Downregulation of protein tyrosine phosphatase PTPL1 alters cell cycle and upregulates invasion-related genes in prostate cancer cells

The final publication is available at link.springer.comPTPL1, a non-receptor type protein tyrosine phosphatase, has been involved in the regulation of apoptosis and invasiveness of various tumour cell types, but its role in prostate cancer remained to be investigated. We report here that downregulation of PTPL1 by small interfering RNA in PC3 cells decreases cell proliferation and concomitantly reduces the expression of cell cycle-related proteins such as cyclins E and B1, PCNA, PTTG1 and phospho-histone H3. PTPL1 downregulation also increases the invasion ability of PC3 cells through Matrigel coated membranes. cDNA array of PTPL1-silenced PC3 cells versus control cells showed an upregulation of invasion-related genes such as uPA, uPAR, tPA, PAI-1, integrin α6 and osteopontin. This increased expression was also confirmed in PTPL1-silenced DU145 prostate cancer cells by quantitative real time PCR and western blot. These findings suggest that PTPL1 is an important mediator of central cellular processes such as proliferation and invasion. © 2012 Springer Science+Business Media B.V.This work was supported by Grants from the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, Spain (FIS PI10/02026 and SAF2008-05046-C02-02), ISCIIIRETIC-RD06/0020-FEDER, Consejería de Salud (PI-2009-0589, AI-2010-003 to M.A.J.), and Consejería de Innovación, Ciencia y Empresa (CTS-6243), Junta de Andalucía (06/189, PI-2009-0589, and AI-2010-003 to M. A. J.). C. C. was supported by a pre-doctoral Grant from the Spanish Ministerio de Educación (F.P.I.: BES200612419) co-financiated by Fondo Social Europeo. C. S. was supported by a contract from Instituto de Salud Carlos III/FIS and Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía, Miguel Servet Program.Peer Reviewe

Caracterización de PRPF31

Motivación: La retinitis pigmentosa es un grupo heterogéneo de distrofias retinales caracterizada por una progresiva degeneración de los fotorreceptores, que acaba produciendo deficiencia visual o ceguera. PRPF31 es uno de los genes cuya mutación causa este tipo de distrofia. Aunque la proteína Prpf31 es un factor de splicing presente en todo el organismo, su mutación sólo produce distrofia retinal. Actualmente no se conoce el mecanismo implicado en la enfermadad, por lo que intentaremos discernirlo con ayuda de este proyecto.Métodos: Utilizando la secuencia del gen PRPF31, planteamos clonarla en el vector p3XFLAG y transfectar con la construcción células de mamífero. La fusión con FLAG facilitará los estudios de localización subcelular y de las interacciones por inmunoprecipitación. Transfectaremos con la construcción las líneas celulare RPE1, derivada de epitelio pigmentario de la retina (RPE) humana y 293, para estudiar la localización y las interacciones de Prpf31 con otras proteínas, con el objetivo de estudiar la base molecular de la enfermedad y poder desarrollar terapias génicas, farmacológicas y/o celulares. Resultados: La clonación de la secuencia de PRPF31 aún está en proceso ya que no hemos obtenido la construcción diseñada a pesar de probar diversos métodos. Se han empezado a poner a punto las condiciones de transfección usando otra construcción similar, tanto con la línea 293 como con RPE1. Se ha estudiado la distribución de Prpf31 en retina y RPE de ratón por western blot, mostrando una mayor abundancia en RPE, lo que no es común a otros factores de splicing como el PRPF3 y PRPF8, con los que se ha comparado. Conclusiones: PRPF31 debe tener alguna función específica en RPE, aún por definir, por lo que la degeneración de los fotorreceptores será secundaria a una disfunción del RPE. Hay otros casos en los que un gen presenta una función específicamente diferente en la retina, como en el caso de ATR, que causa síndrome de Seckel. ATR es un controlador de la división celular, pero en la retina este gen es crítico para el desarrollo postnatal de los fotorreceptores, por lo que su mutación causa degeneraciónen la retina

Diseño del instrumento de ayuda para la toma de decisiones: “alternativas de tratamiento para el cáncer de próstata: ¿qué opción prefiero?”

