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Endocrinology Vol. 147, No. 6 3141-3152
Copyright © 2006 by The Endocrine Society

Novel Early Target Genes of Parathyroid Hormone-Related Peptide in Chondrocytes

Jakomijn Hoogendam, Edwin Parlevliet, Razvan Miclea, Clemens W. G. M. Löwik, Jan M. Wit and Marcel Karperien

Departments of Pediatrics (J.H., E.P., R.M., J.M.W., M.K.) and Endocrinology and Metabolic Diseases (C.W.G.M.L., M.K.), Leiden University Medical Center, 2333 ZA Leiden, The Netherlands

Address all correspondence and requests for reprints to: Dr. Marcel Karperien, Department of Endocrinology, Leiden University Medical Center, C4R89, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: karperien{at}lumc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have performed microarray analysis to identify PTHrP target genes in chondrocytes. ATDC5 cells were cultured as micromasses to induce chondrocyte differentiation. On d 8 of culture, the cells had a prehypertrophic appearance. This time point was chosen for isolation of RNA at 0, 1, 2, and 4 h after a challenge with 10–7 M PTHrP. Samples were subjected to a cDNA microarray using competition hybridization. A list of 12 genes (P < 10–3), the expression regulation of which by PTHrP was confirmed by quantitative PCR analysis, was generated. This included seven up-regulated and five down-regulated genes. Three genes were known to be involved in PTHrP regulation, and six were previously found in growth plate chondrocytes. Most of the genes (10 of 12) were implicated in signal transduction and regulation. PTHrP also induced expression of the up-regulated genes in KS483 osteoblasts, suggesting involvement in a more generalized response to PTHrP. The vast majority of the up-regulated genes (six of seven) contained cAMP response element-binding protein- and/or activating protein-1 transcription factor-binding sites in their promoter regions. Remarkably, a number of PTHrP-regulated genes contained signal transducer and activator of transcription factor (Stat)-binding sites in their promoters. In transient transfection assays, we show that PTHrP is able to positively regulate the activity of Stat3-specific and negatively regulate the activity of Stat5-specific promoter-reporter constructs in ATDC5 and UMR106 cells. In combination with the expression regulation of genes involved in Janus kinase/Stat signaling, this data suggest a previously unrecognized interaction between PTHrP and Janus kinase/Stat signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LONGITUDINAL GROWTH results from chondrocyte proliferation and subsequent differentiation in the epiphyseal growth plate by a process called endochondral ossification. During this process, resting chondrocytes in the stem cell zone enter the proliferative zone, start dividing, and arrange in typical columns. These cells are characterized by the secretion of high amounts of collagen II. Subsequently, cells stop proliferating and start to differentiate into prehypertrophic chondrocytes, secreting collagen IX. Prehypertrophic cells further increase in size and become hypertrophic chondrocytes, which secrete high amounts of collagen X. The extracellular matrix becomes calcified, and finally, mature chondrocytes undergo apoptosis, leaving a scaffold for bone formation.

Systemic hormones regulate longitudinal growth, partly via direct actions on growth plate chondrocytes through their receptors (1, 2, 3). The direct and indirect actions of systemic hormones on growth plate chondrocytes were recently reviewed by Van der Eerden et al. (4). The mechanism of action of the various factors involved in regulating the proliferation and differentiation of chondrocytes within the growth plate is still largely unknown. Systemic hormones probably interact with locally acting growth factors present in the growth plate, such as the fibroblast growth factors or members of the Indian Hedgehog/PTHrP negative feedback loop, which was reported to control the pace of chondrocyte differentiation (5, 6).

In this study, we have focused on the actions of PTHrP on growth plate chondrocytes. PTHrP plays a crucial role in controlling the pace of chondrocyte proliferation and differentiation in the growth plate, as recognized by a number of studies (7, 8). Knockout mice for PTHrP show accelerated chondrocyte differentiation leading to dwarfism (7), whereas ectopic expression of PTHrP in chondrocytes inhibited their differentiation, leading to a smaller cartilaginous skeleton (8). In addition, PTHrP, the expression of which is tightly controlled by Indian Hedgehog (5), regulates both the rate and the extent of chondrocyte proliferation by directly regulating cell cycle machinery, partly by down-regulation of the cyclin-dependent kinase inhibitor p57Kip2 (9).

PTHrP signals through the PTH/PTHrP receptor 1 (PTHR1), which is predominantly expressed in prehypertrophic chondrocytes in the transition zone of the growth plate (10). Binding of PTHrP to its receptor activates various signal transduction pathways. The dominant pathways result in activation of adenylate cyclase/protein kinase A (AC/PKA) and phospholipase C/protein kinase C (11). Downstream targets of PTH signaling include the transcription factors cAMP response element-binding protein (CREB) and members of the activating protein-1 (AP-1) family, which are responsible, at least in part, for the genomic response. Indeed, various PTH response genes have binding sites for these transcription factors in their promoters (12, 13). Recently, early response genes of PTH in osteoblasts were identified by microarray analysis (14).

In this study the chondrogenic ATDC5 cell line was used to identify PTHrP target genes in prehypertrophic-like chondrocytes (15). ATDC5 cells reproducibly differentiate into chondrocytes within 4 wk in a monolayer culture (16). This cell line is a representative model for studying the actions of PTHrP on chondrogenesis. During this process, cells become responsive to PTHrP, and in agreement with in vivo studies, PTHrP inhibits hypertrophic chondrocyte differentiation (17, 18).

We selected this cell line for the identification of early response genes of PTHrP. Using cDNA microarray analysis, we identified 12 early response genes and confirmed their regulation by PTHrP using quantitative PCR (qPCR) in different cell culture models. Bioinformatic and functional analyses of a subset of these response genes, using transient transfection assays, revealed a previously unrecognized level of interaction between PTHrP and Janus kinase (Jak)/signal transducer and activator of transcription factor (Stat) signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
ATDC5 cells were grown in DMEM/Ham’s F-12 (DMEM/F12; Invitrogen Life Technologies, Inc., Breda, The Netherlands) containing 100 U/ml penicillin (Invitrogen Life Technologies, Inc.), 100 U/ml streptomycin (Invitrogen Life Technologies, Inc.), 10% charcoal-stripped fetal calf serum (Integro BV, Zaandam, The Netherlands), 10 µg/ml insulin (Sigma-Aldrich Corp., St Louis, MO), 10 µg/ml bovine transferrin (Roche, Almere, The Netherlands), and 3 x 10–8 M sodium selenite (Roche) in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. The micromass culture technique was modified from that described by Ahrens et al. (19). Trypsinized cells were resuspended in medium at a concentration of 2 x 107 cells/ml, and three drops of 10 µl of this cell suspension were placed in a well of a standard 12-well culture plate. The cells were allowed to adhere for 2 h at 37 C in 5% CO2, then 1 ml medium was added to each well. The medium was replaced every other day. KS483 mesenchymal progenitor cells were differentiated into osteoblasts as described previously (20). UMR106 cells were cultured in DMEM (Invitrogen Life Technologies, Inc.) containing 100 U/ml penicillin (Invitrogen Life Technologies, Inc.), 100 U/ml streptomycin (Invitrogen Life Technologies, Inc.), and 10% fetal calf serum (Integro BV).

cAMP enzyme immunoassay
To establish the responsiveness of ATDC5 cells to PTHrP, intracellular cAMP accumulation was measured as previously described (21), using an enzyme immunoassay (Amersham Biosciences, Freiburg, Germany) according to the manufacturer’s protocol. For this purpose, ATDC5 micromasses were challenged with a range of doses of PTHrP-(1–34) on d 7 and 14 of culture.

RNA isolation and amplification
Medium was refreshed after 7 d, ATDC5 micromasses were challenged with 10–7 M PTHrP-(1–34) on d 8 of culture (0 h), and total RNA was extracted at different time points in triplicate using TRIzol LS reagent (Invitrogen Life Technologies, Inc.), followed by RNA cleanup with the RNeasy mini kit (Qiagen, Valencia, CA). RNA concentrations were determined by measuring the absorbance at 260 nm. Next, RNA samples were pooled, and 0 h was chosen as reference sample time point. Total RNA (3 µg/reaction) was amplified as described previously (22), with slight modifications. In short, first-strand cDNA was synthesized by adding 500 ng T7-oligo(deoxythymidine) primer (5'-TCTAGTCGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCG(T)21-3') to 10 µl RNA sample. Samples were incubated for 10 min at 70 C, followed by 60 min at 42 C, in a total volume of 20 µl containing 5x first strand buffer, 10 mM dithiothreitol (DTT), 0.5 mM deoxy-NTPs (dNTPs), 2 U RNasin (Promega Corp., Leiden, The Netherlands), and 200 U SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc.). Next, second-strand cDNA was synthesized for 2 h at 16 C in a total volume of 150 µl containing 5x second-strand buffer, 0.2 mM dNTPs, 10 U DNA ligase (Invitrogen Life Technologies, Inc.), 40 U DNA polymerase I (Invitrogen Life Technologies, Inc.), and 2 U ribonuclease H (Invitrogen Life Technologies, Inc.). This was followed by the addition 2 µl T4 polymerase (5 U/µl; Invitrogen Life Technologies, Inc.) and incubation for 5 min at 16 C. The double-stranded cDNA reaction was stopped, and the remaining RNA in the mixture was degraded by the addition of 7.5 µl 1 M NaOH and 2 mM EDTA and incubation at 65 C for 10 min. Samples were purified by phenol/chloroform/isoamylalcohol (25:24:1) extraction. The volume of the aqueous phase was increased to 450 µl using H2O. For additional purification, samples were transferred to a Centricon-100 microconcentrator column (Millipore, Amsterdam, The Netherlands; prespun with 450 µl H2O) and centrifuged for 12 min at 2,500 rpm. After three wash steps with 450 µl H2O, cDNA was collected in a total volume of 7 µl by inverting the column and centrifuging for 30 sec at 13,000 rpm. Subsequently, cDNA was transcribed into cRNA using the T7 high-yield transcription kit (Epicenter, Madison, WI). The cDNA solution was incubated at 42 C for 3 h in a total volume of 20 µl containing 10x T7 reaction buffer; 7.5 mM ATP, CTP, GTP, and UTP; 10 mM DTT; 2 U RNasin; and 2 µl AmpliScribe T7 enzyme solution, followed by sample concentration using Centricon-100 microconcentrator columns (Millipore Corp.). This method was based on the original protocol described by Van Gelder et al. (23). Finally, the cRNA concentration was determined by measuring absorbance at 260 nm.

