PITX2 (Pituitary Homeobox Gene 2)
PITX2 belongs to the bicoid class of homeodomain transcription factors, which play essential roles in embryonic development and disease. Pioneered evidences reported by Semina et al. (1996) identified 4q25 translocations association with Rieger syndrome, a human pathological condition characterized by underdevelopment of the teeth, mild craniofacial abnormalities, and distinct eye defects, especially glaucoma. These authors isolated an mRNA transcript with a predicted 271-amino acid protein and named it as RIEG gene. RIEG showed high homology with the PITX1 protein, differing by only two residues, and thus was later dubbed PITX2. Soon thereafter, Gage and Camper (1997) identified PITX2 (RIEG) gene in an adult mouse pituitary gland screen for novel homeobox genes and reported two alternatively spliced mRNA products, namely, PITX2A and PITX2B. Soon thereafter, using a differential display method, Arakawa et al. (1998) isolated a gene that is downregulated in All1 double-knockout mouse embryonic stem (ES) cells, i.e., ARP1 (All1-responsive gene-1), which was homologous to PITX2 but display three distinct variants (i.e., an additional PITX2C isoform). Overall, these studies already demonstrated a rather complex pattern of expression for PITX2 variants in developing and adult stages. In line with its putative involvement in Rieger syndrome, PITX2 displayed ocular, cardiac, and craniofacial expression (St Amand et al. 1998; Campione et al. 2001), positioning it as a causative gene for this syndrome.
Cox et al. (2002) noted that in humans PITX2A and PITX2B transcripts are generated by alternative splicing and that PITX2C uses an alternative promoter. An additional splice variant, PITX2D, that uses the same alternate PITX2C promoter but is N-terminally truncated was also reported which acted as a dominant negative isoform. Given the complex expression pattern and the diversity of spliced variants, Martin et al. (2001) hypothesized that the PITX2 mutations may contribute to the multiple phenotypic anomalies present in CHARGE individuals, yet direct sequencing of CHARGE patients failed to identify any PITX2 causative mutation, while multiple evidences demonstrated association of PITX2 mutations in Rieger syndrome patients (Tümer and Bach-Holm 2009). More recently Gudbarjtsson et al. (2007) identified 4q25 risk variants in 4q25 highly associated to atrial fibrillation opening new roles for PITX2 in human diseases.
At the experimental level, PITX2 expression and function have progressively been investigated during embryonic development, demonstrating a highly dynamic expression pattern and a highly relevant contribution in multiple aspects of embryogenesis. Yoshioka et al. (1998) reported that PITX2 is expressed asymmetrically in the left lateral plate mesoderm in both mouse and chicken developing embryos. They furthermore demonstrated that PITX2 is downstream of Nodal and Lefty-1 in mice, indicating a common pathway from Nodal to PITX2, providing crucial clues for left-right asymmetric determination in vertebrates. Logan et al. (1998), Piedra et al. (1998), and Campione et al. (1999) further supported these findings on the role of PITX2 in left-right embryonic axis formation in distinct experimental models, such as chicken and Xenopus.
Soon thereafter PITX2 genetic deletion experiments in mice demonstrated that PITX2-deficient embryos were characterized by defective body wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning, and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis (Lin et al. 1999). PITX2−/− mice also exhibited a marked hypoplasia of the axial mesoderm, ventral body wall splanchnic mesoderm, and urogenital system, as well as hypoproliferation of the spleen, liver, mandible, periorbital musculature, and lens. These data demonstrated a multifaceted developmental role for PITX2, including therein multiple aspects linked to Rieger syndrome. Lu et al. (1999) independently generated PITX2−/− mice demonstrating that although PITX2−/− embryos had abnormal cardiac morphogenesis, mutant hearts looped into the normal rightward direction. PITX2−/− embryos had correctly oriented, but arrested, embryonic rotation and right pulmonary isomerism. Lu et al. (1999) also observed defective development of the mandibular and maxillary facial prominences, regression of the stomodeum, and arrested tooth development, adding additional links to Rieger syndrome characteristic defects. Overall, these data demonstrated that PITX2 played a pivotal role in multiple defective structures that characterize Axenfeld-Rieger syndrome.
Given the wide spectrum of embryonic abnormalities occurring after embryonic deletion of PITX2, arrays of experiments were designed to unravel the tissue-specific contribution of this homeobox transcription factor in distinct developmental contexts. Yashiro et al. (2007) showed that ablation of unilateral left-sided PITX2 expression in mice impairs asymmetric remodeling of the branchial arch artery system, resulting in randomized laterality of the aortic arches. These data suggest that hemodynamics, generated by a PITX2-induced morphogenetic defect in the outflow tract formation, is responsible for the asymmetric remodeling of the great arteries.
Evans and Gage (2005) generated PITX2 knockout mice in the neural crest precursor pool. Neural crest-specific PITX2 knockout mice were viable, yet exhibited a unique optic nerve phenotype in which the eyes were progressively displaced toward the midline until they were directly attached to the ventral hypothalamus. Evans and Gage (2005) proposed a revised model of optic nerve development and novel mechanisms that may underlie the etiology of glaucoma in Axenfeld-Rieger patients.
Mommersteeg et al. (2007a) found that PITX2c-null mice failed to develop a pulmonary myocardial sleeve due to the absence of pulmonary myocardial cell precursors, concluding that NKX2.5 and PITX2C play critical roles in the formation and identity of the pulmonary myocardium. In addition these authors also demonstrate a pivotal role for PITX2 in sinoatrial node formation (Mommersteeg et al. 2007b).
As previous said, Gudbarjtsson et al. (2007) demonstrated that risk variants in 4q25, adjacent to PITX2 gene, were highly associated to atrial fibrillation. Distinct PITX2 insufficiency experimental model have provided evidences that this transcription factor is crucial controlling atrial electrophysiology (Wang et al. 2010; Kirchhof et al. 2011; Chinchilla et al. 2011; Tao et al. 2014; Lozano-Velasco et al. 2016). Thus, besides the well-described role of PITX2 as causative gene underlying Axenfeld-Rieger syndrome as well as in embryonic left-right determination, new avenues on the role of PITX2 in the adulthood have been opened as it is deregulated in distinct cardiac pathophysiological conditions such as heart failure and cardiac hypertrophy (Torrado et al. 2014), in cardiac and skeletal regeneration (Lozano-Velasco et al. 2015; Tao et al. 2016), and in multiple cancerous processes (Wan Abdul Rahman et al. 2014; Luan et al. 2016). Thus, a novel and intriguing role of this homeobox transcription factor is yet to be discovered in the coming years.
PITX2 in Embryonic Left-Right Signaling
The earliest expression of the homeobox transcription factor PITX2 is observed soon after gastrulation. Gene developmental expression studies demonstrated that PITX2 is asymmetrically expressed in the left lateral plate mesoderm in multiple species, ranging from fish to mice (Logan et al. 1998; Piedra et al. 1998; Campione et al. 1999; Long et al. 2003). Embryonic symmetry break is initiated in the node as a directional leftward flux (Sarmah et al. 2005; Hatayama et al. 2011) which provides instructive signals to initiate left asymmetric expression. Distinct molecules have been implicated in the establishment and propagation of such asymmetric left-sided expression, with distinct species-specific differences, yet importantly a core nodal->PITX2 signaling pathway is conserved in all species (Boorman and Shimeld 2002). Thus, PITX2 is the last effector of the embryonic left-right pathway, providing a link between embryonic left-right signaling and asymmetric organ development. Curiously, a role for PITX2 in flatfish asymmetry development and in particular for eye migration and development has been reported (Suzuki et al. 2009) as well as in lancelets, providing evolutionary left-right links in deuterostomes (Yasui et al. 2000).
Gain-of-function expression in chicken (and Xenopus) embryos further supports a key role of PITX2 conferring asymmetric organogenesis to the developing heart and gut (Logan et al. 1998; Piedra et al. 1998; Campione et al. 1999). Interestingly, left-right asymmetric determination is organ specific and dose dependent (Bisgrove et al. 2000) independently affecting the brain, heart (Campione et al. 1999), gut (Bisgrove et al. 2000), and gonadal (Guioli et al. 2007) development. Surprisingly, PITX2 loss-of-function experiments in mice display normal rightward cardiac looping suggesting that PITX2 function is dispensable for early cardiac asymmetry break (cardiac looping) but pivotal for heart organogenesis as PITX2-deficient mice display right atrial isomerism, double-outlet right ventricle (DORV), and transposition of the great arteries (TGA) (Lin et al. 1999; Lu et al. 1999) as detailed above (Fig. 1).
PITX2 in Striated Muscle Development and Disease
PITX2 expression in the developing heart is observed already at the early bilateral cardiac crescent stage, confined to the left side, and thus prior to any evidence of morphological asymmetry (Campione et al. 2001). Soon thereafter a symmetric cardiac tube is formed, with PITX2 confined to the left side. As soon as cardiac looping initiates, PITX2 expression remains confined to the primitive left side tube serving as a left lineage marker (Campione et al. 2001). At the ballooning stage, PITX2 remains to be expressed at the left side of the inlet and outlet regions, including also the atrial chambers and the atrioventricular region, whereas in the ventricle, PITX2 becomes to be expressed at the ventral but not the dorsal side of the developing ventricles. These findings have been recently corroborated using mouse genetic PITX2 lineage tracing experiments (Furtado et al. 2011). Interestingly, a detailed expression of PITX2 in a laterality defect mouse line, iv, demonstrated that left-right symmetrical expression of PITX2 occurring in both atrial and ventricular chambers displays concordant association with congenital heart defects such as atrial isomerism and double-outlet right ventricle, respectively (Campione et al. 2001). Overall, these data suggest that PITX2 might play pivotal roles in distinct cardiac morphogenetic events (Franco and Campione 2003). Genetic deletion of PITX2 demonstrated a complex embryonic lethal phenotype, including defects in the developing heart (Lin et al. 1999; Liu et al. 2001). Right atrial isomerism was consistently reported, yet other cardiac malformations were less consistently observed. A systemic approach to unravel the role of PITX2 in sinoatrial development demonstrated that PITX2 is essential for pulmonary vein development and SAN determination (Moomersteeg et al. 2007a, b). Myocardial-specific deletion of PITX2 demonstrated a myocardial cell autonomous requirement of PITX2 for correct cardiac development (Tessari et al. 2008), yet intriguingly not cardiac left-right defects were observed. Conditional deletion with an early myocardial-specific deleter Cre mice unravels a critical time window for left-right cardiac determination, including the formation of the sinoatrial node, as cTnTCre-PITX2-deficient mice, but not aMHC-CrePITX2 mice, displayed right atrial isomerism and bilateral functional SANs (Ammirabile et al. 2012).
Importantly, genome-wide association studies (GWAS) identified a novel plausible role for PITX2 in the heart, as Gudbjartsson et al. (2007) demonstrated that several risk variants located in chromosome 4q25, close to PITX2, were highly associated to atrial fibrillation. Given the prominent role of PITX2 conferring tissue specification to the sinoatrial node in the developing heart (Moomersteeg et al. 2007b), a plausible role for this transcription factor was proposed as causative link to atrial fibrillation. Experimental studies of PITX2 genetic deletion demonstrated that PITX2 haploinsufficiency increased the susceptibility to suffer atrial arrhythmogenesis (Wang et al. 2010). An embryonic link to dysregulation of Shox2 and Hcn4 expression was demonstrated in this model. Additional deregulation of several ion channels and gap junctional proteins were reported in a PITX2c haploinsufficiency model which displays similar atrial fibrillation susceptibility (Kirchhof et al. 2011). Atrial-specific deletion of PITX2 resulted in spontaneous ECG abnormalities, including abnormal P wave configuration and impaired cardiac action potential configuration (Chinchilla et al. 2011). At the molecular level, expression of multiple ion channels was impaired, including sodium, potassium, and calcium channels, most of which are controlled by PITX2-dependent Wnt>microRNA signaling pathway (Chinchilla et al. 2011; Lozano-Velasco et al. 2016). These data were further supported by PITX2 conditional ablation in the adult heart as reported by Tao et al. (2014). Overall, these data demonstrate a pivotal role for PITX2 in atrial arrhythmogenesis. In addition to experimental work, PITX2 mutations have been recently reported in AF patients (Yang et al. 2013; Wang et al. 2014), providing novel causative links.
In addition to the role of PITX2 in atrial arrhythmogenesis, novel aspects of PITX2 have been recently reported in other adult cardiac pathophysiologies. PITX2 is abnormally expressed in hypertrophic cardiomyopathy and heart failure patients (Torrado et al. 2014), and PITX2 function is indispensable to provide normal regenerative potential to the myocardium in neonatal stages by controlling redox homeostasis (Tao et al. 2016). In addition, PITX2 loss-of-function mutation contributes to congenital endocardial cushion defects in Axenfeld-Rieger syndrome (Zhao et al. 2015).
Besides the functional role of PITX2 in cardiac muscle, this homeobox transcription factor also regulates key steps of skeletal muscle development. Diehl et al. (2006) firstly described that PITX2 function is crucial for extraocular muscle morphogenesis by regulating the expression of key myogenic determinant factors. Concomitantly, Martinez-Fernandez et al. (2006) demonstrate that overexpression of PITX2c in a myoblast cell line promoted cell proliferation and arrested differentiation, in part by regulating Pax3 expression. Additional evidences on the role of PITX2 on the formation of the branchiomeric and cranial muscles were subsequently provided (Dong et al. 2006) as well as in extraocular muscles (Hebert et al. 2013) by regulating a subset of mature extraocular muscle contraction-related genes (Zhou et al. 2012).
Following the seminal work by Martinez-Fernandez et al. (2006), a role for PITX2 in somite-derived skeletal muscle was reported (Eng et al. 2012). Importantly, the functional role of PITX2 is shared also with Pitx3, demonstrating in part complementary roles during somatic muscle development by regulating Pax3 (Lozano-Velasco et al. 2011) and MyoD as well as affecting the motility of myogenic cells. Furthermore, PITX2-mediated proliferation in skeletal myoblasts is regulated by directly influencing cell cycle regulators (Lozano-Velasco et al. 2011). Besides its role in skeletal muscle development, PITX2 is also redeployed during adult myogenesis promoting myogenic differentiation by a complex PITX2>microRNA signaling pathway (Lozano-Velasco et al. 2015) as well as controlling redox homeostasis (L’Honoré et al. 2014). These observations envision a key role for PITX2 in skeletal muscle regeneration (Fig. 2).
PITX2 in Ocular Development and Disease
Anterior segment dysgenesis (ASD) is a failure of the normal development of the tissues of the anterior segment of the eye. Anomalies leading to ASD are associated with increased risk of glaucoma and corneal opacity. Currently, it is believed that specification of a population of mesenchymal progenitor cells, mainly of neural crest origin, migrates anteriorly around the optic cup. PITX2 was isolated and characterized during lens development (Semina et al. 1996, 1997; Gage and Camper 1997). Several transcription factors and among them PITX2 regulate mesenchymal cell differentiation giving rise to distinct anterior segment tissues. Six3 regulates expression of PITX2 in periocular mesenchymal cells, and Six3 loss of function leads to anterior segment dysgenesis (Hsieh et al. 2002). Retinoic acid (RA) and PITX2 regulate early neural crest cell survival providing a means for correct craniofacial and ocular development (Chawla et al. 2016). In addition, PITX2 is also required for providing correct cell fate and establishing an angiogenic privilege to the developing cornea (Gage et al. 2014).
Upstream regulation of PITX2 in this context is also provided by canonical Wnt/beta-catenin signaling, yet it provides maintenance but not activation in the perioptic mesenchyme in interplay with RA signaling (Gage and Zacharias 2009). Genetic deletion of Lgr4 leads to severe ocular PITX2 downregulation and leads to a wide spectrum of anterior segment dysgenesis such as microphthalmia, iris hypoplasia, defective iris myogenesis, cornea dysgenesis, and cataract. Thus, PITX2, along with other key transcription factors and morphogens, is crucial for the correct specification of a subset of neural crest-derived mesenchymal progenitors that migrate and give rise to distinct anterior segment tissues (Acharya et al. 2011; Evans and Gage 2005).
Importantly, PITX2 zebra fish mutants display abnormal ocular as well as craniofacial development, small heads and eyes, jaw abnormalities, and pericardial edema (Liu and Semina 2012). In zebra fish, Pax6b is upstream of PITX2, provoking an ectopic corneal endothelial expression and thus impaired eye development. In addition to Pax6, lens-derived tfg-beta-Smad7 signaling is also crucial to direct PITX2 expression in the corneal endothelium (Silla et al. 2014; Takamiya et al. 2015).
Overall, these data demonstrate a crucial role for PITX2 in ocular development, and multiple evidences demonstrate a complex interplay between distinct transcription factors that if impaired lead to anterior segment dysgenesis. In line with these evidences, mutations on PITX2 are associated with eye defects including therein iris, cornea, and trabecular meshwork impairment as well as glaucoma (Reis and Semina 2011) and abnormal extraocular development in the context of Axenfeld-Rieger syndrome (Park et al. 2009) and in Axenfeld-Rieger syndrome with glaucoma (Strungaru et al. 2007).
