Prostate cancer (PCa) is the most common male cancer in term of worldwide incidence and represents one of the major causes from cancer death in men (World Health Organization, Geneva, Switzerland; Globocan 2018, estimated age-standardized rates, all ages). This androgen-sensitive cancer is characterized by a significant rise in case number with age and a distinct feature of human PCa is the net propensity to disseminate towards bone and to develop metastasis. Bone metastases, present in more than 70% of prostate cancer patients in the advanced stages, are associated with dramatic complications mainly skeletal-related events (SRE) linked to both osteogenesis and osteolysis [1
]. To date, only palliative treatments for these bone metastases and androgen-deprivation therapies with high potential side effects are mainly available. Moreover, the prediction of PCa cells aggressiveness based on biomarkers to distinguish highly aggressive cells remains challenging due to the well-described histological and cellular heterogeneity of this multifocal cancer [2
The bone privileged metastatic site is particularly due to the vicious dialogue between the cancer cell and the bone microenvironment present in two coexisting niches, namely endosteal and hematopoietic niches. Indeed, soluble factors, cell surface proteins, matrix-associated components and cancer-associated cells exhibit pivotal roles in the regulation of proliferation, survival, adhesion, dormancy and migration of disseminating PCa cells in the skeletal context. These factors facilitate the survival and adaptation of PCa cells to their new environment. In addition to these extensively investigated aspects, components of direct cell-cell interactions were also postulated to play a role in the aggressiveness and dissemination of cancer cells. Among them, intercellular channels composed of connexins (Cx) leading to a gap junction intercellular communication (GJIC) between cytoplasm of adjacent cells were soundly involved [3
Because of their unique function in tissue homeostasis by GJIC, a major role of Cxs loss in the onset and the development of cancer was historically demonstrated [4
]. These transmembrane proteins belong to a multigene family of 21 members in human and share a common primary structure with four transmembrane and two extracellular domains and three cytosolic portions corresponding to N-term, intracellular loop and a long C-term part [5
]. This latter domain also called carboxy terminal (CT) domain accounts for the difference between Cxs, notably in regulatory processes with potential phosphorylation and interaction sites. In addition to the well-known GJIC linked to formation of intercellular channels, recent data demonstrated two non-canonical alternative functions for Cxs [6
]. Hemichannels or connexons, formed by hexamerization of Cxs outside the junctional plaques, induce a communication with the extracellular space [8
]. The CT domain is considered as a controller of signaling networks inside the cells via an “interactome” function [9
]. Although each connexin isoform exhibits a distinct tissue distribution, many cell types express more than one connexin. In normal prostatic epithelium, experimental evidences reported the presence of 3 distinct Cx subtypes, Cx32, Cx43, and Cx26, encoding by genes named GJB1, GJA1, and GJB2, respectively (for review, see Reference [10
During cancer progression, an undeniable but ambivalent role was demonstrated for Cx43 in different types of carcinomas. For many years, Cx43 was considered as a tumor suppressor with accumulating evidences demonstrating anti-proliferative effects in most of cancer cells. Thus, Connexin 43 expression was significantly reduced or lost in prostate cancer tissues from patients with clinically localized prostate cancer [11
]. In addition to the modulation of Cx43 expression level, an alteration of the traffic to the plasma membrane was also demonstrated especially in androgen-insensitive PCa cells [12
]. However, more recent data highlighted a pro-aggressive action of Cx43 during most of dissemination steps leading to colonization of secondary sites [6
]. During prostate cancer progression, the intrinsic promigratory and proinvasive phenotype of PCa cells was clearly linked to an increase in Cx43 expression [10
]. Thus, Cx43 expression level in PCa cells was correlated with transcription factors and proteolytic activities implicated in the epithelial-mesenchymal transition [15
]. During diapedesis or angiogenesis, Cx43 has been shown to facilitate heterocellular dialogue with endothelial cells [18
]. Moreover, increasing number of publications have shown that Cx43 is implicated in the control of cell migration during cancer dissemination process [14
In the bone metastatic context, our previous data using Cx43-overexpressing PCa cell lines clearly demonstrated in vitro and in vivo that Cx43 enhanced the metastatic behavior of tumor cells [15
]. This increased aggressiveness was linked to Cx43 expression level and was restricted to cellular model presenting a functional exportation to the plasma membrane. The impact on bone integrity was due to both alteration of proliferation rate and differentiation abilities of the bone-forming cells (osteoblasts) leading to an osteolytic impact through both GJIC-independent and -dependent actions. More recently, Wang et al. (2018), reported that osteoblastic cells in the osteogenic niche could serve as calcium reservoir transferable by GJIC to cancer cells and could promote bone metastatic progression [22
To date, only a limited number of controverted studies have analyzed the significance of Cx43 expression level during PCa progression in human samples [11
]. No immunohistochemical study performed on bone metastatic tissues really focused on Cx43 expression. Moreover, the mechanisms underlying the implication of Cx43 in the chemotaxis of PCa cells to bone tissue and in the development of bone metastasis are incompletely understood. In the context of a crosstalk established during the formation of bone metastasis, the present work focused on the roles played by Cx43 expression level and its localization in the sensitivity of PCa cells to bone microenvironment.
