The people behind the papers - Junghyun Kim and Sibum Sung.

Plants respond to changes in temperature using complex mechanisms, with decreases in temperature inducing vernalisation and high temperatures causing thermo-morphogenesis. A new paper in Development investigates how VIL1, a PHD finger-containing protein, functions in plants during thermo-morphogenesis. To find out more about this research, we spoke with co-first author of the study, Junghyun Kim, and corresponding author Sibum Sung (Associate Professor of Molecular Bioscience at the University of Texas in Austin, USA). Co-first author Yogendra Bordiya was not available to interview, having now moved to a different sector.

Caenorhabditis elegans sine oculis/SIX-type homeobox genes act as homeotic switches to define neuronal subtype identities.

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.

BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

BarH-like homeobox 2 represses the transcription of keratin 16 and affects Ras signaling pathway to suppress nasopharyngeal carcinoma progression.

Nasopharyngeal carcinoma (NPC) refers to a malignancy initiating from the superior mucosal epithelium of the nasopharynx. Optimal therapies for NPC are still needed. In this investigation, we attempted to explore whether BarH-like homeobox 2 (BARX2), a well-known tumor suppressor, had anti-cancer properties on NPC, and the possible mechanisms. After searching for NPC-related databases, we determined BARX2 as one of the core genes in NPC. The results of RT-qPCR and immunohistochemistry or Western blot demonstrated that BARX2 was reduced in NPC patients and cells. Ectopic expression of BARX2 reverted the malignant phenotype of NPC cells. Mechanistically, BARX2 bound to the keratin 16 (KRT16) promoter to downregulate its expression. In addition, BARX2 was found to reduce the phosphorylation levels of MEK and ERK. Further KRT16 upregulation in cells overexpressing BARX2 promoted malignant aggressiveness of C666-1 and HNE3 cells and activated the Ras signaling pathway. BARX2 inhibited the growth and metastasis of tumors and suppressed the Ras signaling pathway in vivo. In conclusion, our findings indicate that BARX2 reverts malignant phenotypes of NPC cells by downregulating KRT16 in a Ras-dependent fashion. BARX2 might act as a possible therapeutic regulator for NPC.

Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders.

BACKGROUND: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. METHODS: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse. RESULTS: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. CONCLUSION: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.

Circular RNA circCPA4 promotes tumorigenesis by regulating miR-214-3p/TGIF2 in lung cancer.

BACKGROUND: Lung cancer is the most prevalent malignancy in adults. Circular RNA (circRNA) circCPA4 (hsa_circ_0082374) is highly expressed in non-small cell lung cancer (NSCLC). The purpose of this study was to explore the role and mechanism of circCPA4 in lung cancer. METHODS: CircCPA4, linear CPA4, TGF-ß-induced factor homeobox 2 (TGIF2), and microRNA-214-3p (miR-214-3p) levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The protein levels of TGIF2, Beclin1, and p62 were assessed by western blot assay. Colony numbers, migration, invasion, apoptosis, and cell cycle progression were examined by colony formation, wound-healing, transwell, and flow cytometry assays, respectively. The binding relationship between miR-214-3p and circCPA4 or TGIF2 was predicted by StarBase or TargetScan and then verified by a dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pulldown assays. The biological role of circCPA4 on lung tumor growth was assessed by a xenograft tumor model in vivo, and TGIF2 and ki-67 expression was assessed by immunohistochemistry. RESULTS: We determined that CircCPA4 and TGIF2 were increased, and miR-214-3p was decreased in lung cancer tissues and cells. Functionally, circCPA4 knockdown could suppress colony formation, migration, invasion, cell cycle progression, and expedite apoptosis of lung cancer cells in vitro. Mechanically, circCPA4 could regulate TGIF2 expression by sponging miR-214-3p. In addition, circCPA4 deficiency inhibited the tumor growth in lung cancer in the mouse model. CONCLUSIONS: CircCPA4 could act as a sponge of miR-214-3p to upregulate TGIF2 expression, thereby promoting the progression of lung cancer cells. These findings suggested underlying therapeutic targets for the treatment of lung cancer.

HOXD10 expression as a prognostic factor for hepatocellular carcinoma treated with curative resection.

PURPOSE: HOXD10 downregulation resulting from epigenetic changesas well as its role as a tumor suppressor have been reported in several cancers including hepatocellular carcinomas (HCCs). However, the prognostic role of HOXD10 expression in HCC tissue samples has not been evaluated. METHODS: HOXD10 expression was investigated in 278 curatively resected HCC samples using immunohistochemistry and its effectiveness in predicting patient outcome was analyzed. RESULTS: Low expression of HOXD10 was observed in 82.7% of HCC samples, and this was associated with increased age, large tumor size and advanced stage.HOXD10 was an independent predictive factor for early tumor recurrence at less than 2 years. Patients with low HOXD10 expression showed shorter recurrence-free survival (RFS) (p=0.024) and disease-specific survival (DSS) (p=0.016) than those with high expression. Multivariate analysis confirmed that low HOXD10 expression was an independent predictor of shorter RFS (hazard ratio 1.873, p=0.006) and DSS (hazard ratio2.504, p=0.012) than high HOXD10 expression. CONCLUSIONS: The present study provides clinical evidence supporting the use of HOXD10 as a prognostic biomarker in curatively resected HCCs, and suggests that HOXD10 could also be a potential therapeutic target in HCC.

mTORC1 Activation in Chx10-Specific Tsc1 Knockout Mice Accelerates Retina Aging and Degeneration.

Age-associated decline in retina function is largely responsible for the irreversible vision deterioration in the elderly population. It is also an important risk factor for the development of degenerative and angiogenic diseases. However, the molecular mechanisms involved in the process of aging in the retina remain largely elusive. This study investigated the role of mTORC1 signaling in aging of the retina. We showed that mTORC1 was activated in old-aged retina, particularly in the ganglion cells. The role of mTORC1 activation was further investigated in Chx10-Cre;Tsc1fx/fx mouse (Tsc1-cKO). Activation of mTORC1 was found in bipolar and some of the ganglion and amacrine cells in the adult Tsc1-cKO retina. Bipolar cell hypertrophy and Müller gliosis were observed in Tsc1-cKO since 6 weeks of age. The abnormal endings of bipolar cell dendritic tips at the outer nuclear layer resembled that of the old-aged mice. Microglial cell activation became evident in 6-week-old Tsc1-cKO. At 5 months, the Tsc1-cKO mice exhibited advanced features of old-aged retina, including the expression of p16Ink4a and p21, expression of SA-ß-gal in ganglion cells, decreased photoreceptor cell numbers, decreased electroretinogram responses, increased oxidative stress, microglial cell activation, and increased expression of immune and inflammatory genes. Inhibition of microglial cells by minocycline partially prevented photoreceptor cell loss and restored the electroretinogram responses. Collectively, our study showed that the activation of mTORC1 signaling accelerated aging of the retina by both cell autonomous and nonautonomous mechanisms. Our study also highlighted the role of microglia cells in driving the decline in retina function.

Inhibitor of growth protein 3 epigenetically silences endogenous retroviral elements and prevents innate immune activation.

Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells.

Interleukin 6 reduces vascular smooth muscle cell apoptosis via Prep1 and is associated with aging.

Aging exacerbates neointimal formation by reducing apoptosis of vascular smooth muscle cells (VSMCs) and induces inflammation within vascular wall. Prep1 is a homeodomain transcription factor which stimulates the expression of proinflammatory cytokines in aortic endothelial cell models and plays a primary role in the regulation of apoptosis. In this study, we have investigated the role of Prep1 in aorta of Prep1 hypomorphic heterozygous mice (Prep1i/+ ) and in VSMCs, and its correlation with aging. Histological analysis from Prep1i/+ aortas revealed a 25% reduction in medial smooth muscle cell density compared to WT animals. This result paralleled higher apoptosis, caspase 3, caspase 9 and p53 levels in Prep1i/+ mice and lower Bcl-xL. Prep1 overexpression in VSMCs decreased apoptosis by 25% and caspase 3 and caspase 9 expression by 40% and 37%. In parallel, Bcl-xL inhibition by BH3I-1 and p53 induction by etoposide reverted the antiapoptotic effect of Prep1. Experiments performed in aorta from 18 months old WT mice showed a significant increase in Prep1, p16INK4 , p21Waf1 and interleukin 6 (IL-6) compared to youngest animals. Similar results have been observed in H2 O2 -induced senescent VSMCs. Interestingly, the synthetic Prep1 inhibitory peptide Prep1 (54-72) reduced the antiapoptotic effects mediated by IL-6, particularly in senescent VSMCs. These results indicate that IL-6-Prep1 signaling reduces apoptosis, by modulating Bcl-xL and p53 both in murine aorta and in VSMCs. In addition, age-dependent increase in IL-6 and Prep1 in senescent VSMCs and in old mice may be involved in the aging-related vascular dysfunction.

FTO-stabilized lncRNA HOXC13-AS epigenetically upregulated FZD6 and activated Wnt/ß-catenin signaling to drive cervical cancer proliferation, invasion, and EMT.

PURPOSE: Cervical cancer (CC) is the third most prevalent malignancy in women. Frizzled class receptor 6 (FZD6) is demonstrated to either activate or repress the activity of Wnt/ß-catenin pathway, a crucial signaling involved in cancer development. However, the role of FZD6 in CC is unknown. The present study explored the function of FZD6 and its mechanism in CC. METHODS: The levels of FZD6, HOXC13-AS were detected in CC specimens and CC cell lines via qRT-PCR. Cell proliferation and invasion was explored via CCK-8 assay, colony formation assay and transwell assay. Luciferase reporter analysis, FISH, subcellular fractionation, chromatin immunoprecipitation and RNA immunoprecipitation were performed for investigating the molecular mechanism. RESULTS: FZD6 was up-regulated in CC. FZD6 silence retarded proliferation, invasion, and epithelial-to-mesenchymal transition (EMT), and inactivated Wnt/ß-catenin. HOXC13 antisense RNA (HOXC13-AS) was up-regulated in CC and positively correlated with FZD6. Mechanistically, HOCX13-AS1 augmented FZD through cAMP-response element binding protein-binding protein (CBP)-modulated histone H3 on lysine 27 acetylation (H3K27ac). Additionally, fat mass and obesity-associated protein (FTO) reduced N6-methyladenosine (m6A) and stabilized HOXC13-AS in CC. CONCLUSIONS: In conclusion, this study firstly showed that FTO-stabilized HOXC13-AS epigenetically up-regulated FZD6 and activated Wnt/ß-catenin signaling to drive CC proliferation, invasion, and EMT, suggesting HOXC13-AS as a potential target for CC treatment.

Role of the HOXA cluster in HSC emergence and blood cancer.

Hematopoiesis, the process of blood formation, is controlled by a complex developmental program that involves intrinsic and extrinsic regulators. Blood formation is critical to normal embryonic development and during embryogenesis distinct waves of hematopoiesis have been defined that represent the emergence of hematopoietic stem or progenitor cells. The Class I family of homeobox (HOX) genes are also critical for normal embryonic development, whereby mutations are associated with malformations and deformity. Recently, members of the HOXA cluster (comprising 11 genes and non-coding RNA elements) have been associated with the emergence and maintenance of long-term repopulating HSCs. Previous studies identified a gradient of HOXA expression from high in HSCs to low in circulating peripheral cells, indicating their importance in maintaining blood cell numbers and differentiation state. Indeed, dysregulation of HOXA genes either directly or by genetic lesions of upstream regulators correlates with a malignant phenotype. This review discusses the role of the HOXA cluster in both HSC emergence and blood cancer formation highlighting the need for further research to identify specific roles of these master regulators in normal and malignant hematopoiesis.

Elimination of glutamatergic transmission from Hb9 interneurons does not impact treadmill locomotion.

The spinal cord contains neural circuits that can produce the rhythm and pattern of locomotor activity. It has previously been postulated that a population of glutamatergic neurons, termed Hb9 interneurons, contributes to locomotor rhythmogenesis. These neurons were identified by their expression of the homeobox gene, Hb9, which is also expressed in motor neurons. We developed a mouse line in which Cre recombinase activity is inducible in neurons expressing Hb9. We then used this line to eliminate vesicular glutamate transporter 2 from Hb9 interneurons, and found that there were no deficits in treadmill locomotion. We conclude that glutamatergic neurotransmission by Hb9 interneurons is not required for locomotor behaviour. The role of these neurons in neural circuits remains elusive.

Cdx2 regulates immune cell infiltration in the intestine.

The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.

Understanding PITX2-Dependent Atrial Fibrillation Mechanisms through Computational Models.

Atrial fibrillation (AF) is a common arrhythmia. Better prevention and treatment of AF are needed to reduce AF-associated morbidity and mortality. Several major mechanisms cause AF in patients, including genetic predispositions to AF development. Genome-wide association studies have identified a number of genetic variants in association with AF populations, with the strongest hits clustering on chromosome 4q25, close to the gene for the homeobox transcription PITX2. Because of the inherent complexity of the human heart, experimental and basic research is insufficient for understanding the functional impacts of PITX2 variants on AF. Linking PITX2 properties to ion channels, cells, tissues, atriums and the whole heart, computational models provide a supplementary tool for achieving a quantitative understanding of the functional role of PITX2 in remodelling atrial structure and function to predispose to AF. It is hoped that computational approaches incorporating all we know about PITX2-related structural and electrical remodelling would provide better understanding into its proarrhythmic effects leading to development of improved anti-AF therapies. In the present review, we discuss advances in atrial modelling and focus on the mechanistic links between PITX2 and AF. Challenges in applying models for improving patient health are described, as well as a summary of future perspectives.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

Prrx1 promotes stemness and angiogenesis via activating TGF-ß/smad pathway and upregulating proangiogenic factors in glioma.

Glioma is one of the most lethal cancers with highly vascularized networks and growing evidences have identified glioma stem cells (GSCs) to account for excessive angiogenesis in glioma. Aberrant expression of paired-related homeobox1 (Prrx1) has been functionally associated with cancer stem cells including GSCs. In this study, Prrx1 was found to be markedly upregulated in glioma specimens and elevated Prrx1 expression was inversely correlated with prognosis of glioma patients. Prrx1 potentiated stemness acquisition in non-stem tumor cells (NSTCs) and stemness maintenance in GSCs, accompanied with increased expression of stemness markers such as SOX2. Prrx1 also promoted glioma angiogenesis by upregulating proangiogenic factors such as VEGF. Consistently, silencing Prrx1 markedly inhibited glioma proliferation, stemness, and angiogenesis in vivo. Using a combination of subcellular proteomics and in vitro analyses, we revealed that Prrx1 directly bound to the promoter regions of TGF-ß1 gene, upregulated TGF-ß1 expression, and ultimately activated the TGF-ß/smad pathway. Silencing TGF-ß1 mitigated the malignant behaviors induced by Prrx1. Activation of this pathway cooperates with Prrx1 to upregulate the expression of stemness-related genes and proangiogenic factors. In summary, our findings revealed that Prrx1/TGF-ß/smad signal axis exerted a critical role in glioma stemness and angiogeneis. Disrupting the function of this signal axis might represent a new therapeutic strategy in glioma patients.

Molecular mechanism of MdWUS2-MdTCP12 interaction in mediating cytokinin signaling to control axillary bud outgrowth.

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis, indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin.

Paired-like homeodomain transcription factor 2 affects endometrial cell function and embryo implantation through the Wnt/ß-catenin pathway.

The successful implantation of embryos is crucial for pregnancy in mammals. This complex process is inevitably dependent on the development of the endometrium. The paired-like homeodomain transcription factor 2 (PITX2) is involved in a variety of biological processes, but whether it is involved in embryo implantation has not been reported. In this study, we aimed to investigate uterine expression and regulation of PITX2 during implantation. We found that PITX2 was elevated in the human endometrium in the secretory phase. The results of the pregnant mouse models showed that PITX2 expression was spatiotemporal in mouse endometrial tissue throughout peri-implantation period, and it was significantly upregulated at the time of implantation. Interestingly, PITX2 was mainly localized to the glandular epithelium cells on D2.5-3.5 of pregnancy, while D5.5-6.5 was largely expressed in stromal cells. In vitro, PITX2 regulated endometrial cells proliferation, migration, invasion, and other functions through the Wnt/ß-catenin signaling pathway. In addition, a significant decrease in the rate of embryo implantation was observed after injecting PITX2 small interfering RNA into the uterine horn. These results demonstrate the effects of PITX2 on the physiological function of endometrial cells and embryo implantation, suggesting a role in the endometrial regulatory mechanism during implantation.

Nkx2-3 induces autophagy inhibiting proliferation and migration of vascular smooth muscle cells via AMPK/mTOR signaling pathway.

Vascular remodeling and restenosis are common complications after percutaneous coronary intervention. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in intimal hyperplasia-induced vascular restenosis. NK2 Homeobox 3 (Nkx2-3), a critical member of Nkx family, is involved in tissue differentiation and organ development. However, the role of Nkx2-3 in VSMCs proliferation and migration remains unknown. In this study, we used carotid balloon injury model and platelet-derived growth factor-BB (PDGF)-treated VSMCs as in vivo and in vitro experimental models. EdU assay and CCK-8 assay were used to detect cell proliferation. Migration was measured by scratch test. Hematoxylin and eosin staining and immunohistochemistry staining were used to evaluate the intimal hyperplasia. The autophagy level was detected by fluorescent mRFP-GFP-LC3 in vitro and by transmission electron microscopy in vivo. It was shown that Nkx2-3 was upregulated both in balloon injured carotid arteries and PDGF-stimulated VSMCs. Adenovirus-mediated Nkx2-3 overexpression inhibited intimal hyperplasia after balloon injury, and suppressed VSMCs proliferation and migration induced by PDGF. Conversely, silencing of Nkx2-3 by small interfering RNA exaggerated proliferation and migration of VSMCs. Furthermore, we found that Nkx2-3 enhanced autophagy level, while the autophagy inhibitor 3-MA eliminated the inhibitory effect of Nkx2-3 on VSMCs proliferation and migration both in vivo and in vitro. Moreover, Nkx2-3 promoted autophagy in VSMCs by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. These results demonstrated for the first time that Nkx2-3 inhibited VSMCs proliferation and migration through AMPK/mTOR-mediated autophagy.