Probe labeling
cRNA (1.2 µg) was reverse transcribed with random hexamer primers and labeled by incorporation of cyanine 5-dUTP (Cy5) or cyanine 3-dUTP (Cy3; NEN Life Science Products, Boston, MA) according to the protocols used by Ross et al. (24) with slight modifications. In short, cRNA and 8 µg random primers (Roche) in a total volume of 15 µl were incubated for 10 min at 70 C. Subsequently, 6 µl 5x first-strand buffer, 3 µl 0.1 M DTT, 0.6 µl low-thymine dNTPs, 3 µl Cy3 dUTP (0 h) or Cy5-dUTP (other time points), and 1 µl SuperScript II RT (200 U/µl) were added, incubated for 10 min at room temperature, followed by incubation at 42 C for 90 min. After 60 min, fresh SuperScript II reverse transcriptase (1 µl) was added. Next, RNA was degraded by the addition of 15 µl 0.1 M NaOH and incubation for 10 min at 70 C, after which the solution was neutralized by the addition of 15 µl 0.1 M HCl. The labeled samples, supplemented with 180 µl 10 mM Tris/1 mM EDTA (pH 8; TE), and 10 µl mouse Cot-1 DNA (10 mg/ml; Invitrogen Life Technologies, Inc.), were pooled and purified using a Centricon-30 microconcentrator column (Millipore Corp.; prespun with 450 µl TE for 8 min at 13,000 rpm). Polyadenylated RNA (20 µg; Amersham Biosciences) and yeast tRNA (20 µg; Invitrogen Life Technologies, Inc.) were added to 450 µl TE during the second wash step. The purified product was collected by inverting the column and centrifuging for 1 min at 13,000 rpm, and finally was resuspended in a total volume of 45 µl hybridization solution containing 7.65 µl 20x standard saline citrate (SSC) and 1.35 µl 10% sodium dodecyl sulfate.

(Pre)hybridization
For the hybridization experiments, microarrays on which the NIA 15K mouse cDNA clone set (25) was spotted were purchased from the Leiden Genome Technology Center (Leiden, The Netherlands). DNA was cross-linked by UV irradiation at 65 mJ/cm2 (Stratalinker mode 1800 UV illuminator, Stratagene, La Jolla, CA). To prevent nonspecific hybridization, the slides were incubated in 45 µl hybridization solution [400 ng/µl yeast tRNA, 400 ng/µl polyadenylated RNA, 400 ng/µl herring sperm DNA (Invitrogen Life Technologies, Inc.), 100 ng/µl mouse Cot1 DNA, 5x Denhardt’s solution, 3.2x SSC, and 0.4% sodium dodecyl sulfate] at 65 C for 30 min. Before hybridization, the slides containing the prehybridization mixture were incubated for 2 min at 80 C to denature the spotted DNA. After prehybridization, the slides were washed twice in 2x SSC for 5 min each time at room temperature and dehydrated with subsequent steps of 5 min at 70%, 5 min at 90%, and 5 min at 100% ethanol (five times each). For hybridization, the probes were denatured by heating for 2 min at 100 C, left at room temperature for 15 min, centrifuged for 10 min, and placed under a 24 x 60-mm glass coverslip. The slides were incubated overnight at 65 C in a hybridization chamber (Corning, Amsterdam, The Netherlands), washed the next day in 2x SSC for 5 min at room temperature, and dehydrated using graded ethanols.

Microarray design and statistical analysis
The reference array experiments, 0 vs. 0 h, were hybridized in duplicate, 1 vs. 0 h and 2 vs. 0 h in triplicate, and 4 vs. 0 h in quadruplicate. After hybridization, slides were scanned in the Agilent DNA microarray scanner (Agilent Technologies, Amstelveen, The Netherlands). Genepix 3.0 software (Axon Instruments, Inc., Union City, CA) was used to quantify the resulting images. Subsequently, normalization and gene expression analysis were performed with Rosetta Resolver (Rosetta Biosoftware, Seattle, WA). Due to the overall poor quality of the cDNA spots on the microarray, stringent selection criteria were used for inclusion of spots to minimize the risk of false positive signals. A spot was only included if the spot settled the selection criteria at all time points. Spots showing an absolute fold change of 2, spots showing significant regulation (P < 0.01) in the reference array, and flagged spots in all arrays were excluded from analysis. For additional analysis, spots were selected from the remaining list if the signal intensity of Cy3 or Cy5 was above a cutoff level (0.05), and the absolute fold change was less than 50. ANOVA was performed between the remaining spots of the four different hybridizations. Spots showing significant (P < 0.001) differential expression regulation, with an intensity above the background value and a fold change greater than 1.8 for up-regulated genes and less than 0.55 for down-regulated genes, were selected for additional analysis. To identify the selected spots, the PCR-amplified cDNA of each spot (500 ng) was sequenced by the Leiden Genome Technology Center using 12 pmol M13 primers in a total volume of 24 µl.

RT-PCR
RNA was isolated from ATDC5 micromasses, cultured for 7 and 14 d, using TRIzol LS reagent (Invitrogen Life Technologies, Inc.), and reverse transcribed into cDNA using random hexamer primers (Amersham Biosciences). Semiquantitative PCR was performed for ß2-microglobulin (ß2m), collagen II, collagen IX, collagen X, and PTHR1 under the following conditions: cDNA was denatured at 94 C for 5 min, followed by cycles of 30 sec at 94 C, 30 sec at 56 C, and 30 sec at 72 C, and a final extension at 72 C for 10 min (Table 1Go).


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  		TABLE 1. RT-PCR primer sets

 
RNA isolation from KS483 osteoblasts
After 11 d of culture, KS483 cells formed bone nodules, and the cells within the nodules started to mineralize the extracellular matrix. This time point was chosen to stimulate the cells with 10–7 M PTHrP-(1–34). RNA was isolated after 1, 3, and 6 h using TRIzol LS reagent (Invitrogen Life Technologies, Inc.) and reverse transcribed into cDNA using random hexamer primers (Amersham Biosciences).

qPCR
To validate the expression patterns of PTHrP target genes, qPCR was performed using the iCycler (Bio-Rad Laboratories, Veenendaal, The Netherlands). For each gene, a set of primers was designed (Table 2Go), which spanned at least one intron-exon boundary and had an optimal annealing temperature of 60 C, using the software program Primer Express (Applied Biosystems, Foster City, CA). cDNA (5 ng) was amplified in triplicate using the qPCR core kit for SYBR Green 1 (Eurogentec, Maastricht, The Netherlands) under the following conditions: cDNA was denatured for 10 min at 95 C, followed by 40 cycles consisting of 30 sec at 95 C, 20 sec at 60 C, and 40 sec at 72 C. From each sample, a melting curve was generated to test for the absence of primer dimer formation and DNA contamination. Each reaction contained 5 µl cDNA (1 ng/µl), 10x reaction buffer, 3 or 4 mM MgCl2 (Table 2Go), 40 µM dNTPs, 300 nM primer, 0.75 µl SYBR Green, and 0.1 µl HotGoldStar polymerase in a total volume of 25 µl. Fold changes, adjusted for the expression of ß2m, were calculated and log transformed using the comparative method (26).


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  		TABLE 2. qPCR primer sets

 
Identification of transcription factor-binding sites
To find evolutionary conserved transcription factor-binding sites in promoters of target genes, sequences surrounding the transcription start site, from –1000 to +100 bp, were extracted from the National Center for Biotechnology Information database and aligned to either the human or rat genome using the evolutionary conserved regions browser (27). rVista 2.0 was used to predict conserved regulatory elements and define transcription factor-binding sites, with similarity predefined as 0.85 (complete match has a value of 1) (28). Consensus binding sequences, which were predicted in four or more promoters, were selected.

Transient transfection
ATDC5 cells were seeded at a density of 10,000 cells/cm2, and UMR106 cells were seeded at a density of 30,000 cells/cm2 in a 24-well plate. The cells were kept in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. The second day, the cells were transiently cotransfected with 1 µg reporter construct and 100 ng hemagglutinin-Stat3 expression vector (provided by T. Hirano, Osaka University, Osaka, Japan) (29), 100 ng Stat5a, 100 ng Stat5b expression vector (provided by Warren J. Leonard, National Institutes of Health, Bethesda, MD) (30), or 100 ng pcDNA3.1 expression construct using FuGene 6 transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s protocol. The following reporter constructs were used: a Stat3-specific reporter promoter construct (provided by Dr. I. Touw, Erasmus Medical Center, Rotterdam, The Netherlands) (31) and a Stat5-specific reporter promoter construct (provided by Peter Storz, University of Stuttgart, Stuttgart, Germany) (32).

To correct for transfection efficiency, 25 ng Renilla cytomegalovirus was included in all transfection experiments. The next morning, the medium was changed, and at the end of the day, cells were treated with a range of doses of PTHrP (1–34) (10–9, 10–8, and 10–7 M). After 20 h, luciferase assays were performed using the Dual-Luciferase Reporter assay system (Promega Corp.) according to the manufacturer’s protocol. Luciferase activity was measured using the Wallac 1450 Microbeta Trilux luminescence counter (PerkinElmer, Boston, MA). Firefly luciferase activity was corrected for Renilla luciferase activity.

Statistics
Values represent the mean ± SEM. Differences were examined by ANOVA, followed by the post hoc least significant difference test. Results were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of ATDC5 micromass cultures
To induce chondrocyte differentiation, ATDC5 cells were cultured as micromasses. After 7 d, a homogeneous cell pellet was formed containing an Alcian blue-positive cartilage matrix. Both the size of the micromasses and the intensity of the Alcian blue staining increased further after 14 d of culture (Fig. 1AGo). Histological analysis at 7 d showed rounded chondrocytes embedded in an Alcian blue-positive extracellular matrix (Fig. 1BGo). RNA was isolated and used for PCR analysis to study the expression of typical cartilage markers (Fig. 1CGo). Collagen II was expressed at 7 d and tended to decline at 14 d. Markers for prehypertrophic (collagen IX) and hypertrophic chondrocytes (collagen X) were present at 7 d and increased at 14 d. PTHR1 mRNA did not change in the differentiation process (Fig. 1CGo), but the responsiveness of ATDC5 cells to PTHrP increased with differentiation (Fig. 2Go).


Figure 1
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  		FIG. 1. Phenotypic characterization of ATDC5 micromass cultures. A, ATDC5 micromass cultures were cultured for 7 and 14 d and stained for Alcian blue. B, Section of an ATDC5 micromass, cultured for 9 d and stained for Alcian blue. C, Expression patterns of collagen II (coll II), coll IX, coll X, PTHR1, and ß2m mRNAs during ATDC5 micromass differentiation.

 

Figure 2
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  		FIG. 2. Responsiveness of ATDC5 cells to PTHrP during differentiation. The intracellular cAMP concentration corrected for protein was measured on d 7 and 14 of culture after stimulation with a range of doses of PTHrP.

 
Culturing the ATDC5 cells as micromasses efficiently induced chondrocyte differentiation. After 1 wk of culture, the cells responded to PTHrP and expressed markers of prehypertrophic chondrocytes, which are the main target cells for PTHrP in the growth plate. For this reason, this time point was chosen for identification of PTHrP target genes.