Purpose: To design a Decision-making Aid within the ‘Benign Prostatic Hyperplasia’ healthcare process modelling of the Andalusian Public Health System (SSPA) for the therapeutic approach of early-stage disease. Methods: The Decision Aid design was conducted in four phases: 1) Explore the receptiveness of professionals in the mainstream of the SSPA Decision Aid “Benign Prostatic Hyperplasia” process; 2) Select a Decision Aid from international experiences; 3) Transcultural adaptation of above selected Decision Aid; 4) Decision Aid Validation in the SSPA. Results: The results of the validation of Decision Aid “Alternative treatment for prostate cancer: What option do I prefer?” have shown that the document is well taken by patients, their design is attractive and the quality of clinical information it contains is high. The instrument meets the concerns of patients (95%), the language is simple and suitable (92%) and summarizes the essential information to make the decision (92%). The Decision Aid offers relevant information that help the patient in the decision making process (lack of decisional confl ict: 88.93), generates a sense of support (92.82), concerning the decision (86.88) and a sense of availability of information (90.51). Conclusion: Patients and professionals agree to recommend the use of Decision Aid. To improve information and enhance the tranquillity of the patient, the Decision Aid facilitates communication doctor patient consultation and the involvement of patients during the decision-making process.Objetivo: Diseñar un Instrumento de Ayuda para la Toma de Decisiones (IATD) en el Proceso Asistencial Integrado ‘Hipertrofia benigna de próstata. Cáncer de próstata’ del Sistema Sanitario Público de Andalucía (SSPA) para el abordaje terapéutico de esta enfermedad en estadio inicial. Método: El diseño del IATD se realizó en cuatro fases: 1) Explorar la receptividad de los profesionales del SSPA sobre la incorporación de IATD en el proceso “Cáncer de próstata”. 2. Seleccionar un IATD entre las experiencias internacionales. 3. Adaptar transculturalmente del IATD seleccionado al entorno del SSPA. 4. Validar el IATD en el SSPA. Resultado: Los resultados de la validación del IATD “Alternativas de tratamiento para el cáncer de próstata: ¿Qué opción prefiero?” han mostrado que el documento es bien cogido por los pacientes, su diseño resulta atractivo y la calidad en la información clínica que contiene es elevada. El Instrumento resuelve las dudas de los pacientes (95%), el lenguaje resulta sencillo y asequible (92%) y resume la información esencial para tomar la decisión (92%). El IATD ofrece información relevante que prepara al paciente para la toma de decisiones (ausencia de conflicto decisional: 88,93), genera sentimiento de apoyo (92,82), seguridad en la decisión (86,88) y sensación de disponibilidad de información (90,51). Conclusiones: Pacientes y profesionales coinciden en recomendar la utilización del Instrumento. Al mejorar la información y aumentar la tranquilidad del paciente, el IATD facilita la comunicación médico-paciente en la consulta y la participación en la toma de decisiones

Design a decision-making aid: "alternative treatment for prostate cancer: what option do you prefer?"

Purpose: To design a Decision-making Aid within the ‘Benign Prostatic Hyperplasia’ healthcare process modelling of the Andalusian Public Health System (SSPA) for the therapeutic approach of early-stage disease. Methods: The Decision Aid design was conducted in four phases: 1) Explore the receptiveness of professionals in the mainstream of the SSPA Decision Aid “Benign Prostatic Hyperplasia” process; 2) Select a Decision Aid from international experiences; 3) Transcultural adaptation of above selected Decision Aid; 4) Decision Aid Validation in the SSPA. Results: The results of the validation of Decision Aid “Alternative treatment for prostate cancer: What option do I prefer?” have shown that the document is well taken by patients, their design is attractive and the quality of clinical information it contains is high. The instrument meets the concerns of patients (95%), the language is simple and suitable (92%) and summarizes the essential information to make the decision (92%). The Decision Aid offers relevant information that help the patient in the decision making process (lack of decisional confl ict: 88.93), generates a sense of support (92.82), concerning the decision (86.88) and a sense of availability of information (90.51). Conclusion: Patients and professionals agree to recommend the use of Decision Aid. To improve information and enhance the tranquillity of the patient, the Decision Aid facilitates communication doctor patient consultation and the involvement of patients during the decision-making process

Bcl-xL is overexpressed in hormone-resistant prostate cancer and promotes survival of LNCaP cells via interaction with proapoptotic Bak.

