Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells

RESEARCH ARTICLE Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells Marı́a Leiza Vitale*, Christopher J. Garcia, Casimir D. Akpovi, R.-Marc Pelletier Département de pathologie et biologie cellulaire, Faculté de Médecine, Université de Montréal, Montreal, Québec, Canada * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Vitale ML, Garcia CJ, Akpovi CD, Pelletier R-M (2017) Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells. PLoS ONE 12(7): e0182495. https://doi.org/ 10.1371/journal.pone.0182495 Editor: Arantxa Tabernero, Universidad de Salamanca, SPAIN Received: March 8, 2017 Accepted: July 19, 2017 Published: July 31, 2017 Copyright: © 2017 Vitale et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper’s files. Funding: This work was supported by Natural Sciences and Engineering Research Council of Canada Grants OGP0194652 (to M. L. Vitale) and OGP0041653 (to R.-M. Pelletier): www.nserccrsng.ca. R.-M Pelletier and ML Vitale received start up funds from Diabète Québec: www.diabete. qc.ca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Folliculostellate cell gap junctions establish a network for the transmission of information within the anterior pituitary. Connexins make up gap junction channels. Changes in connexin (Cx) turnover modify gap junction-mediated intercellular communication. We have reported that cytokines and hormones influence Cx43 turnover and coupling in folliculostellate cells and in the folliculostellate cell line TtT/GF. In addition, the expression of different connexins alters intercellular communication and connexins may have functions besides cell coupling. Here we assessed the expression, turnover and subcellular localization of Cx46 and Cx50 in the anterior pituitary and TtT/GF cells. Then, we assessed the impact of various natural (lactation, annual reproductive cycle, bFGF) and pathological (autoimmune orchitis, diabetes/obesity) conditions associated with altered anterior pituitary hormone secretion on Cx46 and Cx50. Anterior pituitary Cx46 and Cx50 expression and subcellular distribution were cell-dependent. Cx46 was expressed by folliculostellate, TtT/GF and endocrine cells. In the cytoplasm, Cx46 was chiefly associated with lysosomes. Variously sized Cx46 molecules were recovered exclusively in the TtT/GF cell nuclear fraction. In the nucleus, Cx46 co-localized with Nopp-140, a nucleolar factor involved in rRNA processing. Neither cytoplasmic nor nuclear Cx46 and Cx43 co-localized. Cx50 localized to folliculostellate and TtT/GF cells, and to the walls of blood capillaries, not to endocrine cells. Cx50 was cytoplasmic and associated with the cell membrane, not nuclear. Cx50 did not co-localize with Cx46 but it co-localized in the cytoplasm and co-immunoprecipitated with Cx43. Cx46 and Cx50 responses to various physiological and pathological challenges were different, often opposite. Cx46 and Cx43 expression and phosphorylation profiles differed in the anterior pituitary, whereas Cx50 and Cx43 were similar. The data suggest that Cx46 participates to cellular growth and proliferation and that Cx50, together with Cx43, contributes to folliculostellate cell coupling. PLOS ONE | https://doi.org/10.1371/journal.pone.0182495 July 31, 2017 1 / 30 Cx46 and Cx50 in anterior pituitary folliculostellate cells Competing interests: The authors have declared that no competing interest exist. Introduction The folliculostellate (FS) cells together with endocrine cells constitute the anterior pituitary gland parenchyma. The FS cells control several anterior pituitary activities [1]. Specifically, FS cells produce cytokines and growth factors that regulate anterior pituitary hormone secretion [2;3]. At variance with the anterior pituitary endocrine cells, FS cells contain no secretory granules [4] and express the protein S-100 [5]. In addition, by enclosing endocrine cells in clusters, the FS cell cytoplasmic processes organize the anterior pituitary parenchyma into follicles [6–8]. We and others have shown that the extent of FS cell cytoplasmic processes is responsive to the hormonal milieu [8;9] and to serum-borne molecules [10] such as the basic fibroblast growth factor (bFGF) [11;12]. The gap junction-mediated cell-to-cell communication allows the sharing of information and thus, the coordination and synchronization of responses in connected cells. Within the anterior pituitary, gap junctions join FS-to-FS cells [7;13–15], FS cells-to-endocrine cells [16– 18] and endocrine cells-to-endocrine cells [19]. Experimental evidence shows that regulators of anterior pituitary function modulate FS cell connectivity by acting on FS cell gap junctions [12;15;20–25]. The gap junction channels are made up of proteins named connexins (Cxs). The rodents and human have been found to express approximately 20 Cx variants classified into subgroups based on sequence homology and oligomerization [26;27]. The regulation of gap junctionmediated intercellular communication is achieved by modifying Cx turnover [28] and/or the expression of individual Cx species [29]. Cx43 is expressed by the FS cells [15;17;30] and the cells of the FS cell line TtT/GF [23]. Here, we assessed the expression of Cx46 and Cx50, two α-Cxs known to interact with Cx43 [26], in anterior pituitary FS cells and TtT/FG cells. Cx46 and Cx50 have been extensively studied in the ocular lens [31]. In addition, bone [32–34], lung [35], retinal pigmented epithelial cells [36], heart [37], astrocytes [38] and human breast tumour [39] all express Cx46. Cx50 expression has been described in the retina [40] and corneal endothelial cells [41]. Recently, we reported the expression of Cx46 and Cx50 in cells of the developing and adult testes [42]. Cxs are critically involved in strategic steps of tissue and cell actions. Mutated Cx genes, deregulation of Cx turnover, and/or aberrant localization of Cxs have been documented in pathological disorders [43–46]. Cxs also contribute to other cellular functions besides cell coupling [43;47;48]. To evaluate Cx46 and Cx50 involvement in the anterior pituitary function, the behavior of Cx46 and Cx50 was assessed in physiological and pathological conditions that display changes in anterior pituitary hormone secretion. Specifically, the annual seasonal reproductive cycle offers a unique opportunity to evaluate the influence of natural and reversible hormonal changes on the expression, phosphorylation status and localization of Cx46 and Cx50 in the anterior pituitary. We have established the mink (Mustela (...truncated)