphorylation was detected already after 15 min and was maximal after 1 h. Later, the amount of phosphorylated Smad2 decreased slightly but was still substantial after 6 h. Phosphorylation of the highly homologous Smad3 generally parallels that of Smad2 in response to TGF-ß1 (19, 20) and was not assessed in these experiments, because no specific antiphospho-Smad3 antibody is available to date.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Time-dependent effect of TGF-ß1 on Smad 2 phosphorylation in ATDC5 cells. ATDC5 cells were incubated with 5 ng/ml TGF-ß1 or with its vehicle, for the indicated times, and cell lysates were analyzed by Western blotting, using anti-phospho-Smad2 (upper panel) and anti-Smad2/3 antibodies (lower panel). The position of molecular mass standards is indicated on the right. A representative experiment is shown.

 
Interestingly, TGF-ß1 also induced activation of ERK and p38 MAPKs, as reflected by their phosphorylation (Fig. 8Go). In contrast, we could not detect any significant increase in JNK activity in response to TGF-ß1 in ATDC5 cells (data not shown). The effect of TGF-ß1 on ERK phosphorylation, which, in our experimental conditions, is already high in vehicle-treated controls, was modest and transient (Fig. 8AGo). Like Smad2 phosphorylation, ERK activation was already detectable after 15 min and maximal after 1 h. However, ERK phosphorylation then returned to basal levels within 3 h. Phosphorylation of p38 MAPK occurred with slower kinetics and was first detectable after 30 min (Fig. 8BGo). The response was maximal after 1 h; but, although it decreased slightly thereafter, p38 MAPK phosphorylation was still higher than baseline after 6 h.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 8. Time-dependent effect of TGF-ß1 on ERK and p38 MAPK phosphorylation in ATDC5 cells. A, ATDC5 cells were incubated with 5 ng/ml TGF-ß1 or with its vehicle, for the indicated times, and cell lysates were analyzed by Western blotting, using antiphospho-ERK (upper panel) and anti-ERK2 antibodies (lower panel). The anti-ERK2 polyclonal antibody also reacts, to a lesser extent, with ERK1. The position of molecular mass standards is indicated on the right. A representative experiment is shown. B, The membrane shown in A was reblotted with antiphospho-p38 MAPK (upper panel) and anti-p38 MAPK antibodies (lower panel). The anti-p38 MAPK antibody also reacts, to a lesser extent, with p38ß. The position of molecular mass standards is indicated on the right. A representative experiment is shown.

 
Pi transport stimulation by TGF-ß1 in the presence of protein kinase inhibitors
To examine a potential role of ERK and p38 MAPK in the stimulation of Pi transport by TGF-ß1, we used the MEK inhibitor PD98059 to block activation of ERK and the p38 MAPK inhibitor SB203580 to block p38 MAPK activity (21, 22). At concentrations commonly used to observe specific effects of these inhibitors, neither of them blocked the stimulation of Pi transport induced by TGF-ß1, although SB203580 slightly diminished the response (Table 3Go). In fact, a dose response analysis of the effect of SB203580 on Pi uptake stimulation by TGF-ß1 showed half-maximal and complete inhibition at, respectively, 50 and 100 µM (data not shown). This observation likely corresponds to a nonspecific effect of this agent, independent of p38 MAPK inhibition, because, at these very high concentrations, SB203580 was previously reported to inhibit types I and II TGF receptors (23). Consistently, we observed that the most proximal signaling event described downstream of TGF receptor activation, namely Smad2 phosphorylation, is impaired in the presence of 50 µM SB203580 in TGF-ß1-treated cells (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of MAPK and PKC inhibitors on Pi uptake stimulation by TGF-ß1 in ATDC5 cells

 
Protein kinase C (PKC) was previously suggested to mediate the stimulation of Pi uptake by platelet-derived growth factor and basic fibroblast growth factor (bFGF) in MC3T3-E1 osteoblast-like cells (24, 25). We thus tested the effect of various PKC inhibitors, such as calphostin C, Rö320432, Gö6983, and Gö6976, which target different subsets of PKC family members, on Pi transport stimulation by TGF-ß1 in ATDC5 cells (26, 27). None of these agents blocked the increase in Pi uptake induced by TGF-ß1, although Gö6976 slightly diminished the response (Table 3Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TGF-ß1 induces a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 chondrocytes. To our knowledge, this is the first study showing that a member of the TGF-ß family influences Pi handling in bone-forming cells. Interestingly, we also recently observed a stimulatory effect of TGF-ß1 on Pi transport in human SaOS-2 osteosarcoma cells, indicating that TGF-ß1 regulates Pi uptake in osteoblast-like cells, as well as in chondrocytes (28). The response to TGF-ß1 is dependent on RNA and protein synthesis and reflects a change in the Vmax of the transport system. These characteristics are similar to those previously reported for the regulation of Pi uptake by several hormones or growth factors in different types of bone-forming cells and suggest that the observed increase in Pi uptake may require the synthesis of new Pi carriers and their insertion into the plasma membrane (6, 14, 24, 25).

Consistently, Northern blotting analysis showed a dose-dependent increase in the expression of transcripts encoding the type III Pi transporter Glvr-1 in response to TGF-ß1 in ATDC5 cells. The induction of Glvr-1 expression preceded the maximal expression of Pi transport stimulation by several hours and seemed to be dependent on gene transcription. Furthermore, at least part of the effect of TGF-ß1 on Glvr-1 mRNA expression seemed to be direct, because the response was, to some extent, independent of new protein synthesis. The parallel between the changes in Glvr-1 expression and in Pi uptake strongly suggests that Glvr-1 mediates at least part of the stimulatory effect of TGF-ß1 on Pi transport in ATDC5 cells.

Parallel increases in Glvr-1 expression and Pi transport were also observed in SaOS-2 cells, in response to insulin-like growth factor-1 and TGF-ß1 (Ref. 6 and unpublished observations). Changes in Glvr-1 expression may thus represent a mechanism used by different growth factors to regulate Pi handling in bone-forming cells. Interestingly, although the related type III Pi transporter Ram-1 is also expressed in ATDC5 cells, its expression level was not affected by TGF-ß1. This observation reveals a distinct regulation of Glvr-1 and Ram-1 expression in ATDC5 cells and further suggests a specific role for Glvr-1 in the cellular response to TGF-ß1.

The present in vitro studies were conducted in a population of proliferating and maturing chondrocytes that are less differentiated than the hypertrophic cells in which Glvr-1 expression is observed in vivo, and it remains to be proven that our observations also apply to more differentiated cells (8). Nevertheless, the colocalization of TGF receptors and Glvr-1 expression in the growth plate suggests that a stimulatory effect of TGF-ßs on Pi transport in chondrocytes might also occur in vivo (8, 9, 10).

As in many other cell types, TGF-ß1 induced the activation of the Smad-signaling pathway in ATDC5 chondrocytes, as illustrated by sustained Smad2 phosphorylation. In addition, TGF-ß1 also activated two different types of MAPKs, namely ERK and p38, whereas JNK activity was not increased. A similar selective activation of both ERK and p38 MAPK by TGF-ß1 has been recently observed in semiconfluent, undifferentiated ATDC5 cells, which were serum-starved overnight before addition of growth factors (29). Despite some minor differences, which are most likely attributable to the different experimental conditions, our data obtained in confluent, type II collagen expressing ATDC5 cells confirm the results of this study and show a rapid and transient activation of ERK, as well as a slower and more prolonged activation of p38 MAPK. In addition, we show that Smad2 and ERK are activated with similar kinetics but that, in contrast to ERK activation (which is only transient), Smad2 phosphorylation is maintained for several hours, as is p38 MAPK activation.

The signaling mechanisms involved in the regulation of Pi transport in bone-forming cells are not well understood. PKCs have been suggested to mediate the stimulation of Pi uptake by growth factors acting through receptor tyrosine kinases in MC3T3-E1 osteoblast-like cells (24, 25). In contrast, none of the PKC inhibitors tested markedly impaired the stimulation of Pi uptake induced by TGF-ß1 in ATDC5 cells, although Gö6976 slightly diminished the response. These observations suggest that the regulation of Pi transport by TGF-ß1 in ATDC5 chondrocytes is essentially PKC independent, even though a minor contribution of a Gö6976-sensitive PKC isoform cannot be excluded. The p38 MAPK pathway was also recently suggested to contribute to the regulation of Pi uptake by bFGF in MC3T3-E1 cells (25). However, results obtained with the MEK1 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 suggest that neither ERK nor p38 MAPK are essential for the stimulation of Pi uptake by TGF-ß1 in ATDC5 cells (21, 22). The signaling mechanisms involved in the regulation of Pi transport by TGF-ß1 in ATDC5 chondrocytes thus seem to differ from those previously described in osteoblast-like cells stimulated with receptor tyrosine kinase agonists. As for many transcriptional responses induced by TGF-ßs in various cell types, Smads are, of course, very likely candidates to mediate the effect of TGF-ß1 on Glvr-1 expression and Pi transport in this system, although the specific roles of both Smad2 and Smad3 remain to be investigated.