Journal Article; Research Support, Non-U.S. Gov't;Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.This work was supported by Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III (PI020105), Fundación para la Investigación en Urología, Fundación Reina Mercedes, Consejería de Salud (7/01), and Plan Andaluz de Investigación.Ye

Regulation of chloride transport by regulatory components of nitrate homeostasis

Chloride (Cl−is an essential micronutrient that has traditionally been considered harmful to agriculture. Both Cl− and nitrate (NO3−), an essential nitrogen source, are the most abundant inorganic anions in plants. While it is currently believed that plants prioritize the uptake of NO3 over that of Cl−, it is now evident that plants use a large amount of metabolic energy to accumulate Cl− at macronutrient levels, allowing more efficient uses of nitrogen, carbon and water in plants (Colmenero Flores et al, 2019; Cakmak et al, 2023). Presently, plasma membrane proteins involved in root Cl- uptake are still unknown in Arabidopsis thaliana. Only the NO3 − transceptor (transporter + receptor) AtNPF6.3 has been shown to mediate Cl− uptake when NO3- is very scarce or absent from the rhizosphere (Wen et al, 2018; Xiao et al, 2021). We hypothesize that reciprocal regulation of NO3 vs Cl− homeostasis in plants is more complex than currently believed. We aim to study how the availability of NO3 affects Cl− uptake, and whether the transporters and regulatory components involved in nitrate NO3- transport are also involved in Cl−uptake. In addition, we want to accurately quantify what percentage of the Cl− taken up by the plant depends on AtNPF6.3. Chloride uptake has been quantified in mutant lines of Arabidopsis thaliana with lack of function for different transporters and regulatory components of NO3− uptake mechanisms. Applying different concentrations of NO3 in the irrigation solutions, and Cl− at levels required to fulfil micro‐ or macro nutrient requirements. Our results showed that high‐affinity NO3- transporters did not participate in Cl− transport, while the double‐affinity AtNPF6.3 transporter mediated Cl− transport only under conditions of low NO3- availability. In addition, mutant lines with reduced NO3- content showed an increase in Cl− accumulation. We propose that this responds to a compensatory mechanism that allows a more efficient use of NO3- when this relevant nitrogen source limits plant growth. On the other hand, our results point to the occurrence of an intracellular NO3− sensor that negatively regulates Cl− uptake when there is an adequate availability of NO3− in the rhizosphere. Finally, it should be noted that Cl− uptake mediated by AtNPF6.3 only represents 20% of the total Cl− accumulated in A. thalina, indicating that the most important Cl- uptake mechanisms are yet to be identified in plants References Colmenero‐Flores et al, 2019 Cakmak et al, 2023 Wen et al, 2018 Xiao et al, 2021We acknowledge funding by MICINN‐FEDER Projects PID2021‐125157OB‐I00 and RTI2018‐094460‐B‐100Resumen de la comunicación oral presentado en XVIII Spanish-Portuguese Congress on Plant Biology y XXV Meeting of the Spanish Society of Plant Biology (IPB 2023). Braga (Portugal). 9 al 12 de julio de 2023N

