Thyroid hormone antagonizes tumor necrosis factor-α signaling in pituitary cells through the induction of dual specificity phosphatase 1

Pituitary function has been shown to be regulated by an increasing number of factors, including cytokines and hormones, such as TNF alpha and T-3. Both the proinflammatory cytokine TNF alpha and T-3 have been suggested to be involved in the maintenance of tissue homeostasis in the anterior pituitary gland. In this report we show that T-3 negatively interferes with MAPK p38 and nuclear factor-kappa B (NF-kappa B) activation by TNF alpha in GH4C1 cells. Our data demonstrate that MAPK p38 is specifically activated upon exposure to TNF alpha and that T-3 abolishes this activation in a time-dependent manner by a mechanism that involves the induction of the MAPK phosphatase, DUSP1. Our data show that the pool of up-regulated DUSP1 by T-3 is mainly localized to the cytosol, and that TNF alpha does not affect this localization. On the other hand, we show that T-3 impairs the activation of the NF-kappa B pathway induced by TNF alpha, producing a significant decrease in NF-kappa B-dependent transcription, phosphorylation of I kappa B alpha, translocation of p65/NF-kappa B to the nucleus, and p65/NF-kappa B transactivation potential. Interestingly, the overexpression of DUSP1 inhibits the NF-kappa B activation achieved by either TNF alpha or ectopic expression of the upstream inducer of MAPK p38. Conversely, DUSP1 depletion abrogates the inhibitory effect of T-3 on the induction of NF-kappa B-dependent transcription by TNF alpha. Overall, our results indicate that T-3 antagonizes TNF alpha signaling in rat pituitary tumor cells through the induction of DUSP1.This work was supported by grants from the Fundación Mutua Madrileña (2005X0615), from Fondo de Investigaciones Sanitarias (PI070832), from Ministerio de Educación y Ciencia (BFU2007-62402), from Fondo de Investigaciones Sanitarias (RD06/0020/0036) and the European grant CRESCENDO (FP-018652). M.L. is recipient of a grant from the Spanish Ministerio de Educación y Ciencia (“Ramón y Cajal” Program).Peer reviewe

Thyroid hormone-mediated activation of the ERK/dual specificity phosphatase 1 pathway augments the apoptosis of GH4C1 cells by down-regulating nuclear factor-κB activity

14 pages, 5 figures.Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-kappaB (NF-kappaB)-dependent transcription, IkappaBalpha phosphorylation, translocation of p65/NF-kappaB to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition, T3 increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-kappaB-dependent transcription and p65/NF-kappaB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in rat pituitary tumor cells by down-regulating NF-kappaB activity through a mechanism dependent on the ERK/DUSP1 pathway.This work was supported by grants from the Fundación Mutua Madrileña (2005X0615), from Fondo de Investigaciones Sanitarias (PI070832), from Ministerio de Educación y Ciencia (BFU2004 3465), from Fondo de Investigaciones Sanitarias (RD06/0020/0036), and the European grant CRESCENDO (FP-018652). A.S.-P. and M.L. are recipients of grants from the Spanish MEC (“Ramón y Cajal” Program).Peer reviewe

V600e braf inhibition induces cytoprotective autophagy through ampk in thyroid cancer cells

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, whereV600E BRAF is a main oncogene. Here, we analyse the effect ofV600E BRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition ofV600E BRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show thatV600E BRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells toV600E BRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects ofV600E BRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy byV600E BRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targetingV600E BRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment ofV600E BRAF-positive thyroid tumours.This research was supported by grants from the Ministerio de Ciencia, Innovación y Universidades of Spain (SAF 2014-53639-R) and from UAH-Comunidad de Madrid (CAM) (CM/JIN/2019-019

Downregulation of snail by DUSP1 impairs cell migration and invasion through the inactivation of JNK and ERK and is useful as a predictive factor in the prognosis of prostate cancer

Dual specificity phosphatase 1 (DUSP1) is crucial in prostate cancer (PC), since its expression is downregulated in advanced carcinomas. Here, we investigated DUSP1 effects on the expression of mesenchymal marker Snail, cell migration and invasion, analyzing the underlying mechanisms mediated by mitogen‐activated protein kinases (MAPKs) inhibition. To this purpose, we used different PC cells overexpressing or lacking DUSP1 or incubated with MAPKs inhibitors. Moreover, we addressed the correlation of DUSP1 expression with Snail and activated MAPKs levels in samples from patients diagnosed with benign hyperplasia or prostate carcinoma, studying its implication in tumor prognosis and survival. We found that DUSP1 downregulates Snail expression and impairs migration and invasion in PC cells. Similar results were obtained following the inhibition of c‐Jun N‐terminal kinase (JNK) and extracellular‐signal‐regulated kinase (ERK). In clinical samples, we evidenced an inverse correlation between DUSP1 expression and Snail levels, which are further associated with JNK and ERK activation. Consequently, the pattern DUSP1high/activated JNKlow/activated ERKlow/Snaillow is associated with an overall extended survival of PC patients. In summary, the ratio between DUSP1 and Snail expression, with additional JNK and ERK activity measurement, may serve as a potential biomarker to predict the clinical outcome of PC patients. Furthermore, DUSP1 induction or inhibition of JNK and ERK pathways could be useful to treat PCD.M.-M. was recipient of grants from UAM (“Post-Master Program of Dpt. Biochemistry) and from Comunidad de Madrid (“Ayudas para la contratación de investigadores predoctorales y postdoctorales, ref. PEJD-2018-PRE/BMD-8987). P.B. was recipient of a grant from Comunidad de Madrid (“Atracción al Talento Investigador”, ref. 2017-T1/BMD-5704

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.Fil: Vega, Celina Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vlasova, Anastasia N.. Ohio State University; Estados UnidosFil: Chattha, Kuldeep S.. Ohio State University; Estados UnidosFil: Gómez Sebastián, Silvia. Universidad Politécnica de Madrid; EspañaFil: Nuñez, Carmen. Universidad Politécnica de Madrid; EspañaFil: Alvarado, Carmen. Universidad Politécnica de Madrid; EspañaFil: Lasa, Rodrigo. Universidad Politécnica de Madrid; EspañaFil: Escribano, José M.. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Departamento Mejora Genética y Biotecnología; EspañaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Bok, Karin. National Institutes of Health; Estados UnidosFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Saif, Linda J.. Ohio State University; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Protein kinase D activity is a risk biomarker in prostate cancer that drives cell invasion by a Snail/ERK dependent mechanism

Protein kinase D (PKD) family members play controversial roles in prostate cancer (PC). Thus, PKD1 is nearly absent in advanced tumours, where PKD2 and PKD3 are upregulated. Additionally, consequences of activation of these kinases on PC progression remain largely unclear. Here, we first investigated PKD function on PC cell motility, analysing the underlying molecular mechanisms. We find a striking decrease of Snail levels after PKD inhibition followed by cell migration and invasion impairment, demonstrating an unprecedented role of PKD activity on the regulation of this key transcription factor in PC progression. Specifically, we show that PKD2 activity mediates the effects of MEK/ERK pathway on Snail expression, establishing a joint function of ERK/ PKD2/Snail cascade in PC cell invasion regulation. These results led us to address the clinical relevance of the correlation between PKD2 and ERK activities with Snail abundance in samples from PC patients at different stages, analysing its impact on tumour prognosis and patients´survival. Importantly, this is the first study defining a direct correlation between active PKD2 and Snail levels, further linked to ERK activity. We also evidence that PKD2 activity is associated with important poor prognostic factors. Thus, PC patients with the expression pattern: active PKD2high/active ERKhigh/Snailhigh exhibit increased invasiveness and metastasis, and decreased survival. Our findings provide new insights for understanding the molecular mechanisms involved in PC progression, pinpointing the combination of active PKD2 and Snail levels, with the additional measurement of active ERK, as a confident biomarker to predict clinical outcome of patients with advanced PCD.C.-R., D.M.-M., and P.B. were recipients of grants from Comunidad de Madrid, Spain (grant numbers PEJ-2019-AI/BMD-14294, PEJD2018-PRE/BMD-8987, and 2017-T1/BMD-5704, respectively). T.I. received funding from Centro de Investigacion ´ Biom´edica en Red de Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Spai

Dual specificity phosphatase 1 expression inversely correlates with NF-κB activity and expression in prostate cancer and promotes apoptosis through a p38 MAPK dependent mechanism.

Dual specificity phosphatase 1 (DUSP1) and the transcription factor NF-κB are implicated in prostate cancer since their expression levels are altered along this disease, although there are no evidences up to date demonstrating a crosstalk between them. In this report, we show for the first time that DUSP1 over-expression in DU145 cells promotes apoptosis and decreases NF-κB activity by blocking p65/NF-κB nuclear translocation. Moreover, although DUSP1 impairs TNF-α-induced p38 MAPK and JNK activation, only the specific inhibition of p38 MAPK exerts the same effects than DUSP1 over-expression on both apoptosis and NF-κB activity. Consistently, DUSP1 promotes apoptosis and decreases NF-κB activity in cells in which p38 MAPK is induced by TNF-α treatment. These results demonstrate that p38 MAPK is specifically involved in DUSP1-mediated effects on both apoptosis and NF-κB activity. Interestingly, we show an inverse correlation between DUSP1 expression and activation of both p65/NF-κB and p38 MAPK in human prostate tissue specimens. Thus, most of apparently normal glands, benign prostatic hyperplasia and low-grade prostatic intraepithelial neoplasia samples show high DUSP1 expression and low levels of both nuclear p65/NF-κB and activated p38 MAPK. By contrast, DUSP1 expression levels are low or even absent in high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma samples, whereas nuclear p65/NF-κB and activated p38 MAPK are highly expressed in the same samples. Overall, our results provide evidence for a role of DUSP1 in the apoptosis of prostate cancer cells, through a mechanism involving the inhibition of p38 MAPK and NF-κB. Furthermore, our findings suggest that the ratio between DUSP1 and p65/NF-κB expression levels, rather than the individual expression of both molecules, is a better marker for diagnostic purposes in prostate cancer.Fondo de Investigaciones Sanitaria

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-kappa B/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-kappa B/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response

Prevalencia de genotipos del virus del papiloma humano de alto riesgo no vacunables dentro del programa de Detección Precoz de Cáncer de Cérvix en Cantabria

Estimar la prevalencia de infección por genotipos del virus del papiloma humano(VPH) de alto riesgo no vacunables.Dise˜no: Estudio descriptivo transversal.Emplazamiento: Siete centros de salud de Cantabria seleccionados aleatoriamente.Participantes: Se incluyó a todas las mujeres con una citología vaginal valorable (n = 3.359)entre 2010-2011.Mediciones principales: Se recogieron diagnóstico citológico, resultado de PCR y método anti-conceptivo. Los resultados de las citologías se clasificaron con el sistema Bethesda. Para latipificación de VPH según el riesgo oncogénico se utilizó la clasificación de Mu˜noz et al. Seestimaron proporciones y odds ratio (OR) con sus correspondientes intervalos de confianza al95% (IC95%).Resultados: La prevalencia de infección por VPH fue del 2,71% (IC95%: 2,15-3,27). La prevalen-cia de genotipos de VPH de alto riesgo oncogénico fue del 2,26%; (IC95%: 1,75-2,78). El genotipomás frecuente fue el 16 (28,89%). Más de la mitad de las mujeres fueron positivas para algúngenotipo de alto riesgo no vacunable: 51 (18,89%) o 58 (13,33%) o 68 (12,22%) o 31 (11,11%). Enel 23,33% de las mujeres coexistieron al menos 2 genotipos de alto riesgo no vacunables. Lasmujeres más jóvenes (≤ 30 a˜nos) tuvieron 2 veces más riesgo de infección por cualquier VPH:OR 2,01; (IC95%: 1,02-3,96); y 2 veces más probabilidad de usar anticonceptivos hormonalesfrente al preservativo: OR 2,09; (IC95%: 1,64-2,67).Conclusiones: Atendiendo al alto porcentaje de VPH de alto riesgo oncogénico no vacunable,habría que replantear la estrategia de prevención en la población, que podría tener una falsasensación de protección