Person: Retamal, Mauricio A.
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Retamal
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Mauricio A.
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Publication Connexins in Cancer, the Possible Role of Connexin46 as a Cancer Stem Cell-Determining Protein(2023) León, Isidora; Salgado, María; Novoa, María; Retamal, Mauricio A.Cancer is a widespread and incurable disease caused by genetic mutations, leading to uncontrolled cell proliferation and metastasis. Connexins (Cx) are transmembrane proteins that facilitate intercellular communication via hemichannels and gap junction channels. Among them, Cx46 is found mostly in the eye lens. However, in pathological conditions, Cx46 has been observed in various types of cancers, such as glioblastoma, melanoma, and breast cancer. It has been demonstrated that elevated Cx46 levels in breast cancer contribute to cellular resistance to hypoxia, and it is an enhancer of cancer aggressiveness supporting a pro-tumoral role. Accordingly, Cx46 is associated with an increase in cancer stem cell phenotype. These cells display radio- and chemoresistance, high proliferative abilities, self-renewal, and differentiation capacities. This review aims to consolidate the knowledge of the relationship between Cx46, its role in forming hemichannels and gap junctions, and its connection with cancer and cancer stem cells.Publication Lack of canonical activities of connexins in highly aggressive human prostate cancer cells(2024) Asencio, Catalina; Véliz, Loreto; Flores-Faúndes, Emilia; Azócar, Lorena; Echeverría, Carolina E.; Torres-Estay, Verónica; Orellana Villena, Viviana; Ramírez-Santelices, Catalina; Sotomayor, Paula; Cancino, Jorge; Kerr, Bredford; Fernández-Olivares, Ainoa; Retamal, Mauricio A.; Sáez, Juan C.; Godoy, Alejandro S.Connexins (Cxs) have the ability to form channels that allow the exchange of ions/metabolites between adjacent cells (gap junction channels, GJC) or between the intra- and extra-cellular compartments (hemichannels, HC). Cxs were initially classified as tumor suppressors. However, more recently, it has been shown that Cxs exert anti- and pro-tumorigenic effects depending on the cell and tissue context. In prostate cancer (PCa), the expression and functionality of Cxs remain highly controversial. Here, we analyzed the expression pattern of Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 in PCa cell lines with increasing levels of tumor aggressiveness (LNCaP < LNCaP-C4-2 < Du-145 < PC-3). In addition, GJ and HC activities were evaluated in the PCa cell lines using dye coupling and dye uptake assays, respectively. Lastly, the cellular localization of Cx26, Cx32, and Cx43 was analyzed in LNCaP and PC-3 cell lines using immunofluorescence analyses. Our results showed a positive association between the mRNA levels of Cx26, Cx37 and Cx45 and the degree of aggressiveness of PCa cells, a negative association in the case of Cx32 and Cx43, and no clear pattern for Cx40. At the protein level, a negative relationship between the expression of Cx26, Cx32 and Cx43 and the degree of aggressiveness of PCa cell lines was observed. No significant differences were observed for the expression of Cx37, Cx40, and Cx45 in PCa cell lines. At the functional level, only LNCaP cells showed moderate GJ activity and LNCaP and LNCaP-C4-2 cells showed HC activity. Immunofluorescence analyses confirmed that the majority of Cx26, Cx32, and Cx43 expression was localized in the cytoplasm of both LNCaP and PC3 cell lines. This data indicated that GJ and HC activities were moderately detected only in the less aggressive PCa cells, which suggest that Cxs expression in highly aggressive PCa cells could be associated to channel-independent roles.Publication Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model(2024) Rozas Villanueva, María Fernanda; Orellana Villena, Viviana; Alarcón, Rodrigo; Maripillan, Jaime; Martínez, Agustín; Alfaro, Ivan; Retamal, Mauricio A.Background: Extravillous trophoblasts (EVTs) form stratified columns at the placenta–uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. Methods: We developed two cellular models, one with “low Cx40” (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with “high Cx40” (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. Conclusions: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NOPublication Editorial: Free Fatty Acids as Signaling Molecules: Role of Free Fatty Acid Receptors and CD36(2022) Puebla, Carlos; Morselli, Eugenia; Khan, Naim Akhtar; Retamal, Mauricio A.Publication Assessing Pulmonary Epithelial Damage in Hantavirus Cardiopulmonary Syndrome: Challenging the Predominant Role of Vascular Endothelium through sRAGE as a Potential Biomarker(2023) Meza, Gabriela; López, René; Vial Cox, María Cecilia; Cortes, Lina; Retamal, Mauricio A.; Delgado, IrisHantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory illness primarily associated with microvascular endothelial changes, particularly in the lungs. However, the role of the pulmonary epithelium in HCPS pathogenesis remains unclear. This study explores the potential of soluble Receptors for Advanced Glycation End-products (sRAGE) as a biomarker for assessing pulmonary epithelial damage in severe HCPS, challenging the prevailing view that endothelial dysfunction is the sole driver of this syndrome. We conducted a cross-sectional study on critically ill HCPS patients, categorizing them into mild HCPS, severe HCPS, and negative control groups. Plasma sRAGE levels were measured, revealing significant differences between the severe HCPS group and controls. Our findings suggest that sRAGE holds promise as an indicator of pulmonary epithelial injury in HCPS and may aid in tracking disease progression and guiding therapeutic strategies. This study brings clarity on the importance of investigating the pulmonary epithelium's role in HCPS pathogenesis, offering potential avenues for enhanced diagnostic precision and support in this critical public health concern.Publication KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells(2023) Chang, Cheng-Wei; Poudyal, Naveena; Peña, Francisca; Verdugo, Daniel; Stehberg, Jimmy; Retamal, Mauricio A.Background: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. Methods: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). Results: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. Results: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.Publication Activation of Intra-nodose Ganglion P2X7 Receptors Elicit Increases in Neuronal Activity(2023) Alcayaga, Julio; Vera, Jorge; Reyna, Mauricio; Covarrubias, Alejandra; Coddou, Claudio; Díaz, Esteban; Del Rio, Rodrigo; Retamal, Mauricio A.Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.Publication Acute activation of hemichannels by ethanol leads to Ca2+ -dependent gliotransmitter release in astrocytes(2024) Gómez, Gonzalo I.; García-Rodríguez, Claudia; Marillán, Jesús E.; Vergara, Sergio A.; Alvear, Tanhia F.; Farias-Pasten, Arantza; Sáez, Juan C.; Retamal, Mauricio A.; Rovegno, Maximiliano; Ortiz, Fernando C.; Orellana, Juan A.Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms.Publication SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics(2023) Prieto, Juan; Lucero, Claudia; Rovegno, Maximiliano; Gómez, Gonzalo I.; Retamal, Mauricio A.; Orellana, JuanBackground: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. Results: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. Conclusions: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.Publication Protocol for the study of Connexin and DNA interactions(2024) Fernández-Olivares, Ainoa; Retamal, Mauricio A.Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, “noncanonical” functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells