RESUMO
Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.
Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Disco Intervertebral , Camundongos , Animais , Degeneração do Disco Intervertebral/genética , Proteínas Hedgehog/genética , FenótipoRESUMO
Development of mammalian auditory epithelium, the organ of Corti, requires precise control of both cell cycle withdrawal and differentiation. Sensory progenitors (prosensory cells) in the cochlear apex exit the cell cycle first but differentiate last. Sonic hedgehog (Shh) signaling is required for the spatiotemporal regulation of prosensory cell differentiation, but the underlying mechanisms remain unclear. Here, we show that suppressor of fused (Sufu), a negative regulator of Shh signaling, is essential for controlling the timing and progression of hair cell (HC) differentiation. Removal of Sufu leads to abnormal Atoh1 expression and a severe delay of HC differentiation due to elevated Gli2 mRNA expression. Later in development, HC differentiation defects are restored in the Sufu mutant by the action of speckle-type PDZ protein (Spop), which promotes Gli2 protein degradation. Deletion of both Sufu and Spop results in robust Gli2 activation, exacerbating HC differentiation defects. We further demonstrate that Gli2 inhibits HC differentiation through maintaining the progenitor state of Sox2+ prosensory cells. Along the basal-apical axis of the developing cochlea, the Sox2 expression level is higher in the progenitor cells than in differentiating cells and is down-regulated from base to apex as differentiation proceeds. The dynamic spatiotemporal change of Sox2 expression levels is controlled by Shh signaling through Gli2. Together, our results reveal key functions of Gli2 in sustaining the progenitor state, thereby preventing HC differentiation and in turn governing the basal-apical progression of HC differentiation in the cochlea.
Assuntos
Células Ciliadas Auditivas , Proteínas Hedgehog , Animais , Diferenciação Celular/genética , Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Proteínas Hedgehog/metabolismo , Mamíferos/genética , RNA Mensageiro/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Autism spectrum disorder (ASD) is a highly variable neurodevelopmental disorder that typically manifests childhood, characterized by a triad of symptoms: impaired social interaction, communication difficulties, and restricted interests with repetitive behaviors. De novo variants in related genes can cause ASD. We present the case of a 6-year-old Chinese boy with autistic behavior, including language communication impairments, intellectual disabilities, stunted development, and irritability in social interactions. Using Sanger sequencing, we confirmed a pathogenic in the RERE gene (NM_012102.4) (c.3732delC, p.Tyr1245Thrfs*12; EX21; Het). Subsequently, we generated an RERE point mutation cell line (ReMut) using CRISPR/Cas9 Targeted Genome Editing. Immunofluorescence was conducted to determine the location of the mutant RERE. RNA-sequencing and mass spectrometry analyses were performed to elucidate the ASD-related genes and signaling pathways disrupted by this variant in RERE. We identified 3790 differentially expressed genes and 684 differentially expressed proteins. The SHH signaling pathway was found to be downregulated, and the Hippo pathway was upregulated in ReMut. Genes implicated in autism, such as CNTNAP2, STX1A, FARP2, and GPC1, were significantly downregulated. Simultaneously, we noted alterations in HDAC1 and HDAC2, which are members of the WHHERE complex, suggesting their role in the pathogenesis of this patient. In conclusion, we report a de novo variant in RERE associated with autistic behavior. The finding that ASD is associated with RERE variants underscore the role of genetic factors in ASD and provides insights regarding the mechanisms underlying RERE variants in disease onset.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Masculino , Humanos , Criança , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Transdução de Sinais/genética , Mutação Puntual , Expressão Gênica , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genéticaRESUMO
Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that is critical for hydration of the airways and mucociliary clearance. However, the cellular mechanisms that govern ionocyte specification and function remain unclear. We observed that increased abundance of ionocytes in cystic fibrosis (CF) airway epithelium was associated with enhanced expression of Sonic Hedgehog (SHH) effectors. In this study, we evaluated whether the SHH pathway directly impacts ionocyte differentiation and CFTR function in airway epithelia. Pharmacological HPI1-mediated inhibition of SHH signaling component GLI1 significantly impaired human basal cell specification of ionocytes and ciliated cells but significantly enhanced specification of secretory cells. By contrast, activation of the SHH pathway effector smoothened (SMO) with the chemical agonist SAG significantly enhanced ionocyte specification. The abundance of CFTR+ BSND+ ionocytes under these conditions had a direct relationship with CFTR-mediated currents in differentiated air-liquid interface (ALI) airway cultures. These findings were corroborated in ferret ALI airway cultures generated from basal cells in which the genes encoding the SHH receptor PTCH1 or its intracellular effector SMO were genetically ablated using CRISPR-Cas9, causing aberrant activation or suppression of SHH signaling, respectively. These findings demonstrate that SHH signaling is directly involved in airway basal cell specification of CFTR-expressing pulmonary ionocytes and is likely responsible for enhanced ionocyte abundance in the CF proximal airways. Pharmacologic approaches to enhance ionocyte and reduce secretory cell specification after CFTR gene editing of basal cells may have utility in the treatment of CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Proteínas Hedgehog , Animais , Humanos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Furões , Proteínas Hedgehog/metabolismoRESUMO
Medulloblastoma is the most common pediatric malignant brain tumor composed of four molecular subgroups. Recent intensive genomics has greatly contributed to our understanding of medulloblastoma pathogenesis. Sequencing studies identified novel mutations involved in the cyclic AMP-dependent pathway or RNA processing in the Sonic Hedgehog (SHH) subgroup, and core-binding factor subunit alpha (CBFA) complex in the group 4 subgroup. Likewise, single-cell sequencing provided detailed insights into the cell of origin associated with brain development. In this review, we will summarize recent findings by sequencing analyses for medulloblastoma.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Humanos , Criança , Meduloblastoma/genética , Proteínas Hedgehog/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/patologia , Neoplasias Cerebelares/genéticaRESUMO
BACKGROUND: The primary cilia (PC) is a microtubule-based and nonmotile organelle which protrudes from the surface of almost all mammalian cells. At present, PC has been found to be a deficiency or loss in multiple cancers. Restoring PC could be a novel targeting therapy strategy. Our research showed that PC was reduced in human bladder cancer (BLCA) cells, and PC deficiency promotes cell proliferation. However, the concrete mechanisms remain unknown. SCL/TAL1 interrupting locus (STIL), a PC-related protein, was screened in our previous study and could influence the cell cycle by regulating PC in tumor cells. In this study, we aimed to elucidate the function of STIL for PC to explore the underlying mechanism of PC in BLCA. METHODS: Public database analysis, western blot, and enzyme-linked immunosorbent assay (ELISA) were used to screen genes and explore gene expression alteration. Immunofluorescence and western blot were utilized to investigate PC. Wound healing assay, clone formation assay, and CCK-8 assay were used to explore cell migration, growth, and proliferation. The co-immunoprecipitation and western blot were employed to reveal the interaction of STIL and AURKA. RESULTS: We found that high STIL expression is correlated with poor outcomes of BLCA patients. Further analysis revealed that STIL overexpression could inhibit PC formation, activate SHH signaling pathways, and promote cell proliferation. In contrast, STIL-knockdown could promote PC formation, inactivate SHH signaling, and inhibit cell proliferation. Furthermore, we found that the regulatory functions of STIL for PC depend on AURKA. STIL could influence proteasome activity and maintain AURKA stabilization. AURKA-knockdown could reverse PC deficiency caused by STIL overexpression for PC in BLCA cells. We observed that co-knockdown in STIL and AURKA significantly enhanced PC assembly. CONCLUSION: In summary, our result provides a potential therapy target for BLCA based on the restoration of PC.
Assuntos
Aurora Quinase A , Neoplasias da Bexiga Urinária , Animais , Humanos , Aurora Quinase A/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cílios/metabolismo , Proliferação de Células/genética , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , MamíferosRESUMO
Skin and soft tissue expansion is a widely used technique in plastic surgery. However, the regulatory mechanisms associated with cellular processes involved in skin expansion are not well elucidated. In the present study, we aimed at exploring the transcriptome changes associated with skin expansion and profiling the difference in gene expression between the skin tissue in the top of the dilator and the skin tissue in the side of the dilator. A mouse model of skin expansion was established and RNA sequencing (RNA-Seq) was performed on samples collected at different time points. Differential expression analysis was performed using the DESeq2 package while STEM was used for time series clustering profiling. The regulatory networks were established and the functions of sets of genes were analyzed. The mRNA expression levels of candidate genes were validated by the quantitative RT-PCR. Among the skin tissue in the top of the dilator and normal samples at days 1, 3, 7, 14 and 28, 53 commonly upregulated and 7 commonly downregulated genes were identified while among the skin tissue in the side of the dilator and normal samples, 98 downregulated and 255 upregulated genes were identified. Genes differentially expressed among the skin tissue in the top of the dilator and normal samples were involved in coagulation and proliferation-associated pathways while those among the skin tissue in the side of the dilator and normal samples were involved in the inflammation, immune response, and defense response. Among the skin tissue in the top of the dilator and the skin tissue in the side of the dilator samples, 161 were constantly upregulated while 27 were constantly downregulated; these genes were enriched in the biological processes of cell adhesion and regulation of cell proliferation (n = 11). Furthermore, we identified that SHH signaling genes formed a coexpression regulatory network with cellular proliferation, apoptosis, autophagy and angiogenesis-related genes in the expanded skin. In conclusion, our findings can promote research and understanding of the mechanism of skin expansion and will find application in plastic surgery.
Assuntos
Perfilação da Expressão Gênica , Pele , Animais , Camundongos , Apoptose/genética , Autofagia/genética , Redes Reguladoras de Genes , Pele/metabolismo , TranscriptomaRESUMO
BACKGROUND: Tumor-associated macrophages (TAMs) play a dual role in tumors. However, the factors which drive the function of TAMs in cholangiocarcinoma remain largely undefined. METHODS: SHH signaling pathway and endoplasmic reticulum stress (ERS) indicators were detected in clinical tissues and cholangiocarcinoma cell lines. TAMs were co-cultured with cholangiocarcinoma cells under conditions of hypoxia/normoxia. Polarized TAMs were counted by flow cytometry, and TGF-ß1 levels in cell supernatants were detected by ELISA. The effects of glioma-associated oncogene GLI2 on TAMs themselves and cholangiocarcinoma cells were examined by conducting interference and overexpression assays. RESULTS: The SHH signaling pathway and ERS were both activated in tumor tissues or tumor cell lines under conditions of hypoxia. In co-culture experiments, the presence of cholangiocarcinoma cells increased the proportion of M2-polarized TAMs and the secretion of TGF-ß1 by TAMs, while knockdown of SHH expression reversed those increases. Overexpression of GLI2 in TAMS or stimulation of TAMS with Hh-Ag1.5 increased their levels of TGF-ß1 expression. Furthermore, under co-culture conditions, interference with GLI2 expression in TAMs reduced the tumor cell migration, invasion, and ER homeostasis induced by Hh-Ag1.5-pretreated TAMs. Under conditions of hypoxia, the presence of cholangiocarcinoma cells promoted the expression of GLI2 and TGF-ß1 in Tams, and in turn, TAMs inhibited the apoptosis and promoted the migration and invasion of cholangiocarcinoma cells. In vivo, an injection of cholangiocarcinoma cells plus TAMs contributed to the growth, EMT, and ER homeostasis of tumor tissue, while an injection of TAMs with GLI2 knockdown had the opposite effects. CONCLUSION: Cholangiocarcinoma cells regulated TAM polarization and TGF-ß1 secretion via a paracrine SHH signaling pathway, and in turn, TAMs promoted the growth, EMT, and ER homeostasis of cholangiocarcinoma cells via TGF-ß1.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Transição Epitelial-Mesenquimal , Proteínas Hedgehog , Fator de Crescimento Transformador beta1 , Macrófagos Associados a Tumor , Proteína Gli2 com Dedos de Zinco , Humanos , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colangiocarcinoma/patologia , Proteínas Hedgehog/metabolismo , Proteínas Nucleares , Macrófagos Associados a Tumor/metabolismoRESUMO
PURPOSE: Gliomagenesis and resistance of glioblastoma (GBM) are believed to be mediated by glioma stem cells (GSC). Evidence suggests that SHH signaling promotes GSC proliferation and self-renewal. METHODS: ABTC-0904 was a two-arm, multicenter phase 0/II study of GDC-0449, an oral inhibitor of Smoothened (SMO) in patients undergoing resection for recurrent GBM. All patients (Arms I and II) had surgery and received drug post-operatively. Only patients in Arm I received drug prior to surgery. The primary objective was to determine 6-month progression free survival (PFS-6). Secondary endpoints include median PFS (mPFS) and overall survival (mOS), response rate, and toxicity. Correlative studies included bioanalysis of GDC-0449, and inhibition of SHH signaling, GSC proliferation and self-renewal. RESULTS: Forty-one patients were enrolled. Pharmacokinetics of GDC-0449 in plasma demonstrated levels within expected therapeutic range in 75% of patients. The proportion of tumorcells producing CD133+ neurospheres, neurosphere proliferation, self-renewal, and expression of the SHh downstream signaling was significantly decreased in Arm I following GDC-0449 treatment (p < 0.005; p < 0.001 respectively) compared to Arm II (no drug pre-op). Treatment was well tolerated. There were no objective responders in either arm. Overall PFS-6 was 2.4% (95% CI 0.9-11.1%). Median PFS was 2.3 months (95% CI 1.9-2.6) and mOS was 7.8 months (95% CI 5.4-10.1). CONCLUSIONS: GDC-0449 was well tolerated, reached tumor, and inhibited CD133+ neurosphere formation, but had little clinical efficacy as a single agent in rGBM. This suggests growth and maintenance of rGBM is not solely dependent on the SHH pathway thus targeting SMO may require combined approaches.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/patologia , Proteínas Hedgehog/metabolismo , Recidiva Local de Neoplasia/patologia , Glioma/patologia , Antineoplásicos/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Encefálicas/patologiaRESUMO
BACKGROUND: Despite their high repair capability, bone defects still present a major challenge in orthopedic tissue engineering. Osteoblast differentiation is central to the treatment of bone defects. METHODS AND RESULTS: We used nasal mucosal-derived ectoderm mesenchymal stem cells (EMSCs) to promote osteogenic differentiation by co-culturing MC3T3-E1 cells. Our results showed that MC3T3-E1/EMSCs co-culture upregulated bone-related proteins and transglutaminase 2 (TG2) and increased alkaline phosphatase (ALP) activity and bone nodule formation relative to controls. Furthermore, our results showed that EMSC-derived sonic hedgehog (Shh) accounted for the enhanced MC3T3-E1 differentiation because inhibiting Shh signaling substantially reduced osteogenic differentiation. CONCLUSION: Altogether, these results suggest that EMSCs differentiated into osteoblast cells and supported MC3T3-E1 differentiation. Thus, EMSCs may be a promising cell source for treating bone-related diseases.
Assuntos
Proteínas Hedgehog , Células-Tronco Mesenquimais , Proteínas Hedgehog/metabolismo , Osteogênese , Ectoderma/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismoRESUMO
OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.
Assuntos
Proteínas do Esmalte Dentário , Esmalte Dentário , Camundongos , Animais , Humanos , Amelogenina/genética , Amelogenina/metabolismo , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Amelogênese/genética , Proteínas Supressoras de Tumor , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismoRESUMO
BACKGROUND: Human papillomavirus (HPV) infected keratinocyte dysfunction results in the formation of genital warts, and the specific role of Sonic hedgehog (SHh) signaling in genital warts remains elusive. Thus, this study aimed to identify the correlation between wart formation and SHh signaling. MATERIALS AND METHODS: In this study, nine male patients with genital warts were recruited, and the expression of SHh and its downstream signal molecules Patched-1 and GLI family zinc finger 1 (Ptch1 and Gli1) was detected. Moreover, G2-phase cells in the collected genital warts samples were assessed with normal foreskin samples as a comparison. HPV6/11 were detected via in situ hybridization (ISH), and SHh expression of the corresponding paraffin sections was determined via immunohistochemical staining (IHC). In addition, an in vitro down-regulated SHh model was constructed by siRNA transfection of the HaCaT cell line, and the cell cycle was detected at 36 h by flow cytometry with propidium iodide staining. RESULTS: SHh, Ptch1, and Gli1 in warts were significantly downregulated in the condyloma acuminatum (CA) group compared to the normal foreskin group. G2-phase cells in the middle section of the spinous layer of CA wart tissues were significantly increased. Moreover, the expression of HPV-DNA was amplified and negatively correlated with SHh activity in CA wart tissues. Lastly, the downregulation of SHh-induced G2 arrest in vitro. CONCLUSIONS: The downregulation of the SHh signaling promotes HPV replication and the formation of warts by inducing G2/M arrest in the keratinocytes of CA.
Assuntos
Condiloma Acuminado , Infecções por Papillomavirus , Verrugas , Humanos , Masculino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Regulação para Baixo , Apoptose , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo CelularRESUMO
CAG is a burdensome and progressive disease. Numerous studies have shown the effectiveness of RUT in digestive system diseases. The therapeutic effects of RUT on MNNG-induced CAG and the potential mechanisms were probed. MNNG administration was employed to establish a CAG model. The HE and ELISA methods were applied to detect the treatment effects. WB, qRT-PCR, immunohistochemistry, TUNEL, and GES-1 cell flow cytometry approaches were employed to probe the mechanisms. The CAG model was successfully established. The ELISA and HE staining data showed that the RUT treatment effects on CAG rats were reflected by the amelioration of histological damage. The qRT-PCR and WB analyses indicated that the protective effect of RUT is related to the upregulation of the SHH pathway and downregulation of the downstream of apoptosis to improve gastric cellular survival. Our data suggest that RUT induces a gastroprotective effect by upregulating the SHH signaling pathway and stimulating anti-apoptosis downstream.
Assuntos
Gastrite Atrófica , Proteínas Hedgehog , Camundongos , Ratos , Animais , Gastrite Atrófica/induzido quimicamente , Gastrite Atrófica/tratamento farmacológico , Metilnitronitrosoguanidina , Quinazolinas , Nitrosoguanidinas , Transdução de SinaisRESUMO
Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.
Assuntos
Proteínas Hedgehog , Mesencéfalo , Diferenciação Celular , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Neurônios/metabolismo , Transdução de SinaisRESUMO
The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.
Assuntos
Cóclea/embriologia , Cóclea/metabolismo , Genoma , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Animais , Sequência de Bases , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Transgênicos , Reprodutibilidade dos TestesRESUMO
Metformin has potential anti-inflammatory properties and accelerates wound healing by enhancing vascular development. In this study, we aimed to investigate the effects of metformin on pulmonary vascular development and the underlying mechanism. Newborn mice were subcutaneously injected with metformin from day 2 after exposure to hyperoxia. Pulmonary vascular development, inflammation, and Shh signaling pathway-related protein expression were evaluated by western blotting and immunofluorescence staining. M2 macrophage polarization was measured by flow cytometry. The effect of metformin on macrophage polarization was determined using RAW264.7 macrophages exposed to 90% oxygen in vitro. The role of metformin and purmorphamine on M1 and M2 polarization was observed by flow cytometry. M2 polarization of pulmonary macrophages was inhibited after hyperoxic exposure, and metformin increased the number of M2 macrophages in the lung on postnatal day 14. Metformin upregulated CD31 expression and suppressed inflammation in the lung of mice exposed to hyperoxia on postnatal days 7 and 14. Metformin downregulated the Gli1 expression in macrophages in the lung after exposure to hyperoxia on postnatal day 14. In vitro studies showed that metformin inhibited the Gli1 expression in RAW264.7 macrophages exposed to 90% oxygen, which was reversed after purmorphamine pretreatment. Exposure to 90% oxygen inhibited the polarization of M2 macrophages, whereas metformin increased the number of M2 macrophages. Purmorphamine reversed the effects of metformin on M2 polarization and vascular endothelial growth factor (VEGF) upregulation in RAW264.7 macrophages exposed to hyperoxia. In conclusion, metformin regulates macrophage polarization via the Shh signaling pathway to improve pulmonary vascular development in bronchopulmonary dysplasia.
Assuntos
Displasia Broncopulmonar , Proteínas Hedgehog , Hiperóxia , Macrófagos , Metformina , Animais , Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/genética , Polaridade Celular , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hiperóxia/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metformina/farmacologia , Camundongos , Oxigênio/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Ubiquitin modification plays important roles in many cellular processes that are fundamental for vertebrate embryo development, such as cell division, differentiation, and migration. Aberrant function or deregulation of ubiquitination enzymes can cause developmental disorders, cancer progression, and neurodegenerative diseases in humans. RING finger protein 220 (RNF220) is an evolutionarily conserved RING-type ubiquitin E3 ligase. Recent studies have revealed the roles and mechanisms of RNF220 and its partner protein, zinc finger C4H2-type containing protein (ZC4H2), in embryonic development and human diseases. Using mouse and zebrafish models, it has been shown that RNF220 regulates sonic hedgehog (Shh) signaling via Gli and embryonic ectoderm development (EED), a polycomb repressive complex 2 (PRC2) component, during ventral neural patterning and cerebellum development. In addition, RNF220 also regulates the development and functions of central noradrenergic and motor neurons in mice. By stabilizing ß-catenin and signal transducer and activator of transcription 1 (STAT1), RNF220 is also involved in Wnt and interferon (IFN)-STAT1 signaling and thus the regulation of tumorigenesis and immune response, respectively. In humans, both RNF220 and ZC4H2 mutations have been reported to be associated with diseases accompanied by complicated neural defects. In this review, we summarize the current knowledge of RNF220 with special emphasis on its roles and mechanisms of action in signal transduction, vertebrate neural development, and related human disorders.
Assuntos
Ubiquitina-Proteína Ligases , Animais , Proteínas Hedgehog/genética , Humanos , Camundongos , Neurogênese , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Peixe-ZebraRESUMO
Sonic Hedgehog (Shh) signaling cascade is one of the complex signaling pathways that control the accurately organized developmental processes in multicellular organisms. This pathway has fundamental roles in the tumor formation and induction of resistance to conventional therapies. Numerous non-coding RNAs (ncRNAs) have been found to interact with Shh pathway to induce several pathogenic processes, including malignant and non-malignant disorders. Many of the Shh-interacting ncRNAs are oncogenes whose expressions have been increased in diverse malignancies. A number of Shh-targeting miRNAs such as miR-26a, miR-1471, miR-129-5p, miR-361-3p, miR-26b-5p and miR-361-3p have been found to be down-regulated in tumor tissues. In addition to malignant conditions, Shh-interacting ncRNAs can affect tissue regeneration and development of neurodegenerative disorders. XIST, LOC101930370, lncRNA-Hh, circBCBM1, SNHG6, LINC-PINT, TUG1 and LINC01426 are among long non-coding RNAs/circular RNAs that interact with Shh pathway. Moreover, miR-424, miR-26a, miR-1471, miR-125a, miR-210, miR-130a-5p, miR-199b, miR-155, let-7, miR-30c, miR-326, miR-26b-5p, miR-9, miR-132, miR-146a and miR-425-5p are among Shh-interacting miRNAs. The current review summarizes the interactions between ncRNAs and Shh in these contexts.
RESUMO
Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Krüppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing fetuses died in utero at approximately 10.5 days post-coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant fetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signaling pathway, leading to the upregulation of patched 1, GLI-Krüppel family member GLI1, hedgehog-interacting protein, etc., whereas FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signaling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future.
Assuntos
Ácido Fólico/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Defeitos do Tubo Neural/genética , Transdução de Sinais/genética , Animais , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , CamundongosRESUMO
BACKGROUND: Leiomyosarcoma (LMS), the most common soft tissue sarcoma, exhibits heterogeneous and complex genetic karyotypes with severe chromosomal instability and rearrangement and poor prognosis. METHODS: Clinical variables associated with NKX6-1 were obtained from The Cancer Genome Atlas (TCGA). NKX6-1 mRNA expression was examined in 49 human uterine tissues. The in vitro effects of NXK6-1 in LMS cells were determined by reverse transcriptase PCR, western blotting, colony formation, spheroid formation, and cell viability assays. In vivo tumor growth was evaluated in nude mice. RESULTS: Using The Cancer Genome Atlas (TCGA) and human uterine tissue datasets, we observed that NKX6-1 expression was associated with poor prognosis and malignant potential in LMS. NKX6-1 enhanced in vitro tumor cell aggressiveness via upregulation of cell proliferation and anchorage-independent growth and promoted in vivo tumor growth. Moreover, overexpression and knockdown of NKX6-1 were associated with upregulation and downregulation, respectively, of stem cell transcription factors, including KLF8, MYC, and CD49F, and affected sphere formation, chemoresistance, NOTCH signaling and Sonic hedgehog (SHH) pathways in human sarcoma cells. Importantly, treatment with an SHH inhibitor (RU-SKI 43) but not a NOTCH inhibitor (DAPT) reduced cell survival in NKX6-1-expressing cancer cells, indicating that an SHH inhibitor could be useful in treating LMS. Finally, using the TCGA dataset, we demonstrated that LMS patients with high expression of NKX6-1 and HHAT, an SHH pathway acyltransferase, had poorer survival outcomes compared to those without. CONCLUSIONS: Our findings indicate that NKX6-1 and HHAT play critical roles in the pathogenesis of LMS and could be promising diagnostic and therapeutic targets for LMS patients.