Identification and selection of PTHrP target genes
ATDC5 cells were cultured as micromasses and stimulated on d 8 with 10–7 M PTHrP for 1, 2, and 4 h, and cDNA microarray analysis was performed. After applying stringent selection criteria, 7843 of the 15,442 spots were taken in the analysis. A list of 31 spots that exhibited significant (P < 0.001) differential expression 1, 2, or 4 h after PTHrP stimulation was generated. Sequence analysis revealed that three genes were present in two different spots, and one gene was present in three spots. The expression patterns for the duplicate and triplicate spots were identical. This reduced the number of response genes to 26 (Table 3Go). From this list, 16 genes were chosen for validation experiments in ATDC5 cells. Exclusion criteria were insufficient information on gene identity, for example, established sequence tags, and insufficient information for the design of qPCR primer sets that span intron-exon boundaries.


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  		TABLE 3. Genes regulated by PTHrP in chondrocytes revealed by statistical analysis of the microarray data

 
The expression patterns of the selected genes were first validated by studying expression profiles using qPCR on the same RNA samples subjected to microarray analysis (Fig. 3Go, B, D, and F). As internal standard, ß2m, which was not regulated by PTHrP in microarray analysis (data not shown), was used. Four of 16 genes could not be validated and were discarded from additional study, leaving 12 genes for more detailed analysis (Table 4Go). In qPCR experiments, regulator of G protein signaling 2 (RGS2), Stat3, cysteine- and glycine-rich protein 2 (Csrp2), urokinase plasminogen activator receptor (Upar), and IER3 showed the same expression profile compared with data from microarray analysis (Fig. 3Go, B and D). The expression pattern of early up-regulated target genes, serum glucocortioid-regulated kinase (SGK) and protein tyrosine phosphatase 4a1 (Ptp4a1), were slightly different. SGK was only up-regulated after 1 h, and Ptp4a1 was up-regulated after 2 and 4 h, instead of the continuous up-regulation of both target genes revealed by microarray analysis. The down-regulated target genes, splicing factor 3a, subunit 2 (Sf3a2), activin receptor IIb (Acvr2b), growth factor receptor-bound protein 2-associated protein 1 (Gab1), laminin receptor I (LamRI), and dymeclin (Dym), showed data down-regulation at all time points in the qPCR instead of the transient down-regulation 1 h after PTHrP stimulation found by microarray analysis (Fig. 3FGo). The expression patterns were confirmed in RNA samples from ATDC5 cells isolated in a new independent experiment (data not shown).


Figure 3
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  		FIG. 3. Expression profiles of PTHrP target genes in ATDC5 chondrocytes revealed by microarray analysis and qPCR. RNA was isolated from ATDC5 micromass cultures 0, 1, 2, and 4 h after PTHrP stimulation; amplified; and labeled as described in the text. Samples were hybridized against the 0 h sample. Based on the expression profiles revealed by microarray analysis, the genes were divided over three clusters. A, Cluster 1 contained five immediate-early up-regulated genes, i.e. RGS2, SGK, Ptp4a1, UPAR, and IER3. The expression pattern revealed by qPCR is shown in B. C, Cluster 2 contained two immediate-early up-regulated genes, i.e. STAT3 and Csrp2. The expression pattern revealed by qPCR is shown in D. E, Cluster 3 contained five immediate down-regulated genes, i.e. Sf3a2, Acvr2b, Gab1, LamRI, and Dym. The expression pattern revealed by qPCR is shown in F.

 

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  		TABLE 4. List of validated PTHrP target genes

 
Validation of PTHrP target genes by qPCR
Among these 12 identified PTHrP target genes (Table 3Go), seven were up-regulated, and five were down-regulated. Most of the genes were implicated in signal transduction and regulation and were intracellular mediators or receptors. Three genes were previously known target genes of PTHrP in osteoblasts, and six genes had previously been found in growth plate chondrocytes. Based on the expression patterns, the genes were divided into three groups, two groups for up-regulated genes and one group for down-regulated genes (Fig. 3Go, A, C, and E). Genes in expression pattern 1 were up-regulated after 1 h, and genes in expression pattern 2 were up-regulated after 2 and 4 h. The genes in expression pattern 3 were down-regulated after 1 h. Expression pattern 1 contained RGS2, SGK, Upar, IER3, and Ptp4a1; Stat3 and Csrp2 formed expression pattern 2; and expression pattern 3 contained Sf3a2, Gab1, Dym, LamRI, and Acvr2b.

To test whether the responses of these target genes were restricted to chondrocytes or were part of a more generalized response to PTHrP, the expression patterns were analyzed in differentiated KS483 osteoblasts using qPCR (Fig. 4Go). For this purpose, RNA was isolated 0, 1, 3, and 6 h after challenge with PTHrP. The expression patterns of all up-regulated genes were comparable with the expression patterns found in chondrocytes. The expression patterns of the down-regulated genes in KS483 osteoblasts were less clear. All genes responded to PTHrP. Only Gab1 was down-regulated, although this regulation was transient instead of continuous as in ATDC5 cells. Remarkably, Sf3a2 and Dym were up-regulated instead of down-regulated. No consistent pattern of regulation was found for Acvr2b and LamRI.


Figure 4
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  		FIG. 4. Expression patterns of PTHrP target genes in KS483 osteoblasts. The genes were grouped according to expression profiles revealed by microarray analysis in chondrocytes, as described in Fig. 3Go. RNA was isolated from KS483 osteoblasts and stimulated with PTHrP on d 11 and after 0, 1, 3, and 6 h, and qPCR was performed. The expressions of the genes in expression pattern 1 in chondrocytes (A), in expression pattern 2 in chondrocytes (B), and in expression pattern 3 in chondrocytes (C) were determined by qPCR.

