RESUMEN
Anorectal malformation (ARM) is the most common congenital digestive tract anomaly in newborns, and children with ARM often have varying degrees of underdevelopment of the pelvic floor muscles (PFMs). To explore the effects of RARα and Pitx2 on the development of rat PFMs, we constructed a rat ARM animal model using all-trans retinoic acid (ATRA), and verified the expression of RARα and Pitx2 in the PFMs of fetal rats. Additionally, we used rat myoblasts (L6 cells) to investigate the regulatory roles of RARα and Pitx2 in skeletal muscle myoblast differentiation and their interactions. The results indicated a significant decrease in the expression of RARα and Pitx2 in the PFMs of fetal rats with ARM. ATRA can also decrease the expression of RARα and Pitx2 in the L6 cells, while affecting the differentiation and fusion of L6 cells. Knocking down RARα in L6 cells reduced the expression of Pitx2, MYOD1, MYMK, and decreased myogenic activity in L6 cells. When RARα is activated, the decreased expression of Pitx2, MYOD1, and MYMK and myogenic differentiation can be restored to different extents. At the same time, increasing or inhibiting the expression of Pitx2 can counteract the effects of knocking down RARα and activating RARα respectively. These results indicate that Pitx2 may be downstream of the transcription factor RARα, mediating the effects of ATRA on the development of fetal rat PFMs.
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Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.
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Ferroptosis , Regulación Neoplásica de la Expresión Génica , Proteína del Homeodomínio PITX2 , Proteínas de Homeodominio , Neoplasias Pancreáticas , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Especies Reactivas de Oxígeno , Factores de Transcripción , Animales , Humanos , Ratones , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Ferroptosis/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Peroxidación de Lípido , Potencial de la Membrana Mitocondrial/genética , Ratones Desnudos , Mitocondrias/metabolismo , Mitocondrias/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Vía de Señalización Wnt/genéticaRESUMEN
Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.
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Labio Leporino , Fisura del Paladar , MicroARNs , Síndrome de Wolf-Hirschhorn , Animales , Ratones , MicroARNs/genética , Factores de Transcripción , Síndrome de Wolf-Hirschhorn/genética , Síndrome de Wolf-Hirschhorn/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Ovarian cancer (OC) is the deadliest gynecological malignancy, having a high mortality rate due to its asymptomatic nature, chemoresistance, and recurrence. However, the proper mechanistic knowledge behind these phenomena is still inadequate. Cancer recurrence is commonly observed due to cancer stem cells which also show chemoresistance. We aimed to decipher the molecular mechanism behind chemoresistance and stemness in OC. Earlier studies suggested that PITX2, a homeobox transcription factor and, its different isoforms are associated with OC progression upon regulating different signaling pathways. Moreover, they regulate the expression of drug efflux transporters in kidney and colon cancer, rendering chemoresistance properties in the tumor cell. Considering these backgrounds, we decided to look for the role of PITX2 isoforms in promoting stemness and chemoresistance in OC cells. In this study, PITX2A/B has been shown to promote stemness and to enhance the transcription of ABCB1. PITX2 has been discovered to augment ABCB1 gene expression by directly binding to its promoter. To further investigate the regulatory mechanism of PITX2 gene expression, we found that TGFß signaling could augment the PITX2A/B expression through both SMAD and non-SMAD signaling pathways. Collectively, we conclude that TGFß1-activated PITX2A/B induces stem-like features and chemoresistance properties in the OC cells.
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The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Morfogénesis , Estómago/embriología , Animales , Anuros , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Embrión no Mamífero , Endodermo/embriología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.
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Segmento Anterior del Ojo , Anomalías del Ojo , Enfermedades Hereditarias del Ojo , Proteína del Homeodomínio PITX2 , Femenino , Humanos , Segmento Anterior del Ojo/anomalías , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/patología , Factores de Transcripción Forkhead/genética , Proteínas de Homeodominio/genéticaRESUMEN
Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is no less important, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways governing left-right asymmetry are highly conserved and involve multiple components of the TGF-ß superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left, and BMP signaling on the right. In addition, a plethora of other cell signaling pathways including Shh, FGF, and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia that mediate the left-sided propagation of Nodal signaling, followed by left-sided activation of Lefty and then Pitx2, a transcription factor that specifies visceral organ asymmetry. While this overall scheme is well conserved, there are striking species differences, including the finding that motile cilia do not play a role in left-right patterning in some vertebrates. Surprisingly, the direction of heart looping, one of the first signs of organ left-right asymmetry, was recently shown to be specified by intrinsic cell chirality, not Nodal signaling, possibly a reflection of the early origin of Nodal signaling in radially symmetric organisms. How this intrinsic chirality interacts with downstream molecular pathways regulating visceral organ asymmetry will need to be further investigated to elucidate how disturbance in left-right patterning may contribute to complex CHD.
