RESUMEN
Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. ßPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and postwounding restitution of rat intestinal epithelial monolayers. We hypothesized that ßPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined ßPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective ßPix deletion [ßPixflox/flox/Tg(villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 conditional knockout (CKO) and control mice, we investigated the consequences of ßPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear ßPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile, with normal gross intestinal architecture but reduced small intestinal villus height, villus-to-crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite addition of Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared with littermate control mice, dextran sodium sulfate-treated ßPix-deficient mice lost more weight and had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal ßPix expression is important for enterocyte development, intestinal homeostasis, and resistance to toxic injury.NEW & NOTEWORTHY To explore the role of ßPix, a guanine nucleotide exchange factor encoded by ARHGEF7, in intestinal development and physiology, we created mice with intestinal epithelial cell Arhgef7/ßPix deficiency. We found ßPix essential for normal small intestinal epithelial cell proliferation, villus development, and mucosal resistance to injury. Moreover, Rho kinase signaling mediated developmental arrest observed in enteroids derived from ßPix-deficient small intestinal crypts. Our studies provide insights into the role Arhgef7/ßPix plays in intestinal epithelial homeostasis.
Asunto(s)
Proliferación Celular , Colitis/metabolismo , Colon/metabolismo , Enterocitos/metabolismo , Mucosa Intestinal/metabolismo , Microvellosidades/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/deficiencia , Animales , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Enterocitos/patología , Femenino , Eliminación de Gen , Humanos , Mucosa Intestinal/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/patología , Organoides , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Técnicas de Cultivo de Tejidos , Quinasas Asociadas a rho/metabolismoRESUMEN
Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M3 muscarinic receptors (M3R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M3R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M3R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Biomarcadores , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Manejo de la Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Metaloproteinasas de la Matriz/metabolismo , Terapia Molecular Dirigida , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/clasificación , Receptores Muscarínicos/genéticaRESUMEN
Cancers arising from gastrointestinal epithelial cells are common, aggressive, and difficult to treat. Progress in this area resulted from recognizing that the biological behavior of these cancers is highly dependent on bioactive molecules released by neurocrine, paracrine, and autocrine mechanisms within the tumor microenvironment. For many decades after its discovery as a neurotransmitter, acetylcholine was thought to be synthesized and released uniquely from neurons and considered the sole physiological ligand for muscarinic receptor subtypes, which were believed to have similar or redundant actions. In the intervening years, we learned this former dogma is not tenable. (1) Acetylcholine is not produced and released only by neurons. The cellular machinery required to synthesize and release acetylcholine is present in immune, cancer, and other cells, as well as in lower organisms (e.g., bacteria) that inhabit the gut. (2) Acetylcholine is not the sole physiological activator of muscarinic receptors. For example, selected bile acids can modulate muscarinic receptor function. (3) Muscarinic receptor subtypes anticipated to have overlapping functions based on similar G protein coupling and downstream signaling may have unexpectedly diverse actions. Here, we review the relevant research findings supporting these conclusions and discuss how the complexity of muscarinic receptor biology impacts health and disease, focusing on their role in the initiation and progression of gastric, pancreatic, and colon cancers.
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Neoplasias del Colon/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores Muscarínicos/metabolismo , Neoplasias Gástricas/metabolismo , Acetilcolina/metabolismo , Ácidos y Sales Biliares/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal , Microambiente TumoralRESUMEN
M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/ß alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/ß inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/ß and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 1 de la Matriz/metabolismo , Receptor Muscarínico M3/genética , Transducción de Señal/genética , Acetilcolina/farmacología , Células CACO-2 , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HT29 , Humanos , Metaloproteinasa 1 de la Matriz/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Muscarínico M3/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Although epidemiological evidence in humans and bile acid feeding studies in rodents implicate bile acids as tumor promoters, the role of endogenous bile acids in colon carcinogenesis remains unclear. In this study, we exploited mice deficient in the ileal apical sodium-dependent bile acid transporter (ASBT, encoded by SLC10A2) in whom fecal bile acid excretion is augmented more than 10-fold. Wild-type and Asbt-deficient (Slc10a2 (-/-) ) male mice were treated with azoxymethane (AOM) alone to examine the development of aberrant crypt foci, the earliest histological marker of colon neoplasia and a combination of AOM and dextran sulfate sodium to induce colon tumor formation. Asbt-deficient mice exhibited a 54% increase in aberrant crypt foci, and 70 and 59% increases in colon tumor number and size, respectively. Compared to littermate controls, Asbt-deficient mice had a striking, 2-fold increase in the number of colon adenocarcinomas. Consistent with previous studies demonstrating a role for muscarinic and epidermal growth factor receptor signaling in bile acid-induced colon neoplasia, increasing bile acid malabsorption was associated with M3 muscarinic and epidermal growth factor receptor expression, and activation of extracellular signal-related kinase, a key post-receptor signaling molecule.
Asunto(s)
Ácidos y Sales Biliares/toxicidad , Neoplasias del Colon/metabolismo , Íleon/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Animales , Ácidos y Sales Biliares/genética , Ácidos y Sales Biliares/metabolismo , Neoplasias del Colon/etiología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Heces , Humanos , Íleon/patología , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transducción de Señal/efectos de los fármacos , Simportadores/metabolismoRESUMEN
BACKGROUND: Compromised colonic butyrate production resulting from low dietary fiber or altered gut microbiota may promote colon neoplasia. Previous reports indicate these actions are mediated in part by altered levels of miRNAs, including suppressed expression of the oncogenic miR-17-92a cluster. Here, we sought to identify the mechanisms underlying these effects of butyrate in colon cancer. METHODS: miR-92a levels were measured in archived human colon cancer and adjacent normal colon specimens by microarray and quantitative RT-PCR (qPCR). The effects of butyrate and other histone deacetylase inhibitors (suberoylanilide hydroxamic acid (SAHA) and valproic acid) on primary (pri-miR17-92a), precursor and mature miR-92a were analyzed in HCT-116 and HT-29 human colon cancer cells using qPCR. The effects of butyrate, SAHA and valproic acid on protein levels of c-Myc, Drosha and p57 were measured in HCT-116 cells using immunoblotting. Regulation of C13orf25 promoter activity by butyrate was analyzed by luciferase reporter assay using modified pGL3 constructs containing a wild-type or mutated c-Myc binding site. Expression of c-Myc was modulated using siRNA or adenovirus vectors. p57 mRNA and protein were measured before and after transfection with miR-92a-mimic molecules. Following butyrate treatment and miR-92a-mimic transfection, apoptosis was analyzed by TUNEL staining and caspase-3 immunoblotting. RESULTS: Microarray, confirmed by qPCR, revealed a seven-fold increase in miR-92a levels in sporadic human colon cancer tissue compared to adjacent normal colon. Treating human colon cancer cells with butyrate reduced the levels of pri-miR17-92a, precursor and mature miR-92a, as well as c-Myc. SAHA and valproic acid had similar effects. Mutation of the c-Myc binding site diminished butyrate's inhibitory effects on C13orf25 promoter activity. Silencing c-Myc expression reduced miR-92a levels. c-Myc over-expression neutralized butyrate-induced attenuation of pri-miR17-92a. Exogenous miR-92a inhibited butyrate-induced p57 expression and reversed the beneficial actions of butyrate on colon cancer cell proliferation and apoptosis. CONCLUSIONS: Our findings identify a novel cellular mechanism whereby butyrate inhibits miR-92a transcription by reducing c-Myc, thus augmenting p57 levels. These actions diminish colon cancer cell proliferation and stimulate apoptosis. This newly described regulation of oncogenic miRNA biogenesis expands our understanding of colon cancer cell biology and identifies novel therapeutic targets.
Asunto(s)
Butiratos/farmacología , Proliferación Celular/fisiología , Neoplasias del Colon/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , MicroARNs/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proliferación Celular/genética , Neoplasias del Colon/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Genes myc/genética , Células HCT116 , Células HT29 , Inhibidores de Histona Desacetilasas/farmacología , HumanosRESUMEN
Gastrinomas are the most common neuroendocrine tumors worldwide and cause a clinical syndrome known as Zollinger-Ellison Syndrome (ZES). Increased acid production resulting from elevated gastrin levels contributes to symptoms such as abdominal pain, heartburn, and diarrhea. However, the non-specificity and overlap in the symptoms with idiopathic peptic ulcer disease and gastroesophageal reflux disease (GERD) can lead to delayed diagnosis. In this case, we describe a patient with a past medical history of GERD and a perforated gastric ulcer who continued to experience symptoms of dyspepsia and had a subsequent esophageal perforation, despite H. pylori eradication and high-dose proton pump inhibitor (PPI) therapy. Multiple ulcers were visualized in the first portion of the duodenum, and metastatic lesions were demonstrable in the liver. Serum gastrin level was elevated to 433 pg/mL. Histology of liver biopsy showed a well-differentiated neuroendocrine tumor, supporting the diagnosis of ZES. This article underscores the significance of considering ZES in the differential in cases of refractory gastric hyperacidity and the importance of early diagnosis with serum gastrin testing to prevent complications such as gastric obstruction, perforation, hemorrhage, esophageal strictures, or rupture and to minimize the risk of metastasis. It is noteworthy that while perforations in cases of ZES typically occur in the duodenum, this particular case is atypical as it had involved the stomach. Furthermore, it was associated with esophageal perforation, likely resulting from forceful and prolonged vomiting caused by persistent dyspepsia.
RESUMEN
M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.
RESUMEN
M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.
Asunto(s)
Neoplasias del Colon , beta Catenina , Ratones , Humanos , Animales , beta Catenina/metabolismo , Ciclooxigenasa 2/metabolismo , Neoplasias del Colon/patología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Receptores Muscarínicos/metabolismo , Ratones Noqueados , Regulación Neoplásica de la Expresión GénicaRESUMEN
Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.
Asunto(s)
Neoplasias del Colon , MicroARNs , Acetilcolina/metabolismo , Acetilcolina/farmacología , Derivados de Atropina , Colinérgicos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Humanos , Cinética , MicroARNs/genética , Antagonistas Muscarínicos/farmacología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Preterm infants face many challenges in transitioning from the in utero to extrauterine environment while still immature. Failure of the preterm gut to successfully mature to accommodate bacteria and food substrate leads to significant morbidity such as neonatal necrotizing enterocolitis. The intestinal epithelial barrier plays a critical role in gut protection. Heat shock protein 70 (Hsp70) is an inducible cytoprotective molecule shown to protect the intestinal epithelium in adult models. To investigate the hypothesis that Hsp70 may be important for early protection of the immature intestine, Hsp70 expression was evaluated in intestine of immature rat pups. Data demonstrate that Hsp70 is induced by exposure to mother's milk. Hsp70 is found in mother's milk, and increased Hsp70 transcription is induced by mother's milk. This Hsp70 colocalizes with the tight junction protein ZO-1. Mother's milk-induced Hsp70 may contribute to maintenance of barrier function in the face of oxidant stress. Further understanding of the means by which mother's milk increases Hsp70 in the ileum will allow potential means of strengthening the intestinal barrier in at-risk preterm infants.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Leche/metabolismo , Animales , Animales Recién Nacidos , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/microbiología , Citoprotección , Modelos Animales de Enfermedad , Proteínas HSP70 de Choque Térmico/genética , Hipoxia/metabolismo , Íleon/microbiología , Íleon/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Proteínas de la Membrana/metabolismo , Estrés Oxidativo , Permeabilidad , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Uniones Estrechas/metabolismo , Regulación hacia Arriba , Proteína de la Zonula Occludens-1RESUMEN
Mucosal inflammation, through cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), has many effects on the intestinal epithelium, including selective translational inhibition of the cytoprotective protein heat shock protein 70 (Hsp70). To further elucidate the mechanisms underlying this effect, we examined the role of stress granules in mediating the actions of these proinflammatory cytokines. Using conditionally immortalized young adult mouse colonic epithelial cells, we demonstrate that IFN-gamma and TNF-alpha, which upregulate eukaryotic initiation factor-alpha (eIF-2alpha) phosphorylation and reduce Hsp70 translation, significantly enhance stress granule formation in heat-shocked intestinal epithelial cells. The IFN-gamma and TNF-alpha effects in upregulation of stress granule formation and downregulation of Hsp70 were eIF-2alpha dependent, and the effect could be negated by blocking eIF-2alpha phosphorylation with use of an RNA-dependent protein kinase inhibitor. Correspondingly, IFN-gamma and TNF-alpha increased binding of cytoplasmic proteins to the 3'-untranslated region of Hsp70 mRNA, suggesting specific recruitment of Hsp70 to stress granules as the mechanism of IFN-gamma and TNF-alpha inhibition of Hsp70 translation. We thus report a novel linkage between inflammatory cytokine production, stress granule formation, and Hsp70 translation inhibition, providing additional insights into the response of intestinal epithelial cells to inflammatory stress.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Interferón gamma/farmacología , Biosíntesis de Proteínas/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Regiones no Traducidas 3'/genética , Animales , Línea Celular Transformada , Colon/citología , Gránulos Citoplasmáticos/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Proteínas HSP70 de Choque Térmico/genética , Ratones , Modelos Biológicos , Fosforilación/efectos de los fármacos , Unión Proteica/genética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno Intracelular 1 de las Células T , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
Cytoprotective heat shock proteins (Hsps) are critical for intestinal homeostasis and are known to be decreased in inflammatory bowel diseases. Signals responsible for maintenance of Hsp expression are incompletely understood. In this study, we find that Hsp25/27 and Hsp70 protein expressions are differentially regulated along the longitudinal length of the large intestine, being highest in the proximal colon and decreasing to the distal colon. This longitudinal gradient was similar in both conventionally colonized mouse colon as well as biopsies of human proximal and distal colon but was abolished in the colon of germ-free mice, suggesting a role of intestinal microbiota in the Hsp regional expression. Correspondingly, analysis of 16S ribosomal RNA genes of bacteria from each colonic segment indicated increased bacterial richness and diversity in the proximal colon. The mechanism of regulation is transcriptional, as Hsp70 mRNA followed a similar pattern to Hsp70 protein expression. Lysates of mucosa-associated bacteria from the proximal colon stimulated greater Hsp25 and Hsp70 mRNA transcription and subsequent protein expression in intestinal epithelial cells than did lysates from distal colon. In addition, transrectal administration of cecal contents stimulated Hsp25 and Hsp70 expression in the distal colon. Thus host-microbial interactions resulting in differential Hsp expression may have significant implications for the maintenance of intestinal homeostasis and possibly for development of inflammatory diseases of the bowel.
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Colon/microbiología , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/metabolismo , Mucosa Intestinal/microbiología , Animales , Biopsia , Ciego/microbiología , Células Cultivadas , Colon/anatomía & histología , Colon/citología , Colon/metabolismo , Vida Libre de Gérmenes , Proteínas de Choque Térmico/genética , Humanos , Yeyuno/metabolismo , Yeyuno/microbiología , Ratones , Ratones Endogámicos C57BL , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
AA amyloidosis, previously known as secondary amyloidosis, has been associated with multiple chronic inflammatory conditions, including various autoimmune diseases and rarely chronic infection. Hereby, we present a case of AA amyloidosis secondary to chronic infection which initially presented with nausea and hematemesis. Endoscopic biopsies revealed diffuse AA amyloid deposition in the stomach, but not the esophagus. AA Amyloidosis presumably compromised gastric motility, promoted reflux related esophageal ulcers and erosions, and caused his cardiac and renal insufficiency. Therefore, endoscopic biopsies could be practical investigation to identify AA amyloidosis in the setting of chronic inflammatory diseases, especially with multi-organ involvement.
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Amiloidosis , Hematemesis , Amiloidosis/complicaciones , Amiloidosis/diagnóstico , Biopsia , Enfermedad Crónica , Hematemesis/etiología , Humanos , Proteína Amiloide A Sérica , EstómagoRESUMEN
BACKGROUND: Heat shock protein 70 (Hsp70) regulates protein biosynthesis and refolding of denatured proteins. Since Hsp70 participates in recovery from stress injury, we examined the effect of Hsp70 genetic deletion in the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of inflammation and colon cancer. METHODS: Hsp70 mutant mice (Hsp70.1(-/-)/70.3(-/-)) and wild-type (WT) littermates received AOM and three cycles of DSS and were killed 24 weeks later. Tumors were graded for histology and immunostained for p53, adenomatous polyposis coli, beta-catenin, cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) and sequenced for p53 mutations. RESULTS: Elevated adenomas developed in 4/10 WT mice with no dysplasia in adjacent mucosa. In contrast, 7/8 Hsp70 knock out (KO) mice developed chronic mucosal inflammation and multifocal areas of flat dysplasia and 4/8 progressed to invasive carcinomas arising in a background of flat dysplastic mucosa. These differences in the incidence of flat dysplasia and invasive cancers were significant (P < 0.05). Nuclear p53 was stronger in Hsp70 KO tumors compared with WT tumors, and sequencing confirmed p53 mutations in 2/5 tumors from Hsp70(-/-) versus 0/5 in WT mice. In Hsp70 WT tumors, beta-catenin was predominantly nuclear, compared with membranous beta-catenin in Hsp70(-/-) tumors, suggesting that Hsp70 regulates beta-catenin in colonic tumorigenesis. Cox-2 and iNOS levels were increased in tumors from Hsp70(-/-) mice compared with Hsp70 WT tumors. CONCLUSIONS: Hsp70-deleted mice treated with AOM/DSS develop flat invasive colonic tumors that mimic many histological and molecular features of ulcerative colitis colon cancer. This model will be useful to dissect the role of Hsp70 in inflammatory bowel disease colon cancer.
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Colitis/patología , Neoplasias del Colon/patología , Proteínas HSP70 de Choque Térmico/fisiología , Animales , Secuencia de Bases , Western Blotting , Neoplasias del Colon/metabolismo , Cartilla de ADN , Proteínas HSP70 de Choque Térmico/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Invasividad Neoplásica , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Despite a reduction in incidence over the past decade, colon cancer remains the second most common cause of cancer death in the United States; recent demographics suggest this disease is now afflicting younger persons. M3 muscarinic receptor (M3R) mRNA and protein are over-expressed in colon cancer, and M3R can be activated by both traditional (e.g., acetylcholine) and non-traditional (e.g., bile acids) muscarinic ligands. In this review, we weigh the data supporting a prominent role for key protein kinases downstream of M3R activation in promoting colon cancer progression and dissemination. Specifically, we explore the roles that downstream activation of the mitogen activated protein kinase/extracellular signal-related kinase (MAPK/ERK), protein kinase C, p38 MAPK, and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways play in mediating colon cancer cell proliferation, survival, migration and invasion. We assess the impact of M3R-stimulated induction of selected matrix metalloproteinases germane to these hallmarks of colon cancer progression. In this context, we also critically review the reproducibility of findings derived from a variety of in vivo and in vitro colon cancer models, and their fidelity to human disease. Finally, we summarize the therapeutic potential of targeting various steps from ligand-M3R interaction to the activation of key downstream molecules.
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BACKGROUND & AIMS: Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. METHODS: iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10(-/-) mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF-2alpha) was determined by Western blot. RESULTS: Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10(-/-) mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-gamma + tumor necrosis factor (TNF)-alpha also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-gamma+TNF-alpha inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-gamma+TNF-alpha cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2alpha, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2alpha were also observed in active IBD tissues. CONCLUSIONS: Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury.
