Retinoic Acid Down-Regulates Bone Morphogenetic Protein 7 Expression in Rat with Cleft Palate

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Chin Med Sci J March 2008

CHINESE MEDICAL SCIENCES JOURNAL RETINOIC ACID DOWN-REGULATES BONE MORPHOGENETIC PROTEIN 7 EXPRESSION IN RAT WITH CLEFT PALATE' Lei Guo'* , Yu-yan Zhao2, Shi-liang Zhang' , Kui Liu' , and Xiao-yu Gaol 1

Department of Orthopedic Surgery, 2 Department of Endocrinology, the First Affiliated Hospital, China Medical University, Shenyang 110001

Key words: bone morphogenetic protein 7 ; retinoic acid; cleft palate; osteoblast Objective To evaluate the effects of retinoic acid (RA) on expression of bone morphogenetic protein 7 (BMP-7) in rat fetus with cleft palate, and the effects of RA on proliferation and apoptosis of osteoblasts. Methods All-trans RA ( ATRA) was used to induce congenital cleft palate in Wistar rat. BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis. Flow cytometry and M'IT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-El cells. BMP-7 mRNA and protein expressions in ATRA-treated MC-3"3-E1 cells were detected by RT-PCR and Western blotting analysis.

Results ATRA could induce cleft palate of rat fetus. The incidence rate of cleft palate induced by 100 mg/kg ATRA (45.5% ) was significantly higher than 50 mg/kg ATRA ( 12.5% , P < 0.05 ). BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate. MC-3T3-El cells proliferation treated with 1 x

moVL

ATRA decreased by 60% , the cell apoptosis increased by 2 times. BMP-7 mRNA and protein levels in MC-3n-El cells treated with 1 x

moVL ATRA decreased by 60% and 80% , respectively, compared with ATRA-untreated

cells ( P < 0 . 0 5 ) . Concluaione BMP-7 may play an important role in embryonic palate development. RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.

I

SOLATED cleft palate is a common congenital structural defect in humans with a prevalence of approximate 1%0-2%0live births. Cleft palate usually displays a multifactor mode of inheritance. However, the mechanism of cleft palate is not well understood. In this study, we investigated the effects of retinoic acid ( RA) on expression of bone morphogenetic protein 7 ( BMP-7 ) in rat

Received for publication April 9 , 2007. 'Corresponding author Tel: 13386861958, E-mail: g572@sinacom ASuppocted by National Natural Science Foundation of China (30500414) and Scientific Research Project in Department of Education of Liaoning Province (05L508, 20061010).

fetus with cleft palate, to elucidate the role of BMP-7 in congenital jaw malformation.

MATERIALS AND METHODS Animal model Fifteen female Wistar rats weighing 220-240 g provided by Animal Center of China Medical University were divided into 3 groups, 5 in each group. Animal rooms maintained temperature and relative humidity at approximately'22"C and 60%. A 12-hour lightldark photocycle was maintained in all animal rooms. Pregnant time was decided by checking sperm in vagina within 12 hours after putting female rat and male rat together. All-

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trans retinoic acid ( ATRA , 40 mg/mL, Sigma) dissolved in mineral oil was poured into stomach of pregnant rats through gastric tube on the gestation day 10. Totally 50 mg/kg and 100 mg/kg ATRA was administered to rats in the second and the third group, respectively. The rats who received the same dosage of mineral oil were used as the control group. On the gestation day 20, fetus were taken from uterus for observing and recording fetal development, and kept in - 70°C for use. Total RNA extraction and Noahern blotting analysis Total RNA of the maxillary bone tissue of fetal rats was extracted with TRIzol Reagent ( Gibco) . RNA was fractionated by 1.5 % agarose-formaldehyde gel electrophoresis in 3 - ( N-morpholino ) propane sulfonic acid buffer and transferred to a nylon membrane using an ammonium acetate rapid blotting procedure.' Rat BMP-7 probe was labeled P I d-CTP (6000 CVmmol) using the random with [ primer. Hybridizations were carried out at 42°C for 24 hours in 50% formamide, 10% dextran sulfate, 1% SDS, 1 x Denhardt Solution, and 100 pg/mL salmon sperm DNA. After hybridization, nylon membrane was washed by 1 x saline-sodium citrate ( SSC) at 65Q . Membranes were exposed to Kodak XAR-2 film for 24 hours. The band intensities were quantified by densitometry. The expression levels of each mRNA were divided by levels of mRNA of p-actin. Cell culture and medication MC-3T3 -El cell strains (mouse fetus calvaria fibroblast-like cells) provided by the Institute of Cell Biology of Peking Union Medical College were cultured in 10% DMEM ( Gibco) containing 10% fetal bovine serum, 100 IWmL penicillin, 100 pg/mL streptomycin under 37°C and 5 % CO, . When cells reached the logarithm growth phase after 24 hours culture, they were incubated with 0, 1 x 10-', and 1 x 10 -'moVL ATRA. ATRA was dissolved in dimethylsulfoxide, the final concentration of dnethylsulfoxide was 1 x 10 - 4 moVL. After 24-hour incubation with ATRA, cells were harvested. How cybmetry MC-3T3-El cells were cenmfugated at 3000 rpm for 5 minutes at 4°C , and washed twice with PBS. After incubated with 70% cold ethanol over night at 4°C , precipitate cells were resuspended by cold PBS, and centrifugated at 3000 rpm for 5 minutes. After cells were incubated with 200 ng/mL RNase for 30 minutes at 37°C , 40 ng/mL PI ( Sigma) was added. Cells