PITX2 in Cancer
- Chinchilla A, Daimi H, Lozano-Velasco E, Dominguez JN, Caballero R, Delpón E, et al. PITX2 insufficiency leads to atrial electrical and structural remodeling linked to arrhythmogenesis. Circ Cardiovasc Genet. 2011;4:269–79. doi:10.1161/CIRCGENETICS.110.958116.PubMedCrossRefPubMedCentralGoogle Scholar
- Harbeck N, Nimmrich I, Hartmann A, Ross JS, Cufer T, Grützmann R, et al. Multicenter study using paraffin-embedded tumor tissue testing PITX2 DNA methylation as a marker for outcome prediction in tamoxifen-treated, node-negative breast cancer patients. J Clin Oncol. 2008;26:5036–42. doi:10.1200/JCO.2007.14.1697.PubMedCrossRefPubMedCentralGoogle Scholar
- Kirchhof P, Kahr PC, Kaese S, Piccini I, Vokshi I, Scheld HH, et al. PITX2c is expressed in the adult left atrium, and reducing PITX2c expression promotes atrial fibrillation inducibility and complex changes in gene expression. Circ Cardiovasc Genet. 2011;4:123–33. doi:10.1161/CIRCGENETICS.110.958058.PubMedCrossRefPubMedCentralGoogle Scholar
- Liu C, Liu W, Lu MF, Brown NA, Martin JF. Regulation of left-right asymmetry by thresholds of Pitx2c activity. Development. 2001;128(11):2039–48. PubMed PMID: 11493526.Google Scholar
- Lozano-Velasco E, Contreras A, Crist C, Hernández-Torres F, Franco D, Aránega AE. PITX2c modulates Pax3+/Pax7+ cell populations and regulates Pax3 expression by repressing miR27 expression during myogenesis. Dev Biol. 2011;357:165–78. doi:10.1016/j.ydbio.2011.06.039.PubMedCrossRefPubMedCentralGoogle Scholar
- Lozano-Velasco E, Vallejo D, Esteban FJ, Doherty C, Hernández-Torres F, Franco D, et al. A PITX2-microRNA pathway modulates cell proliferation in myoblasts and skeletal-muscle satellite cells and promotes their commitment to a myogenic cell fate. Mol Cell Biol. 2015;35:2892–909. doi:10.1128/MCB.00536-15.PubMedPubMedCentralCrossRefGoogle Scholar
- Luan ZM, Zhang H, Qu XL. Prediction efficiency of PITX2 DNA methylation for prostate cancer survival. Genet Mol Res. 2016;15. doi:10.4238/gmr.15026750.Google Scholar
- Nimmrich I, Sieuwerts AM, Meijer-van Gelder ME, Schwope I, Bolt-de Vries J, Harbeck N, et al. DNA hypermethylation of PITX2 is a marker of poor prognosis in untreated lymph node-negative hormone receptor-positive breast cancer patients. Breast Cancer Res Treat. 2008;111:429–37.PubMedCrossRefGoogle Scholar
- Schayek H, Bentov I, Jacob-Hirsch J, Yeung C, Khanna C, Helman LJ, et al. Global methylation analysis identifies PITX2 as an upstream regulator of the androgen receptor and IGF-I receptor genes in prostate cancer. Horm Metab Res. 2012;44:511–9. doi:10.1055/s-0032-1311566.PubMedCrossRefPubMedCentralGoogle Scholar
- Semina EV, Reiter RS, Murray JC. Isolation of a new homeobox gene belonging to the Pitx/Rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19. Hum Mol Genet. 1997;6(12):2109–16. PubMed PMID: 9328475.Google Scholar
- Takamiya M, Weger BD, Schindler S, Beil T, Yang L, Armant O, et al. Molecular description of eye defects in the zebrafish Pax6b mutant, sunrise, reveals a Pax6b-dependent genetic network in the developing anterior chamber. PLoS One. 2015;10:e0117645. doi:10.1371/journal.pone.0117645.PubMedPubMedCentralCrossRefGoogle Scholar
- Torrado M, Franco D, Hernández-Torres F, Crespo-Leiro MG, Iglesias-Gil C, Castro-Beiras A, et al. Pitx2c is reactivated in the failing myocardium and stimulates myf5 expression in cultured cardiomyocytes. PLoS One. 2014;9:e90561. doi:10.1371/journal.pone.0090561.PubMedPubMedCentralCrossRefGoogle Scholar
- Weiss G, Cottrell S, Distler J, Schatz P, Kristiansen G, Ittmann M, et al. DNA methylation of the PITX2 gene promoter region is a strong independent prognostic marker of biochemical recurrence in patients with prostate cancer after radical prostatectomy. J Urol. 2009;181:1678–85. doi:10.1016/j.juro.2008.11.120.PubMedCrossRefPubMedCentralGoogle Scholar