Connexins the structural proteins of gap junctions are a

Connexins, the structural proteins of gap junctions, are a family of transmembrane proteins that oligomerize into hemichannels containing six connexin subunits [9]. Upon reaching the cell surface, two hemichannels pair to complete an intercellular gap junction channel, which directly links the cytoplasm of neighboring MK-8745 mg and mediate the exchange of low-molecular-mass molecules (<1000Da), including cAMP, inositol trisphosphate, and Ca [10]. The permeability of gap junctions is finely regulated at the transcriptional [11], [12] and posttranslational level via cycles of connexin phosphorylation [13], intracellular Ca or H+ concentrations [14], [15] (for reviews, see Refs. [16], [17], [18]). A critical role in the progression of a variety of tumors has been attributed to gap junction intercellular communication (GJIC) [19], [20]. Reduced or loss of gap junctional activity through different mechanisms including reduced expression has been implicated in various human cancers [21]. The crucial role of altered GJIC in tumor progression was further demonstrated by the exogenous expression of connexins in gap junction-deficient cell lines, which was shown to restore functional communication and to delay tumor growth [22], [23]. The number of possible mechanisms responsible for connexin-mediated tumor suppression has grown with insights into the possible functional roles of hemichannels [24], cross-talk between gap junctions and other adhesion-based junctional complexes [25] and the discovery of novel connexin-binding proteins [26]. Thus, increasing evidence suggests that GJ-independent mechanisms are also involved in connexin-mediated tumor suppression [20], [21]. Despite many studies having demonstrated the importance of connexin43 (Cx43), the most abundant connexin in bone cells, for bone development and turnover during the last decade [27], little is currently known about GJIC and Cx43 in primary bone tumors. In this report, we analyzed the specific role of Cx43-driven GJIC in ES tumor growth. Using a combination of in vitro and in vivo experimental approaches, we demonstrated: i) a lack of Cx43 gene expression in ES cells, ii) that the expression level of Cx43 is associated with that of EWS–FLI1, iii) that Cx43 inhibits ES tumor growth via modulation of cell proliferation, and iv) Cx43 reduces tumor cell-driven osteoclast activity.
Materials and methods
Results
Discussion Observations indicating a role of connexin in tumorigenesis are supported by many in vitro analyses demonstrating the down-regulation or loss of connexin expression in a wide range of neoplastic cells and primary tumors [35], [36], [37]. Here, we demonstrated a loss of Cx43 gene expression in ES cell lines. Early works using the NIH3T3 cell model have demonstrated that EWS–FLI1 acts as a transcriptional activator that allows oncogenic transformation [38], [39]. Current opinion holds that EWS–FLI1 functions as an aberrant transcription factor supported by works which demonstrated that EWS–FLI1 localizes to the nucleus, binds DNA in a site-specific manner and possesses a powerful transcriptional activator that is more potent than the native FLI1 [7]. Gene expression studies have further demonstrated that EWS–FLI1 is able to enhance the expression of many genes implicated in transformation and/or tumor progression, including MYC [40], ID2 [41], CCND1 [42] and PDGFC [43]. Other studies have revealed that EWS–FLI1 is able to decrease the expression of many genes including those encoding p21 [44], p57kip [45], TGFβRII [46] and IGFBP3 [47]. Among these, only the TGFβRII and IGFBP3 genes, which are down-regulated by EWS–FLI1, have been identified as direct EWS–FLI1 targets [48]. Here, using a EWS–FLI1 knock-down approach, we demonstrated that EWS–FLI1 affects the Cx43 gene expression, suggesting that Cx43 is a EWS–FLI1 target gene. However, we cannot exclude that Cx43 induction in response to EWS–FLI1 silencing is a consequence of modified cell differentiation for example. The exact mechanisms underlying the down-regulation of Cx43 gene by EWS–FLI1 remain to be elucidated.