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were analyzed on Becton Dickinson FACScan flow cytometer to determine the DNA content using CELLQuest software. M'IT After an initial overnight incubation, 0 , 1 x 10 -', and 1 x 10 - 6 moYL ATRA were added to MC-3T3El cells, respectively. After 24 hours of ATRA administration, MTT ( 5 mg/mL ) were added and the mixture was cultured at 37°C for 3 hours. Formazan extraction was performed using isopropanol, and the quantity was determined using a spectrophotometer at 490 nm. Total RNA extraction and RT-PCR Total RNA was extracted from MC-3T3-El cells with TRIzol Reagent (Gibco) as described by the manufacturer. RNA was reverse transcribed with M-MLV reverse transcription agent kit ( Promega ) according to manufacturer's instructions. PCR agent was from Promega Company. The following primers were used : mouse BMP-7 primers : 5 I-TCTGGACGATAGCATGGTTG-3' and 5 'CGG'ITTGACAACGAGACCTT-3' (592 bp) ; p-actin: 5 I-AGAGCTACGAGCTGCCTGAC-3 and 5 'AGTACTTGCGCTCAGGAGGA-3 (298 bp) . The PCR reactions were cycles 30 times after initial denaturation ( 9 4 t , 4 minutes) with the following parameters: denaturation 94°C for 1 minute, annealing 56°C for 1 minute, extension 72°C for 1 minute. PCR products were electrophoresed on 1% agarose gel. We used p-actin as intra-contrast parameter. Total protein extracton and Western blotting analysis After 24 hours of ATRA administration, MC-3T3-El cells were harvested with trypsinization and resuspended in 10 mmoVL Tris-HC1 (pH 7 . 4 ) , 100 mmoVL NaCl, 0.5% desoxycholic acid, and 1 mmoYL dithiothreitol containing 10 g/mL aprotinin at 4°C for 30 minutes for total protein extraction. Protein concentration was measured by Bio-Rad protein assay ( Munich, Germany). The protein ( 10 pg ) was loaded onto 12% SDS-PAGE, transferred onto nitrocellulose membrane ( Amersham , Freiburg , Germany) after electrophoresis, and incubated with the rabbit antimouse polyclonal BMP-7 antibody ( 1:1000) ( Santa Cruz Biotechnology, Inc., CA, USA) and the polyclonal goat anti-mouse p-actin antibody ( 1:400) ( Lab Vision Corporation, CA, USA) respectively for 1 hour. The bands were visualized using electrochemiluminescence ( ECL ) detection ( Amersham Pharmacia Biotech Inc., NJ, USA) , and the band intensity was analyzed using Chemilmager-5500 V 2. 03 electrophoresis gel image system.

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p-actin was used as intra-contrast parameter. Stahtical analysis Data were expressed as mean k SD and compared by ANOVA and Student's c-test. P < 0.05 was considered significant.

10 -6 moVL ATRA were significantly lower than untreated c e l l s ( l 6 . 5 * 3 . 4 , 3 . 5 * 0 . 6 , P c 0 . 0 5 ) . However, l x 10 -'moVL ATRA did not significantly decrease BMP-7 mRNA ( 1 2 . 4 * 3 . 6 ) andproteinexpression ( 3 . 4 k 0 . 6 , P>0.05,Figs.2, 3 ) .