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Tipificación del Cuerpo , Transducción de Señal , Animales , Humanos , Ratones , Tipificación del Cuerpo/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Factores de Determinación Derecha-Izquierda/genética , Factores de Determinación Derecha-Izquierda/metabolismoRESUMEN
Formation of the vertebrate heart with its complex arterial and venous connections is critically dependent on patterning of the left-right axis during early embryonic development. Abnormalities in left-right patterning can lead to a variety of complex life-threatening congenital heart defects. A highly conserved pathway responsible for left-right axis specification has been uncovered. This pathway involves initial asymmetric activation of a nodal signaling cascade at the embryonic node, followed by its propagation to the left lateral plate mesoderm and activation of left-sided expression of the Pitx2 transcription factor specifying visceral organ asymmetry. Intriguingly, recent work suggests that cardiac laterality is encoded by intrinsic cell and tissue chirality independent of Nodal signaling. Thus, Nodal signaling may be superimposed on this intrinsic chirality, providing additional instructive cues to pattern cardiac situs. The impact of intrinsic chirality and the perturbation of left-right patterning on myofiber organization and cardiac function warrants further investigation. We summarize recent insights gained from studies in animal models and also some human clinical studies in a brief overview of the complex processes regulating cardiac asymmetry and their impact on cardiac function and the pathogenesis of congenital heart defects.
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Tipificación del Cuerpo , Cardiopatías Congénitas , Corazón , Humanos , Animales , Corazón/embriología , Corazón/fisiología , Tipificación del Cuerpo/genética , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/fisiopatología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Transducción de Señal , Regulación del Desarrollo de la Expresión Génica , Proteína Nodal/metabolismo , Proteína Nodal/genéticaRESUMEN
Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.
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Procesamiento Postranscripcional del ARN , ARN no Traducido , Animales , Humanos , Empalme Alternativo/genética , Regulación de la Expresión Génica , Edición de ARN , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.
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Defectos del Tabique Interventricular , Humanos , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , Predisposición Genética a la Enfermedad/genética , Defectos del Tabique Interventricular/genética , Mutación , Factores de Transcripción/genéticaRESUMEN
Paired homologous domain transcription factor 2 (PITX2) is critically involved in ocular and cardiac development. Mutations in PITX2 are consistently reported in association with Axenfeld-Rieger syndrome, an autosomal dominant genetic disorder and atrial fibrillation, a common cardiac arrhythmia. In this study, we have mined missense mutations in PITX2 gene from NCBI-dbSNP and Ensembl databases, evaluated the pathogenicity of the missense variants in the homeodomain and C-terminal region using five in silico prediction tools SIFT, PolyPhen2, GERP, Mutation Assessor and CADD. Fifteen homeodomain mutations G42V, G42R, R45W, S49Y, R53W, E53D, E55V, R62H, P65S, R69H, G75R, R84G, R86K, R87W, R91P were found to be highly pathogenic by both SIFT, PolyPhen2 were further functionally characterized using I-Mutant 2.0, Consurf, MutPred and Project Hope. The findings of the study can be used for prioritizing mutations in the context of genetic studies.
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Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.
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Fibrilación Atrial , Depsipéptidos , Canal Liberador de Calcio Receptor de Rianodina , Animales , Humanos , Ratones , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.
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Amelogénesis , Regulación de la Expresión Génica , Proteína HMGN2 , Proteínas de Homeodominio , Factor de Unión 1 al Potenciador Linfoide , Factores de Transcripción , Transcripción Genética , Amelogénesis/genética , Amelogenina/genética , Animales , Cromatina/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Proteínas de Homeodominio/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.
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Ácido Láctico , Neoplasias Ováricas , Humanos , Femenino , Animales , Ratones , Ácido Láctico/metabolismo , Neoplasias Ováricas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Oncogenes , Lactato Deshidrogenasa 5/genética , Carcinogénesis/genética , Epigénesis Genética , Glucólisis/genética , L-Lactato Deshidrogenasa/metabolismoRESUMEN
Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.
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Células Epiteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proliferación Celular , Esmalte Dental/metabolismo , Embrión de Mamíferos/metabolismo , Células Epiteliales/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Ratones Noqueados , Odontogénesis , Factores de Transcripción SOXB1/genética , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Diente/citología , Diente/crecimiento & desarrollo , Diente/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , Proteína del Homeodomínio PITX2RESUMEN
PITX2 and FOXC1 are the most common pathogenic genes associated with Axenfeld-Rieger syndrome (ARS). In this study, we aimed to explore the variation spectrum of PITX2 and FOXC1 and their associated phenotype based on data from our study and previously reported literatures. Whole exome sequencing was performed on eight probands in our study. Multistep bioinformatic and co-segregation analyses were performed to detect pathogenic variants. Genotype-phenotype correlations of PITX2 and FOXC1 and the differences between them were determined. We detected three variants of FOXC1 and two variants of PITX2 in five unrelated families with ARS. Macular retinoschisis had been observed in AR1 with variant in PITX2 and it is not reported before. Additionally, a review of published literature and our study led to the identification of 593 families with variants of PITX2 or FOXC1, including 316 families with heterozygous variants in FOXC1, 251 families with heterozygous variants in PITX2, 13 families with variants in double genes, seven families with homozygous or compound heterozygous variants in FOXC1, and six families with variants in ADAMTS17, PRDM5, COL4A1 or CYP1B1. Significant differences were observed between the prevalence of missense and in-frame, truncation, and large deletion variants in PITX2 (32.00%, 42.67%, and 25.33%, respectively) and FOXC1 (34.49%, 35.13%, 30.38%, respectively) (p = 1.16E-43). Enrichment and frequency analyses revealed that missense variants were concentrated in the forkhead domain of FOXC1 (76.14%) and homeodomain of PITX2 (87.50%). The percentage of Caucasians with variants in FOXC1 was significantly higher than that of PITX2 (p = 2.00E-2). Significant differences between PITX2 and FOXC1 were observed in glaucoma (p = 3.00E-2), corectopia (p = 3.050E-6), and polycoria (p = 5.21E-08). Additionally, we observed a significant difference in best-corrected visual acuity (BCVA) between FOXC1 and PITX2 (p = 3.80E-2). Among all the family members with PITX2 or FOXC1 variants, the prevalence of systemic abnormalities was significantly higher in PITX2 than in FOXC1 (89.16% vs. 58.77%, p = 5.44E-17). In conclusion, macular retinoschisis as a novel phenotype had been observed in patient with variant in PITX2. Significant differences were detected in phenotypes and genotypes between PITX2 and FOXC1.
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Anomalías del Ojo , Enfermedades Hereditarias del Ojo , Proteínas de Homeodominio , Humanos , Segmento Anterior del Ojo , Anomalías del Ojo/genética , Enfermedades Hereditarias del Ojo/genética , Factores de Transcripción Forkhead/genética , Estudios de Asociación Genética , Proteínas de Homeodominio/genética , Mutación , Linaje , Retinosquisis , Proteína del Homeodomínio PITX2RESUMEN
OBJECTIVES: To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld-Rieger syndrome (ARS) with a heterozygous microdeletion involving paired-like homeodomain 2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects. SUBJECTS AND METHODS: Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis (CMA) were used to screen the disease-causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro-computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/ß-catenin pathway in proliferation of ameloblast precursor cells was investigated. RESULTS: An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stifmulation partially reversed the proliferation ability after Pitx2 knockdown. CONCLUSIONS: Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/ß-catenin signaling pathway.
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Enfermedades Dentales , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Segmento Anterior del Ojo , Esmalte DentalRESUMEN
We aimed to characterize the genetic basis and craniofacial and dental features of Finnish patients with Axenfeld-Rieger syndrome (ARS). Mutational analyses of seven patients in five families were performed by sequencing or comparative genomic hybridization. Phenotypic analysis was based on both clinical and radiographic examinations, as well as on medical data. Lateral cephalometric radiographs of five patients were analysed using Viewbox 3.1-Cephalometric Software. The cephalometric values were compared to Finnish population-standard values of the same age and gender. Two frameshift mutations and three whole gene deletions were detected in five families. Class III skeletal relationship with retrognathic maxilla and mildly retrognathic mandible were detected in all five patients studied. Significant differences compared with the control values were in SNA (P = .0014), ANB (P = .0043) and SNB angles (P = .013). Five patients had anterior crossbite. Six patients showed tooth agenesis. The average number of missing teeth (third molars excluded) was 9 (range 0-15). The tooth agenesis rate was 52% in maxilla and 26% in mandible. Maxillary central and lateral permanent incisors were most often missing (rate 71% equally) while no one lacked canines or first molars in mandible. Two patients had a supernumerary mandibular permanent incisor. Six patients had either taurodontic and/or single-rooted molars. Our results suggest that class III skeletal relationship with maxillary and mandibular retrognathism, anterior crossbite, maxillary incisor agenesis and taurodontic, even pyramidal, roots are common determinants of ARS caused by PITX2 mutations.
Asunto(s)
Anodoncia , Maloclusión , Humanos , Hibridación Genómica Comparativa , Anodoncia/diagnóstico por imagen , Anodoncia/genética , Mutación , MaxilarRESUMEN
Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/ß-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/ß-catenin signaling.
Asunto(s)
Neoplasias Pancreáticas , beta Catenina , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Vimentina/genética , Vimentina/metabolismo , Células Estrelladas Pancreáticas/metabolismo , Movimiento Celular/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Vía de Señalización Wnt/genética , Línea Celular Tumoral , Proliferación Celular/genéticaRESUMEN
Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas.