Person: Retamal, Mauricio A.
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Retamal
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Mauricio A.
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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 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 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 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