RESULTS Effect of ATRA on cleft palate Fifteen female rats produced 215 fetal rats, 48 of which showed cleft palate: short palatine shelf and absence of the palatine shelf. The incidence rate of cleft palate induced by 100 mg/kg ATRA (45.5% ) was significantly higher than 50 mg/kg ATRA ( 12.5% , P < 0.05 ) . No cleft palate occurred in control group. BMP-7 mRNA expression of rat maxillary bone tissue treated with 100 mg/kg ATRA (0.8 k 0.3 ) was significantly lower than control rats (2.2 k 0.5, P < 0.05 ) . BMP-7 mRNA expression had no significant difference between control group and 50 mg/kg ATRA treated group ( 2 . 4 k 0 . 6 , P > 0 . 0 5 , Fig. 1).

pisUn 2. RT-PCR analysis of BMP-7 mRNA expression in MC3"3-E1 cells. M. marker; 1. control group; 2 . 1 x RA; 3 . 1 x moVL ATRA.

F Northern blotting analysis of BMP-7 mRNA expression in rat maxillary bone tissue. 1. control group; 2.50 mg/kg ATRA; 3. 100 mg/kg ATRA. BMP-7 : bone morphogenetic protein 7 ; ATRA: alltrans retinoic acid.

Effect of ATRA on cell apoptosis and proliferation Incubation with 1 x 10 -'moVL ATRA for 24 hours, MC3T3-El cells proliferation (32. 5 % k4. 5 % ) and apoptosis ( 18.2% k 4. 1% ) had no significant difference with untreated cells (31.4% k 5 . 2% , 13.4% 3. 1% , P > 0.05 ) . When the concentration of ATRA increased to 1 x mol/L, MC-3n-El cells proliferation ( 12.5% k 3.6% ) was significantly inhibited, and the cell apoptosis (28.6% k 3.5% ) was significantly increased ( P < 0.05) . Effect of ATRA on BMP-7 expression of MC-3T3-El cells The expressions of BMP-7 mRNA (6. 5 k 2 . 2 ) and protein (0. 8 * O . 3 ) in MC-3T3-El cells treated with 1 x

*

moVL AT-

i 3. Western blotting analysis of BMP-7 expression in MC3T3-El cells. M. marker; 1. control group; 2. 1 x moVL ATRA. RA; 3 . 1 x

moVL AT-

DISCUSSION Cleft palate is a congenital hereditary disease caused by combination of hereditary and environmental factors. The episode of cleft palate has close relation to bone development in embryonic stage.' The animal model provides feasible research mode for study of pathogenesis of cleft palate. RA , a metabolite of vitamin A , has extensive biological effects on proliferation, differentiation, growth, metabolism of tissues and cells, and stabilization of internal environment.' Vitamin A generates ATRA through a series of dehydrogenase-catalyzed reactions in cell, where ATRA regulates gene expression via combination with its receptor. Delgado et a13 administered RA to pregnant rats

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via stomach tube, and found the deformity of calvaria bone, malformation of neural tube, palate, and hand. In our study, the female rats were exposed to 100 mg/kg RA on the gestation day 10, which is the crucial stage of embryonic skeletal development. We found that the fetal rats were with short palatine shelf and absence of the palatine shelf induced by 100 mg/kg ATRA. It suggested that 100 mg/kg of ATRA can seriously interfere normal cell proliferation and differentiation during orthogenesis of rat embryo and induce congenital cleft palate. BMPs belong to the transforming growth factor-beta superfamily. Members of the BMP family were originally cloned and characterized by their ability to regulate osteogene~is.~ Nishihara et al’ recorded BMP-7 can stimulate differentiation of chondrocytes. Cuervo’s research6 showed that during secondary palate shelf fusion in the mouse, programmed cell death appeared in the medial edge epithelia of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the medial edge epithelia, whereas exogenous RA increased cell death.’ BMP-7 , however, was unable to activate cell death within the palate tissue. Dupe et a1* recorded that RA might increase the amount of cell death in the interdigital necrotic zones through down-regulating BMP-7 expression. In this study, we found that expression of BMP-7 mRNA in osteoblast was significantly down-regulated by higher concentration of RA ( 1 x 10 -6 mol/L ) in v i m , but 1 x 10 mol/L RA did not decrease it significantly. Moreover, the expression of BMP-7 decreased upon RA treatment in maxillary bone tissue of fetal rats with cleft palate. It suggested that RA might alter the cell proliferation and differentiation through regulation of BMP-7 gene expression. Therefore, we concluded that BMP-7, as an impor-

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tant cell factor of osteogenesis, plays important regulative function in embryonic palate development. Moreover, RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene transcription and translation.

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