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1.
Hum Mol Genet ; 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38881369

RESUMEN

The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.

2.
FASEB J ; 35(5): e21371, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33811694

RESUMEN

Studies demonstrate a role for neurotensin (NT) in obesity and related comorbidities. Bile acid (BA) homeostasis alterations are associated with obesity. We determined the effect of NT on BA metabolism in obese and non-obese conditions. Plasma and fecal BA profiles were analyzed by LC-MS/MS in male and female NT+/+ and NT-/- mice fed low-fat (LFD) or high-fat diet (HFD) for 6 weeks (early stage of obesity) or greater than 20 weeks (late stage of obesity). The nuclear farnesoid X receptor (FXR) and BA transporter mRNA expression were assessed in ileum, mouse enteroids, and human cell lines. HFD decreased plasma primary and secondary BAs in NT+/+ mice; HFD-induced decrease of plasma BAs was improved in NT-deficient mice. In NT+/+ mice, HFD inhibited ileal FXR and BA transporter expression; HFD-decreased expression of FXR and BA transporters was prevented in NT-/- mice. Compared with LFD-fed NT+/+ mice, LFD-fed NT-/- mice had relatively lower levels of ileal FXR and BA transporter expression. Moreover, NT stimulates the expression of FXR and BA transporters in Caco-2 cells; however, stimulated expression of BA transporters was attenuated in NT-/- enteroids. Therefore, we demonstrate that HFD disrupts the BA metabolism and ileal FXR and BA transporter axis which are improved in the absence of NT, suggesting that NT contributes to HFD-induced disruption of BA metabolism and plays an inhibitory role in the regulation of ileal FXR and BA transporter signaling under obese conditions. Conversely, NT positively regulates the expression of ileal FXR and BA transporters under non-obese conditions. Therefore, NT plays a dual role in obese and non-obese conditions, suggesting possible therapeutic strategies for obesity control.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Intestinos/fisiología , Neurotensina/fisiología , Nutrientes/metabolismo , Obesidad/fisiopatología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Células CACO-2 , Dieta Alta en Grasa , Femenino , Humanos , Masculino , Ratones
3.
Nature ; 533(7603): 411-5, 2016 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-27193687

RESUMEN

Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually and are among the most prevalent and challenging conditions confronting the medical profession. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine and released by fat ingestion, facilitates fatty acid translocation in rat intestine, and stimulates the growth of various cancers. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively). Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality; however, a role for NT as a causative factor in these diseases is unknown. Here we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Neurotensina/metabolismo , Obesidad/inducido químicamente , Obesidad/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Drosophila melanogaster/metabolismo , Células Enteroendocrinas/metabolismo , Activación Enzimática , Cuerpo Adiposo/metabolismo , Ácidos Grasos/metabolismo , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Femenino , Humanos , Resistencia a la Insulina/fisiología , Mucosa Intestinal/metabolismo , Intestinos/citología , Metabolismo de los Lípidos , Masculino , Ratones , Persona de Mediana Edad , Neurotensina/sangre , Neurotensina/deficiencia , Neurotensina/genética , Obesidad/sangre , Obesidad/prevención & control , Precursores de Proteínas/sangre , Precursores de Proteínas/metabolismo
4.
Prostate ; 81(10): 667-682, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33956349

RESUMEN

The surface expression of Na/K-ATPase α1 (NKA) is significantly reduced in primary prostate tumors and further decreased in bone metastatic lesions. Here, we show that the loss of cell surface expression of NKA induces epithelial-mesenchymal transition (EMT) and promotes metastatic potential and tumor growth of prostate cancer (PCa) by decreasing the expression of E-cadherin and increasing c-Myc expression via the activation of Src/FAK pathways. Mechanistically, reduced surface expression of NKA in PCa is due to increased endocytosis through the activation of NKA/Src receptor complex. Using a high-throughput NKA ligand-screening platform, we have discovered MB5 as an inverse agonist of the NKA/Src receptor complex, capable of blocking the endocytosis of NKA. MB5 treatment increased NKA expression and E-cadherin in PCa cells, which reversed EMT and consequently decreased the invasion and growth of spheroid models and tumor xenografts. Thus, we have identified a hitherto unrecognized mechanism that regulates EMT and invasiveness of PCa and demonstrated for the first time the feasibility of identifying inverse agonists of receptor NKA/Src complex and their potential utility as anticancer drugs. We, therefore, conclude that cell surface expression of α1 NKA can be targeted for the development of new therapeutics against aggressive PCa and that MB5 may serve as a prototype for drug development against EMT in metastatic PCa.


Asunto(s)
Agonismo Inverso de Drogas , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Neoplasias de la Próstata/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ouabaína/farmacología , Tiamina/análogos & derivados , Tiamina/farmacología , Tiamina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
5.
FASEB J ; 34(6): 8596-8610, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32359121

RESUMEN

We previously reported that high levels of plasma neurotensin (NT), a gut hormone released from enteroendocrine cells of the small bowel, contribute to obesity and comorbid conditions. Gut microbiota has been implicated in the obesity development. Paneth cells are critical in maintaining gut microbiota composition and homeostasis by releasing antimicrobial proteins including α-defensins. The purpose of our current study was to determine the possible role of NT in gut microbiota composition and α-defensin gene expression associated with obesity. Here we show that the ratio of Firmicutes/Bacteroidetes (F/B ratio) and intestinal proinflammatory cytokines is significantly increased in NT+/+ mice fed with a high-fat diet (HFD) which were improved in NT-deficient mice. HFD disrupted the intestinal Mmp7/α-defensin axis, which was completely prevented in NT-/- mice. In addition, NT treatment inhibited DEFA5 expression and concurrent NF-κB activity, which was blocked by a pan PKC inhibitor (Gö6983) or an inhibitor for atypical PKCs (CRT0066854). More importantly, the shRNA-mediated knockdown of atypical PKCτ reversed NT-attenuated DEFA5 expression and increased NF-κB activity. NT contributes to the HFD-induced disruption of gut microbiota composition and α-defensin expression. PKCτ/λ plays a central role in NT-mediated α-defensin gene expression which might be mediated through the inhibition of NF-κB signaling pathways in Paneth cells.


Asunto(s)
Disbiosis/metabolismo , Inflamación/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Neurotensina/metabolismo , alfa-Defensinas/metabolismo , Tejido Adiposo/metabolismo , Animales , Citocinas/metabolismo , Dieta Alta en Grasa/efectos adversos , Disbiosis/patología , Microbioma Gastrointestinal/fisiología , Inflamación/patología , Resistencia a la Insulina/fisiología , Intestinos/patología , Masculino , Ratones , Ratones Obesos , FN-kappa B/metabolismo , Obesidad/metabolismo , Células de Paneth/metabolismo , Transducción de Señal/fisiología
6.
Langmuir ; 37(40): 11737-11749, 2021 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-34597055

RESUMEN

Capillary forces of a shearing liquid bridge can significantly affect the friction and adhesion of interacting surfaces, but the underlying mechanisms remain unclear. We custom built a surface force apparatus (SFA, ±2 µN) equipped with in situ optical microscopy and performed normal and lateral force measurements on a reciprocating water bridge formed between two flat plates. A modified wedge method was developed to correct the unique force measurement errors caused by the changing bridge geometry and position. The results found (1) strong linear relations among the bridge shear displacement, the cosine difference between the left and right contact angles, and the lateral adhesion force and (2) the normal adhesion force increased monotonically up to 13% as the bridge geometry approached its axisymmetric state. Quasi-static force analyses based on a newly developed decahedral model showed good agreement with the experiments and improved accuracy compared with that of cylindrical or rectangular column models previously proposed in the literature. Although limited in certain aspects, this study may (1) prove helpful to the design and analysis of liquid bridge force experiments on platforms similar to the SFA used in this study and (2) help to bridge the gap between friction and liquid bridge physics in the literature.

7.
Int J Cancer ; 145(10): 2767-2780, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31008533

RESUMEN

Although integrin α9 (ITGA9) is known to be involved in cell adhesion and motility, its expression in cancer and its role in tumor growth and metastasis remain largely unknown. Our study was designed to investigate the role of ITGA9 in triple-negative breast cancer (TNBC). ITGA9 expression in TNBC cells was knocked out (KO) using CRISPR/Cas9 technology. Four orthotopic mouse mammary xenograft tumor models coupled with cell culture studies were performed to determine the effect of ITGA9 depletion on TNBC tumor growth and metastasis and the underlying mechanism. Bioinformatics analysis showed that ITGA9 level is significantly higher in TNBC than other breast cancer subtypes, and higher ITGA9 level is associated with significantly worse distant metastasis-free survival and recurrence-free survival in TNBC patients. Experimentally, ITGA9 KO significantly reduced TNBC cell cancer stem cell (CSC)-like property, tumor angiogenesis, tumor growth and metastasis by promoting ß-catenin degradation. Further mechanistic studies revealed that ITGA9 KO causes integrin-linked kinase (ILK) relocation from the membrane region to the cytoplasm, where it interacts with protein kinase A (PKA) and inhibits PKA activity leading to increased activity of glycogen synthase kinase 3 (GSK3) and subsequent ß-catenin degradation. Overexpressing ß-catenin in ITGA9 KO cells reversed the inhibitory effect of ITGA9 KO on tumor growth and metastasis. Furthermore, ITGA9 downregulation in TNBC tumors by nanoparticle-mediated delivery of ITGA9 siRNA drastically decreased tumor angiogenesis, tumor growth and metastasis. These findings indicate that ITGA9 depletion suppresses TNBC tumor growth and metastasis by promoting ß-catenin degradation through the ILK/PKA/GSK3 pathway.


Asunto(s)
Integrinas/metabolismo , Recurrencia Local de Neoplasia/patología , Neoplasias de la Mama Triple Negativas/patología , beta Catenina/metabolismo , Animales , Mama/patología , Línea Celular Tumoral , Proliferación Celular , Biología Computacional , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Conjuntos de Datos como Asunto , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Técnicas de Inactivación de Genes , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Integrinas/genética , Ratones , Recurrencia Local de Neoplasia/epidemiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/mortalidad
8.
J Biol Chem ; 291(48): 25167-25178, 2016 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-27760826

RESUMEN

The proper establishment of epithelial polarity allows cells to sense and respond to signals that arise from the microenvironment in a spatiotemporally controlled manner. Atypical PKCs (aPKCs) are implicated as key regulators of epithelial polarity. However, the molecular mechanism underlying the negative regulation of aPKCs remains largely unknown. In this study, we demonstrated that PH domain leucine-rich repeat protein phosphatase (PHLPP), a novel family of Ser/Thr protein phosphatases, plays an important role in regulating epithelial polarity by controlling the phosphorylation of both aPKC isoforms. Altered expression of PHLPP1 or PHLPP2 disrupted polarization of Caco2 cells grown in 3D cell cultures as indicated by the formation of aberrant multi-lumen structures. Overexpression of PHLPP resulted in a decrease in aPKC phosphorylation at both the activation loop and the turn motif sites; conversely, knockdown of PHLPP increased aPKC phosphorylation. Moreover, in vitro dephosphorylation experiments revealed that both aPKC isoforms were substrates of PHLPP. Interestingly, knockdown of PKCζ, but not PKCι, led to similar disruption of the polarized lumen structure, suggesting that PKCζ likely controls the polarization process of Caco2 cells. Furthermore, knockdown of PHLPP altered the apical membrane localization of aPKCs and reduced the formation of aPKC-Par3 complex. Taken together, our results identify a novel role of PHLPP in regulating aPKC and cell polarity.


Asunto(s)
Polaridad Celular/fisiología , Isoenzimas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células CACO-2 , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/enzimología , Membrana Celular/genética , Humanos , Isoenzimas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon/genética
9.
J Biol Chem ; 291(8): 4166-77, 2016 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-26565021

RESUMEN

Wnt/ß-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated ß-catenin (pß-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in ß-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with ß-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial ß-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces ß-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the ß-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Colitis/metabolismo , Neoplasias del Colon/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Animales , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Colitis/genética , Colitis/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Humanos , Ratones , Ratones Transgénicos , Complejos Multiproteicos/genética , Proteínas de Neoplasias/genética , beta Catenina/genética
10.
J Biol Chem ; 291(49): 25729-25741, 2016 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-27780861

RESUMEN

Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion. However, it is unknown how upstream signals control PIPKIγ90 ubiquitination or degradation. Here we show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin (mTOR), phosphorylates PIPKIγ90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKIγ90 phosphorylation is essential for cell migration and invasion. Moreover, PIPKIγ90 phosphorylation is required for the development of focal adhesions and invadopodia, key machineries for cell migration and invasion. Surprisingly, substitution of Thr-553 and Ser-555 with Ala promoted PIPKIγ90 ubiquitination but enhanced the stability of PIPKIγ90, and depletion of S6K1 also enhanced the stability of PIPKIγ90, indicating that PIPKIγ90 ubiquitination alone is insufficient for its degradation. These data suggest that S6K1-mediated PIPKIγ90 phosphorylation regulates cell migration and invasion by controlling PIPKIγ90 degradation.


Asunto(s)
Movimiento Celular/fisiología , Adhesiones Focales/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteolisis , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Ubiquitinación/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Adhesiones Focales/genética , Humanos , Fosforilación/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo
11.
Proc Natl Acad Sci U S A ; 111(45): E4842-50, 2014 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-25349414

RESUMEN

Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Proteína Quinasa C/biosíntesis , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Animales , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Hedgehog/genética , Fosforilación/fisiología , Proteína Quinasa C/genética , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Factores de Transcripción/genética , Regulación hacia Arriba/fisiología , Alas de Animales/metabolismo
12.
Proc Natl Acad Sci U S A ; 111(38): E3957-65, 2014 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-25201979

RESUMEN

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.


Asunto(s)
Receptores ErbB/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Modelos Biológicos , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Línea Celular Transformada , Receptores ErbB/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Secuencias Repetitivas de Aminoácido , Transcripción Genética/fisiología
13.
Biochim Biophys Acta ; 1852(10 Pt A): 2013-23, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26187040

RESUMEN

A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.

14.
EMBO Rep ; 15(2): 191-8, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24375676

RESUMEN

Protein kinase Cζ (PKCζ) is phosphorylated at the activation loop and the turn motif (TM). However, the TM kinase and functional relevance of TM phosphorylation remain largely unknown. We demonstrate that PKCζ TM is phosphorylated directly by the mTORC2 complex, and this phosphorylation is required for maintaining PKCζ kinase activity and stability. Functionally, mTORC2 regulates the activity of Rho family of GTPases, and therefore the organization of the actin cytoskeleton, through the control of PKCζ activity. Taken together, our findings identify PKCζ as a novel substrate and downstream effector of mTORC2 signaling.


Asunto(s)
Complejos Multiproteicos/metabolismo , Proteína Quinasa C/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Estabilidad de Enzimas , Células HEK293 , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Complejos Multiproteicos/genética , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética
15.
Gastroenterology ; 146(5): 1301-12.e1-10, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24530606

RESUMEN

BACKGROUND & AIMS: Hyperactivation of the RAS-RAF signaling pathway in colorectal tumors is associated with metastasis and poor outcomes of patients. Little is known about how RAS-RAF signaling is turned off once activated. We investigated how the pH domain and leucine-rich repeat protein phosphatases (PHLPPs) control RAS-RAF signaling and colorectal cancer (CRC) development. METHODS: We used co-immunoprecipitation assays to identify substrates of PHLPP1 and PHLPP2. We studied phosphorylation of RAF1 in CRC cells that express exogenous PHLPP1 or PHLPP2, or lentiviral-based small hairpin RNAs against their transcripts; we measured effects on cell motility, migration, and invasion in vitro. Tumor progression and survival were analyzed in Phlpp1(-/-) Apc(Min) and Apc(Min)/Phlpp1(-/-) mice. Microarray datasets of colorectal tumor and nontumor tissues were analyzed for PHLPP gene expression. RESULTS: PHLPP1 and 2 were found to dephosphorylate RAF1 at S338, inhibiting its kinase activity in vitro and in CRC cells. In cells, knockdown of PHLPP1 or PHLPP2 increased the amplitude and duration of RAF-MEK-ERK signaling downstream of epidermal growth factor receptor and KRAS, whereas overexpression had the opposite effect. In addition, knockdown of PHLPP1 or PHLPP2 caused CRC cells to express markers of the epithelial-mesenchymal transition, and increased cell migration and invasion. Apc(Min)/Phlpp1(-/-) mice had decreased survival and developed larger intestinal and colon tumors compared to Apc(Min) mice. Whereas Apc(Min) mice developed mostly low-grade adenomas, 20% of the tumors that developed in Apc(Min)/Phlpp1(-/-) mice were invasive adenocarcinomas. Normal villi and adenomas of Apc(Min)/Phlpp1(-/-) mice had significantly fewer apoptotic cells than Apc(Min) mice. Human CRC patient microarray data revealed that the expression of PHLPP1 or PHLPP2 is positively correlated with CDH1. CONCLUSIONS: PHLPP1 and PHLPP2 dephosphorylate RAF1 to reduce its signaling, increase the invasive and migratory activities of CRC cells, and activate the epithelial-mesenchymal transition. In Apc(Min) mice, loss of PHLPP1 promotes tumor progression.


Asunto(s)
Adenocarcinoma/enzimología , Adenoma/enzimología , Movimiento Celular , Neoplasias Colorrectales/enzimología , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/genética , Adenoma/patología , Animales , Antígenos CD , Apoptosis , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Genes APC , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Invasividad Neoplásica , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/deficiencia , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Carga Tumoral
16.
Carcinogenesis ; 35(6): 1341-51, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24510238

RESUMEN

Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC-EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in 'normalization' of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF(189), upregulation of antiangiogenic isoform VEGF(165b) and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC.


Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células Endoteliales/metabolismo , Ácido Graso Sintasas/genética , Neovascularización Patológica/genética , Animales , Línea Celular Tumoral , Embrión de Pollo , Modelos Animales de Enfermedad , Ácido Graso Sintasas/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Neovascularización Patológica/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
17.
J Biol Chem ; 288(32): 23225-33, 2013 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-23814053

RESUMEN

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Humanos , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas/genética , Fosforilación/fisiología , Proteína Fosfatasa 2C , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo
18.
Am J Transl Res ; 16(5): 2122-2131, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38883360

RESUMEN

OBJECTIVE: To evaluate the efficacy of minocycline hydrochloride combined with metronidazole versus metronidazole alone in treating peri-implantitis and their impact on specific inflammatory markers. METHODS: A retrospective review was undertaken of 107 patients with peri-implantitis from January 2018 to January 2021. Patients were treated either with metronidazole alone (Con group, n = 57) or with additional minocycline hydrochloride (Exp group, n = 50). Inflammatory markers, including interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase-8 (MMP-8) were determined before and after treatment. Clinical outcomes were determined using the plaque index (PLI), gingival sulcus bleeding index (SBI), and periodontal probing depth (PD). Furthermore, receiver operator characteristic (ROC) curves analyzed the clinical relevance of the markers. Logistic regression was conducted to analyze the risk factors affecting efficacy in patients. RESULTS: The Exp group exhibited more favorable clinical outcomes and showed lower levels of IL-6, IL-1ß, TNF-α, and MMP-8 than the Con group. IL-1ß, TNF-α, and MMP-8 levels were significantly correlated with treatment success (P < 0.05), but IL-6 was not (P > 0.05). The ROC curves for IL-1ß and TNF-α significantly outperformed those for IL-6 and MMP-8 (P < 0.05). Logistic regression analysis showed that only IL-1ß and TNF-α were independent risk factors affecting efficacy in patients. CONCLUSION: Combining minocycline hydrochloride with metronidazole yields better outcomes for peri-implantitis compared to metronidazole alone. Of the factors analyzed, only IL-1ß and TNF-α emerged as dependable independent efficacy indicators.

19.
Environ Pollut ; 347: 123586, 2024 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-38467368

RESUMEN

Inorganic arsenic (iAs) causes cancer by initiating dynamic transitions between epithelial and mesenchymal cell phenotypes. These transitions transform normal cells into cancerous cells, and cancerous cells into metastatic cells. Most in vitro models assume that transitions between states are binary and complete, and do not consider the possibility that intermediate, stable cellular states might exist. In this paper, we describe a new, two-hit in vitro model of iAs-induced carcinogenesis that extends to 28 weeks of iAs exposure. Through week 17, the model faithfully recapitulates known and expected phenotypic, genetic, and epigenetic characteristics of iAs-induced carcinogenesis. By 28 weeks, however, exposed cells exhibit stable, intermediate phenotypes and epigenetic properties, and key transcription factor promoters (SNAI1, ZEB1) enter an epigenetically poised or bivalent state. These data suggest that key epigenetic transitions and cellular states exist during iAs-induced epithelial-to-mesenchymal transition (EMT), and that it is important for our in vitro models to encapsulate all aspects of EMT and the mesenchymal-to-epithelial transition (MET). In so doing, and by understanding the epigenetic systems controlling these transitions, we might find new, unexpected opportunities for developing targeted, cell state-specific therapeutics.


Asunto(s)
Arsénico , Neoplasias , Humanos , Arsénico/toxicidad , Factores de Transcripción/metabolismo , Epigénesis Genética , Carcinogénesis/inducido químicamente
20.
EMBO Rep ; 12(8): 818-24, 2011 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-21701506

RESUMEN

PHLPP1 (PH domain leucine-rich-repeats protein phosphatase) is a Ser/Thr protein phosphatase that acts as a tumour suppressor by negatively regulating Akt. Here, we show that PHLPP1 is recruited to the cell membrane by binding to a scaffolding protein: Scribble. Knockdown of Scribble (Scrib) results in redistribution of PHLPP1 from the membrane to the cytoplasm and an increase in Akt phosphorylation, whereas overexpression of Scrib has the opposite effect. Furthermore, PHLPP1-dependent inhibition of cell proliferation is facilitated by the formation of a Scrib, PHLPP1 and Akt trimeric complex. Thus, our findings identify a functional interaction between PHLPP1 and Scrib in negatively regulating Akt signalling.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Células CACO-2 , Procesos de Crecimiento Celular/fisiología , Membrana Celular/metabolismo , Citoplasma/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Células HCT116 , Humanos , Dominios PDZ , Fosforilación , Unión Proteica , Transducción de Señal , Células Tumorales Cultivadas
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