Retinoic Acid Promotes Retinoic Acid Signaling by Suppression of Pitx1 In Tendon Cells: A Possible Mechanism of a Clubfoot-Like Phenotype Induced by Retinoic Acid

BACKGROUND: The pathogenesis of idiopathic congenital clubfoot (CCF) is unknown. Although some familial patients have Pitx1 mutations, and the Pitx1+/− genotype causes a clubfoot-like phenotype in mice, the mechanism of Pitx1-induced CCF is unknown. MATERIAL/METHODS: We used tibialis anterior tendon samples to detect the expression of Pitx1 in idiopathic and neurogenic clubfoot patients. After obtaining Sprague-Dawley (SD) rat Achilles tendon cells, the expression of Pitx1 was knocked down by SiRNA. After 48 h of culture, mass spectrometry was used to quantitatively analyze proteins. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to assess the downstream pathway of PITX1. The relationship between Pitx1 and the promoter region of deacetylase 1 (Sirtuin-1 and Sirt1) was examined by luciferase and ChIP assays. RESULTS: We found that Pitx1 expression in the tendon samples of idiopathic CCF patients was downregulated. Mass spectrometry analysis revealed that the inhibition of Pitx1 induced the downregulation of Sirt1 expression in tendon cells. Luciferase and ChIP assays confirmed that Pitx1 binds to the promoter region of SIRT1 and promotes Sirt1 gene transcription. Further results showed that, after the inhibition of Pitx1 in tendon cells, CRABP2 acetylation increased, the nuclear import of CRABP2 was enhanced, and the expression of RARβ2 increased. After the inhibition of Pitx1, RARβ2 expression was further increased by RA treatment in tendon cells. In the presence of retinoic acid, the expression of Pitx1 was inhibited in tendon cells. CONCLUSIONS: Pitx1 binds to the promoter region of SIRT1 and promotes the transcription of SIRT1. Positive feedback occurs between RA signaling and Pitx1.