RESUMEN
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Transdiferenciación Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Glándulas Mamarias Animales/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Células MCF-7 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción HES-1/metabolismo , TransfecciónRESUMEN
The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool.
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Regulación del Desarrollo de la Expresión Génica/genética , Proteína MioD/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factor de Transcripción HES-1/metabolismo , Animales , Células Cultivadas , Ratones , Proteína MioD/genética , Receptores Notch/metabolismo , Transducción de Señal , Factor de Transcripción HES-1/genéticaRESUMEN
Somatic stem/progenitor cells are active in embryonic tissues but quiescent in many adult tissues. The detailed mechanisms that regulate active versus quiescent stem cell states are largely unknown. In active neural stem cells, Hes1 expression oscillates and drives cyclic expression of the proneural gene Ascl1, which activates cell proliferation. Here, we found that in quiescent neural stem cells in the adult mouse brain, Hes1 levels are oscillatory, although the peaks and troughs are higher than those in active neural stem cells, causing Ascl1 expression to be continuously suppressed. Inactivation of Hes1 and its related genes up-regulates Ascl1 expression and increases neurogenesis. This causes rapid depletion of neural stem cells and premature termination of neurogenesis. Conversely, sustained Hes1 expression represses Ascl1, inhibits neurogenesis, and maintains quiescent neural stem cells. In contrast, induction of Ascl1 oscillations activates neural stem cells and increases neurogenesis in the adult mouse brain. Thus, Ascl1 oscillations, which normally depend on Hes1 oscillations, regulate the active state, while high Hes1 expression and resultant Ascl1 suppression promote quiescence in neural stem cells.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Encéfalo/citología , Regulación de la Expresión Génica , Células-Madre Neurales , Neurogénesis/genética , Factor de Transcripción HES-1/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Silenciador del Gen , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Optogenética , Regiones Promotoras Genéticas , Factor de Transcripción HES-1/metabolismoRESUMEN
Adult neural stem cells are mostly quiescent and only rarely enter the cell cycle to self-renew and generate neuronal or glial progenies. The Notch signaling pathway is essential for both the quiescent and proliferative states of neural stem cells. However, these are mutually exclusive cellular states; thus, how Notch promotes both of these programs within adult neural stem cells has remained unclear. In this issue of Genes & Development, Sueda and colleagues (pp. 511-523) use an extensive repertoire of mouse genetic tools and techniques to demonstrate that it is the levels and dynamic expression of the Notch transcriptional effector Hairy and Enhancer of Split 1 that enables this dual role.
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Células Madre Adultas/citología , Células-Madre Neurales/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular , Ratones , Sistema Nervioso , Transducción de Señal , Factor de Transcripción HES-1RESUMEN
Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.
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Desarrollo Óseo , Huesos , Cartílago , Diferenciación Celular , Linaje de la Célula , Condrocitos , Células Madre Mesenquimatosas , Factor de Transcripción HES-1 , Animales , Ratones , Huesos/citología , Cartílago/citología , Cartílago/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Factor de Transcripción HES-1/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Receptores Notch/metabolismoRESUMEN
Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neuroblastoma , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores Notch/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiones Promotoras Genéticas/genética , Línea Celular , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
Neural stem cells (NSCs) gradually alter their characteristics during mammalian neocortical development, resulting in the production of various neurons and glial cells, and remain in the postnatal brain as a source of adult neurogenesis. Notch-Hes signaling is a key regulator of stem cell properties in the developing and postnatal brain, and Hes1 is a major effector that strongly inhibits neuronal differentiation and maintains NSCs. To manipulate Hes1 expression levels in NSCs, we generated transgenic (Tg) mice using the Tet-On system. In Hes1-overexpressing Tg mice, NSCs were maintained in both embryonic and postnatal brains, and generation of later-born neurons was prolonged until later stages in the Tg neocortex. Hes1 overexpression inhibited the production of Tbr2+ intermediate progenitor cells but instead promoted the generation of basal radial glia-like cells in the subventricular zone (SVZ) at late embryonic stages. Furthermore, Hes1-overexpressing Tg mice exhibited the expansion of NSCs and enhanced neurogenesis in the SVZ of adult brain. These results indicate that Hes1 overexpression expanded the embryonic NSC pool and led to the expansion of the NSC reservoir in the postnatal and adult brain.
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Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Expresión Génica , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Factor de Transcripción HES-1/genética , Animales , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Proliferación Celular , Células Cultivadas , Electroporación , Células Madre Embrionarias/citología , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Ratones , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción HES-1/metabolismoRESUMEN
The release of host mitochondrial cardiolipin is believed to be the main factor that contributes to the production of anti-cardiolipin antibodies in syphilis. However, the precise mechanism by which mitochondria release cardiolipin in this context remains elusive. This study aimed to elucidate the mechanisms underlying mitochondrial cardiolipin release in syphilis. We conducted a cardiolipin quantitative assay and immunofluorescence analysis to detect mitochondrial cardiolipin release in human microvascular endothelial cells (HMEC-1), with and without Treponema pallidum (Tp) infection. Furthermore, we explored apoptosis, a key mechanism for mitochondrial cardiolipin release. The potential mediator molecules were then analyzed through RNA-sequence and subsequently validated using in vitro knockout techniques mediated by CRISPR-Cas9 and pathway-specific inhibitors. Our findings confirm that live-Tp is capable of initiating the release of mitochondrial cardiolipin, whereas inactivated-Tp does not exhibit this capability. Additionally, apoptosis detection further supports the notion that the release of mitochondrial cardiolipin occurs independently of apoptosis. The RNA-sequencing results indicated that microtubule-associated protein2 (MAP2), an axonogenesis and dendrite development gene, was up-regulated in HMEC-1 treated with Tp, which was further confirmed in syphilitic lesions by immunofluorescence. Notably, genetic knockout of MAP2 inhibited Tp-induced mitochondrial cardiolipin release in HMEC-1. Mechanically, Tp-infection regulated MAP2 expression via the MEK-ERK-HES1 pathway, and MEK/ERK phosphorylation inhibitors effectively block Tp-induced mitochondrial cardiolipin release. This study demonstrated that the infection of live-Tp enhanced the expression of MAP2 via the MEK-ERK-HES1 pathway, thereby contributing to our understanding of the role of anti-cardiolipin antibodies in the diagnosis of syphilis.
RESUMEN
Adipose tissue-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential, which mainly restore tissue repair function and promote cell regeneration. It can be directionally differentiated into Schwann-like cells to promote the repair of peripheral nerve injury. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the repair of nerve injury, but the underlying mechanism remains unclear, which seriously limits its further application.The study aimed to identify the molecular mechanism by which overexpression of glial cell line-derived neurotrophic factor (GDNF) facilitates the differentiation of ADSCs into Schwann cells, enhancing nerve regeneration after injury. In vitro, ADSCs overexpressing GDNF for 48 h exhibited changes in their morphology, with 80% of the cells having two or more prominences. Compared with that of ADSCs, GDNF-ADSCs exhibited increased expression of the Schwann cell marker S100, nerve damage repair-related factors.ADSC cells in normal culture and ADSC cells were overexpressing GDNF(GDNF-ADSCs) were analysed using TMT-Based Proteomic Analysis and revealed a significantly higher expression of MTA1 in GDNF-ADSCs than in control ADSCs. Hes1 expression was significantly higher in GDNF-ADSCs than in ADSCs and decreased by MTA1 silencing, along with a simultaneous decrease in the expression of S100 and nerve damage repair factors. These findings indicate that GDNF promotes the differentiation of ADSCs into Schwann cells and induces factors that accelerate peripheral nerve damage repair.
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Factor Neurotrófico Derivado de la Línea Celular Glial , Proteómica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Regeneración Nerviosa , Tejido Adiposo , Diferenciación Celular , Células de SchwannRESUMEN
RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors.
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Receptores Notch , Transducción de Señal , Factor de Transcripción HES-1 , Proteínas Supresoras de Tumor , Humanos , Proteínas Nucleares/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Recent studies have shown that the expression of bHLH transcription factors Hes1, Ascl1, and Oligo2 has an oscillating balance in neural stem cells (NSCs) to maintain their self-proliferation and multi-directional differentiation potential. This balance can be disrupted by exogenous stimulation. Our previous work has identified that electrical stimulation could induce neuronal differentiation of mouse NSCs. METHODS: To further evaluate if physiological electric fields (EFs)-induced neuronal differentiation is related to the expression patterns of bHLH transcription factors Hes1, Ascl1, and Oligo2, mouse embryonic brain NSCs were used to investigate the expression changes of Ascl1, Hes1 and Oligo2 in mRNA and protein levels during EF-induced neuronal differentiation. RESULTS: Our results showed that NSCs expressed high level of Hes1, while expression of Ascl1 and Oligo2 stayed at very low levels. When NSCs exited proliferation, the expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level. Oligo2 showed irregular changes in low expression level. EF-stimulation significantly increased the expression of Ascl1 at mRNA and protein levels accompanied by an increased percentage of neuronal differentiation. What's more, over-expression of Hes1 inhibited the neuronal differentiation induced by EFs. CONCLUSION: EF-stimulation directed neuronal differentiation of NSCs by promoting the continuous accumulation of Ascl1 expression and decreasing the expression of Hes1.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo , Factor de Transcripción 2 de los Oligodendrocitos , Factor de Transcripción HES-1 , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Estimulación Eléctrica , ARN Mensajero/genética , Factor de Transcripción HES-1/genética , Factor de Transcripción 2 de los Oligodendrocitos/genéticaRESUMEN
Here, we study the dynamical expression of endogenously labeled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in estrogen receptor (ER)+ breast cancer cells. We find that Hes1 shows oscillatory expression with â¼25 h periodicity and during each cell cycle has a variable peak in G1, a trough around G1-S transition, and a less variable second peak in G2/M. Compared to other subpopulations, the cell cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave, but preceding mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration, and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations by enforcing sustained expression slows down the cell cycle, impairs proliferation, abolishes the dynamic expression of p21, and increases the percentage of CD44HighCD24Low cells. Reciprocally, blocking the cell cycle causes an elongation of Hes1 periodicity, suggesting a bidirectional interaction of the Hes1 oscillator and the cell cycle. We propose that Hes1 oscillations are functionally important for the efficient progression of the cell cycle and that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations.
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Neoplasias de la Mama/metabolismo , Ciclo Celular , Ritmo Circadiano , Receptores de Estrógenos/metabolismo , Factor de Transcripción HES-1/metabolismo , Humanos , Células MCF-7 , Células Madre Neoplásicas/fisiologíaRESUMEN
BACKGROUND: Ischemic stroke is the most common form of stroke and the second most common cause of death and incapacity worldwide. Its pathogenesis and treatment have been the focus of considerable research. In traditional Chinese medicine, the root of Mongolian astragalus has been important in the treatment of stroke since ancient times. Astragalus polysaccharide (APS) is a key active ingredient of astragalus and offers therapeutic potential for conditions affecting the neurological system, the heart, cancer, and other disorders. However, it is not yet known how APS works to protect against ischemic stroke. METHODS: Rats were subjected to middle cerebral artery occlusion (MCAO) to imitate localized cerebral ischemia. Each of four experimental groups (normal, sham, MCAO, and MCAO+APS) contained 12 adult male Sprague-Dawley (SD) rats selected randomly from a total of 48 rats. Following successful establishment of the model, rats in the MCAO+APS group received intraperitoneal injection of APS (50 mg/kg) once daily for 14 days, whereas all other groups received no APS. The Bederson nerve function score and the forelimb placement test were used to detect motor and sensory function defects, while Nissl staining was used to investigate pathological defects in the ventroposterior thalamic nucleus (VPN). Immunohistochemical staining and Western blot were used to evaluate the expression of Neurogenic locus notch homolog protein 1 (Notch1), hairy and enhancer of split 1 (Hes1), phospho-nuclear factor-κB p65 (p-NFκB p65), and nuclear factor-κB p65 (NFκB p65) proteins in the VPN on the ischemic side of MCAO rats. RESULTS: APS promoted the recovery of sensory and motor function, enhanced neuronal morphology, increased the number of neurons, and inhibited the expression of Notch1/NFκB signaling pathway proteins in the VPN of rats with cerebral ischemia. CONCLUSION: After cerebral ischemia, APS can alleviate symptoms of secondary damage to the VPN, which may be attributed to the suppression of the Notch1/NFκB pathway.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Ratas , Masculino , Animales , Ratas Sprague-Dawley , FN-kappa B/metabolismo , Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Transducción de Señal , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular Isquémico/complicaciones , Receptor Notch1/metabolismo , Receptor Notch1/uso terapéuticoRESUMEN
Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2-50 µM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders.
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Indicán , Células Madre Mesenquimatosas , Osteogénesis , Receptores de Hidrocarburo de Aril , Transducción de Señal , Factor de Transcripción HES-1 , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Osteogénesis/efectos de los fármacos , Ratones , Factor de Transcripción HES-1/metabolismo , Factor de Transcripción HES-1/genética , Transducción de Señal/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.
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Factor 2 de Diferenciación de Crecimiento , Células Madre Mesenquimatosas , Animales , Ratones , Diferenciación Celular/genética , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.
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Neoplasias Óseas , Dolor en Cáncer , Ratas , Animales , Dolor en Cáncer/etiología , Neoplasias Óseas/complicaciones , Transducción de Señal/fisiología , Dolor , Médula EspinalRESUMEN
Serious intestinal side-effects that target the NOTCH-HES1 pathway in human cancer differentiation therapy make it necessary to understand the pathway at the human organ level. Herein, we endogenously introduced HES1-/- mutations into human embryonic stem cells (hESCs) and differentiated them into human intestinal organoids (HIO). The HES1-/- hESCs retained ES cell properties and showed gene expression patterns similar to those of wild-type hESCs when they differentiated into definitive endoderm and hindgut. During the formation of the HES1-/- lumen we noted an impaired development of mesenchymal cells in addition to the increased differentiation of secretory epithelium. RNA-Seq revealed that inhibited development of the mesenchymal cells may have been due to a downregulation of WNT5A signaling. Overexpression of HES1 and silencing of WNT5A in the intestinal fibroblast cell line CCD-18Co indicated that HES1 was involved in the activation of WNT5A-induced fibroblast growth and migration, suggesting the likelihood of the Notch pathway in epithelial-mesenchymal crosstalk. Our results facilitated the identification of more precise underlying molecular mechanisms displaying distinct roles in HES1 signaling in stromal and epithelial development in human intestinal mucosa.
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Mucosa Intestinal , Intestinos , Humanos , Diferenciación Celular/genética , Mucosa Intestinal/metabolismo , Transducción de Señal/fisiología , Células Madre Embrionarias , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMEN
The expression of the transcriptional repressor Hes1 oscillates in many cell types, including neural progenitor cells (NPCs), but the significance of Hes1 oscillations in development is not fully understood. To examine the effect of altered oscillatory dynamics of Hes1, we generated two types of Hes1 knock-in mice, a shortened (type-1) and an elongated (type-2) Hes1 gene, and examined their phenotypes focusing on neural development. Although both mutations affected Hes1 oscillations, the type-1 mutation dampened Hes1 oscillations more severely, resulting in much lower amplitudes. The average levels of Hes1 expression in type-1 mutant NPCs were also lower than in wild-type NPCs but similar to or slightly higher than those in Hes1 heterozygous mutant mice, which exhibit no apparent defects. Whereas type-2 mutant mice were apparently normal, type-1 mutant mice displayed smaller brains than wild-type mice and upregulated proneural gene expression. Furthermore, proliferation of NPCs decreased and cell death increased in type-1 mutant embryos. When Hes3 and Hes5 were additionally deleted, neuronal differentiation was also accelerated, leading to microcephaly. Thus, robust Hes1 oscillations are required for maintenance and proliferation of NPCs and the normal timing of neurogenesis, thereby regulating brain morphogenesis.
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Encéfalo/embriología , Neuronas/fisiología , Oscilometría , Factor de Transcripción HES-1/fisiología , Animales , Muerte Celular , Diferenciación Celular , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Procesamiento de Imagen Asistido por Computador , Intrones , Masculino , Ratones , Modelos Teóricos , Mutación , Células-Madre Neurales/citología , NeurogénesisRESUMEN
Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.
Asunto(s)
Cartílago Articular , Síndrome Metabólico , Osteoartritis , Humanos , Femenino , Leptina/farmacología , Condrocitos/metabolismo , Síndrome Metabólico/metabolismo , Osteoartritis/metabolismo , Fenotipo , Cartílago Articular/metabolismo , Células CultivadasRESUMEN
Methotrexate (MTX)-induced hepatotoxicity is a serious adverse effect that may limit its use. Therefore, eligible drugs to ameliorate MTX-induced hepatotoxicity are required. l-Carnitine (LC) is a natural molecule with beneficial metabolic effects and infliximab (INF) is an anti-inflammatory monoclonal antibody against tumor necrosis factor-alpha (TNF-α). Recently, Notch1/Hes-1 signaling was found to play a key role in the pathogenesis of liver injury. However, its role in MTX-induced hepatotoxicity is unclear. This study aimed to evaluate the modulatory effects of LC or INF on MTX-induced hepatotoxicity and to explore the underlying mechanism with emphasis on the Notch1/Hes-1 signaling pathway. Sixty rats were randomized into six groups (n = 10): (1) control (saline); (2) MTX (20 mg/kg MTX, intraperitoneal [ip], once); (3) LC group (500 mg/kg ip, 5 days); (4) INF (7 mg/kg INF ip, once); (5) MTX+LC (20 mg/kg ip, once, 500 mg/kg ip, 5 days, respectively); (6) MTX+INF (20 mg/kg ip, once, 7 mg/kg INF ip, once, respectively). Oxidative stress, inflammatory markers, and Notch1/Hes-1 were investigated. MTX induced the expression of Notch1 and Hes-1 proteins and increased the levels of TNF-α, interleukin (IL)-6, and IL-1ß in the liver. Cotreatment with LC or INF showed apparent antioxidant and anti-inflammatory effects. Interestingly, the downregulation of Notch1 and Hes-1 expression was more prominent in LC cotreatment as compared with INF. In conclusion, LC or INF attenuates MTX-induced hepatotoxicity through modulation of Notch1/Hes-1 signaling. The LC ameliorative effect against MTX-induced hepatotoxicity is significantly better than that of INF. Therefore, LC cotreatment may present a safe and therapeutically effective therapy in alleviating MTX-induced hepatotoxicity.