In conclusion, our data indicate that TGF-ß1 stimulates both sodium-dependent Pi transport and Glvr-1 expression in ATDC5 cells. In addition to proliferation, differentiation, and matrix synthesis, TGF-ß1 thus also influences Pi handling in bone-forming cells, most likely by up-regulating the expression of the Glvr-1 Pi transporter. The signaling mechanisms mediating these responses and, in particular, the potential role of Smads remain to be investigated.


   Acknowledgments
 
We are indebted to Sabina Troccaz and Pierre Apostolides for excellent technical help.


   Footnotes
 
1 Presented, in part, in abstract form at the 21st Annual Meeting of The American Society of Bone and Mineral Research, St. Louis, September 30-October 4, 1999 (30 ). This work was supported by the Swiss National Science Foundation (Grant 32–49839.96) and by a grant from the Sir Jules Thorne Charitable Trust (to G.P.). Back

Received November 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Heldin CH, Miyazono K, Ten Dijke P 1997 TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471[CrossRef][Medline]
  2. Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E, Matsumoto K 1995 Identification of a member of the MAPKKK family as a potential mediator of TGF-beta signal transduction. Science 270:2008–2011[Abstract/Free Full Text]
  3. Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, Matsumoto K, Nishida E 1999 Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-ß-induced gene expression. J Biol Chem 274:27161–27167[Abstract/Free Full Text]
  4. Caverzasio J, Bonjour JP 1996 Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism. Kidney Int 49:975–980[Medline]
  5. Caverzasio J, Montessuit C, Bonjour JP 1996 Functional role of Pi transport in osteogenic cells. News Physiol Sci 11:119–125[Abstract/Free Full Text]
  6. Palmer G, Bonjour JP, Caverzasio J 1997 Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology 138:5202–5209[Abstract/Free Full Text]
  7. Chien ML, Foster JL, Douglas JL, Garcia JV 1997 The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion. J Virol 71:4564–4570[Abstract]
  8. Palmer G, Zhao J, Bonjour JP, Hofstetter W, Caverzasio J 1999 In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development. Bone 24:1–7[Medline]
  9. Kabasawa Y, Ejiri S, Matsuki Y, Hara K, Ozawa H 1998 Immunoreactive localization of transforming growth factor-beta type II receptor-positive cells in rat tibiae. Bone 22:93–98[Medline]
  10. Sakou T, Onishi T, Yamamoto T, Nagamine T, Sampath T, Ten Dijke P 1999 Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification. J Bone Miner Res 14:1145–1152[CrossRef][Medline]
  11. Shukunami C, Shigeno C, Atsumi T, Ishizeki K, Suzuki F, Hiraki Y 1996 Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol 133:457–468[Abstract/Free Full Text]
  12. Shukunami C, Ishizeki K, Atsumi T, Ohta Y, Suzuki F, Hiraki Y 1997 Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro. J Bone Miner Res 12:1174–1188[CrossRef][Medline]
  13. Guicheux J, Palmer G, Shukunami C, Hiraki Y, Bonjour JP, Caverzasion J 1999 A novel in vitro culture system for the analysis of the functional role of Pi transport in endochondral ossification. J Bone Miner Res 14:S311 (Abstract)
  14. Selz T, Caverzasio J, Bonjour JP 1989 Regulation of Na-dependent Pi transport by parathyroid hormone in osteoblast-like cells. Am J Physiol 256:E93–E100
  15. O’Hara B, Johann SV, Klinger HP, Blair DG, Rubinson H, Dunn KJ, Sass P, Vitek SM, Robins T 1990 Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus. Cell Growth Differ 1:119–127[Abstract]
  16. Miller DG, Edwards RH, Miller AD 1994 Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc Natl Acad Sci USA 91:78–82[Abstract/Free Full Text]
  17. Haendler B, Hofer-Warbinek R, Hofer E 1987 Complementary DNA for human T-cell cyclophilin. EMBO J 6:947–950[Medline]
  18. Caverzasio J, Palmer G, Suzuki A, Bonjour JP 1997 Mechanism of the mitogenic effect of fluoride on osteoblast-like cells: evidences for a G protein-dependent tyrosine phosphorylation process. J Bone Miner Res 12:1975–1983[CrossRef][Medline]
  19. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, Ten Dijke P 1997 TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16:5353–5362[CrossRef][Medline]
  20. Dennler S, Huet S, Gauthier JM 1999 A short amino-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3. Oncogene 18:1643–1648[CrossRef][Medline]
  21. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR 1995 A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689[Abstract/Free Full Text]
  22. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229–233[CrossRef][Medline]
  23. Eyers PA, Craxton M, Morrice N, Cohen P, Goedert M 1998 Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution. Chem Biol 5:321–328[CrossRef][Medline]
  24. Zhen X, Bonjour JP, Caverzasio J 1997 Platelet-derived growth factor stimulates sodium-dependent Pi transport in osteoblastic cells via phospholipase Cgamma and phosphatidylinositol 3'-kinase. J Bone Miner Res 12:36–44[CrossRef][Medline]
  25. Suzuki A, Palmer G, Bonjour JP, Caverzasio J 2000 Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3–E1 osteoblast-like cells. J Bone Miner Res 15:95–102[CrossRef][Medline]
  26. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ 1996 Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett 392:77–80[CrossRef][Medline]
  27. Wilkinson SE, Parker PJ, Nixon JS 1993 Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J 294:335–337
  28. Palmer G, Suzuki A, Bonjour JP, Caverzasio J 1998 Transforming growth factor-ß stimulates inorganic phosphate transport in SaOS-2 osteosarcoma cells. Bone 23:S240 (Abstract)
  29. Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K, Makishima F 1999 P38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. Exp Cell Res 250:351–363[CrossRef][Medline]
  30. Palmer G, Guicheux J, Bonjour JP, Caverzasio J 1999 Transforming growth factor-§ stimulates inorganic phosphate (Pi) transport and expression of the type III Pi transporter Glvr-1 in chondrogenic ATDC5 cells. J Bone Miner Res 14:S426 (Abstract)



