All-trans-retinoic acid inhibits chondrogenesis of rat embryo hindlimb bud mesenchymal cells by downregulating p53 expression.
Despite the well-established role of all-trans-retinoic acid (ATRA) in congenital clubfoot (CCF)-like deformities in in vivo models, the essential cellular and molecular targets and the signaling mechanisms for ATRA-induced CCF-like deformities remain to be elucidated. Recent studies have demonstrated that p53 and p21, expressed in the hindlimb bud mesenchyme, regulate cellular proliferation and differentiation, contributing to a significant proportion of embryonic CCF-like abnormalities. The objective of the present study was to investigate the mechanisms for ATRA-induced CCF, by assessing ATRA-regulated chondrogenesis in rat embryo hindlimb bud mesenchymal cells (rEHBMCs) in vitro. The experimental study was based on varying concentrations of ATRA exposure on embryonic day 12.5 rEHBMCs in vitro. The present study demonstrated that ATRA inhibited the proliferation of cells by stimulating apoptotic cell death of rEHBMCs. It was also observed that ATRA induced a dose-dependent reduction of cartilage nodules compared with the control group. Reverse transcription-polymerase chain reaction and western blotting assays revealed that the mRNA and protein expression of cartilage-specific molecules, including aggrecan, Sox9 and collagen, type II, α 1 (Col2a1), were downregulated by ATRA in a dose-dependent manner; the mRNA levels of p53 and p21 were dose-dependently upregulated from 16 to 20 h of incubation with ATRA, but dose-dependently downregulated from 24 to 48 h. Of note, p53 and p21 were regulated at the translational level in parallel with the transcription with rEHBMCs treated with ATRA. Furthermore, the immunofluorescent microscopy assays indicated that proteins of p53 and p21 were predominantly expressed in the cartilage nodules. The present study demonstrated that ATRA decreases the chondrogenesis of rEHBMCs by inhibiting cartilage-specific molecules, including aggrecan, Sox9 and Col2al, via regulating the expression of p53 and p21.
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Tao-Gen
TG
Zhang
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Xue-Dong
XD
Li
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Guo-Yong
GY
Yu
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Peng
P
Xie
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Yun-Guo
YG
Wang
Department of Orthopedics, The First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
Zhao-Yong
ZY
Liu
Department of Orthopedics, The First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
Quan
Q
Hong
Department of Orthopedics, The First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
De-Zhong
DZ
Liu
Department of Orthopedics, The Affiliated Luohu Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China.
Shi-Xin
SX
Du
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental; D006614:Hindlimb; D018878:Limb Buds / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D061251:Primary Cell Culture; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014212:Tretinoin / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D053674:Aggrecans; C511513:COL2A1 protein, rat; D024043:Collagen Type II; D050759:Cyclin-Dependent Kinase Inhibitor p21; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D016159:Tumor Suppressor Protein p53; D014212:Tretinoin