Increased Expression of Protein Kinase Cα, Interleukin-1α, and RhoA Guanosine 5′-Triphosphatase in Osteoblasts Expressing the Ser252Trp Fibroblast Growth Factor 2 Apert Mutation: Identification by Analysis of Complementary DNA Microarray
Institut National de la Santé et de la Recherche Médicale Unit 349, Lariboisiere Hospital, Paris Cedex 10, France
Address reprint requests to: A. Lomri, Ph.D., INSERM U349, Lariboisiere Hospital, 2 rue Ambroise Paré, 75475 Paris Cedex 10, France
Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Cα (PKC-α), interleukin-1α (IL-1α), and the small guanosine-5′-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-α, IL-1α, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1α and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1α and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1α and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-α, IL-1α, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.
COMPARING THE PATTERN of gene expression in cells and tissues has important applications in a variety of biological systems. Recent years have seen an explosion in tools for analyzing differential gene expression. Numerous nucleic acid arrays and their applications have been described previously in the literature.(1–7) Bone pathology is a field in which progress depends heavily on discovery and understanding regulated gene expression and therefore can benefit from these technologies, particularly in pathologies associated with genetic mutations. Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis).(8) More than 98% of previously reported cases were ascribed to two recurrent mutations in the fibroblast growth factor receptor 2 (FGFR-2) gene.(9) Recently, we showed that the Ser252Trp FGFR-2 mutation in Ap syndrome results in increased osteoblast differentiation, leading to increased subperiosteal bone matrix formation and premature calvaria ossification.(10,11) However, the receptor signaling mechanisms involved in the increased differentiation pathways remain unknown. Recently, it was reported that the Ap Pro253Arg FGFR-2 mutation is associated with activation of protein kinase Cα (PKC-α) and ϵ-isoenzymes in osteoblasts.(12) However, it was unclear whether this finding is the cause or the consequence of the mutation. Furthermore, the cellular and molecular pathways that are up- or downstream of PKC signaling events remain unknown. Thus, no data are available on the molecular mechanisms by which the Ser252Trp FGFR-2 substitution may induce premature suture closure.
This article presents a study in which the powerful technology of the complementary DNA (cDNA) microarrays was applied for the first time to bone pathology, namely, in the Ap syndrome. In considering candidate genes involved in Ap syndrome, we decided to use the human cDNA expression array membrane containing 588 known genes. Many of the cDNA fragments in the Atlas array have been selected carefully from regions thought to be unique to the individual genes. Unlike other nucleic acid arrays, the Atlas human expression arrays we used include genes that are under tight transcriptional control (Co). Moreover, all these genes have been reported to play key roles in many different biological processes. Using this array system, we show here that PKC-α, the small guanosine-5′-triphosphatase (GTPase) RhoA, and cytokine interleukin-1α (IL-1α) transcripts among other genes are increased in FGFR-2 mutant Ap cells. The differential and quantitative expression analyses of these genes in normal and Ser252Trp FGFR-2 mutant osteoblastic cells provide new insight into the underlying mechanisms involved in the disease.
MATERIALS AND METHODS
Subjects and bone samples
Calvaria bone samples were obtained from two fetuses aged 27 weeks with clinical evidence of Ap syndrome. Mutation analysis revealed the Ser252Trp FGFR-2 mutation. Normal fetal calvaria bone samples were obtained from two age-matched aborted fetuses with no evidence of bone disease, according to the French Ethical Committee recommendation.(13)
Cell cultures and treatments
Mutant and normal calvaria osteoblastic cells were isolated from the coronal samples as previously described.(13) To evaluate the cellular and molecular events induced by the Ser252Trp FGFR-2 mutation, mutant and normal calvaria cells from one Ap and one normal fetus were immortalized using the SV40 large T-antigen as previously described.(10) Immortalized Co and Ap cells were cultured at confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Eurobio, Les Ulis, France). Inhibition of the PKC pathway was done in the presence of 1–2 μM of calphostin C (Sigma, St. Louis, MO, USA) for 24 h for RNA expression and 48 h for protein production.
Preparation of amplified cDNA
Total cellular RNA was extracted from confluent Co and Ap cells by using the Extract-All solution according to the manufacturer's instructions (Eurobio). Residual genomic DNA in RNA preparations was removed by incubating total RNA in the presence of RNAse-free DNAse I (Gibco BRL, Gaithersburg, MD, USA) for 1 h at 37°C. After ethanol precipitation, the pellet was dissolved in deionized H2O followed with oligo-dT purification of polyA+ RNA by using the messenger RNA (mRNA) isolation kit (Roche, Meylan, France) following the manufacturer's recommendations. We checked the quality of the polyA+ RNA for genomic DNA contamination by using polyA+ RNA as a template in a polymerase chain reaction (PCR) with primers for a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]).
Microarray hybridization and analysis
The gene arrays used here consist of a single 80 mm × 120 mm nylon filter membrane spotted with 588 PCR-amplified cDNA fragments (200–500 base pairs [bp]). There are 1176 spots per filter or 588 individual cDNAs divided into 6 groups of genes (3 families of genes per group). All the reagents for the first-strand cDNA synthesis, hybridization, and washing solutions were provided with the Atlas Human Expression Array Kit (Clontech, Palo Alto, CA, USA). One microgram of polyA+ RNA from Co and Ap cells was converted into [32P]-labeled first-strand cDNA at 50°C for 20 minutes. We also performed a parallel labeling reaction with the positive Co polyA+ RNA that was provided with the kit. cDNA synthesis and hybridization steps were performed according to the manufacturer's instructions. Labeled cDNA was purified from unincorporated [32P]-labeled nucleotides and small cDNA fragments by using a Chroma Spin-200 diethylpyrocarbonate (DEPC)-H2O column (Roche). Before hybridizing cDNA probes to the Atlas arrays, we performed test hybridization using the probes and a blank piece of membrane. Because no background hybridization was obtained with the blank membrane, we applied the purified cDNA probes to the Atlas arrays. Each Atlas membrane was hybridized overnight with continuous agitation at 68°C with 2 × 106 cpm. Next day, the Atlas arrays were washed four times in 2× SSC/0.5% sodium dodecyl sulfate (SDS) for 20 minutes at 68°C followed by two additional 20-minute washes in 0.1× SSC/0.5% SDS at 68°C. The Atlas arrays were wrapped immediately in plastic wrap and exposed to Kodak BioMax MS X-ray film (Kodak, Rochester, NY, USA) with intensifying screen at −70°C. To detect high-, medium-, and low-abundance transcripts, we performed a trial run exposure of the Atlas array membranes.
Analysis and quantification
The quantification of signals was conducted using autoradiographs of the Atlas arrays scanned (ARCUS II scanner; Agfa, Ridgefield Park, NJ, USA) into a Power Macintosh 4400/200 computer (Apple Computer, Cupertino, CA, USA) and analyzed using Adobe Photoshop (version 3.0; Adobe Systems, San Jose, CA, USA) and the public domain National Institutes of Health (NIH) Image program (version 1.61; developed at the NIH). Results were transferred from NIH Image into Cricket Graph (version 1.3.2; Computer Associates International, Malvern, PA, USA) for quantification.
Northern blot analysis
To confirm the data generated by Atlas arrays, Co and Ap cells were grown in DMEM plus 5% FCS in the presence or absence of 1 μM of calphostin C. After 24 h, total cellular RNA was extracted as described previously. Twenty micrograms of RNA per lane was separated on 1.2% formaldehyde/agarose gel and transferred to hybond N+ nylon membranes (Amersham, Les Ulis, France) by alkaline transfer. Blots were prehybridized 30 minutes at 68°C in ExpressHyb hybridization solution (Clontech). The same membrane was probed with 358, 422, and 473-bp fragments encoding PKC- α, IL-1α, and RhoA, respectively. The human PKC-α, RhoA, IL-1α, and GAPDH probes were cloned by PCR from human osteoblast cDNAs. The cloned probes were confirmed by sequencing. Inserts were labeled with deoxycytosine triphosphate (dCTP) using a nick translation kit (Gibco BRL). Hybridization was carried out overnight at 68°C in the same buffer with the addition of 106 cpm of each radiolabeled probe per milliliter. Filters were washed twice at 68°C in 2× SSC/0.1% SDS for 15 minutes and then twice at 68°C in 0.1× SSC/0.1% SDS for 45 minutes. To confirm equal loading, the blot was stripped and then reprobed with a radiolabeled human GAPDH cDNA. Hybridizing bands were visualized and quantified using a phosphor Imager (Molecular Dynamics, Sunnyvale, CA, USA). For PKC signal, we measured only the upper (8.5 kilobases [kb]) of the two transcripts, although both are increased in Ap cells.
Immunodetection of IL-1α in mutant cells in vitro
Immunocytochemistry of IL-1α was performed in Co and Ap cells cultured at preconfluence. The cells were fixed in 4% paraformaldehyde, incubated in 0.1% bovine serum albumin (BSA)/3% goat serum to block nonspecific binding and then processed for immunocytochemistry. Briefly, after washing in phosphate-buffered saline (PBS)/0.01 Triton X-100 and incubation in 0.1% BSA/3% goat serum to block nonspecific binding, cells were exposed to a rabbit anti-human IL-1α polyclonal antibody (1:100; Genzyme Co., Cambridge, MA, USA) or a nonimmune rabbit immunoglobulin G (IgG; Dako, Glostrup, Denmark) at room temperature for 1 h. After washing, the cells were incubated with a goat anti-rabbit antibody linked to colloidal gold particles enlarged by precipitation of metallic silver before visualization.
Co and Ap cells were grown in 100-mm culture dishes in standard condition until confluence and then serum-starved overnight before treatment with calphostin C (1–2 μM), SB203580 (10 μM), a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), or PD-98059 (10 μM), a specific inhibitor of MAPK kinase (MEKK). At the end of the incubation time, the cells were washed twice with cold PBS and scraped in ice-cold lysis buffer (10 mM of Tris-HCl, 5 mM of EDTA, 150 mM NaCl, 30 mM of sodium pyrophosphate, 50 mM NaF, and 1 mM Na3VO4) containing 10% glycerol and protease inhibitors (Roche). Protein samples were solubilized in 2× Laemmli SDS loading buffer and boiled at 95°C for 5 minutes. Cellular proteins, determined using the detergent compatible (DC) protein assay (Bio-Rad Laboratories, Hercules, CA, USA), were resolved on 4–15% gradient minigels (Bio-Rad Laboratories) or 12% acrylamide gel and transferred onto polyvinylidene difluoride (PVDF)-Hybond-P membranes (Amersham). Blots were saturated overnight with 1% blocking solution (Roche) in Tris-buffered saline (TBS) buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20). Membranes were then incubated with anti-human PKC (1:250; StressGen, Victoria, BC, Canada), anti-human RhoA (2 μg/ml; Santa Cruz, CA, USA), or antiactin (1 μg/ml; Genzyme Co., Cambridge, MA, USA) for 1 h at room temperature. After incubation with the secondary antibodies and washes, the signals were visualized with Boehringer Mannheim (BM) chemiluminescence blotting substrate (Roche) and quantified as described previously for the array signals.
Differential gene expression
To identify genes that are expressed differentially in mutant and normal cells, confluent Co and Ap cell cultures were harvested and lysed, and polyA+ mRNA from the two cell samples was labeled by reverse transcriptase (RT) incorporation of with [α-32P]deoxyadenosine triphosphate (dATP). Labeled cDNA probes were hybridized to one of the two identical Atlas array membranes. The membranes were washed at high stringency and scanned to quantify hybridization signals. Hybridization signals were observed to more than 40% of the human cDNA array elements after a 24-h exposure time, but not to any of the negative controls (Fig. 1). Comparative expression analysis of mutant Ap compared with Co cells revealed 20 genes not shown previously to be expressed in osteoblastic cells. Twenty-two genes were up-regulated in Ap cells, and five were negatively regulated (Table 1). FGFR-2 mutation affected gene expression for a diverse range of cellular pathways and functions, including oncogenes, tumor suppressors, intracellular transducers, apoptosis, cell surface antigens and adhesion, cytokines, and chemokines. Among these genes, PKC-α, the small GTPase RhoA and IL-1α expression was markedly increased in FGFR-2 mutated Ap cells compared with Co cells. The validity of the hybridization array results was confirmed by RT-PCR (not shown) and Northern blot (Fig. 2A) analyses using mRNAs derived from separate Ap and Co cultures. The quantitative analysis showed that the levels of expression of RhoA, PKC-α, and IL-1α genes were 3.6-, 2.9-, and 3-fold higher in Ap compared with Co cells, respectively (Fig. 2B), confirming the validity of the microarray assay data.
Table Table 1.. List of Genes Differentially Expressed in Ap Mutant Osteoblasts Determined by cDNA Microarray Analyses
Expression of IL-1α in vitro
Next, we examined whether the overexpression of IL-1α protein can be detected in Co and Ap cells. As shown in Fig. 3, both cell lines expressed IL-1α protein. IL-1α staining was higher in Ap cells (Fig. 3B) compared with Co cells (Fig. 3A). Co cells with nonimmune serum showed no specific staining (not shown). These in vitro data at the protein level confirm the finding of increased IL-1α mRNA in mutant cells determined by Atlas arrays and Northern blot analysis.
Western blot analysis of PKC and RhoA
We then examined PKC and RhoA protein expression by immunoblot analysis in immortalized Co and mutant Ap cells. As shown in Fig. 4A, RhoA and PKC protein levels were increased in mutant Ap cells compared with Co cells. Densitometric analysis of the corresponding bands corrected for actin indicated a 300% and 75% increase in RhoA and PKC proteins, respectively (Fig. 4B). Similar results were confirmed in repeated experiments.
Effects of specific inhibitors on PKC, RhoA, and IL-1α
To determine the relationship between the up-regulation of PKC, RhoA, and IL-1α proteins and transcripts in Ap cells, we treated Co and Ap cells with calphostin C, a PKC selective inhibitor. Treatment of Co and Ap cells with calphostin C (1 μM) decreased PKC-α protein levels in Ap mutated cells (Fig. 4). Inhibition of PKC pathway by calphostin C also induced down-regulation of the small GTPase RhoA protein (Fig. 4). At the transcript level, calphostin C treatment resulted in 60% inhibition of PKC-α mRNA expression, as determined by Northern blot analysis (Fig. 2). Furthermore, inhibition of PKC induced down-regulation of IL-1α (80%) and RhoA (62%) transcripts in Ap cells (Fig. 2). Altogether, the results indicate that the Ser252 Trp mutation constitutively increases the expression of PKCα, IL-1α, and small GTPase RhoA in mutant osteoblasts, and that PKC is involved in the RhoA and IL-1α overexpression in Ap immortalized cells.
We also repeated these experiments with another dose of calphostin C (2 μM) to further determine the role of PKC in the increased expression IL-1α and RhoA GTPase. As shown in Fig. 5A, calphostin C (2 μM) suppressed RhoA protein levels in Ap cells but not in Co cells. Quantification of signals showed that at this dose of calphostin C, RhoA protein levels were almost completely suppressed and IL-1α levels were restored to normal levels in Ap cells (Fig. 5B).
We finally hypothesized that a portion of RhoA expression may be regulated by a mechanism distinct from PKC and we addressed this question by using specific inhibitors of MAPKs. As shown in Fig. 5, we found that SB203580, a specific inhibitor of p38 MAPK, and PD-98059, a specific inhibitor of MEKK, decreased IL-1α and RhoA expression in Ap cells, suggesting that these pathways also may be involved together with PKC in RhoA and IL-1α regulation. Furthermore, these inhibitory effects were not observed in Co cells, suggesting a specific effect in mutant cells.
We previously showed that the Ser252Trp FGFR-2 mutation in the Ap syndromic craniosynostosis leads to accelerated osteoblast differentiation in human calvaria cells in vivo and in vitro.(10–12) In the present study, we used the microarray system to determine signaling molecules that may be involved in the abnormal osteoblast phenotype induced by the mutation. Here, we provide experimental evidence indicating that PKC-α, IL-α, and RhoA are increased by the Ser252Trp FGFR-2 mutation in Ap craniosynostosis.
The genes that are modified in mutant calvaria cells were analyzed by cDNA microarrays. We choose the Atlas human expression arrays that include a series of genes that are transcriptionally controlled and that play key roles in biological processes. Using this array system, and after analyzing the data from both membranes, we were able to detect up- and down-regulated transcripts in the Ser252Trp FGFR-2 mutant osteoblastic cells. Among the genes that were expressed differentially, we selected three that may regulate osteoblast function. The analysis of genes that were selectively overexpressed in mutant cells revealed that the PKC-α gene expression was higher in Ap cells compared with Co cells. This overexpression in Ap mutant cells was confirmed at the mRNA level by RT-PCR and Northern blot analyses and at the protein level by Western blotting. In separate experiments, we found that treatment of Ap cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, resulted in a dramatic increase in alkaline phosphatase (ALP) activity, suggesting that the PKC pathway may be involved in ALP overexpression in mutant cells (data not shown). This is consistent with our recent finding that PKC activity is increased and that the PKC inhibitor calphostin C represses the expression of osteoblast-specific genes in Ap cells.(14) The Ap Pro253Arg FGFR-2 mutation also was found to be associated with activation of PKC-α in human osteoblasts.(12) The present data provide novel evidence that the Ser252Trp FGFR-2 mutation induces PKC-α gene overexpression in human osteoblasts.
The microarray system allowed the genes that are downstream of the PKC pathway in Ap FGFR-2 mutant cells to be determined. Among the up-regulated transcripts in the Ser252Trp FGFR-2 mutated osteoblast cells, we identified the small GTPase RhoA and IL-1α. To confirm these microarray data, we used RT-PCR and Northern blot to analyze these genes in both cell lines, and we found increased RhoA and IL-1α gene expression in FGFR-2 mutant cells compared with Co cells. Western blotting and immunocytochemistry showed that RhoA and IL-1α mRNA overexpression in mutant cells resulted in increased RhoA and IL-1α proteins. To determine the relationship between the increased PKC-α and IL-1α overexpression in mutant cells, we examined the effects of the potent and selective PKC inhibitor calphostin C on IL-1α mRNA expression. Inhibition of the PKC pathway decreased the expression of IL-1α gene, suggesting that the PKC pathway is upstream of IL-1α gene expression in mutant cells. G protein activities have previously been linked to IL-1 activated signaling. For example, IL-1-mediated stimulation of cyclic AMP production in a pre-B-cell line requires a GTPase activity that is sensitive to pertussis toxin.(15) The Rho family GTPase Rac-1 and Cdc42 also were shown to be necessary for IL-1-induced p38 activation.(16) In Ap mutant cells, which overexpressed IL-1α, RhoA mRNA and protein levels were increased compared with Co cells, indicating that the mutation increased both IL-1α and RhoA expression. Furthermore, inhibition of the PKC pathway with calphostin C decreased IL-1α but also down-regulated RhoA protein, suggesting that PKC-α may be required for subsequent increased expression of IL-1α and RhoA. These data indicate that IL-1α and RhoA are involved in the PKC downstream-signaling pathway induced by the Ser252Trp FGFR-2 mutation in Ap osteoblasts. However, other signaling pathways may be involved because we found that inhibition of p38 and MEKK MAPKs also reduced RhoA and IL-1α in Ap cells. Thus, it is likely that more than one signaling mechanism may be involved in the increased RhoA and IL-1α expression in mutant cells.
Our data suggest that IL-1α might play a role in the altered cellular phenotype in Ap syndrome. IL-1 is known to influence a large number of osteoblastic activities. IL-1 induces cell proliferation and collagenase production and reduces collagen and osteocalcin synthesis in osteoblasts.(17–20) However, we previously showed that Ap mutant cells display a constitutive increase in type 1 collagen and osteocalcin, with no change in cell proliferation.(10,11) Thus, it is unlikely that the increased IL-1α expression was responsible for the premature osteoblast differentiation observed in mutant cells. IL-1 is an important regulator of components of the extracellular matrix as the cytokine regulates matrix turnover by acting on several effectors.(21) Thus, the alteration of IL-1α mRNA and protein levels in mutant cells may play a role in the balance between bone matrix synthesis and degradation during premature formation of the extracellular matrix. Alternatively, it is possible that IL-1 affected cell survival in osteoblasts.(22) Because apoptosis may play a role in suture development,(23) we can hypothesize that IL-1α overexpression in mutant osteoblasts may lead to abnormal apoptosis and premature suture closure.
Multiple and complex mechanisms may be involved in the RhoA downstream events in mutant osteoblasts. Rho proteins are critical elements of signal transduction pathways and have central roles in cell morphology, proliferation, differentiation, and apoptosis by interacting with multiple target proteins.(24–26) The human Rho proteins RhoA, Rho C, and Rac 1 were found to induce apoptosis by regulating signal pathways that are required for cell survival.(27) Previously, RhoA has been shown to regulate the activities of both nuclear factor κB (NF-κB) and C-Jun N-terminal kinase (JNK),(28,29) signaling molecules known to be stimulated by IL-1. The hypothesis that RhoA and IL-1α overexpression in mutant Ap cells may result in alteration of apoptosis in vitro and in vivo is currently under investigation in our laboratory.
In summary, we have shown that PKC-α, IL-1α, and RhoA are up-regulated by the Ap Ser252Trp FGFR-2 mutation in human osteoblasts. The differential and quantitative expression analyses of these genes in normal and mutant osteoblastic cells provide new insight into the effects of the Ser252Trp FGFR-2 mutation in calvaria osteoblasts in Ap syndrome.
We thank Dr. Richard I. Weiner (University of California, San Francisco, CA) for reviewing the manuscript.