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Proteínas de Choque Térmico/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interferón gamma/inmunología , Mucosa Intestinal/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Apoptosis/inmunología , Células Cultivadas , Colitis Ulcerosa/inmunología , Colon/inmunología , Enfermedad de Crohn/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares , Proteínas de Neoplasias/inmunología , Biosíntesis de Proteínas/inmunologíaRESUMEN
BACKGROUND: Expression and activation of subtype-3 muscarinic receptors (M3R) plays an important role in the progression of colorectal neoplasia. METHOD: Herein, we describe the role of muscarinic receptors in colon cancer, focusing specifically on M3R, illustrate how M3R over-expression and activation of post-receptor signaling pathways potentiates tumor progression, and explore the efficacy and safety of a variety of therapeutic approaches that can target the molecules involved. RESULTS: Colon cancers overexpress M3R mRNA (CHRM3) and protein, and post-M3R signaling stimulates cell proliferation. Post-M3R signal transduction is complex, involving interplay between epidermal growth factor receptors (EGFR)/ERK and protein kinase C (PKC)/p38 mitogen-activated protein (MAP) kinase signaling pathways. In particular, the development of an invasive and metastatic phenotype requires that these signaling interactions augment cellular release of a key collagenase, matrix metalloproteinase-1 (MMP1). Blocking either M3R activation or post-M3R signaling attenuates MMP1 release and colon cancer invasiveness. CONCLUSION: Parsing the complexities of these signaling interactions is important, not only to understand these mechanisms of cancer initiation and progression, but also to develop novel treatment modalities. Since the vast majority of persons with colon cancer die from disseminated disease, preventing or reversing metastatic spread of cancer cells by targeting M3R, post-M3R signaling, or MMP1 has therapeutic potential.
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Neoplasias del Colon/terapia , Terapia Molecular Dirigida , Receptor Muscarínico M3/antagonistas & inhibidores , Neoplasias del Colon/patología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Receptor Muscarínico M3/metabolismo , Transducción de SeñalRESUMEN
AIM: Strong evidence reveals important differences between cancers in the proximal vs. distal colon. Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations. We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences. METHODS: We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer. In survival surgery experiments, we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1nu or NOD.Cg-PrkdcscidIl2rgTim1Wji/SzJ mice and compared the development of primary tumors and metastases. RESULTS: Within 7-17 weeks after intramural cecal injection of HT-29 cells, eight mice failed to develop solid primary tumors or metastases. In contrast, within four weeks after cell injection into the distal colon, 13 mice developed metastases - 12 mice developed subcutaneous metastases; of these, four developed liver metastases and one developed both liver and lung metastases. One mouse developed liver metastases only. Histological examination confirmed these lesions were adenocarcinomas. CONCLUSION: Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon. The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans. This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.
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PURPOSE: To report a case of Alport syndrome with recurrent herpes simplex virus (HSV) keratitis. METHODS: Case report and review of the literature. RESULTS: A 29-year-old man with Alport syndrome suffered from 4 consecutive episodes of HSV keratitis within 2 years. A type IV collagen defect in basement membranes plays an important role in the pathogenesis of Alport syndrome. The relationship between HSV keratitis and Alport syndrome is discussed. After remission from HSV keratitis, the patient underwent bilateral phacoemulsification and posterior-chamber intraocular lens implantation for anterior lenticonus and an anterior polar cataract. After surgery, the uncorrected visual acuity was 20/20 in both eyes. CONCLUSIONS: We assume that the underlying basement membrane defects in Alport syndrome may account for the recurrent episodes of HSV keratitis in this patient. In addition, phacoemulsification and posterior-chamber intraocular lens implantation, rather than correction of refractive errors, provide a safe and efficient therapeutic choice for the management of anterior lenticonus, with or without associated cataract in patients with Alport syndrome.