Regulation of chloride homeostasis in plants by nitrate availability

Poster presentado en el XVI Meeting of Plant Molecular Biology (RPMB) 14-16 sept. 2022, SevillaChloride (Clˉ) and nitrate (NO₃ˉ) are the most abundant inorganic anions in plants, sharing physical properties in solution and showing strong dynamic interactions. Thus, a higher tissue concentration of Clˉ leads to a lower concentration of NO₃ˉ and vice versa. This has been explained by the fact that both anions share and compete for similar transport mechanisms, meaning that Clˉ reduces the plants ability to take up NO₃ˉ from the soil. For this reason, Clˉ has been traditionally considered harmful to agriculture. Since NO₃ˉ is an essential nitrogen source, it is currently considered that plants strongly prioritize the uptake of NO₃ˉ over that of Clˉ. NO₃ˉ preference has been explained as: i) the occurrence of NO₃ˉ -selective transporters (e.g. AtNPF6.3 and MtNPF6.7) that mediate Clˉ only when NO₃ˉ is not available (Wen et al., 2018; Xiao et al., 2021); and ii) the occurrence of a signaling mechanism activated by NOзˉ supply that induces selective NO₃ˉ transporters (e.g. MtNPF6.7) while represses selective Clˉ transporters (e.g. MtNPF6.5; Xiao et al., 2021). The signaling cascade involves the AtNPF6.3 transceptor (transporter + receptor), the calcium transporter AtCNGC15 and the transcription factor AtNLP7. We have recently shown that Clˉ has a reduced impact on NO₃ˉ transport while signifcantly improving NO₃ˉ utilization and nitrogen-use efciency (NUE) in several plant species (Rosales et al., 2020). We hypothesize that regulation of NO₃ˉ and Clˉ homeostasis is more complex than currently believed, and other transporters diferent to those of the NPF6 subclade are involved in Clˉ uptake. To better understand the regulation of Clˉ uptake in plants and its interaction with NO₃ˉ availability, Clˉ accumulation has been quantifed in Arabidopsis mutant lines with lack of function for a number of relevant NO₃ˉ transport and NO₃ˉ signaling proteins. References • Rosales et al (2020). Front Plant Sci 11(442) • Wen et al (2018). Front Plant Sci 9(973) • Xiao et al (2021) EMBO J 40:e106847.We acknowledge funding by MICINN-FEDER Projects PID2021-125157OB-I00 and RTI2018-094460-B-100, and the CSIC-2021-JAE-INTRO-21 fellowship.N

Chloride uptake is regulated by nitrate availability in plants: identification of regulatory components

Resumen del póster presentado en XVIII Spanish-Portuguese Congress on Plant Biology y XXV Meeting of the Spanish Society of Plant Biology (IPB 2023). Braga (Portugal). 9 al 12 de julio de 2023Chloride (Cl−) and nitrate (NO3−) are the most abundant inorganic anions in plants, showing strong dynamic interactions, in part because both anions compete for common transport mechanisms. Since NO3− is an essential nitrogen source, it is currently considered that plants strongly prioritize the uptake of NO3− over that of Cl−. However, plants also accumulate Cl− to content levels which are typical of a macronutrient, inducing beneficial roles that improve the efficiency in the use of water, Nitrogen (NUE) and Carbon/energy (Colmenero‐Flores et al, 2019; Cakmak et al, 2023). The NO3− transceptor (transporter + receptor) AtNPF6.3, and its orthologs in other species, is the only root Cl− uptake mechanism identified in plants so far (Xiao et al, 2021). We hypothesize that reciprocal regulation of NO3− vs Cl− homeostasis in plants is more complex than currently believed. We aim to assess whether components that regulate NO3− uptake, including plasma membrane transporters, also regulate Cl− uptake, and to what extent AtNPF6.3 is the only mechanism involved in Cl− uptake in higher plants. Chloride uptake has been quantified in mutant lines of Arabidopsis thaliana affected in: i) high‐ and lowaffinity NO3− uptake transporters; ii) regulatory components that signal NO3− availability in plants. Quantifications were performed under high, mid and low NO3− availability.The dual‐affinity AtNPF6.3, but not the high‐affinity NO3− transporters tested in this work, mediated Cl− uptake, but only when NO3− availability was low. This strategy allows plants to improve NUE under NO3− limitation. Mutant lines dealing to a reduction in plant NO3− content gave rise to increased Cl− content, indicating the occurrence of an intracellular sensor that down‐regulates Cl− uptake when NO3− nutrition is adequate. A new regulatory component was also identified that participates in the induction of Cl− uptake under low NO3− availability. In A. thaliana, Cl− uptake mediated by AtNPF6 represented 20% of total Cl− accumulated, so other transporters with relevant roles in Cl− nutrition have yet to be identified in plants. References Cakmak et al (2022) Micronutrients. In Marschner’s Mineral Nutrition of Higher Plants (4th Ed) Colmenero‐Flores et al (2019) IJMS 20, 4686. Xiao et al (2021) EMBO J 40: e106847.N