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

Abstract

BackgroundThe 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/MethodsWe 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.ResultsWe 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.ConclusionsPitx1 binds to the promoter region of SIRT1 and promotes the transcription of SIRT1. Positive feedback occurs between RA signaling and Pitx1.

What is New in Pediatric Orthopaedic: Basic Science

“…Interestingly, HOX gene expression and oxidative damage were increased in the tissue of TEV when compared with tissue obtained in control children 49 . Pitx1 expression, a homeobox gene known to be mutated in some familial clubfoot patients and causes a similar phenotype in mice, was downregulated from the tibialis anterior tendon of idiopathic clubfoot patients 35. Among the multiple etiologies contributing to clubfoot, the proliferation of extracellular matrix proteins is suggested to contribute to pathogenesis.…”

“…Population studies continue to demonstrate a strong genetic component to the development of talipes equinovarus (TEV) 33,34. Genes involved in limb development such as PITX1B-Tbx4 , homeobox genes have been associated with clubfoot 35–38. In addition to these developmental genes, mutations in other pathways including matrix proteins, sulfation genes, GLI3 (a transcription repressor), N-acetylation genes, and TGF-β signaling seem possibly contributory 39–42…”

“…33,34 Genes involved in limb development such as PITX1B-Tbx4, homeobox genes have been associated with clubfoot. [35][36][37][38] In addition to these developmental genes, mutations in other pathways including matrix proteins, sulfation genes, GLI3 (a transcription repressor), N-acetylation genes, and TGF-β signaling seem possibly contributory. [39][40][41][42] Lim-domain kinase 1 (LIMK1) is a known regulator in embryologic actin organization and cell migration.…”

“…49 Pitx1 expression, a homeobox gene known to be mutated in some familial clubfoot patients and causes a similar phenotype in mice, was downregulated from the tibialis anterior tendon of idiopathic clubfoot patients. 35 Among the multiple etiologies contributing to clubfoot, the proliferation of extracellular matrix proteins is suggested to contribute to pathogenesis. Mass spectroscopy analysis identified 11 upregulated proteins, 7 of which were on the medial side and 4 on the lateral side of cadaver specimens with clubfoot.…”