 
Transcription factor-binding sites
We subsequently analyzed promoter regions (sequences from –1000 to + 100 bp from the transcription start site) of target genes to identify common regulatory elements involved in their expression regulation. To decrease the number of false positive results, we searched for transcription factor-binding sites that were conserved in human and rat genomes. The promoter regions of RGS2, SGK, Ptp4a1, Stat3, Acvr2b, Gab1, and LamRI were aligned to the human genome, and the promoter regions of IER3, Upar, and Csrp2 were aligned to the rat genome. Insufficient information was available in the database to align the promoter regions of Sf3a2 and Dym with the human and rat genomes, preventing the selection of evolutionary conserved binding elements in these genes. Table 5Go shows transcription factors for which binding sequences have been found in four or more promoters of the identified PTHrP target genes.


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  		TABLE 5. Transcription factor-binding sites

 
Activating transcription factor (ATF)/CREB-binding sites were predicted in the promoter regions of six of seven up-regulated genes, four times in combination with an AP-1-binding site. ATF/CREB-binding sites were also predicted in two down-regulated genes, but never in combination with an AP-1-binding site. Remarkably, one of these genes was Dym, which was up-regulated in KS483 osteoblasts. Another interesting finding was the identification of Stat-binding sites, predominantly in the promoter regions of up-regulated PTHrP target genes. Binding sites for the transcription factor ELK1 (member of ETS oncogene family), which is, like ATF/CREB, AP-1, and Stat, involved in immediate-early responses, were predicted in seven promoter regions. Binding sites for the transcription factors E2F and SP1, both of which are facilitating factors involved in gene transcription, were distributed over up- and down-regulated target genes and were predicted in 10 of 12 and six of 12 promoter regions, respectively.

Stat regulation by PTHrP
To investigate the biological significance of the interactions between PTHrP and Stat signaling, transient transfection experiments were performed. In contrast to undifferentiated cells, differentiated ATDC5 (and KS483) cells could not be transfected. Because undifferentiated ATDC5 cells express very low amounts of PTHR1, experiments were also performed in UMR106 cells, which can easily be transfected and have higher PTHR1 expression levels.

In line with the up-regulation of Stat3 mRNA revealed by microarray analysis, PTHrP induced activity of a Stat3 reporter in both cell types dose dependently, with maximal fold inductions of 1.4 in ATDC5 cells (Fig. 5AGo) and 2.5 in UMR cells (Fig. 5BGo). In the presence of Stat3 expression vector, luciferase activity of the Stat3 reporter was enhanced in ATDC5 cells, but not in UMR106 cells (Fig. 5Go). PTHrP also increased Stat3 reporter activity in the presence of Stat3 expression vector in both cell lines.


Figure 5
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  		FIG. 5. Regulation of the activity of Stat3 protein by PTHrP. ATDC5 cells (A) and UMR106 cells (B) were transiently transfected with the Stat3-specific promoter-reporter construct, cotransfected with 100 ng pcDNA3.1 or 100 ng stat3 expression vectors, and treated with a range of doses of PTHrP (0, 10–9, 10–8, and 10–7 M). Data are expressed as the fold induction compared with the control value after correction for transfection efficiency. Experiments were performed in quadruplicate and were repeated at least twice. *, P < 0.05 compared with vehicle stimulation; #, P < 0.05 compared with vehicle stimulation in pcDNA3.1 cotransfections.

 
To investigate whether the regulation of Stat proteins by PTHrP is part of a more generalized interaction between PTHrP and Jak/Stat signaling, we subsequently studied the regulation of Stat5a and Stat5b by PTHrP. Comparable with Stat3, the mRNA expression of Stat5a (Fig. 6AGo) and Stat5b (Fig. 6BGo) was up-regulated after 4 h of PTHrP treatment in ATDC5 cells, as determined by qPCR. Transient transfection experiments were performed in ATDC5 and UMR106 cells using a Stat5 promoter-reporter construct in the absence of Stat5 expression vector. PTHrP inhibited luciferase activity of the Stat5 reporter, with a maximal fold change of 0.5, in UMR106 cells (Fig. 6DGo), but had no effect in ATDC5 cells (Fig. 6CGo). This observation is in contrast to the increase in mRNA expression of Stat5a and Stat5b. Addition of Stat5a or Stat5b enhanced reporter activity in ATDC5 and UMR106 cells. In both cell lines, PTHrP inhibited Stat5 reporter activity in the presence of Stat5b expression vector, whereas in the presence of Stat5a expression vector, reporter activity was only inhibited in UMR106 cells.


Figure 6
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  		FIG. 6. Regulation of Stat5 mRNA expression and Stat5 protein activity by PTHrP. RNA was isolated on d 11 from ATDC5 micromass cultures 0, 1, 2, and 4 h after PTHrP stimulation, and qPCR was performed for Stat5a (A) and Stat5b (B). ATDC5 cells (C) and UMR106 cells (D) were transiently transfected with the Stat5-specific promoter-reporter construct, cotransfected with 100 ng pcDNA3.1, 100 ng Stat5a, or 100 ng Stat5b expression vectors, and treated with a range of doses of PTHrP (0, 10–9, 10–8, and 10–7 M). Data are expressed as the fold induction compared with the control value after correction for transfection efficiency. Experiments were performed in quadruplicate and were repeated at least twice. *, P < 0.05 compared with vehicle stimulation; #, P < 0.05 compared with vehicle stimulation in pcDNA3.1 cotransfections.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we have identified early response genes of PTHrP in chondrocytes using the chondrogenic ATDC5 cell line. Disadvantages of the ATDC5 cell line differentiated under standard conditions in monolayer are the formation of a heterogeneous culture and the necessity for prolonged culture periods of at least 28 d (15). In monolayer cultures, chondrogenesis is initiated when multicell layers appear, suggesting that cell-cell contacts are crucial for chondrocyte differentiation. These cell-cell contacts are the initial trigger for mesenchymal stem cells to form chondrocytes in embryogenesis. To accelerate the formation of cell-cell contacts, we used the micromass culture technique, resembling the condensation of mesenchymal stem cells in vivo by formation of a three-dimensional spheroid structure (19, 33). Aggregation of ATDC5 cells resulted in more homogeneous cultures. By 7 d, type II collagen was abundantly expressed compared with expression after 14 d in monolayer culture (16). Types IX and X collagen were expressed at low levels after 7 d compared with expression after 14 and 21 d, respectively, in monolayer culture (16). In both cultures, the responsiveness of ATDC5 cells to PTHrP increased during differentiation (18). We decided to stimulate the micromasses on d 8 with PTHrP. At this time point, chondrocytes respond to PTHrP and have prehypertrophic characteristics, as demonstrated by the expression of collagen type IX. Late-proliferating and prehypertrophic cells are the main target cells of PTHrP in the growth plate (34). The higher levels of collagen X expression on d 14 of culture suggested that more cells had undergone chondrocyte hypertrophy at this time point.

To identify early response genes of PTHrP, the NIA 15K mouse cDNA bank was used. This bank was amplified and spotted by the Leiden Genome Technology Center. The quality control of custom-made cDNA microarray, compared with commercially available microarrays, is a well-known problem. The quality of the spots of the microarray used in this study was low due to heterogeneous spot morphologies ("doughnuts"), deposition inconsistencies, and oversized spots (35). In addition, identification of the spots was only possible by direct sequencing of the cDNAs used in the spotting process, because of contamination. This contamination is most likely introduced during multiple rounds of replication of the bank by PCR, as previously suggested (36). Because of the uncertainty of the identity of the spots, our results could not be used for pathway screening or genome-wide analysis. By applying very stringent selection criteria, the microarray could still be used to identify a subset of PTHrP target genes. The validity of this approach was subsequently shown by qPCR in different cell models and by bioinformatics analysis. We were able to classify 12 of 16 genes as bonafide target genes of PTHrP. qPCR analysis is an established method for validation of microarray data. However, due to the distinct methodologies and, in our case, the relative poor quality of the custom-made arrays, the overlap was not 100%. Others have also reported this, particularly with respect to the fold changes (37, 38, 39).

Bioinformatic analysis revealed that RGS2, Upar, and SGK were already identified as PTH target genes in osteoblasts (14, 40, 41). Indeed, in this study, RGS2, Upar, and SGK were induced in osteoblasts as well as in chondrocytes by PTHrP. Six of 12 target genes have not previously been demonstrated in growth plate chondrocytes. The majority of the identified PTHrP target genes (10 of 12) were involved in signal transduction pathways and modulation of these pathways. These include RGS2, SGK, Ptp4a1, immediate-early response 3 (Ier3), Stat3, Csrp2, and Gab1. These factors are involved in various signal transduction pathways, like AC/PKA, ERK, Jak/Stat, and phosphotidylinositol 3-kinase/AKT pathways, suggesting that PTHrP signaling could influence these signaling cascades (41, 42, 43, 44, 45, 46). In addition, PTHrP could influence other pathways via regulation of expression of receptors, such as Upar, Acvr2b, and LamRI.

qPCR resulted in validation of the up-regulated PTHrP target genes identified by microarray analysis in chondrocytes as well in osteoblasts. These data suggest that the up-regulated genes are part of a more generalized response to PTHrP, which is not restricted to chondrocytes. Verification of the down-regulated target genes revealed a more cell type-dependent picture. For instance, two genes, Dym and Sf3a2, were down-regulated in chondrocytes, but up-regulated in osteoblasts.

The dominant pathway activated by PTHrP is the AC/PKA pathway, which results in activation of the transcription factors CREB and/or AP-1 (47). Indeed, in various early response genes of PTH or PTHrP, functional CREB and AP-1 response elements have been identified (48). In line with this, promoter analysis revealed CREB transcription factor-binding sites predominantly in the up-regulated genes (six of seven), four times in combination with an AP-1 site. A less consistent picture was found for the down-regulated genes. In the promoter regions of two down-regulated genes (LamRI and Dym), CREB transcription factor-binding sites were predicted. Remarkably, Dym was induced by PTHrP in KS483 osteoblasts. In addition, the presence of SP1, E2F, and ELK1 was predicted in both up- and down-regulated genes. Recently, Qin et al. (14) used a statistical approach to identify transcription factor-binding sites used by PTH signaling in osteoblasts instead of enrichment for the evolutionarily conserved binding sites applied in this paper. Comparable to our study were the predictions of CREB- and AP-1 transcription factor-binding sites predominantly in up-regulated genes. Also, the presence of Sp1 sites in up- and down-regulated genes was predicted by both methods. The validity of our approach was underscored by previous data showing the presence of a CREB transcription factor-binding site in the Stat3 promoter (49). Furthermore, RGS2 is induced by cAMP, suggesting a CREB transcription factor-binding site in its promoter (50). In addition, SP1-binding sites have been described previously in the promoter regions of IER3 and Csrp2 (51, 52).

A remarkable finding in our study was the regulation of expression of proteins involved in the Jak/Stat signaling cascade, such as Stat3 and Csrp2. Csrp2 is a binding partner of Pias1, which is an inhibitor of Stat1 (42). In addition, a novel observation was the prediction of several Stat transcription factor-binding sites in the promoter regions of genes induced by PTHrP (six of seven), often in combination with CREB and AP1 sites. These observations were of biological significance, because we also observed that PTHrP induced the activity of a Stat3 reporter construct in the presence or absence of extra Stat3. This result is comparable with those of another study, in which Stat3 reporter activity was induced by activation of the AC/PKA pathway, by increasing the posttranslational activation of Stat3 proteins in rat thyroid cells (53). In addition, we showed that the regulation of Stat proteins by PTHrP was not only restricted to Stat3, but also included other members of the Jak/Stat family, such as Stat5a and Stat5b. Despite the induction of Stat5a and Stat5b mRNA, PTHrP inhibited Stat5 reporter activity in the absence and presence of exogenous Stat5a and Stat5b. This observation is most likely explained by an effect of PTHrP on the posttranslational actions of Stat5a and Stat5b, mediated by the AC/PKA pathway. Indeed, the inhibiting effect on Stat5 activity after activation of the AC/PKA pathway has been described in T lymphocytes (54). It was shown that AC/PKA signaling inhibited tyrosine phosphorylation of Stat5a and Stat5b, thereby preventing their activation. Our results suggest that this mechanism may also be operating in chondrocytes and osteoblasts after activation of PTHR1 signaling.

The effects of PTHrP on Stat3 and Stat5 reporters were observed in ATDC5 and UMR106 cells with only slight differences. Generally, the responses in ATDC5 cells were lower than those in UMR cells. This is most likely explained by low PTHR1 responses of the undifferentiated ATDC5 cells. Due to the excessive formation of cartilage matrix, which prevented efficient transfections, the transfection experiment could not be repeated in differentiated ATDC5 cells and KS483 cells, which express higher levels of PTHR1. The data suggest, however, that cross-talk between PTHR1 and Jak/Stat signaling is a more generalized mechanism. Taken together, we provide evidence for interactions between PTHrP and Jak/Stat signaling, not only at the level of mRNA expression regulation, but also at the level of posttranslational modification, resulting in either activation of Stat3 or repression of Stat5a- and Stat5b-mediated gene transcription.

The involvement of Jak/Stat proteins in signaling cascades of other growth factors in chondrocyte differentiation has been described (55, 56). Stat1 and Stat3 are involved in the effects of fibroblast growth factor on chondrocyte proliferation within the growth plate (55). In addition, the Jak/Stat cascade is involved in GH signaling. Stat5b is the most important Stat protein with respect to the actions of GH on growth and is responsible for the induction of IGF-I (56). Because Jak/Stat signaling plays an important role in chondrocyte proliferation, modulation of this pathway by PTHrP might be an essential mechanism involved in the actions of PTHrP in keeping growth plate chondrocytes in a proliferation-competent state.

In summary, we have applied new culture conditions to induce chondrogenic differentiation of ATDC5 cells. In addition, we have identified 12 PTHrP target genes. Among them were several genes involved in distinct signaling pathways operational within the growth plate, suggesting the presence of cross-talk with PTHrP signaling. In addition, we report for the first time the presence of a previously unrecognized interaction between PTHrP and Jak/Stat signaling.


   Acknowledgments
 
We express our gratitude to Dr. Touw (Erasmus Medical Center, Rotterdam, The Netherlands) for providing us with Stat3-specific promoter reporter construct, and to Dr. Hirano (Department of Molecular Oncology, Osaka University, Osaka, Japan) for supplying us with hemagglutinin-Stat3 expression vector. We are grateful to Dr. Storz (University of Stuttgart, Stuttgart, Germany) for giving us the Stat5-specific reporter promoter construct, and to Dr. Leonard (National Institutes of Health, Bethesda, MD) for providing us with Stat5a and Stat5b expression vectors.


   Footnotes
 
This work was supported by the Center for Medical Systems Biology, a center of excellence approved by The Netherlands Genomics Initiative/Netherlands Organization for Scientific Research, and a grant from Netherlands Organization for Scientific Research.

The authors have nothing to disclose.

First Published Online February 23, 2005

Abbreviations: AC/PKA, Adenylate cyclase/protein kinase A; Acvr2b, activin receptor IIb; AP-1, activating protein-1; ATF, activating transcription factor; CREB, cAMP response element-binding protein; Csrp2, cysteine- and glycine-rich protein 2; d, deoxy; DTT, dithiothreitol; Dym, dymeclin; Gab1, growth factor receptor-bound protein 2-associated protein 1; Ier3, immediate-early response 3; Jak, Janus kinase; LamRI, laminin receptor I; ß2m, ß2-microglobulin; PTHR1, PTHrP receptor 1; Ptp41a, protein tyrosine phosphatase 4a1; qPCR, quantitative PCR; RGS2, regulator of G protein signaling 2; Sf3a2, splicing factor 3a, subunit 2; SGK, serum glucocorticoid-regulated kinase; SSC, standard saline citrate; Stat, signal transducer and activator of transcription factor; Upar, urokinase plasminogen activator receptor.

Received January 19, 2006.

Accepted for publication February 13, 2006.


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 Introduction
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 Discussion
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