title: All-trans-retinoid acid (ATRA) suppresses chondrogenesis of rat primary hind limb bud mesenchymal cells by downregulating p63 and cartilage-specific molecules.
abstract: P63 null mice have no or truncated limbs and mutations in human p63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in bone development. In the current study, we investigated the effect of ATRA on chondrogenesis using mesenchymal cells from rat hind limb bud and further examined the mRNA and protein expression of Sox9 and Col2a1 and p63 in rat hind limb bud cells. Limb buds were isolated from embryos from euthanized female rats. Growth of hind limb bud mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. Formation of cartilage nodules was examined by Alcian blue-nuclear fast red staining. The expression of Sox9, Col2al and p63 was determined by Real-time RT-PCR and immunoblotting assays, respectively. Our MTT assays revealed that ATRA at 1 and 10μM significantly suppressed the growth of mesenchymal cells from rat hind limb bud at 24 and 48h (P<0.01 vs. controls). Alcian blue staining further showed that ATRA caused a significant dose-dependent reduction in the area of cartilage nodules (P<0.05 in all vs. controls). At 1μM ATRA, the area of cartilage nodules from hind limb bud cells was reduced to 0.05±0.03mm from 0.15±0.01mm in controls. Real-time RT-PCR assays further indicated that 1 and 10μM ATRA markedly reduced the mRNA expression of Sox9, Col2al and p63 in hind limb bud cells (P<0.05 in all vs. controls). In addition, ATRA time-dependently inhibits the mRNA expression of p63, Sox9 and Col2al. Western blotting assays additionally showed that ATRA dose-dependently reduced the expression of Sox9, Col2al and p63 (P<0.05 in all vs. controls). Together, our results suggest that ATRA suppresses chondrogenesis by modulating the expression of Sox9, Col2al and p63 in primary hind limb bud mesenchymal cells.
authors.affiliation: Department of Orthopedics, the Second Hospital of Tianjin Medical University, Tianjin, 300211, China.
authors.forename: Yun-Guo
authors.initials: YG
authors.lastname: Wang
authors.affiliation: Department of Orthopedics, the Second Hospital of Tianjin Medical University, Tianjin, 300211, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xie
authors.affiliation: Wang Chuanshan College, University of South China, Hengyang, Hunan, 421001, China.
authors.forename: Yun-Gong
authors.initials: YG
authors.lastname: Wang
authors.affiliation: Department of Orthopedics, the First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Xue-Dong
authors.initials: XD
authors.lastname: Li
authors.affiliation: Department of Orthopedics, the First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Tao-Gen
authors.initials: TG
authors.lastname: Zhang
authors.affiliation: Department of Orthopedics, the First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Zhao-Yong
authors.initials: ZY
authors.lastname: Liu
authors.affiliation: Department of Orthopedics, the First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Hong
authors.affiliation: Department of Orthopedics, the Second Hospital of Tianjin Medical University, Tianjin, 300211, China. Electronic address: 8760715@163.com.
authors.forename: Shi-Xin
authors.initials: SX
authors.lastname: Du
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006614:Hindlimb / Q000196:embryology* / Q000378:metabolism; D018878:Limb Buds / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000008:administration & dosage* / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
chemical_list: C511513:COL2A1 protein, rat; D024043:Collagen Type II; D055758:SOX9 Transcription Factor; C108581:Tp63 protein, rat; D016159:Tumor Suppressor Protein p53; D014212:Tretinoin
keywords: Chondrogenesis; Col2a1; Hind limb bud mesenchymal cells; Retinoic acid; Sox9; p63