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Originally published In Press as doi:10.1074/jbc.M605467200 on September 8, 2006

J. Biol. Chem., Vol. 281, Issue 45, 33997-34008, November 10, 2006
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Identification and Characterization of a Novel Gene, Mcpr1, and Its Possible Function in the Proliferation of Embryonic Palatal Mesenchymal Cells*

Dong-Ying Xuan{ddagger}§, Xin Li{ddagger}§, Zhi-Hong Deng, Hua-Li Zhang{ddagger}§, Pei-xun Feng{ddagger}§, Xiao-Yan Duan{ddagger}§, and Yan Jin{ddagger}§1

From the {ddagger}Department of Oral Histology and Pathology, College of Stomatology, the §Research and Development Center for Tissue Engineering, and the Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China

Received for publication, June 7, 2006 , and in revised form, September 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We cloned a novel mouse cDNA, Mcpr1 (mouse cleft palate-related gene 1), between retinoic acid (RA)-treated murine embryonic palatal and control shelves by improved subtractive hybridization. Its transcript was identified by Northern blotting. The open reading frame encodes 132 amino acids and shows almost no identity to other genetic products. Mcpr1 expression could be detected extensively in adult mouse tissues and during murine embryonic development. It was identified to be significantly stimulated by RA in murine palatal shelves at embryonic day 12 and in palatal mesenchymal cells in vitro. We demonstrate that MCPR1 protein was localized primarily in the cytoplasm and could be synthesized and secreted by transfected COS-7 cells. Both the secretory and recombinant proteins of Mcpr1 inhibited proliferation of murine embryonic palatal mesenchymal cells and impeded the progression from the G1 to S phase in the cell cycle. The cells were prone to apoptosis after exposure to glutathione S-transferase-MCPR1. Furthermore, knockdown of MCPR1 protein levels by antisense oligodeoxynucleotides promoted progression of cells from the G1 to S phase and completely abolished the RA-induced block of the cell cycle from the G1 to S phase. These findings suggest that Mcpr1 might function as one of the RA-up-regulated genes involved in inhibiting cell proliferation during palatogenesis and RA-induced cleft palate by regulating proliferation and apoptosis of embryonic palatal mesenchymal cells and might even play a role in the development of many other organs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid (RA)2 is teratogenic in many species, producing multiple malformations. Many reports have demonstrated that RA, especially all-trans-RA (atRA), plays an important role in embryogenesis, including palatogenesis, by regulating morphogenesis, cell proliferation and differentiation, and extracellular matrix production (1–4). The induction of cleft palate by RA varies depending on the developmental stage exposed. Studies in vivo have indicated that, after exposure of embryonic mice to RA on gestation day 10, small palatal shelves that do not contact each other are produced and that the medial epithelial cells differentiate into an oral-like epithelium; after exposure on gestation day 12, shelves of normal size form but fail to fuse as the medial cells proliferate, and the normal growth and differentiation process of palatal mesenchymal cells is inhibited (5, 6).

The pleiotropic effects of RA are mediated through ligand-dependent transcription regulators that belong to the nuclear receptor superfamily: the retinoic acid receptors (RAR{alpha}, RARbeta, and RAR{gamma}) and the retinoid X receptors (RXR{alpha}, RXRbeta, and RXR{gamma}), which bind as RXR/RAR heterodimers to response elements located in RA-responsive genes. A genetic dissection by targeted mutagenesis performed in the mouse has revealed that RXR{alpha}/RAR ({alpha}, beta, and {gamma}) heterodimers represent the main functional units of the RA signaling pathway during embryonic development (7). It has long been established that retinoids exert their action by regulating the expression of specific subsets of genes within target tissues. However, it is only during the last 15 years that the understanding of retinoid action has rapidly increased, and subsequently, research began to involve the cloning of nuclear retinoid receptors and the identification of elements (within the promoters of retinoid-responsive genes) exhibiting a high affinity for these receptors. These nuclear receptors have been shown to work as ligand-activated transcription activators in a spatiotemporal-specific manner during embryonic development (8–10).

Despite these valuable insights into the molecular mechanism of RA-induced cleft palate, the RA-responsive signaling pathway during palatogenesis remains unclear. Moreover, research on cleft palate has focused on known candidate genes, e.g. transforming growth factor (TGF)-beta3, transcription factor AP-2, and DLX-2 (11–15). No novel gene has been reported to be responsible for RA-induced cleft palate. During the last decade, the cloning of multiple differentially expressed genes without knowledge of their sequences or protein characteristics has been accomplished by several techniques (16–19), and an improved technique based on the PCR subtractive hybridization method has been designed (20).

This study aimed to identify novel genes that may function as regulatory molecules involved in RA-induced teratogenic alteration during palatal development. In this study, a novel gene (named Mcpr1) was identified to be up-regulated by RA. We demonstrate that Mcpr1 may be involved in murine embryonic palatal mesenchymal (MEPM) cell proliferation and even contribute to apoptosis. Moreover, the inhibition of cell proliferation can be antagonized by interfering with the expression of the Mcpr1 gene. The novel gene is expressed not only in palatal shelves, but also extensively during murine embryonic development. We propose that Mcpr1 may play an important role in palatogenesis and cleft palate formation by regulating proliferation of MEPM cells as a component of the RA signaling pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatment—C57BL/6N mice (12 weeks old and ~25 g) were purchased from the Central Animal Laboratory Unit of the Fourth Military Medical University. Females were crossed with fertile males overnight. The presence of vaginal plugs was designated as embryonic day (E) 0. Pregnant females at day 10 were randomly divided into two groups.

atRA (Sigma) was prepared as described by Abbott et al. (6). All mice in one group were administered atRA dissolved in corn oil by gavage at 80 mg/kg, whereas mice in the other group received only corn oil as a control. The mice were killed on E12, and palatal shelves were dissected from fetuses using microscissors (21) and collected for further analysis.

Construction of a Subtractive cDNA Library—Total RNA and poly(A)+ mRNA were isolated using total RNA isolation and Poly(A) Tract System 1000 kits (Promega Corp.), respectively, from the palatal processes of both RA-exposed E12 murine embryos and controls. mRNA extracted from the RA-exposed group was designated tester mRNA, and that from the control group was designated driver mRNA. Driver and tester mRNAs were reverse-transcribed using a SMARTTM PCR cDNA synthesis kit (Clontech). Subtraction was performed according to the improved techniques based on the PCR subtractive hybridization method (20). Briefly, 5 µl of tester reverse-transcribed products was added to 100 µl of driver cDNA PCR products. This mixture was then denatured at 98 °C for 5 min and hybridized at 65 °C for 12 h in 6x SSC and 0.5% SDS. After hybridization, 20 µl of the hybridization liquid was loaded onto a Sephacryl S-400 spin column (Promega Corp.), and the recovered liquid (20 µl) was added to 0.1 ml of streptavidin-coated MagnetSphere paramagnetic particles (Promega Corp.) to prepare the PCR template. The PCR product could be used to repeat subtractive hybridization.

The efficiency of subtraction was estimated by electrophoresis on agarose gel, and the recovered DNA fragments were inserted into the pGEM®-T Easy vector (Promega Corp.) to construct a subtractive cDNA library for sequencing. Dot blotting was used to exclude the false positive and negative clones according to procedures described previously (20).

Bioinformatics Analysis—Searches for DNA and protein homology were performed using the BLASTN and BLASTX programs of the NCBI Databank (www.ncbi.nlm.nih.gov). The amino acid sequences deduced from cDNA were aligned with other similar protein sequences using the open reading frame finder (NCBI). Signal peptides and transmembrane fragments were predicted using the ExPASy proteomics server. The mapping of chromosomal localization was performed using the NCBI Map Viewer (www.ncbi.nlm.nih.gov/mapview/maps.cgi).

Northern Blot Analysis—To identify the expression of the novel gene, total RNAs from the palatal processes of both RA-exposed E12 murine embryos and controls were analyzed by Northern blotting. A nonradioactive digoxigenin-labeled system (Roche Applied Science) was used to label a cDNA probe specific for the Mcpr1 gene based on a standard method (22). Purified RNA (up to 25 µg) was diluted with 5x Mops, formaldehyde, and formamide; heated to 65 °C; and chilled on ice. Samples were then run on 1.2% agarose gels containing formaldehyde. When a gel had run for the appropriate distance, it was washed with RNase-free water and bathed with 20x SSC for 30 min. RNA was then transferred to a positively charged nylon membrane (Amersham Biosciences) in a blotting transfer chamber. The membrane was baked at 80 °C for 2 h. Blot hybridization parameters included prehybridization for 2 h at 68 °C and hybridization overnight at 68 °C. The membrane was reprobed with digoxigenin-labeled beta-actin cDNA as an indicator of mRNA loading.

In Situ Hybridization—Mouse palatal sections were fixed in 4% paraformaldehyde following treatment with 1 µg/ml proteinase K in phosphate-buffered saline (PBS) containing 0.1% Tween 20. Hybridization was performed overnight at 65 °C with digoxigenin-labeled cDNA probes specific for Mcpr1 (as mentioned above). The probes were reacted overnight at 4 °C with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:1000 dilution; Roche Applied Science) and then visualized by coloring reaction in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20, and 2 mM levamisole (Sigma) containing 35 mg/ml nitro blue tetrazolium (Roche Applied Science), 17.5 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science).

Reverse Transcription (RT)-PCR—Total RNAs from adult mouse brain, heart, liver, lung, kidney, spleen, and muscle were isolated and reverse-transcribed using the SMARTTM PCR cDNA synthesis kit. Primers specific for the Mcpr1 gene (P1 and P2) were used. Primers P1 (5'-ATG GCC AAG CAT CCG CGG CG-3', upstream) and P2 (5'-AAG AGT TGG ATC TGA GAA AG-3', downstream) yielded an amplicon of 396 bp. A 20-µl volume of reverse-transcribed product was amplified with Taq DNA polymerase. The reaction was performed with a 5-min denaturation at 94 °C, followed by 30 cycles each with denaturation at 94 °C for 50 s, primer annealing at 55 °C for 30 s, and product extension at 72 °C for 1 min. The final cycle, 72 °C extension, was carried out for 6 min. In all PCRs, the amplification of beta-actin cDNA was used as an internal control, and the primers used were as follows: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3' (upstream) and 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3' (downstream).

Expression of Prokaryotic Recombinant Protein and Preparation of Polyclonal Antibodies—The partial coding regions of the Mcpr1 gene were amplified by PCR from the cDNA library obtained as described above using primers P3 (5'-CGC GGA TCC ATG GCC AAG CAT CCG CGG CG-3', upstream; with BamHI sites indicated in italic type) and P4 (5'-CCG CTC GAG TAA GTT GCT ACT CTG GCC TCC-3', downstream; with XhoI sites indicated in italic type). The expression vector pGEX-4T-Mcpr1 was generated by directionally cloning the BamHI-XhoI fragment into the multiple cloning site of the pGEX-4T-1 vector (kindly donated by Prof. Ming Jin). Protein expression was performed according to Sambrook et al. (22). Escherichia coli DH5{alpha} was used as a recombinant expression host. Glutathione S-transferase (GST) protein was expressed in parallel with the pGEX-4T-1 vector as a control. Expressed proteins were purified using a GST affinity column. Purified proteins were subjected to SDS-PAGE on a 12% gel to detect their relative molecular masses and purity.

Purified GST-MCPR1 protein was used for the preparation of polyclonal antibodies (pAbs) against MCPR1 protein. Briefly, purified GST-MCPR1 was subjected to SDS-PAGE on a 12% gel, and the corresponding gel loaded with the protein was cut and stored at –80 °C for the preparation of antigens. The gel (200 mg) was mechanically homogenized in Freund's incomplete adjuvant to obtain antigens, and 200 mg of antigens was injected subcutaneously into a rabbit at six sites every other week. Serum was collected 1 week after the third booster injection and weekly thereafter. pAbs were purified from the sera using protein A-agarose according to the manufacturer's protocol. The specificity and sensitivity of the purified pAbs against MCPR1 protein were evaluated by enzyme-linked immunosorbent assay and Western blotting. The purified pAbs that were identified with a titer of 1:100,000 were used for Western blotting and immunochemical analysis.

Western Blot Analysis—To detect the expression pattern of MCPR1 protein, Western blotting was performed to analyze many tissue extracts. The samples included normal adult mouse liver, heart, brain, spleen, kidney, lung, and muscle; palatal shelves at E12–16 and postnatal day 1; and the whole head at E9–11. The tissue extracts were prepared in 10x radioimmune precipitation assay buffer, and protein in these samples was measured by the Bradford method (23). Briefly, a sample containing 10 µg of the appropriate extract was mixed with an equal volume of 2x sample buffer. These samples were resolved by electrophoresis on 12% denaturing gels containing SDS. The proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% fat-free milk and incubated overnight at 4 °C with primary antibodies (pAbs against MCPR1 protein, 1:1000 dilution). The membranes were washed with PBS and incubated with the corresponding peroxidase-conjugated secondary antibodies (1:200–400 dilution) using an SA2022 Western blotting kit (BOSTER). Complexes of proteins and antibodies were detected using the chromogen diaminobenzidine (Dako). The results were quantitatively analyzed using UN-SCAN-IT software (Silk Scientific, Inc.).

Immunohistochemical Staining of MCPR1 Protein—Sequential cryostat sections (5 µm) were mounted and stained using the standard immunoperoxidase method (Dako). The optimum concentration of antibodies was established. Prior to application of the primary antibodies, endogenous peroxidase was blocked by the addition of hydrogen peroxide. Negative controls were obtained by omission of pAbs against MCPR1 protein. Slides were incubated with the primary antibodies for 1 h at 37 °C and incubated for 40 min at 37 °C with biotinylated goat anti-mouse IgG (1:400 dilution; Dako). A streptavidin-biotin-peroxidase complex was added according to the manufacturer's instructions. Peroxidase activity was demonstrated using diaminobenzidine. Each step was separated by thorough washes with PBS. Finally, slides were mounted with coverslips and viewed under a conventional light microscope.

MEPM Cell Culture and Treatment—Primary MEPM cell cultures were established as described previously (24). Briefly, secondary palatal shelves were collected from pups at E13. The use of E13 MEPM cells to study events occurring on E12 in vivo was justified based on published data (25). MEPM cells between the third and fifth passages were used for analysis.

Once the cultures attained ~40% confluence (~72 h), the cells were exposed to RA (at a final concentration of 3 x 10–4 µg/ml in the medium) or purified GST-MCPR1 (at a final concentration of 0.1, 1, 5, 10, 20, or 40 µg/ml in the medium). Purified GST was used in parallel as a control. Following 48 h of incubation, the media were collected; the cell sheet was washed with PBS; and the cells were treated differently according to different analyses.

To assay the change in Mcpr1 expression in MEPM cells exposed to RA, RT-PCR, Western blotting, immunocytochemical analysis, and in situ hybridization were performed as described above. Flow cytometric analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction were performed to assay the biological effect of MCPR1 protein on MEPM cells. The detailed procedures were described previously (26–28).

After exposure to GST-MCPR1 for 48 h, cells were detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) system (ApopTag Plus in situ apoptosis detection kit, Oncor). Finally, slides were mounted with coverslips and viewed under a conventional light microscope.

All experiments were carried out in duplicate, and the results represent the means ± S.E. of at least three independent experiments. One-way analysis of variance was used to determine significance among groups, after which a post hoc test with Bonferroni's correction were used for comparison between individual groups. A p value ≤0.05 was considered significant.

Subcellular Fractionation and Immunoblot Analysis—To detect subcellular localization of MCPR1 protein, cytoplasmic and nuclear fractionation was performed as described previously (29). MEPM cells were pelleted and resuspended in 300 µl of buffer A (0.25 mol/liter sucrose, 10 mmo/liter Tris-HCl pH 8.0, 3 mmol/liter MgCl2, 0.1 mmol/liter phenylmethyl sulfonylfluoride). After 15 min on ice, the product was centrifuged, and the supernatant (cytoplasmic fraction) was collected and stored. The pellet was washed with 200 µl of buffer B (Buffer A + 0.1% (v/v) Triton X-100) and then resuspended in 100 µl of buffer C (2.2 mol/liter sucrose, 10 mmol/liter Tris-HCl pH 8.0, 3 mmol/liter MgCl2, 0.1 mmol/liter phenylmethyl sulfonylfluoride). After centrifugation, the supernatant (nuclear fraction) was recovered. Cytoplasmic and nuclear fractions were quantitated for protein content by the Bradford method (23) and subjected to Western blot analysis.


Figure 1
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  		FIGURE 1.
Analysis of deduced MCPR1 protein using the ExPASy proteomics server. A, hydrophobicity (Hphob.) plot of MCPR1 protein. Positive values indicate hydrophobic regions, and negative values indicate hydrophilic regions. B, prediction of the transmembrane region. i->o indicates inside -> outside helices, and o->i indicates outside -> inside helices. Only scores above 500 were considered significant. i->o 106–128 (score of 1575) and o->i 105–125 (score of 1401) are the indicated transmembrane helices. C, the hidden Markov model calculates the probability (prop.) of whether the submitted sequence contains a signal peptide or not and also reports the probability of a signal anchor. The signal peptide probability is 0.767, and the signal anchor probability is 0.001. The cleavage site (short red ordinate) between positions 43 and 44 was assigned a probability score (0.231), together with scores for the n-, h-, and c-regions of the signal peptide.

 
Eukaryotic Recombinant Expression and Biological Activity Analysis—COS-7 cells were used as the recombinant expression hosts. A eukaryotic expression vector was generated by subcloning the EcoRI-BamHI fragment into the multiple cloning site of the pEGFP-N3 eukaryotic expression vector (Clontech). Subsequently, COS-7 cells were transfected with the construct pEGFP-N3-Mcpr1 using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's protocol. 48 h after transfection, the cells were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS. Subcellular localization of the enhanced green fluorescent protein (EGFP)-MCPR1 fusion protein was detected using a fluorescence microscope.

To obtain COS-7 cells steadily expressing EGFP-MCPR1 (COS-7/Mcpr1 cells), we selected transiently transfected COS-7 cells using selective medium containing 500 µg/ml G418 (Promega Corp.). After 4 weeks of culture in the selective medium, clonal isolates were expanded, and Mcpr1 expression at the transcriptional level was verified by RT-PCR.

To investigate whether MCPR1 protein could be secreted from cells, the conditioned medium of COS-7/Mcpr1 cells was collected 3 days after the cells were passaged, and protein from the conditioned medium was concentrated with a Centricon-10 concentrator (Millipore Corp., Bedford, MA). Cells were lysed with radioimmune precipitation assay buffer. Western blot assays were performed to identify the occurrence of MCPR1 protein.

Once MCPR1 protein was identified, the conditioned medium was used to treat MEPM cells to analyze possible biological activity of the protein. The medium of COS-7 cells transfected with pEGFP-N3 (COS-7/N3 cells) was used as a control to exclude the effect of other components in the medium. MTT reduction was performed to assay cell viability.

Synthesis of Antisense Mcpr1 Oligodeoxynucleotides (ODNs)—Antisense ODNs for Mcpr1 were selected using the DNASIS program (Hitachi Software, San Bruno, CA). Sense ODNs were used as controls. ODNs were thio-modified for stability and fluorescently labeled so that the transfection efficiency of ODNs could be subsequently evaluated. The sequences of ODNs for Mcpr1 mRNA were as follows: 5'-CGG ATG CTT GGC CAT-3' (antisense) and 5'-ATG GCC AAG CAT CCG-3' (sense). The ODNs were synthesized by Sunbiotech (Peking, China).

Antisense ODNs in MEPM Cells—MEPM cells were seeded at 2 x 104 cells/well in 8-well chamber slides and cultured for 24 h in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 1% penicillin/streptomycin. MEPM cells were serum-starved for 24 h in DMEM containing 0.5% fetal calf serum (HyClone, Logan, UT) and transfected with antisense Mcpr1 ODNs or sense ODNs. Cationic liposomes (LipofectamineTM 2000) were used to facilitate transfection as recommended by Invitrogen. The ODN-liposome complexes were added to each well and incubated at 37 °C for 5 h. Th cells were further incubated in DMEM containing 10% fetal calf serum for 48 h and analyzed. The MEPM cells were washed three times with PBS and fixed in methanol at 4 °C for 10 min. The cells were mounted and observed under a fluorescence microscope. The effect of antisense ODNs on Mcpr1 expression in MEPM cells was investigated by RT-PCR and Western blot analysis. The effect of antisense ODNs on the cell cycle was assessed by flow cytometric analysis as described previously (28).

To further investigate whether the Mcpr1 gene is required for the inhibitory effect of atRA on MEPM cell growth, we exposed the antisense ODN-transfected cells to atRA. The transfected cells were incubated in DMEM containing 10% fetal calf serum for 24 h. atRA (10–8 mol/liter) was added to each well, followed by incubation in DMEM containing 10% fetal calf serum for 24 h. The cell cycle was then examined by flow cytometric analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning and Sequence Analysis of the Mcpr1 cDNA—To identify genes differentially expressed during normal palatogenesis and cleft palate formation, the improved subtractive hybridization technique was applied to the mRNA extracted from palatal shelves with or without atRA treatment, and we selected several clones specifically up-regulated by atRA treatment. We determined the sequences of the isolated partial cDNA clones and carried out homology searches in the Gen-BankTM Data Bank using BLAST. Of four clones (clones 1–4) that had no significant homology to any known genes in the nucleotide sequence data bases, clone 1 was determined to be a full-length sequence, and the complete coding regions was considered to be encompassed by analysis with the GenBankTM open reading frame data base. We therefore focused all further analysis on this clone. The clone 1 gene was revised, and suspected T-vector sequences in the 3' terminus were deleted. The 780-bp cDNA sequence was named Mcpr1 for mouse cleft palate-related gene 1 and submitted to the GenBankTM Data Bank as a novel gene with accession number AY074887 [GenBank] .

The full length of 780 bp obtained was in good agreement with the transcript size of ~800 bp estimated by Northern blotting. An open reading frame extends from nucleotides 200 to 598 and encodes a 132-amino acid polypeptide with an estimated molecular mass of 14.1 kDa and an isoelectric point of 11.06. The predicted polypeptide includes a signal peptide and a hydrophobic segment thought to be a transmembrane-spanning region (Fig. 1, A–C). According to the NCBI Map Viewer, the Mcpr1 gene is assigned to chromosome 9q54 in the mouse.


Figure 2
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  		FIGURE 2.
Mcpr1 expression is markedly induced by RA treatment in E12 murine embryonic palatal shelves and cultured MEPM cells. A, Northern blot analysis confirming the differential expression of Mcpr1 in E12 murine embryonic palatal shelves. An expected transcript of ~800 bp (based on RNA Marker RL1000) was detected. beta-Actin was used as the control for genomic contamination. B, RT-PCR analysis of Mcpr1 expression in RA-treated and untreated MEPM cells in vitro. Mcpr1 expression was increased significantly in RA-treated cells. beta-Actin was used as the control for genomic contamination. C, Western blot analysis of MCPR1 protein in RA-treated and untreated MEPM cells in vitro. MCPR1 protein was induced significantly by RA treatment. D and E, immunocytochemical analysis of MCPR1 protein expression in RA-treated and normal control MEPM cells, respectively (magnification x100). The positive signals were localized primarily in the cytoplasm. F and G, in situ hybridization analysis of Mcpr1 expression in RA-treated and normal control MEPM cells, respectively (magnification x100). Mcpr1 transcripts were significantly induced by RA.