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
L. V. Virkki, J. Biber, H. Murer, and I. C. Forster
Phosphate transporters: a tale of two solute carrier families
Am J Physiol Renal Physiol, September 1, 2007; 293(3): F643 - F654.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
Y. Yoshiko, G. A. Candeliere, N. Maeda, and J. E. Aubin
Osteoblast Autonomous Pi Regulation via Pit1 Plays a Role in Bone Mineralization
Mol. Cell. Biol., June 15, 2007; 27(12): 4465 - 4474.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
C. M. Giachelli, M. Y. Speer, X. Li, R. M. Rajachar, and H. Yang
Regulation of Vascular Calcification: Roles of Phosphate and Osteopontin
Circ. Res., April 15, 2005; 96(7): 717 - 722.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Palmada, M. Dieter, A. Speil, C. Bohmer, A. F. Mack, H. J. Wagner, K. Klingel, R. Kandolf, H. Murer, J. Biber, et al.
Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1
Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G143 - G150.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Pandini, E. Medico, E. Conte, L. Sciacca, R. Vigneri, and A. Belfiore
Differential Gene Expression Induced by Insulin and Insulin-like Growth Factor-II through the Insulin Receptor Isoform A
J. Biol. Chem., October 24, 2003; 278(43): 42178 - 42189.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Yoshiko, N. Maeda, and J. E. Aubin
Stanniocalcin 1 Stimulates Osteoblast Differentiation in Rat Calvaria Cell Cultures
Endocrinology, September 1, 2003; 144(9): 4134 - 4143.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. A. Khadeer, Z. Tang, H. S. Tenenhouse, M. V. Eiden, H. Murer, N. Hernando, E. J. Weinman, M. A. Chellaiah, and A. Gupta
Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions
Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1633 - C1644.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palmer, G.
Right arrow Articles by Caverzasio, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palmer, G.
Right arrow Articles by Caverzasio, J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals