RESUMEN

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent.

RESUMEN

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.

Asunto(s)

Gastrinas , Estómago , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células Enteroendocrinas/metabolismo , Ratones Noqueados

RESUMEN

BACKGROUND AND AIM: Mucosal healing has emerged as a desirable treatment goal for patients with ulcerative colitis (UC). Healing of mucosal wounds involves epithelial cell proliferation and differentiation, and Y-box transcription factor ZONAB has recently been identified as the key modulator of intestinal epithelial restitution. METHODS: We studied the characteristics of UXT-V1 expression in UC patients using immunohistochemistry and qPCR. The functional role of UXT-V1 in the colonic epithelium was investigated using lentivirus-mediated shRNA in vitro and ex vivo. Through endogenous Co-immunoprecipitation and LC-MS/MS, we identified ZONAB as a UXT-V1-interactive protein. RESULTS: Herein, we report that UXT-V1 promotes differentiation of intestinal epithelial cells by regulating the nuclear translocation of ZONAB. UXT-V1 was upregulated in the intestinal epithelia of UC patients compared with that of healthy controls. Knocking down UXT-V1 in NCM-460 cells led to the enrichment of pathways associated with proliferation and differentiation. Furthermore, the absence of UXT-V1 in cultured intestinal epithelial cells and colonic organoids inhibited differentiation to the goblet cell phenotype. Mechanistically, the loss of UXT-V1 in the intestinal epithelial cells allowed nuclear translocation of ZONAB, wherein it regulated the transcription of differentiation-related genes, including AML1 and KLF4. CONCLUSION: Taken together, our study reveals a potential role of UXT-V1 in regulating epithelial cell differentiation, proving a molecular basis for mucosal healing in UC.

Asunto(s)

Colitis Ulcerosa , Humanos , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Mucosa Intestinal/metabolismo , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo

RESUMEN

Objective: To reduce the risk of locoregional recurrence, the addition of neoadjuvant concurrent chemoradiotherapy (CCRT) is recommended before surgical management for rectal cancer patients. However, despite identical tumor histology, individual patient response to neoadjuvant CCRT varies greatly. Accordingly, a comprehensive molecular characterization that is used to predict CCRT efficacy is instantly needed. Methods: Pearson's chi-squared test was utilized to correlate dehydrogenase/reductase 9 (DHRS9) expression with clinicopathological features. Survival curves were created applying the Kaplan-Meier method, and the log-rank test was conducted to compare prognostic utility between high and low DHRS9 expression groups. Multivariate Cox proportional hazards regression analysis was applied to identify independent prognostic biomarkers based on variables with prognostic utility at the univariate level. Results: Utilizing a public transcriptome dataset, we identified that the DHRS9 gene is the most considerably upregulated gene related to epithelial cell differentiation (GO: 0030855) among rectal cancer patients with CCRT resistance. Employing immunohistochemical staining, we also demonstrated that high DHRS9 immunoexpression is considerably associated with an aggressive clinical course and CCRT resistance in our rectal cancer cohort. Among all variables with prognostic utility at the univariate level, only high DHRS9 immunoexpression was independently unfavorably prognostic of all three endpoints (all p ≤ 0.048) in the multivariate analysis. In addition, applying bioinformatic analysis, we also linked DHRS9 with unrevealed functions, such as keratan sulfate and mucin synthesis which may be implicated in CCRT resistance. Conclusion: Altogether, DHRS9 expression may serve as a helpful predictive and prognostic biomarker and assist decision-making for rectal cancer patients who underwent neoadjuvant CCRT.

Asunto(s)

Sulfato de Queratano , Neoplasias del Recto , Humanos , Sulfato de Queratano/uso terapéutico , Inmunohistoquímica , Supervivencia sin Enfermedad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias del Recto/terapia , Quimioradioterapia , Terapia Neoadyuvante , Pronóstico , Mucinas/uso terapéutico , Oxidorreductasas/uso terapéutico , Estudios Retrospectivos

RESUMEN

Delayed and chronic wounds result from the dysregulation of molecular and cellular events associated with wound healing, including migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. Adipose tissue is an abundant, easily accessible, and rich source of mesenchymal stem cells (MSCs) with high therapeutic potential. In addition to their capability to differentiate into various lineages with specialized functions, adipose-derived MSCs (AMSCs) can mediate to the wound repair process through the secretion of different growth factors and mediators rather than making structural contribution alone. Adipose-derived MSCs mediate the formation of blood vessels, recruit progenitor cells, stimulate cell differentiation and ECM formation, and promote wound healing by releasing immune mediators and exosomes. Herein, we discuss and review the therapeutic potential of AMSCs for wound repair via acceleration of wound closure, re-epithelialization, enhancement of angiogenesis and immunomodulation of prolonged inflammatory responses, as well as the current challenges in clinical implementation.

Asunto(s)

Células Madre Mesenquimatosas , Humanos , Células Madre Mesenquimatosas/metabolismo , Cicatrización de Heridas/fisiología , Diferenciación Celular , Células Madre , Matriz Extracelular

RESUMEN

Epithelial cells that form the kidney proximal tubule (PT) rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients-a key requisite for homeostasis. The endolysosome stands at the crossroads of this sophisticated network, internalizing molecules through endocytosis, sorting receptors and nutrient transporters, maintaining cellular quality control via autophagy, and toggling the balance between PT differentiation and cell proliferation. Dysregulation of such endolysosome-guided trafficking pathways might thus lead to a generalized dysfunction of PT cells, often causing chronic kidney disease and life-threatening complications. In this review, we highlight the biological functions of endolysosome-residing proteins from the perspectives of understanding-and potentially reversing-the pathophysiology of rare inherited diseases affecting the kidney PT. Using cystinosis as a paradigm of endolysosome disease causing PT dysfunction, we discuss how the endolysosome governs the homeostasis of specialized epithelial cells. This review also provides a critical analysis of the molecular mechanisms through which defects in autophagy pathways can contribute to PT dysfunction, and proposes potential interventions for affected tissues. These insights might ultimately accelerate the discovery and development of new therapeutics, not only for cystinosis, but also for other currently intractable endolysosome-related diseases, eventually transforming our ability to regulate homeostasis and health.

Asunto(s)

Cistinosis , Enfermedades Metabólicas , Autofagia , Cistinosis/metabolismo , Ecosistema , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Enfermedades Metabólicas/metabolismo

RESUMEN

Long-noncoding RNAs (lncRNAs) have numerous biological functions controlling cell differentiation and tissue development. The knowledge about the role of lncRNAs in human lungs remains limited. Here we found the regulatory role of the terminal differentiation-induced lncRNA (TINCR) in bronchial cell differentiation. RNA in situ hybridization revealed that TINCR was mainly expressed in bronchial epithelial cells in normal human lung. We performed RNA sequencing analysis of normal human bronchial epithelial cells (NHBECs) with or without TINCR inhibition and found the differential expression of 603 genes, which were enriched for cell adhesion and migration, wound healing, extracellular matrix organization, tissue development and differentiation. To investigate the role of TINCR in the differentiation of NHBECs, we employed air-liquid interface culture and 3D organoid formation assay. TINCR was upregulated during differentiation, loss of TINCR significantly induced an early basal-like cell phenotype (TP63) and a ciliated cell differentiation (FOXJ1) in late phase and TINCR overexpression suppressed basal cell phenotype and the differentiation toward to ciliated cells. Critical regulators of differentiation such as SOX2 and NOTCH genes (NOTCH1, HES1, and JAG1) were significantly upregulated by TINCR inhibition and downregulated by TINCR overexpression. RNA immunoprecipitation assay revealed that TINCR was required for the direct bindings of Staufen1 protein to SOX2, HES1, and JAG1 mRNA. Loss of Staufen1 induced TP63, SOX2, NOTCH1, HES1, and JAG1 mRNA expressions, which TINCR overexpression suppressed partially. In conclusion, TINCR is a novel regular of bronchial cell differentiation, affecting downstream regulators such as SOX2 and NOTCH genes, potentially in coordination with Staufen1.

Asunto(s)

Bronquios/metabolismo , Diferenciación Celular , Células Epiteliales/metabolismo , ARN Largo no Codificante/metabolismo , Bronquios/citología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Humanos , Fenotipo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal

RESUMEN

Prenatal smoke exposure is a risk factor for impaired lung development in children. Recent studies have indicated that amphiregulin (AREG), which is a ligand of the epidermal growth factor receptor (EGFR), has a regulatory role in airway epithelial cell differentiation. In this study, we investigated the effect of prenatal smoke exposure on lung epithelial cell differentiation and linked this with AREG-EGFR signaling in 1-day-old mouse offspring. Bronchial and alveolar epithelial cell differentiations were assessed by immunohistochemistry. Areg, epidermal growth factor (Egf), and mRNA expressions of specific markers for bronchial and alveolar epithelial cells were assessed by RT-qPCR. The results in neonatal lungs were validated in an AREG-treated three-dimensional mouse lung organoid model. We found that prenatal smoke exposure reduced the number of ciliated cells and the expression of the cilia-related transcription factor Foxj1, whereas it resulted in higher expression of mucus-related transcription factors Spdef and Foxm1 in the lung. Moreover, prenatally smoke-exposed offspring had higher numbers of alveolar epithelial type II cells (AECII) and lower expression of the AECI-related Pdpn and Gramd2 markers. This was accompanied by higher expression of Areg and lower expression of Egf in prenatally smoke-exposed offspring. In bronchial organoids, AREG treatment resulted in fewer ciliated cells and more basal cells when compared with non-treated bronchiolar organoids. In alveolar organoids, AREG treatment led to more AECII cells than non-treated AECII cells. Taken together, the observed impaired bronchial and alveolar cell development in prenatally smoke-exposed neonatal offspring may be induced by increased AREG-EGFR signaling.

Asunto(s)

Anfirregulina/metabolismo , Anfirregulina/farmacología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/metabolismo , Humo/efectos adversos , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Nicotiana/efectos adversos

RESUMEN

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.

Asunto(s)

Enfermedad de Crohn/genética , Células Epiteliales/fisiología , Íleon/citología , Glicoproteínas de Membrana/metabolismo , Transcriptoma , Adulto , Células CACO-2 , Estudios de Casos y Controles , Diferenciación Celular , Enfermedad de Crohn/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/metabolismo , Glicoproteínas de Membrana/genética , Consumo de Oxígeno

RESUMEN

BACKGROUND: The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. METHODS: We established a novel stepwise protocol with transwell culture and an air-liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARß and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. RESULTS: We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. CONCLUSION: We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

Asunto(s)

Células Epiteliales/citología , Células Madre Pluripotentes Inducidas/citología , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Esófago/citología , Esófago/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones , Receptores de Ácido Retinoico/efectos de los fármacos , Receptor alfa de Ácido Retinoico/efectos de los fármacos , Receptor alfa de Ácido Retinoico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Receptor de Ácido Retinoico gamma

RESUMEN

Zinc finger homeobox 3 (ZFHX3) is a transcription factor that regulates multiple cellular processes including cell proliferation, differentiation and neoplastic development. It is also involved in the function of steroid hormones estrogen and progesterone and the peptide hormone prolactin in mammary epithelial cells. In this study, we investigated whether and how ZFHX3 regulates intracellular calcium homeostasis in mammary epithelial cells. We found that ZFHX3 affected both store operated calcium entry and store independent calcium entry (SOCE and SICE). Simultaneously, the expression of the calcium channel TRPV6 was regulated by ZFHX3, as demonstrated by expression analysis and luciferase reporter assay. In cells with knockdown of ZFHX3, calcium entry was partially rescued by the overexpression of wild type but not the pore mutants of TRPV6. In addition, overexpression of TRPV6 promoted differentiation of the MCF10A mammary epithelial cells in three-dimensional culture, which is consistent with our previous findings that ZFHX3 is essential for mammary gland differentiation. These findings suggest that ZFHX3 plays an important role in intracellular calcium homeostasis in mammary epithelial cells, at least in part, by regulating TRPV6.

Asunto(s)

Canales de Calcio/metabolismo , Calcio/metabolismo , Células Epiteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales de Calcio/genética , Células Cultivadas , Células HEK293 , Humanos , Canales Catiónicos TRPV/genética

RESUMEN

Probiotic lactobacilli and their exopolysaccharides (EPS) are thought to modulate mucosal homeostasis; however, their mechanisms remain elusive. Thus, we tried to clarify the role of exopolysaccharides from Lactobacillus plantarum NCU116 (EPS116) in the intestinal mucosal homeostasis. Our results indicated that EPS116 regulated the colon mucosal healing and homeostasis, enhanced the goblet cell differentiation, and promoted the expression of Muc2 gene in vivo and in vitro. Further experiments showed that EPS116 promoted the expression and phosphorylation of transcription factor c-Jun and facilitated its binding to the promoter of Muc2. Moreover, knocking down c-Jun or inhibiting its function in LS 174T cells treated with EPS116 led to decreased expression of Muc2, implying that EPS116 promoted the colonic mucosal homeostasis and Muc2 expression via c-Jun. Therefore, our study uncovered a novel model where EPS116 enhanced colon mucosal homeostasis by controlling the epithelial cell differentiation and c-Jun/Muc2 signaling.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Lactobacillus plantarum/química , Mucina 2/metabolismo , Polisacáridos Bacterianos/farmacología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Línea Celular Tumoral , Colon/citología , Colon/metabolismo , Colon/fisiopatología , Homeostasis/efectos de los fármacos , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Mucina 2/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal/efectos de los fármacos

RESUMEN

The members of the annexin family of calcium- and phospholipid-binding proteins participate in different cellular processes. Annexin A2 binds to S100A10, forming a functional heterotetrameric protein that has been involved in many cellular functions, such as exocytosis, endocytosis, cell junction formation, and actin cytoskeleton dynamics. Herein, we studied annexin A2 cellular movements and looked for its partners during epithelial cell differentiation. By using immunofluorescence, mass spectrometry (MS), and western blot analyses after S100A10 affinity column separation, we identified several annexin A2-S100A10 partner candidates. The association of putative annexin A2-S100A10 partner candidates obtained by MS after column affinity was validated by immunofluorescence and sucrose density gradient separation. The results show that three proteins are clearly associated with annexin A2: E-cadherin, actin, and caveolin 1. Overall, the data show that annexin A2 can associate with molecular complexes containing actin, caveolin 1, and flotillin 2 before epithelial differentiation and with complexes containing E-cadherin, actin, and caveolin 1, but not flotillin 2 after cell differentiation. The results indicate that actin, caveolin 1, and E-cadherin are the principal protein partners of annexin A2 in epithelial cells and that the serine phosphorylation of the N-terminal domain does not play an essential role during epithelial cell differentiation.

Asunto(s)

Anexina A2/genética , Diferenciación Celular , Células de Riñón Canino Madin Darby/citología , Células de Riñón Canino Madin Darby/metabolismo , Animales , Anexina A2/metabolismo , Células Cultivadas , Perros , Humanos , Mutación , Fosforilación , Serina/metabolismo

RESUMEN

The differentiation of alveolar epithelial type I (AT1) and type II (AT2) cells is essential for the lung gas exchange function. Disruption of this process results in neonatal death or in severe lung diseases that last into adulthood. We developed live imaging techniques to characterize the mechanisms that control alveolar epithelial cell differentiation. We discovered that mechanical forces generated from the inhalation of amniotic fluid by fetal breathing movements are essential for AT1 cell differentiation. We found that a large subset of alveolar progenitor cells is able to protrude from the airway epithelium toward the mesenchyme in an FGF10/FGFR2 signaling-dependent manner. The cell protrusion process results in enrichment of myosin in the apical region of protruded cells; this myosin prevents these cells from being flattened by mechanical forces, thereby ensuring their AT2 cell fate. Our study demonstrates that mechanical forces and local growth factors synergistically control alveolar epithelial cell differentiation.

Asunto(s)

Células Epiteliales Alveolares/citología , Diferenciación Celular , Movimiento Celular/fisiología , Embrión de Mamíferos/citología , Factor 10 de Crecimiento de Fibroblastos/fisiología , Fenómenos Mecánicos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/fisiología , Células Epiteliales Alveolares/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal

RESUMEN

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Asunto(s)

Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Desarrollo Embrionario/efectos de los fármacos , Epitelio/metabolismo , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Progesterona/metabolismo , Útero/metabolismo , Animales , Células Cultivadas , Implantación del Embrión/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Embarazo , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Útero/efectos de los fármacos , Útero/patología

RESUMEN

AIM: To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS: For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS: CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION: Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Asunto(s)

Diferenciación Celular , Colon/metabolismo , Enterocitos/fisiología , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Uniones Adherentes/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Colon/citología , Hormona Liberadora de Corticotropina/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Regulación hacia Abajo , Enterocitos/efectos de los fármacos , Humanos , Enfermedades Inflamatorias del Intestino/etiología , Mucosa Intestinal/efectos de los fármacos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Microscopía Confocal , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Permeabilidad , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Psicológico/complicaciones , Uniones Estrechas/metabolismo , Urocortinas/metabolismo

RESUMEN

Following loss of functional small bowel surface area due to surgical resection for therapy of Crohn's disease, ischemia, trauma or other disorders, the remnant gut undergoes a morphometric and functional compensatory adaptive response which has been best characterized in preclinical models. Increased crypt cell proliferation results in increased villus height, crypt depth and villus hyperplasia, accompanied by increased nutrient, fluid and electrolyte absorption. Clinical observations suggest that functional adaptation occurs in humans. In the immediate postoperative period, patients with substantial small bowel resection have massive fluid and electrolyte loss with reduced nutrient absorption. For many patients, the adaptive response permits partial or complete weaning from parenteral nutrition (PN), within two years following resection. However, others have life-long PN dependence. An understanding of the molecular mechanisms that regulate the gut adaptive response is critical for developing novel therapies for short bowel syndrome. Herein we present a summary of key studies that seek to elucidate the mechanisms that regulate post-resection adaptation, focusing on stem and crypt cell proliferation, epithelial differentiation, apoptosis, enterocyte function and the role of growth factors and the enteric nervous system.

Asunto(s)

Adaptación Fisiológica , Intestino Delgado/fisiopatología , Síndrome del Intestino Corto/fisiopatología , Sistema Nervioso Entérico/fisiopatología , Humanos , Mucosa Intestinal , Nutrición Parenteral

RESUMEN

Non-coding RNAs (ncRNAs) are untranslated RNA molecules that function to regulate the expression of numerous genes and associated biochemical pathways and cellular functions. NcRNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs). They participate in the regulation of all developmental processes and are frequently aberrantly expressed or functionally defective in disease. This Chapter will focus on the role of ncRNAs, in particular miRNAs and lncRNAs, in mammary gland development and disease.

Asunto(s)

Enfermedades de la Mama/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Humanas/crecimiento & desarrollo , ARN no Traducido/metabolismo , Animales , Enfermedades de la Mama/genética , Enfermedades de la Mama/patología , Femenino , Humanos , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , ARN no Traducido/genética

RESUMEN

Undifferentiated metanephric mesenchymes, when grown in transfilter contact with an inductor tissue, differentiate into epithelial kidney tubules. The segregation of these tubules into the different segments of the nephron was studied.In explants grown in continuous transfilter contact with the inductor, immunohistological and histochemical markers specific for the glomerular epithelial, proximal tubule, and distal tubule cells appeared by 4 1/2 to 5 days, 4 days, and 5 days of culture, respectively. Electron microscopy confirmed segmentation of the tubules: Avascular glomeruli with glomerular basement membrane material, proximal tubules with brush border formation, and distal tubules were revealed in the explants after 5 days of culture.A short (18 h) transfilter induction pulse, followed by a prolonged subculture in the absence of the inductor, resulted sulted in the formation of only a small number of tubules in about half of the explants while the rest remained undifferentiated. These scarce tubules showed the markers specific for the proximal tubules only. The segregation of all three aspects of the nephron seems to be programmed during the transfilter culture, but apparently the time needed for the induction of the different segments varies.