 


Figure 3
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  		FIGURE 3.
Tissue expression analysis of the Mcpr1 gene. A, RT-PCR analysis of adult mouse tissues. beta-Actin was used as the control for genomic contamination. B and C, Western blot analysis of MCPR1 protein in adult mouse tissues and in palatal shelves during embryonic development (E9–16 and postnatal day 1 (P1)), respectively. The immunoreaction band at 14.4 kDa was based on protein molecular mass markers. D, immunohistochemical analysis of MCPR1 protein in an E12 murine embryo. The arrow indicates strong expression in embryonic heart and liver (magnification x40). E, negative control omitting the primary pAbs against MCPR1 protein (magnification x40).

 
Identification of the Mcpr1 Gene in the RA Signaling Pathway—Compared with Mcpr1 expression in the normal controls, Mcpr1 expression in RA-treated E12 palatal shelves showed a marked induction as determined by Northern blot analysis (Fig. 2A), and one transcript of ~800 bp was detected. Mcpr1 expression in MEPM cells exposed to RA in vitro was significantly enhanced at the mRNA and protein levels compared with that in the normal controls (Fig. 2, B and C). The results of immunocytochemistry staining and in situ hybridization further indicated that Mcpr1 expression was up-regulated by RA treatment at the cellular level (Fig. 2, D–G), and the positive signals detected by immunocytochemistry were localized primarily in the cytoplasm of MEPM cells (Fig. 2, D and E).

Extensive Tissue Expression Pattern of Mcpr1 in Adult and Embryonic Mice—Mcpr1 expression pattern analyses were carried out by RT-PCR, Western blotting, and immunohistochemistry. The beta-actin housekeeping gene was used as a control to monitor genomic DNA contamination and equality of sample loading. RT-PCR products of beta-actin are shown in Fig. 3A, and none of the RNA samples had genomic contamination. The results of RT-PCR and Western blot assay showed that Mcpr1 was expressed extensively in adult mouse tissues at the transcriptional and translational levels (Fig. 3, A and B). MCPR1 was undetectable in E9 and E10 murine embryos by Western blot analysis; however, it could be detected in developing palates from E11 to the postnatal period (Fig. 3C). Immunohistochemical staining showed its extensive expression in E12 embryos, particularly in embryonic heart and liver (Fig. 3D).

To determine the detailed expression pattern of Mcpr1 in palatogenesis, we investigated its expression in palatal shelves at E12–14 by immunohistochemical and in situ hybridization analyses. At E12, when palatal shelves have elongated and grown vertically, Mcpr1 was expressed in palatal mesenchymal cells (Fig. 4, A and D). At E13, when elevation of the palatal shelves has occurred, Mcpr1 expression showed no significant variation in mesenchymal cells compared with that at E12, and discrete populations of cells in the epithelial component showed Mcpr1 transcripts and protein (Fig. 4, B and E). At E14, just before immediate contact of the palatal shelves, Mcpr1 was still expressed throughout the mesenchymal cells (Fig. 4, C and F).

MCPR1 Protein Localizes Primarily in the Cytoplasm and May Be Secretory—Western blot analysis of the cytoplasmic and nuclear extracts of MEPM cells demonstrated a band at ~14.4 kDa in the cytoplasm rather than the nucleus (Fig. 5A). COS-7/Mcpr1 cells were established after culture in G418 selective medium, and the expression of Mcpr1 was verified by RT-PCR (Fig. 5B). These cells displayed green fluorescence primarily in the cytoplasm and rarely in the nucleus, whereas COS-7 cells transfected with pEGFP-N3 vector showed diffuse green fluorescence throughout the cells (Fig. 5, C and D). These findings demonstrated that MCPR1 protein was localized primarily in the cytoplasm. Western blot analysis showed that MCPR1 could be detected in both the conditioned medium and COS-7/Mcpr1 cells (Fig. 5E). These findings indicated that COS-7/Mcpr1 cells could synthesize MCPR1 protein and secrete it into the medium.


Figure 4
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  		FIGURE 4.
Expression pattern analysis of Mcpr1 in developing palates at E12–14 by in situ hybridization (A–C) and by immunohistochemistry (D–F). A and D, at E12, the Mcpr1 transcript and MCPR1 protein were expressed in palatal mesenchymal cells. B and E, at E13, Mcpr1 expression showed no significant variation in mesenchymal cells compared with that at E12, and discrete populations of cells in the epithelial component showed Mcpr1 transcripts. C and F, at E14, Mcpr1 was still expressed throughout mesenchymal cells. Arrows indicate positive cells.

 
MCPR1 Protein Inhibits Cell Viability—We examined the effects of the conditioned medium on MEPM cell proliferation. As shown in Fig. 6A, MTT assay revealed that MEPM cell viability was repressed by treatment with the conditioned medium, whereas no significant inhibition was found in cells treated with the control medium.

To further examine the biological function of Mcpr1, the GST-MCPR1 fusion protein was expressed with the pGEX-4T-Mcpr1 vector. As shown in Fig. 6B, purified GST protein migrated at the site between 31 and 20.1 kDa, in agreement with its estimated molecular mass (26 kDa), and recombinant GST-MCPR1 purified from the E. coli extracts migrated at the site between 43 and 31 kDa. These results indicated that recombinant GST-MCPR1 was successfully generated and purified.

A concentration-dependent effect of GST-MCPR1 in vitro on the growth of MEPM cells was shown by absorbance values determined by MTT assay, and it inhibited cell proliferation at concentrations of 5–20 µg/ml (Fig. 6C). Cell growth was not affected significantly at 0.1–1 µg/ml. There was no significant change between GST-treated cells and the normal controls. According to the results of flow cytometric analyses, the cell cycle of MEPM cells treated with 10 µg/ml GST-MCPR1 was arrested. As shown in Fig. 6D and Table 1, GST-MCPR1 resulted in a significant increase (85.7%) in the number of cells in the G0/G1 phase compared with GST (74.6%), with a concomitant decrease in the number of cells in the S and G2/M phases. Therefore, we propose that MCPR1 protein resulted in cell cycle arrest of MEPM cells from the G1 to S phase. Furthermore, according to TUNEL assay, the number of MEPM cells undergoing apoptosis increased upon exposure to GST-MCPR1 compared with the controls (Fig. 6, E–G).


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  		TABLE 1
Percentage of cells in each cell cycle stage analyzed by flow cytometric analysis indicating inhibition of the G1-to-S phase transition of MEPM cells by GST-MCPR1 fusion protein

 


Figure 5
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  		FIGURE 5.
Detection of MCPR1 protein in the cytoplasm of MEPM cells and transfected COS-7/Mcpr1 cells and in the conditioned medium. A, shown are the results from Western blot analysis of MCPR1 protein in nuclear and cytoplasmic fractions of MEPM cells. B, shown are the results from RT-PCR analysis of Mcpr1 expression in COS-7 cells transfected with the pEGFP-N3-Mcpr1 (COS-7/Mcpr1 cells) or pEGFP-N3 (COS-7/N3 cells) vector. No signal was found in COS-7/N3 cells. C, the EGFP-MCPR1 fusion protein was detected primarily in the cytoplasm of COS-7/Mcpr1 cells under a fluorescence microscope (magnification x100). D, EGFP was expressed diffusely in the nucleus and cytoplasm of COS-7/N3 cells (used as a transfection control; magnification x400). E, shown are the results from Western blot analysis of MCPR1 protein in the lysates and conditioned medium (CM) of COS-7/Mcpr1 cells (lanes 1 and 3, respectively) or COS-7/N3 cells (lanes 2 and 4, respectively). MCPR1 protein was detected only in the lysates and conditioned medium of COS-7/Mcpr1 cells (lanes 1 and 3, respectively).

 
Antisense ODNs Knock Down Mcpr1 Expression in MEPM Cells and Promote Progression of Cells from the G1 to S Phase of the Cell Cycle—Most of the MEPM cells transfected with fluorescein-labeled ODNs exhibited strong green fluorescence in the nucleus (Fig. 7, A and B). Mcpr1 expression was significantly decreased at the transcriptional and translational levels by antisense ODNs compared with sense ODNs and liposomes alone (Fig. 7, C and D).

The normal culture conditions resulted in cycling of ~27% of the cells in the S and G2/M phases (Table 1). In the antisense ODN group, the number of cycling cells (combined total number of cells in the S and G2/M phases) was increased by >39%, with a concomitant decrease in the number of cells in the G0/G1 phase. A significant increase in the number of cells was seen in the S and G2/M phases (Fig. 8). These findings indicated that proliferation of MEPM cells could be promoted by interfering with Mcpr1 expression.

Down-regulation of the Mcpr1 Gene Can Abolish Cell Cycle Arrest of MEPM Cells Treated with RA—In atRA-treated MEPM cultures, a significant inhibition of cell viability was observed (Fig. 8), in agreement with previous results (30). To further understand the functions of Mcpr1 in atRA-induced inhibition of MEPM cell growth, we knocked down endogenous MCPR1 protein levels in MEPM cells with antisense ODNs and then exposed the cells to atRA. According to the results of flow cytometric analyses, RA caused only 70.7% of the antisense ODN-transfected cells and 88.9% of the untransfected cells to stagnate in the G0/G1 phase (Table 2). The proliferation index of RA-treated antisense ODN-transfected cells was significantly higher than that of RA-treated untransfected cells (Table 2). The results indicated that knockdown of Mcpr1 expression could completely abolish cell cycle arrest from the G1 to S phase caused by atRA; therefore, we propose that the Mcpr1 gene is essential for RA-induced cell cycle arrest and that it might function as one of the negative cell cycle regulators involved in palatogenesis.


View this table:
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  		TABLE 2
Percentage of cells in each cell cycle stage analyzed by flow cytometric analysis indicating that knockdown of Mcpr1 expression by antisense ODNs can promote progression from the G1 to S phase of the MEPM cell cycle and abolish RA-induced cell cycle arrest at the G1 phase

 
To summarize, the novel gene Mcpr1 was cloned between RA-treated and untreated murine embryonic palatal shelves. Mcpr1 was demonstrated to be markedly up-regulated in atRA-treated palatal shelves and MEPM cells. Our results indicated that Mcpr1 had extensive tissue expression patterns in adult mice and during embryonic development. MCPR1 protein was localized primarily in the cytoplasm and could be secreted into the medium by cells. Both secretory and recombinant MCPR1 proteins resulted in cell cycle arrest of MEPM cells. Knockdown of Mcpr1 expression promoted cell proliferation, and more interestingly, the cell cycle arrest caused by RA was completely abrogated in Mcpr1 knockdown cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cloning and Identification of the Novel Gene Mcpr1—During the last 2 decades, differentially expressed novel genes have been detected during development by subtractive hybridization techniques. For instance, the CORS26 gene was cloned by this method in TGF-beta1-treated and untreated C3H10T1/2 cells and shown to play an important role in skeletal development (31). Pfisterer et al. (32) used a combination of suppression subtractive hybridization and high throughput differential screening to identify target genes of transcription factor AP-2 in wild-type and AP-2-deficient embryonic mouse heads, and a set of genes repressed by AP-2 were identified successfully.

No novel genes involved in cleft palate formation in either human or mouse have been reported. In this study, we identified a novel gene (Mcpr1) that was induced markedly by atRA treatment in palatal shelves and in cultured MEPM cells. We were therefore fascinated with this novel gene and investigated its characterization and potential function in the RA-responsive signaling pathway during palatogenesis.

We investigated some of the known genes obtained from our subtractive cDNA library to search for some cues as to function of the Mcpr1 gene. Among the known genes we obtained, many were full-length cDNA, such as the Fau gene (Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene) and ribosomal proteins L8, L27, S16, and S27. Fau is conserved in higher eukaryotes, is expressed extensively in all mammalian tissues, and functions as a tumor suppressor gene by inhibiting cell growth (33, 34). Ribosomal protein families are expressed extensively in both prokaryotes and eukaryotes and are related to apoptosis. It has been reported that ribosomal proteins L3, L7, L16, and L27 are expressed highly in tumors with a high apoptosis index (35–37).

Is Mcpr1 a Direct Target Gene of RA?—This study has demonstrated that Mcpr1 is one of the components of the RA-mediated gene network, i.e. it is a target for RA-mediated transcriptional up-regulation. This conclusion is based on the following observations. 1) Transcription and translation of Mcpr1 were up-regulated by RA treatment in palatal shelves in vivo and in cultured MEPM cells in vitro. 2) Knockdown of MCPR1 protein levels completely abolished the RA-induced inhibition of the MEPM cell cycle from the G1 phase to S phase.


Figure 6
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  		FIGURE 6.
Effect of MCPR1 protein on biological characterization of MEPM cells in vitro. A, MTT assay indicated that the conditioned medium of COS-7/Mcpr1 cells repressed cell viability compared with those of normal and COS-7/N3 controls. Error bars indicate S.D. B, shown are the results from SDS-PAGE analysis of MCPR1 protein expression and purification. Lane 1, purified GST protein; lanes 2 and 5, purified GST-MCPR1 fusion protein; lanes 3 and 4, lysates of E. coli cells transformed with pGEX-4T-Mcpr1; lane 6, protein molecular mass markers. C, MTT analysis showed that ~1µg/ml GST-MCPR1 could not significantly inhibit cell viability; however, dose-dependent inhibitory effects were observed when the concentration ranged from to 40 µg/ml. D, shown are the results from flow cytometric analysis of the inhibitory effect of 10 µg/ml GST-MCPR1 on MEPM cell growth. The percentage of cells in each cell cycle stage is shown in the Table 1. The percentage of cells in the G1 phase was significantly increased in cells treated with GST-MCPR1 compared with GST (p < 0.01). E and F, TUNEL assay showed apoptosis of MEPM cells treated with GST-MCPR1 and GST, respectively (magnification x100). G, the histogram shows the number of TUNEL-labeled MEPM cells treated with GST-MCPR1 or GST (n = 3). Error bars indicate S.D. The number of apoptotic cells was significantly increased by GST-MCPR1 treatment (p < 0.05).

 
Moreover, RA acts by binding to heterodimeric receptors composed of an RAR and an RXR; these receptors bind to RAR and modulate target gene transcription. RAR have been identified in the promoters of a large number of retinoid target genes implicated in a wide variety of functions (10). By bioinformatics analysis, we did not find RAR composed typically of two direct repeats of a core hexameric motif (physical unit G(G/T)TCA) or other known or reported RAR related to the Mcpr1 gene. Therefore, we assume that Mcpr1 may not be a direct target gene of RA and that RA up-regulates Mcpr1 expression via indirectly modulating the expression of an intermediate factor, which in turn up-regulates the Mcpr1 promoter.

RA signaling has been implicated in a variety of development processes, including cardiovascular development and embryonic and early postnatal brain development (38, 39). The Mcpr1 gene was cloned from embryonic murine palatal shelves by administration of excess RA to pregnant mice on gestation day 10, and we have demonstrated that it was extensively expressed in other tissues besides palates, with especially strong expression in embryonic heart and liver at E12. Our results indicate that Mcpr1 is a target of RA-mediated transcriptional up-regulation. Therefore, we propose that Mcpr1 might be implicated in heart and other organ development as one of the RA-regulated downstream genes. Future work might reveal some mechanism of Mcpr1 involvement in embryonic development.

Mcpr1 May Be Involved in RA-induced Cleft Palate by Regulating MEPM Cell Proliferation—It is well established that RA is a teratogenic effector of cleft palate and that RA alters the spatial and temporal expression of a set of growth factors during palatogenesis; however, no known growth factors have been proven to be responsible for RA-induced cleft palate. Here, we cloned a target gene of atRA-mediated transcriptional up-regulation from embryonic palatal shelves.


Figure 7
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  		FIGURE 7.
Effect of antisense Mcpr1 ODNs on the growth of MEPM cells. A and B, MEPM cells were transfected with fluorescein-labeled antisense ODNs and sense ODNs, respectively. Most of the MEPM cells exhibited strong fluorescence in the cytoplasm and nucleus. A, magnification x100; B, magnification x400. C and D, RT-PCR and Western blot analysis showed that antisense ODNs inhibited the expression of Mcpr1 in MEPM cells at the transcriptional and translational levels compared with controls.

 
In this study, we have demonstrated that MCPR1 protein can result in cell cycle arrest of MEPM cells in the G1 phase and that the characteristics are the same as those of atRA because atRA caused a block in the cell cycle at the G1 phase with an increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the S phase. Previous studies demonstrated that atRA inhibits the expression of cyclins D and E, which regulate the G1-to-S phase transition (40, 41). Furthermore, atRA treatment reduces phosphorylated retinoblastoma protein and decreases CDK2 and CDK4 kinase activity. These data suggest that atRA has anti-proliferative activity by modulating G1/S cell cycle regulators and by inhibiting retinoblastoma protein phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by RA. Considering the biological characteristics of Mcpr1, we infer that Mcpr1 might be involved in MEPM cell growth by interacting with these cell cycle-related regulators.

This study has shown that MCPR1 protein was undetectable in murine embryos at E9 and E10; however, it could be detected during palatal development from E11 to the postnatal period, especially in MEPM cells at E12–14 in vivo. Therefore, according to the extensive expression pattern, we infer that the Mcpr1 gene may be required for the proper development of palatal shelves.

Our findings concerning the biological activity of MCPR1 protein indicated that both secretory and recombinant MCPR1 proteins, when added exogenously to cultured MEPM cells, inhibited cell viability. Moreover, because antisense ODNs markedly suppressed the expression of Mcpr1 in MEPM cells, the progression of the cells from the G1 to S phase of the cell cycle was promoted significantly compared with controls. More interestingly, the knockdown of Mcpr1 expression completely abolished RA-induced cell cycle arrest at the G1 phase. These findings suggest that the Mcpr1 gene is essential for RA-induced cell cycle arrest by blocking the G1-to-S phase transition. The differentiation and proliferation of embryonic palatal mesenchymal cells are critical during palatal morphogenesis (24, 25), and the inhibition of embryonic palatal mesenchymal cell proliferation might account for the small palatal shelves in RA-induced cleft palate. Therefore, we propose that Mcpr1 is involved in palatogenesis or RA-induced cleft palate by regulating MEPM cell proliferation as a negative cell cycle regulator.

In addition, when added exogenously to cultured MEPM cells in vitro, recombinant MCPR1 protein appeared to contribute to apoptosis. A previous study demonstrated that atRA inhibits the growth of MEPM cells by inducing apoptosis in a dose-dependent manner, except for a G1 block in the cell cycle (30). Consequently, we assume that Mcpr1 might be involved in RA-induced apoptosis as an up-regulated gene in downstream signaling. Mcpr1 might be involved in palatal development to some extent by inducing apoptosis. The detailed function or molecular mechanism needs to be determined in future investigation.


Figure 8
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  		FIGURE 8.
Flow cytometric analysis (n = 3) of the effect of Mcpr1 ODNs on the distribution of MEPM cells in the various cell cycle stages (G0/G1, S, and G2/M). The percentage of cells in each cell cycle stage is shown in Table 2, along with S.E.

 
Signaling Pathway in RA-induced Cleft Palate—Processes critical to normal palatogenesis, including control of proliferation, differentiation, and extracellular matrix production, are likely to be regulated by the specific spatial and temporal expression of some growth factors or their receptors. Chondrogenesis is a critical step in palatogenesis. It was well established that RA-mediated gene repression is both necessary and sufficient for chondroblast differentiation (42). atRA has been shown to inhibit chondrogenic differentiation of embryonic mesenchymal cells and to cause loss of differentiated chondrocyte phenotype (43–45), suggesting that similar effects on cartilage differentiation in vivo may be responsible for abnormalities in the developing palate (46–48).

There are many RA-related signaling pathways in cleft palate formation, and according to the present investigation we cannot clarify which way the Mcpr1 gene is involved. We wonder whether the Mcpr1 gene is related to some key components during palatal development, e.g. TGF-beta3 or AP-2. It has been recently shown that TGF-beta3 is an absolute requirement for successful palatal fusion; interactive effects between RA and TGF-beta3 have been defined in a number of cell types (49, 50). atRA causes an increase in TGF-beta3 expression at the transcriptional level in MEPM cells, and TGF-beta3 initiates its cellular actions by binding to TGF-beta receptor II and activates the intrinsic serine/threonine kinase activity of TGF-beta receptor I, which phosphorylates transcription factors Smad2 and Smad3 (51, 52). RA and TGF-beta3 inhibit [3H[thymidine incorporation into the DNA of MEPM cells; however, when administered simultaneously, they inhibit proliferative activity to a greater extent (52–54). Our findings indicated that the Mcpr1 gene inhibited MEPM cell viability and that the expression of Mcpr1 was simultaneously increased upon RA-mediated inhibition of MEPM cell viability. Moreover, down-regulation of the expression of the Mcpr1 gene completely abolished atRA-induced cell cycle arrest. These findings indicate that Mcpr1 is essential for atRA-induced inhibition of cell proliferation. Considering the similar activity of Mcpr1 and TGF-beta3, more studies on the relationship between Mcpr1 and TGF-beta3 signaling may contribute to determining the function of the Mcpr1 gene in palatogenesis.

In summary, we have demonstrated that Mcpr1 is a target gene of RA-mediated transcriptional up-regulation and may be essential for RA signaling to inhibit MEPM cell growth. Consequently, we propose that Mcpr1 may be involved in palatogenesis or RA-induced cleft palate by regulating MEPM cell proliferation. Furthermore, the evidence that knockdown of MCPR1 protein levels can completely abolish cell cycle arrest from the G1 to S phase caused by atRA provides strong support for this. The results of this study have raised several important questions that will be the subject of future investigations. For instance, what is the role of Mcpr1 in RA signaling? How does Mcpr1 affect palatal development? A possibility is that RA up-regulates Mcpr1 expression via indirectly modulating the expression of an intermediate factor, which in turn up-regulates the Mcpr1 promoter, and then Mcpr1 functions as a negative regulator of cell growth to affect palatal development. The detailed function or molecular mechanism needs to be determined in the future investigations.


   FOOTNOTES
 
* This work was supported by Grants 2002AA205041 and 2005AA205241 from Development of High and New Science and Technology Project 863 and by Project 30572046 of the Nature Science Foundation of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) AY074887 [GenBank] . Back

1 To whom correspondence should be addressed: Dept. of Oral Histology and Pathology, College of Stomatology, Fourth Military Medical University, 7 Kangfu Rd., Xi'an, Shaanxi 710032, China. Tel.: 86-29-8477-6147; Fax: 86-29-8321-8039; E-mail: yanjin{at}fmmu.edu.cn or yanjinfmmu{at}vip.sina.com.

2 The abbreviations used are: RA, retinoic acid; atRA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; TGF, transforming growth factor; MEPM, murine embryonic palatal mesenchymal; E, embryonic day(s); Mops, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; RT, reverse transcription; GST, glutathione S-transferase; pAbs, polyclonal antibodies; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; EGFP, enhanced green fluorescent protein; ODNs, oligodeoxynucleotides; DMEM, Dulbecco's modified Eagle's medium. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Wen Yue (University of Pittsburgh Cancer Institute) and Drs. Kun Xuan and Faming Chen (Fourth Military Medical University) for generous help during cloning of the Mcpr1 gene and critical comments on the manuscript and Prof. Ming Jin (Fourth Military Medical University) for preparing polyclonal antibodies against the Mcpr1 gene.



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 RESULTS
 DISCUSSION
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