EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction

Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. Conclusion: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction

Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

Funding Information: This research was supported by the Isabel Gemio Foundation (P18–13) and was also partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. E.A.-C. was supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. S.C.-C. and E.U.-C. were supported by FPU fellowships (FPU19/04435 and FPU16/00684, respectively) from the Ministerio de Ciencia, Innovación y Universidades, Spain. M.P.-B. and A.G.-B. received fellowships from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N.-S. was supported by the “Ramon y Cajal” Program (RYC-2016–20883), and P.G.-S., was funded by “Juan de la Cierva Incorporación” Program (IJC2019–039229-I), Spain. S.M.S.Y.-D. was supported by the Isabel Gemio Foundation and CIBERNED (CB06/05/0041). J.M.F received research support from the Isabel Gemio Foundation and the “Instituto de Salud Carlos” III, CIBERNED (CB06/05/0041). Publisher Copyright: © 2022 by the authors.Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.publishersversionpublishe

Aging impairs neurogenic contraction in guinea pig urinary bladder: role of oxidative stress and melatonin

The incidence of urinary bladder disturbances increases with age, and free radical accumulation has been proposed as a causal factor. Here we investigated the association between changes in bladder neuromuscular function and oxidative stress in aging and the possible benefits of melatonin treatment. Neuromuscular function was assessed by electrical field stimulation (EFS) of isolated guinea pig detrusor strips from adult and aged female guinea pigs. A group of adult and aged animals were treated with 2.5 mg x kg(-1) x day(-1) melatonin for 28 days. Neurotransmitter blockers were used to dissect pharmacologically the EFS-elicited contractile response. EFS induced a neurogenic and frequency-dependent contraction that was impaired by aging. This impairment is in part related to a decrease in detrusor myogenic contractility. Age also decreased the sensitivity of the contraction to pharmacological blockade of purinergic and sensitive fibers but increased the effect of blockade of nitrergic and adrenergic nerves. The density of cholinergic and nitrergic nerves remained unaltered, but aging modified afferent fibers. These changes were associated with an increased level of markers for oxidative stress. Melatonin treatment normalized oxidative levels and counteracted the aging-associated changes in bladder neuromuscular function. In conclusion, these results show that aging modifies neurogenic contraction and the functional profile of the urinary bladder plexus and simultaneously increases the oxidative damage to the organ. Melatonin reduces oxidative stress and improves the age-induced changes in bladder neuromuscular function, which could be of importance in reducing the impact of age-related bladder disorders.status: publishe

Monochloramine effects on gallbladder contractility

8 Pág. Departamento de Medio Ambiente y Agronomía​Digestive inflammatory processes induce motility alterations associated with an increase in reactive oxygen species production, including monochloramine (NH2 Cl). The aim of the study was to characterize the effects of the naturally occurring oxidant monochloramine in the guinea pig gallbladder. We used standard in vitro contractility technique to record guinea pig gallbladder strips contractions. NH2 Cl caused a concentration-dependent contraction which was reduced by inhibition of extracellular Ca2+ influx and tyrosine kinase pathways. The PKC antagonist GF109203X also reduced the response but not after previous tyrosine kinase inhibition, suggesting that PKC is activated by tyrosine kinase activity. The NH2 Cl contractile effect was also reduced by inhibitors of mitogen-activated protein kinase (MAPK), nitric oxide synthase, phospholipase A2 and cyclooxygenase. In addition, NH2 Cl impaired the responses to CCK, tissue depolarization and electrical field stimulation. In conclusion, we present new evidence that monochloramine impairs not only the gallbladder response to CCK but also to membrane depolarization and nervous plexus stimulation, and that tyrosine kinase, PKC, MAPK and NO pathways are involved in the contractile direct effect of monochloramine.The study was supported by grants IB18025 and GR18119 from Junta de Extremadura, Spain.Peer reviewe