RESUMO
This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2'-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing.
Assuntos
Proliferação de Células , Epitélio Corneano , Exossomos , Células-Tronco Pluripotentes Induzidas , Organoides , Cicatrização , Exossomos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/metabolismo , Animais , Epitélio Corneano/metabolismo , Camundongos , Retina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de SinaisRESUMO
X-linked juvenile retinoschisis (XLRS) is a hereditary retinal degeneration affecting young males caused by mutations in the retinoschisin (RS1) gene. We generated human induced pluripotent stem cells (hiPSCs) from XLRS patients and established three-dimensional retinal organoids (ROs) for disease investigation. This disease model recapitulates the characteristics of XLRS, exhibiting defects in RS1 protein production and photoreceptor cell development. XLRS ROs also revealed dysregulation of Na/K-ATPase due to RS1 deficiency and increased ERK signaling pathway activity. Transcriptomic analyses of XLRS ROs showed decreased expression of retinal cells, particularly photoreceptor cells. Furthermore, relevant recovery of the XLRS phenotype was observed when co-cultured with control ROs derived from healthy subject during the early stages of differentiation. In conclusion, our in vitro XLRS RO model presents a valuable tool for elucidating the pathophysiological mechanisms underlying XLRS, offering insights into disease progression. Additionally, this model serves as a robust platform for the development and optimization of targeted therapeutic strategies, potentially improving treatment outcomes for patients with XLRS.
Assuntos
Proteínas do Olho , Células-Tronco Pluripotentes Induzidas , Organoides , Retina , Retinosquise , Humanos , Retinosquise/genética , Retinosquise/metabolismo , Retinosquise/patologia , Organoides/metabolismo , Organoides/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Retina/metabolismo , Retina/patologia , Diferenciação Celular/genética , Modelos BiológicosRESUMO
Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.
Assuntos
Exossomos , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Degeneração Retiniana , Cirurgiões , Ratos , Humanos , Animais , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Exossomos/metabolismo , Espécies Reativas de Oxigênio , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.
Assuntos
Receptores CCR2/metabolismo , Degeneração Retiniana/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/metabolismoRESUMO
Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , TransgenesRESUMO
The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.
Assuntos
Chaperonas Moleculares/metabolismo , Neurogênese/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Coenzimas/metabolismo , Células HEK293 , Humanos , CamundongosRESUMO
To investigate the characteristics of regenerated retinal pigment epithelial (RPE) cells after retinal laser photocoagulation in diabetic mice. C57BL/6J mice were used to induce diabetes using intraperitoneal injection of streptozotocin. The proliferation of RPE cells after laser photocoagulation was determined using the 5-ethynyl-2'-deoxyuridine (EdU) assay in both diabetic and wild-type mice. The morphological changes of RPE cells were evaluated by using Voronoi diagram from immunostaining for ß-catenin. Characteristics of regenerated cells were evaluated by quantifying the mRNA and protein levels of RPE and epithelial-mesenchymal transition (EMT) markers. There were significantly less EdU-positive cells in laser-treated areas in diabetic mice than wild-type mice. Hexagonality was extensively lost in diabetic mice. Many EdU-positive cells were co-localized with Otx2-positive cells in the center of the laser-treated areas in wild-type mice, but only EdU-positive cells were widely distributed in diabetic mice. Quantitative analysis of mRNA and protein levels showed that the expression levels of RPE markers, Pax6, Mitf, and Otx2, were significantly decreased in RPE of diabetic mice compared with that of wild-type mice, whereas the expression levels of EMT markers, vimentin and fibronectin, were significantly increased. The proliferation and hexagonality of regenerating RPE cells were impaired after laser photocoagulation, and the regenerated RPE cells lost their original properties in diabetic mice. Further clinical research is needed to elucidate the RPE response after laser photocoagulation in diabetic patients.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Fotocoagulação a Laser , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , Animais , Glicemia/metabolismo , Peso Corporal , Proliferação de Células/efeitos da radiação , Forma Celular/efeitos da radiação , Diabetes Mellitus Experimental/sangue , Transição Epitelial-Mesenquimal , Fibronectinas/metabolismo , Camundongos Endogâmicos C57BL , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/metabolismo , Vimentina/metabolismoRESUMO
To investigate the role of Wnt/ß-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of ß-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by ß-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed ß-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/ß-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/ß-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Fotocoagulação a Laser , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos da radiação , Via de Sinalização Wnt , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Forma Celular/efeitos da radiação , Fluorescência , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores de Tempo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/efeitos da radiação , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/metabolismoRESUMO
PURPOSE: This study investigated microglia and inflammatory cell responses after selective retina therapy (SRT) with microsecond-pulsed laser in comparison to continuous-wave laser photocoagulation (cwPC). METHODS: Healthy C57BL/6 J mice were treated with either a train of short pulses (SRT; 527-nm, Q-switched, 1.7-µs pulse) or a conventional thermal continuous-wave (532-nm, 100-ms pulse duration) laser. The mice were sacrificed and their eyes were enucleated 1, 3, 7, and 14 days after both laser treatments. Pattern of cell death on retinal section was evaluated by TUNEL assay, and the distribution of activated inflammatory cells and glial cells were observed under immunohistochemistry. Consecutive changes for the expression of cytokines such as IL-1ß, TNF-α, and TGF-ß were also examined using immunohistochemistry, and compared among each period after quantification by Western blotting. RESULTS: The numbers of TUNEL-positive cells in the retinal pigment epithelium (RPE) layer did not differ in SRT and cwPC lesions, but TUNEL-positive cells in neural retinas were significantly less on SRT. Vague glial cell activation was observed in SRT-treated lesions. The population of inflammatory cells was also significantly decreased after SRT, and the cells were located in the RPE layer and subretinal space. Proinflammatory cytokines, including IL-1ß and TNF-α, showed significantly lower levels after SRT; conversely, the level of TGF-ß was similar to the cwPC-treated lesion. CONCLUSIONS: SRT resulted in selective RPE damage without collateral thermal injury to the neural retina, and apparently produced negligible glial activation. In addition, SRT showed a markedly less inflammatory response than cwPC, which may have important therapeutic implications for several macular diseases.
Assuntos
Citocinas/biossíntese , Fotocoagulação a Laser/métodos , Lasers de Estado Sólido/uso terapêutico , Neuroglia/patologia , Doenças Retinianas/cirurgia , Epitélio Pigmentado da Retina/patologia , Animais , Apoptose , Western Blotting , Contagem de Células , Modelos Animais de Doenças , Angiofluoresceinografia/métodos , Fundo de Olho , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Doenças Retinianas/diagnóstico , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Tomografia de Coerência Óptica/métodosRESUMO
Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health.
Assuntos
Apoptose/efeitos dos fármacos , Córnea/efeitos dos fármacos , Material Particulado/toxicidade , Animais , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Telencéfalo/citologia , Ácido gama-Aminobutírico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/farmacologia , Proteína Wnt3 , Proteína Wnt3ARESUMO
BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.
RESUMO
Photobiomodulation therapy has been proposed as a promising therapeutic approach for retinal degenerative diseases. However, its effect on the regenerative capacity in mammalian retina and its intracellular signalling mechanisms remain unknown. Here, we show that photobiomodulation with 670 nm light stimulates Müller glia cell cycle re-entry and dedifferentiation into a progenitor-like state in both the uninjured and injured retina. We also find that 670 nm light treatment inhibits the Hippo pathway, which is activated in Müller glia following NaIO3-induced retinal injury. YAP, a major downstream effector of the Hippo signalling pathway was translocated into the nucleus of Müller glia along with YAP dephosphorylation in retina treated with 670 nm light. Deficiency of YAP attenuated Müller glia cell cycle re-entry and dedifferentiation. Our data reveal that the Hippo-YAP signalling pathway is associated with the photostimulatory effect on regenerative response in mammalian retina, and suggest a potential therapeutic strategy for retinal degenerative diseases. [BMB Reports 2023; 56(9): 502-507].
Assuntos
Terapia com Luz de Baixa Intensidade , Doenças Retinianas , Animais , Humanos , Proliferação de Células , Retina/lesões , Retina/metabolismo , Neuroglia/metabolismo , MamíferosRESUMO
Introduction: Metabolism-associated fatty liver disease (MAFLD) is a global health concern because of its association with obesity, insulin resistance, and other metabolic abnormalities. Methylsulfonylmethane (MSM), an organic sulfur compound found in various plants and animals, exerts antioxidant and anti-inflammatory effects. Here, we aimed to assess the anti-obesity activity and autophagy-related mechanisms of Methylsulfonylmethane. Method: Human hepatoma (HepG2) cells treated with palmitic acid (PA) were used to examine the effects of MSM on autophagic clearance. To evaluate the anti-obesity effect of MSM, male C57/BL6 mice were fed a high-fat diet (HFD; 60% calories) and administered an oral dose of MSM (200 or 400 mg/kg/day). Moreover, we investigated the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin complex 1 (mTORC1)/UNC-51-like autophagy-activating kinase 1 (ULK1) signaling pathway to further determine the underlying action mechanism of MSM. Results: Methylsulfonylmethane treatment significantly mitigated PA-induced protein aggregation in human hepatoma HepG2 cells. Additionally, Methylsulfonylmethane treatment reversed the PA-induced impairment of autophagic flux. Methylsulfonylmethane also enhanced the insulin sensitivity and significantly suppressed the HFD-induced obesity and hepatic steatosis in mice. Western blotting revealed that Methylsulfonylmethane improved ubiquitinated protein clearance in HFD-induced fatty liver. Remarkably, Methylsulfonylmethane promoted the activation of AMPK and ULK1 and inhibited mTOR activity. Conclusion: Our study suggests that MSM ameliorates hepatic steatosis by enhancing the autophagic flux via an AMPK/mTOR/ULK1-dependent signaling pathway. These findings highlight the therapeutic potential of MSM for obesity-related MAFLD treatment.
RESUMO
Limbal stem cell deficiency causes conjunctivalization characterized by the covering of the corneal surface with conjunctival epithelium. However, the driving force for the encroachment of these conjunctival cells is unclear. Conjunctival stem cells are bipotent stem cells that can proliferate and differentiate into conjunctival epithelial cells and goblet cells to maintain regeneration of the conjunctival epithelium. Here, we show a robust proliferative response of conjunctival stem cells and upregulation of Wnt2b and Wnt3a gene expression in the conjunctivae of mice with induced limbal stem cell deficiency. Topical application of the Wnt/ß-catenin signaling activator CHIR resulted in increased proliferation of ΔNp63α-positive stem cells in the basal layers of the bulbar and forniceal conjunctivae and enhanced invasion of conjunctival epithelial and goblet cells into the corneal surface. We also found that in cultures of stem cells isolated from the human conjunctiva, Wnt/ß-catenin pathway activation improved the expansion of the ΔNp63α/ABCG2 double-positive cell population by promoting the proliferation and preventing the differentiation of these cells. These expanded stem cells formed a stratified epithelium containing goblet cells under airlift culture conditions. Our data reveal that Wnt/ß-catenin signaling contributes to the pathological process of limbal stem cell deficiency by promoting the self-renewal of conjunctival stem cells and suggest that these cells are a driving force in corneal conjunctivalization.
Assuntos
Células-Tronco , beta Catenina , Animais , Diferenciação Celular , Túnica Conjuntiva , Humanos , Camundongos , Células-Tronco/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.
RESUMO
Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of specific sets of transcription factors. Oct4, Sox2, and Klf4, factors that share many target genes in embryonic stem (ES) cells, are critical components in various reprogramming protocols. Nevertheless, it remains unclear whether these factors function together or separately in reprogramming. Here we show that Klf4 interacts directly with Oct4 and Sox2 when expressed at levels sufficient to induce iPS cells. Endogenous Klf4 also interacts with Oct4 and Sox2 in iPS cells and in mouse ES cells. The Klf4 C terminus, which contains three tandem zinc fingers, is critical for this interaction and is required for activation of the target gene Nanog. In addition, Klf4 and Oct4 co-occupy the Nanog promoter. A dominant negative mutant of Klf4 can compete with wild-type Klf4 to form defective Oct4/Sox2/Klf4 complexes and strongly inhibit reprogramming. In the absence of Klf4 overexpression, interaction of endogenous Klf4 with Oct4/Sox2 is also required for reprogramming. This study supports the idea that direct interactions between Klf4, Oct4, and Sox2 are critical for somatic cell reprogramming.
Assuntos
Reprogramação Celular , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mutação , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOXB1/genéticaRESUMO
Corneal wound healing is essential for the maintenance of corneal integrity and transparency and involves a series of physiological processes that depend on the proliferation of epithelial cells. However, the molecular mechanisms that control corneal epithelial cell proliferation are poorly understood. Here, we show that Sestrin2, a stress-inducible protein, is downregulated in the corneal epithelium during wound healing and that the proliferation of epithelial basal cells is enhanced in Sestrin2-deficient mice. We also show that YAP, a major downstream effector of the Hippo signaling pathway, regulates cell proliferation during corneal epithelial wound repair and that Sestrin2 suppresses its activity. Moreover, increased levels of reactive oxygen species in the Sestrin2-deficient corneal epithelium promote the nuclear localization and dephosphorylation of YAP, activating it to enhance the proliferation of corneal epithelial cells. These results reveal that Sestrin2 is a negative regulator of YAP, which regulates the proliferative capacity of basal epithelial cells, and may serve as a potential therapeutic target for corneal epithelial damage.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Peroxidases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Epitélio Corneano/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peroxidases/genética , Proteínas de Sinalização YAPRESUMO
The importance of modulating the intensity of Wnt signaling has been highlighted in various biological models, but their mechanisms remain unclear. In this study, we found that Ryk-an atypical Wnt receptor with a pseudokinase domain-has a Wnt-modulating effect in bone marrow stromal cells to control hematopoiesis-supporting activities. We first found that Ryk is predominantly expressed in the mesenchymal stromal cells (MSCs) of the bone marrow (BM) compared with hematopoietic cells. Downregulation of Ryk in MSCs decreased their clonogenic activity and ability to support self-renewing expansion of primitive hematopoietic progenitors (HPCs) in response to canonical Wnt ligands. In contrast, under high concentrations of Wnt, Ryk exerted suppressive effects on the transactivation of target genes and HPC-supporting effects in MSCs, thus fine-tuning the signaling intensity of Wnt in BM stromal cells. This ability of Ryk to modulate the HPC-supporting niche activity of MSCs was abrogated by induction of deletion mutants of Ryk lacking the intracellular domain or extracellular domain, indicating that the pseudokinase-containing intracellular domain mediates the Wnt-modulating effects in response to extracellular Wnt ligands. These findings indicate that the ability of the BM microenvironment to respond to extracellular signals and support hematopoiesis may be fine-tuned by Ryk via modulation of Wnt signaling intensity to coordinate hematopoietic activity.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Via de Sinalização Wnt , Animais , Sistemas CRISPR-Cas/genética , Ensaio de Unidades Formadoras de Colônias , Hematopoese , Homozigoto , Ligantes , Camundongos Endogâmicos C57BL , Domínios Proteicos , Receptores Proteína Tirosina Quinases/químicaRESUMO
BACKGROUND: Benzalkonium chloride (BAK), commonly used in glaucoma treatment, is an eye drop preservative with dose-dependent toxicity. Previous studies have observed the multi-functional benefits of angiogenin (ANG) against glaucoma. In our study, we evaluated ANG's cytoprotective effect on the trabecular meshwork (TM) damage induced by BAK. Additionally, we developed a plant-derived ANG fusion protein and evaluated its effect on TM structure and function. METHODS: We synthesized plant-derived ANG (ANG-FcK) by fuzing immunoglobulin G's Fc region and KDEL to conventional recombinant human ANG (Rh-ANG) purified from transgenic tobacco plants. We established a mouse model using BAK to look for degenerative changes in the TM, and to evaluate the protective effects of ANG-FcK and Rh-ANG. Intraocular pressure (IOP) was measured for 4 weeks and ultrastructural changes, deposition of fluorescent microbeads, type I and IV collagen, fibronectin, laminin and α-SMA expression were analyzed after the mice were euthanized. RESULTS: TM structural and functional degeneration were induced by 0.1% BAK instillation in mice. ANG co-treatment preserved TM outflow function, which we measured using IOP and a microbead tracer. ANG prevented phenotypic and ultrastructure changes, and that protective effect might be related to the anti-fibrosis mechanism. We observed a similar cytoprotective effect in the BAK-induced degenerative TM mouse model, suggesting that plant-derived ANG-FcK could be a promising glaucoma treatment.