In this study, we demonstrate that Cx43 potentiates the migration ability of PCa cells and the sensitivity to ObCM. We found that Cx43 membrane localization at the leading edge is required for this promigratory effect and that the carboxyl-terminal domain of Cx43, which interacts with Rac1 and cortactin, is sufficient to sustain cell migration in this bone context. Our data strengthen the protumoral propensity of the carboxy terminal domain of Cx43 and demonstrate that, in the metastatic context, it can modulate the phenotypic response of PCa cells to the osteoblastic environment.
The mechanisms underlying Cx43 action in the progression of prostate cancer cells towards bones are incompletely deciphered. An ambiguous and spatiotemporal role for Cx43 during initiation and progression of solid cancers and dissemination of carcinoma cells is now well documented [6
], with antitumoral features during the first steps mainly linked to antiproliferative effects, whereas later stages are rather associated with protumoral actions. To answer some of the outstanding questions about the role of Cx43 during the development of bone metastases, we first performed immunohistochemical assays on patient samples and we then analyzed the impact of Cx43-overexpression in different prostatic cell lines on their phenotypic features when placed in bone microenvironment reproduced by osteoblastic-conditioned medium.
As observed in most solid tumors and in accordance with previous observations in primary localized prostate tumors [11
], our data showed a loss of Cx43 expression in PCa cells from patients during early stages compared to normal tissue or benign prostatic hypertrophy. The reasons leading to this Cx43 downregulation are not fully deciphered. Androgens, which are linked to the development of prostate cancer [29
], may act by repressing Cx43 expression through androgen receptor pathway [30
]. More generally, GJA1 gene expression could be reduced at both transcriptional and posttranscriptional levels involving transcriptional repressors, promoter methylation and mRNA degradation by miRNA [31
]. We also revealed a Cx43 re-expression in advanced histological grades (notably grade 4), whereas, in a retrospective and a single protein analysis, a decreased expression level was associated with high Gleason score and worse prognosis by Xu et al. (2016) [23
]. The main contribution of our retrospective analysis of published databases was to unveil a specific Cx43 signature in bone metastasis compared to other metastatic sites at the transcript and protein levels. These results confirm the assumption that Cx43 is implicated in pathophysiological processes linked to dissemination of PCa cells toward bones [16
]. Such a Cx43 expression profile has already been demonstrated in bone metastatic breast cancer cells [32
], but in this context Cx43 was considered as an osteoblastic differentiation marker reflecting an osteomimetic phenotype for cancer cells more than an active factor of metastatic acquisition. The Cx43 expression profiles in various PCa cell lines reinforce the hypothesis of an important role of Cx43 in the bone metastatic context as only bone-targeting cell lines (C4-2B and PC-3) exhibit an increased relative expression level compared to normal epithelial cells. Under normal growth culture conditions, Cx43 expression level in the LNCaP model was very low, as recently published [14
] and shown in the present study. In a progression model based on cell lines, Tate et al. (2006) [33
] also demonstrated that PC-3 metastatic cells (PC-3M) expressed higher levels of Cx43 transcripts than parental PC-3 cells.
Using Cx43-overexpressing low aggressive LNCaP cells, we confirmed that Cx43 expression level had no significant effects on LNCaP cells proliferation and apoptosis as previously shown by our group and others [14
]. Interestingly, no influence on these cellular functions could be demonstrated under osteoblast-conditioned medium (ObCM) treatment. Regarding the invasion process, we revealed a stimulating action of ObCM similar whatever Cx43 expression level, while Cx43 seems to be required for invasive phenotype and for invadopodia formation in other cancer types, such as breast and gliomas [28
]. Accumulating evidences have revealed an emerging role of Cx43 in promoting cell migration in normal development [37
], as well as during progression of different types of cancer, including glioma, melanoma, and breast cancer (for review: Reference [38
]. For prostate cancer, we previously demonstrated a correlation between Cx43 expression levels with the metastatic potential of LNCaP cells [15
]. Moreover, Zhang et al. [14
] positively correlated the malignancy potential of PCa cells with Cx43 expression level among the 7 Cxs expressed in prostate tissues. By a sh-RNA approach on PC-3 cells, these authors confirmed the implication of Cx43 in the migration ability of PCa cells.
Nonetheless, the hypotheses of authors working in field of cancer differ regarding the mechanisms employed by Cx43 to potentiate cell migration processes. Indeed, GJIC, hemichannel functionality or intracellular signaling linked to CT-Cx43 have all been suggested to be critical in promoting cell motility. Since the observed effects were obtained using single cells, GJIC cannot account for the Cx43 promigratory action. In our study, no hemichannel activity (ATP and purinergic receptors) could be implicated in the promigratory action as demonstrated by pharmacological treatment using flufenamic acid. Moreover, in the context of direct communication between PCa and osteoblastic cells during bone metastasis development, GJIC was mainly implicated in the dormancy or reactivation of cancer cells [39
]. Interestingly, osteogenic cells can promote bone metastasis from breast or prostate origin by direct calcium influx through gap junctions into cancer cells [22
]. Both in physiological and pathological situations, it becomes now clear that Cx43 expression at the plasma membrane level mainly influences the forward cell movement and the directionality of cellular protrusions by intracellular activities [37
]. As in other models (breast cancer and lymphoma [38
]), our data obtained with PCa cells support a role for Cx43 in the regulation of the actin cytoskeleton network, in the stabilization of cell protrusions and intrinsic directed migration. Abnormal actin organization and reduced directionality have also been observed in neural crest cells derived from Cx43 knockout mice [40
]. Therefore, it is conceivable that Cx43 modulates cell motility by affecting directly or indirectly the cytoskeletal network. A dynamic remodeling of the actin network occurs during cancer cell migration and invasion [41
], during which the small GTPases signaling cascades are the main regulators of actin reorganization [42
]. Typically, actin filament polymerization induced by either the Cdc42 or Rac-mediated cascades results in the emergence of filopodia and lamellipodia at the leading edges [43
]. As in other cell types, a clear correlation exists between migration abilities and activated Rac-1 level in PCa cells. In PC-3 cells, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration as recently demonstrated [44
], and, in the present study, Cx43 overexpression in PC-3 or C4-2B cells was able to enhance concomitantly Rac-1 and migration activities. In the LNCaP model, we also demonstrated a direct link between Cx43 and active Rac-1 together with a decreased dynamic of lamellipodia. Moreover, Cx43 that colocalized with cortactin in protrusive membrane of LNCaP cells optimized the directionality under ObCM stimulation. However, other intracellular proteins implicated in metastatic progression and able to interact with Cx43 (tubulins, ezrin, ZO-1, vimentin, vinculin; for review, see Reference [16
]) cannot be excluded and their roles should be tested.
It is now well documented that the C-tail of Cx43 is the domain that interact directly with various cytoskeleton proteins implicated in cell motility (reviewed in Reference [45
]). Using truncated forms of Cx43, we demonstrated that the CT domain (aa 243–382) is essential and sufficient for the promigratory effects and corroborated the Cx43 CT domain implication in migration previously demonstrated in Hela cells [20
]. The enhanced migration of cells expressing either full length Cx43 or Cx43 CT was associated with an increased activation of the p38 MAP kinase in HeLa cells [48
]. Interestingly, we observed the same result with LNCaP cells in preliminary experiments [49
]. Therefore, the unique functions of Cx43 CT on migration process could be carried out both as an essential component of full-length Cx43 and as an independent signaling hub. Recently, a direct regulation of N-cadherin transcription after translocation of Cx43 carboxy tail to the nucleus was demonstrated to impact neural crest cell migration in vivo [50
]. Moreover, high proportion of prolines and serines has important physiological consequences and makes this part of Cx43 a potential target for extensive post-translational modifications that modulate its intracellular trafficking [9
]. Interestingly, after transfection of the human GJA1 gene in PCa cells, we revealed the presence of a Cx43 isoform at 20 kDa (Gja1-20k) expressed at high level in LNCaP cells, at lower level in PC-3 cells and absent in C4-2b cells. This internally translated Cx43 short isoform was implicated in the full-length Cx43 anterograde traffic to the plasma membrane [51
], in its delivery to cardiac intercalated discs [53
], and in its protection from degradation [54
]. Such a control of Cx43 membrane localization by Gja1-20k was also demonstrated during TGFβ-induced epithelial to mesenchymal transition of mammary cells [55
]. As PC-3 and C4-2b cells present exportation defects of the full-length form of Cx43, our results argue for a potential role played by Gja1-20k and confirm that post-transcriptional mechanisms based on chaperon action or on microtubules stabilization exist to limit the presence of Cx43-FL at the plasma membrane. Moreover, under ObCM stimulation, the promigratory effect in LNCaP cells correlates with an increased expression level of Gja1-20k together with an increase of membranous Cx43. To decipher further the role of Gja1-20k in the proactive effect of Cx43 on directional migration, we plan to overexpress Gja1-20k in traffic-deficient cell lines (PC-3 or C4-2b) and to analyze the impact on Cx43 localization and ObCM sensitivity.
To our knowledge, the present study represents the first demonstration of a role for Cx43 in sensitivity of prostate cancer cell migration to osteoblastic-conditioned medium. Different osteoblastic-secreted factors were already demonstrated to affect PCa cells proliferation and apoptosis processes [56
], dissemination towards bones [58
], their adherence [60
], or their survival/dormancy [61
] in bone context. Among them, bone-specific like osteonectin/SPARC (secreted protein, acidic and rich in cysteine) and nonspecific factors like TGF-beta1 present in ObCM were shown to increase migration of PCa cells and to induce formation of a mesenchymal-like phenotype [62
]. Collectively, these findings indicate that osteoblastic cells produce factors that are able to modify the migration capability of prostate cancer cells and our data suggest that Cx43 may be an important element of the metastatic risk by modulating sensitivity to these bone factors. ObCM sensitivity requires the membrane localization for Cx43 given the absence of promigratory effects of ObCM in PC-3 and C4-2b cells. As a perspective, we obviously plan to identify the main osteoblastic factors involved in the Cx43-dependent promigratory effect by proteomic analyses of ObCM. Among the potential bone factors, Wnt5a and CCL5 were recently implicated in the promotion of PCa cells migration [64
]. In preliminary data, we pointed out periostin or Osteoblast Specific Factor 2 (OSF-2) that presents an expression level correlated with Gleason score and poor prognosis [66
]. This chemo-attractive molecule, produced by reactive stroma of other aggressive carcinomas, was implicated in the control of cell migratory abilities [67
]. This factor, also present in ObCM produced by preosteoblastic cells in our culture conditions, is able to increase expression level and localization of Cx43 at plasma membrane in migratory front of LNCaP Cx43 cells and to induce alone a rise in directional migration [49
]. Extracellular vesicles potentially present in ObCM could also be consider as modulators of cancer microenvironment [69
]. Crosstalk between PCa cells and mesenchymal stem cells via exosomes containing miRNAs have already been demonstrated [70
]. Finally, the differential sensitivity demonstrated between LNCaP cells could be due to a specific surfaceome profile induced by the presence of increased level of Cx43 at the plasma membrane and could be investigated using an RNA-seq and cell surface proteomic approach.
4. Materials and Methods
Mouse monoclonal antibodies directed against Cx43 N-ter (#MABT903, clone P1E11.B19), mouse monoclonal anti-Cortactin (#05-180) and mouse monoclonal antibody anti-Rac1 (#23A8) were purchased from Merck-Millipore (Darmstadt, Germany). Rabbit polyclonal antibody directed against Cx43 C-ter (#C6219) and mouse monoclonal antibody anti-Actin ((#A5441) were supplied by Sigma (St. Louis, MO, USA). Mouse monoclonal antibody anti-β1Integrin was from Abcam (#ab30394, Cambridge, England). Mouse monoclonal antibody directed against Cx43 C-ter was purchased from BD Transduction Laboratories (#610062, San Jose, CA, USA). Mouse monoclonal against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Hytest (#5G4, Turku, Finland).
4.2. Plasmid Constructs
Plasmids with pMSCV-puro backbone (Clonetech laboratories, Palo Alto, CA, USA, containing cDNA for full-length (FL, aa 1-382) or for the two truncated versions (CT, aa 243–382 and ∆CT, aa 1–382) of human Cx43 were a kind gift of Dr Wun-Chey Sin (Vancouver, BC, Canada) and characterized in Reference [43
4.3. Cell Culture
LNCaP, PC-3, DU-145, and RWPE-1 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VI, USA; CRL-1740, CRL-1435, HTB-81, and CRL-11609 respectively) and C4-2B cell line was kindly provided by Dr. Potier-Cartereau (Tours, France). All cells were cultured at 37°C in a 5% CO2 incubator. LNCaP, PC-3, and DU-145 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 200 mM GlutaMAX, 0.1 g/L sodium pyruvate, 4.5 g/L D-glucose and supplemented with 10% Fetal Bovine Serum (FBS), 100 IU/mL penicillin, and 100 μg/mL Streptomycin. C4-2B cells were grown in Roswell Park Memorial Institute medium RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 200 mM GlutaMAX, 2 g/L D-glucose and supplemented as described above. RWPE-1 cells were maintained in Keratinocyte Serum Free Medium (K-SFM) supplemented with 30 μg/mL Bovine Pituitary Extract (BPE), 0.2 ng/mL Epidermal Growth Factor (EGF), and 5 μg/mL Gentamycin.
LNCaP and PC-3 cells stably expressing human Cx43 (LNCaP Cx43 and PC-3 Cx43) were previously established by means of retroviral particles and described in Reference [15
]. C4-2B cells expressing human Cx43 were obtained by transfecting parental cell lines with empty or Cx43 cDNA-containing pMSCV-puro using FuGENE HD transfection reagent according to the manufacturer’s instructions (Promega, Madison, WI, USA). Transfected cells were selected for 2 weeks with 5 μg/mL puromycin (Sigma). LNCaP cells expressing Cx43 FL, CT, and ΔCT forms of human Cx43 were also obtained by transfection as described above.
Murine primary osteoblastic cells (OB) were isolated as previously described [15
]. Briefly, OB cells were isolated from calvaria of 3–5 days-old mice after sequential digestions with trypsin and collagenase. After grown to confluency at 37 °C in phenol-red free DMEM containing 200 mM GlutaMAX, 4.5 g/L D-glucose and supplemented with 20% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin, OB cells were then seeded and maintained for 5 days in differentiating medium (phenol-red free DMEM supplemented with 10% FBS and 10 mM sodium β-glycerophosphate (Sigma). The media was change once, after day 2 of differentiation. Conditioned medium was then collected, centrifuged sequentially at 300 and 14,000 g
and then kept at −80 °C. This medium was referred to ObCM in this study.
4.4. Immunohistochemistry on Patient Samples
Investigations were carried out following the rules of the Declaration of Helsinki. Human samples were obtained from a tissue collection of patients treated at the university hospital of Poitiers (CHU La Milétrie, Poitiers, France) and permission to use this materiel was obtained from the ethical board of the Centre de Ressources Biologiques (CRB, Poitiers, France; ethical code number: BB-0033-00068, date of approval: 9 June 2017). Signed informed consent of patients were obtained by the local Ethic Committee and our request for additional data was approved under Project “Cx43 and bone metastasis” by the CNIL (Commission Nationale de l’Informatique et des Libertés, Paris, France, date of approval: 27 April 2017). To avoid pharmacological bias, patients included in the study never underwent hormonotherapy, chemotherapy or antiresorptive treatments. Blocks from surgical excision were cut 5-μm thick and stained with hematoxylin-eosin. Five-milliliter tissue sections were placed on charged slides, baked at 60 °C overnight, then deparaffinized and rehydrated. After blocking with Bovine Serum Albumin (BSA; Sigma, St. Louis, MO, USA) solutions and permeabilization in 0.5% Triton X-100 diluted in PBS, samples were incubated overnight using the mouse monoclonal antibody directed against Cx43 C-ter (Transduction Laboratories, San Jose, CA, USA) diluted at 1:200. Staining procedure used the ChemMate Detection kit (DakoCytomation, Glostrup, Denmark), based on an indirect biotin-avidin system with a universal biotinylated immunoglobulin secondary antibody, diaminobenzidin (DAB) substrate, and hematoxylin counterstain. Original slides from the 35 samples were reviewed by a pathologist to locate the tumor area before microphotographs acquisition.
4.5. Western Blot
Cells were lysed in ice cold RIPA buffer containing 50 mM Tris HCl pH7.4, 15 mM NaCl, 5 mM EDTA, 0.05% NP-40, 0.5% Sodium deoxycholate, 1% Triton X100, 0.01% SDS, and completed by 1 x protease and phosphatase inhibitors cocktail (Roche Applied Science, Penzberg, Germany). Protein extracts were cleared from cell debris by centrifugation at 12,500× g for 10 min at 4 °C. Protein concentration was measured using DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amount of proteins from different samples were mixed with 5X Laemmli-loading buffer (150 mM Tris-HCl, pH 6.8, 5% SDS, 12.5% 2-mercaptoethanol, 25% glycerol, and 0.025% bromophenol blue) to get a 1× final concentration.
Proteins were resolved using 10% SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked for 2 h at room temperature (RT) with 5% (w/v) non-fat powdered milk in Tris-buffered saline-Tween (TBS-T; 25 mM Tris-HCl, pH 8.0, 15 mM NaCl, 0.01% Tween 20) and incubated overnight at 4°C with primary antibodies diluted in TBS-T containing 5% (w/v) non-fat milk. The primary antibody concentration was as follows: 0.25 μg/mL monoclonal anti-Cx43 CT, 1:50 dilution for monoclonal anti-Cx43 NT, 1 μg/mL dilution for anti-IntegrinB1, 0.5 μg/mL anti-cadherin11, 1:1000 dilution for anti-cortactin, anti-Cx43 CT, anti-Rac1, anti-GAPDH, and anti-Actin. Membranes were then washed with TBS-T and then incubated for 1 h at RT with corresponding secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (1:10,000) for 1 h. Immunodetection was performed using chemiluminescent Luminata Forte substrate (Merck Millipore, Darmstadt, Germany) and LAS-3000 imaging system (Fujifilm, Tokyo, Japan). Densitometric analysis of signals was carried out using ImageJ software (version 1.39, National Institutes of Health, Bethesda, MD, USA).
After grown at low density on glass coverslips, LNCaP cells were treated with or without ObCM for 17 h before fixation in ice-cold 1:1 mix of methanol/acetone for 10 min. Cells were then blocked with 1% BSA, 1% Triton X100 in PBS at room temperature for 1 h. LNCaP cells were incubated overnight at 4 °C with mouse monoclonal anti-Cterm Cx43 or anti-Nterm Cx43, and mouse monoclonal anti-Cortactin antibodies prepared in blocking buffer at 1:250 dilution. A negative control was performed by omitting primary antibodies. LNCaP cells were then incubated with either Alexa Fluor 555 or 488 anti-Mouse secondary antibodies (1:1000) in blocking buffer for 2 h at RT. Coverslips were mounted with vectashield- Di-Aminido-Phenyl-lndol (DAPI) fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA) allowing nuclei staining. Images were captured using a confocal microscope (Olympus FV1000, Tokyo, Japan).
4.7. Cell Surface Biotinylation
To determine cell surface expression of Cx43, 0.8 × 106
LNCaP cells were seeded in 6 cm-diameter culture dishes. After adhesion to plastic, cells were stimulated under the same conditions as for migration. After 17 h of stimulation with or without ObCM, cells were rinsed twice at 4 °C with biotinylation buffer containing 0.1 mM CaCl2
, 1 mM MgCl2
, PBS pH7,4. Cells were then incubated with EZ-LINK sulfo-NHS-SS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 4 °C with gentle rocking. The labelling reaction was quenched for 10 min at 4 °C with biotinylation buffer containing 0.1% BSA. Cells were then washed twice in biotinylation buffer and were lysed in RIPA buffer. Protein concentration of each sample was determined by DC Protein Assay kit at 750 nm. Forty micrograms of the cell lysate was kept for total quantification of Cx43, Integrin-B1, Cadherin-11, and GAPDH expression by immunoblot. In parallel, 100 μg of proteins from the cell lysate were incubated together with 5% (v
) Streptavidin-Agarose beads in RIPA buffer at 4 °C overnight with gentle rocking. Biotin-Streptavidin complexes were then washed twice with RIPA buffer and biotinylated were eluted with 2× Laemmli-sample buffer. Total lysates and biotinylated proteins were analyzed by Western blot with anti-Cx43, Integrin-B1, Cadherin-11, and GAPDH, as described in Section 4.5
4.8. Integral Membrane Protein Extraction
Integral membrane proteins and membrane-associated proteins from LNCaP cells were extracted by using Mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. One hundred micrograms of membrane-protein extracts and 40 μg of proteins from whole cell lysates were prepared in Laemmli sample buffer and resolved by 12% SDS-PAGE prior to immunoblotting with anti-Cx43 and anti-Cadherin11, as previously described in Section 4.5
4.9. Single Cell Migration by Time-Lapse Videomicroscopy
For single cell migration assessment, tumor cell lines were seeded in osteoblastic differentiation medium in 6-well plates at low density (1500 cells/cm2) and were allowed to adhere on plastic. After 7 h, cell proliferation capacities were inhibited by a treatment with a non-cytotoxic concentration (10 μg/mL) of mitomycin-C for 1 h. Cells were then stimulated with either fresh OB differentiation medium or ObCM. Time-lapse recording was performed with JuLITMStage device (NanoEnTek, Seoul, Korea) in optimal cell culture conditions (37 °C, 5% CO2, humidified atmosphere). Images were acquired on 4 random fields in each well with a 10x brightfield objective, at a frame rate of 1 at 3-min intervals for 17 h. Image analysis was performed by manual tracking of cell centroid for all time points of the recording using ‘Manual Tracking’ plugin from ImageJ. All cells of a given field were analyzed excluding those which paths were interrupted during the recording. X; Y coordinates files obtained were then imported with the recommended format into ‘Chemotaxis and Migration Tool’ software (version 2.0, Ibidi GmbH, Martinstried, Germany). Calculated dynamic parameters, such as Euclidian distance, accumulated distance, and directness, were extracted after calibration of the software with appropriate settings (pixel size and time interval). Data represent the mean values of dynamic parameters from 3 independent experiments. After 15 h of recording, three morphological parameters were also quantified for all cells in a given field with ImageJ software: circularity index as defined by 4 × π × area/perimeter (AU), Feret’s maximum diameter (μm), and cell perimeter (μm). Data represent the mean values of morphological parameters from 3 independent experiments.
4.10. Scanning Electron Microscopy (SEM)
LNCaP cells were fixed for 1 h with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.1. After PBS washes, dehydration was carried out using successive incubations of increasing ethanol concentrations (from 50% to 100%). Cells were dried by hexamethyldisilazane treatment and sputter-coated in a vacuum with an electrically conductive 3-nm thick layer of platinum. SEM images were then recorded with a scanning electron microscope, TENEO Volumescope of ThemoFisher scientific at 5 KV voltage.
4.11. Lucifer Yellow Uptake
To evidence the presence of functional hemichannels in Cx43 overexpressing LNCaP cells, we experimentally induced hemichannels opening by using calcium-free extracellular solutions. For this, 4 × 104
LNCaP or U251 cells (serving as a positive control of functional hemichannels) in 1 mL growth medium were seeded in 24-well plates. Twenty-four hours later, cells were rinsed twice with regular extracellular solution (ECS; 142 mM NaCl, 5.4 mM KCl, 1.5 mM MgCl2
, 2 mM CaCl2
, 10 mM HEPES, 25 mM D-glucose, pH 7.35) [71
]. Cells were then incubated at 37 °C for 15 min with either ECS (unstimulated condition) or divalent cation-free solution (DCF, which is ECS without MgCl2
and with 2 mM EGTA; stimulated condition) supplemented with 0.5 mg/mL fluorescent dye Lucifer Yellow (Sigma). This step allowed the forced opening of the Cx43 hemichannels and, consequently, the entry of the fluorescent tracer into the cells. Cells were then washed twice in ECS before low magnification image acquisition using Olympus MVX10 macroscope (objective 1×/1; 22°C; medium: PBS; Camera: Hamamatsu ORCA-03G; cellSens Dimension Version 1.4 software Olympus, Tokyo, Japan).
4.12. ATP Release
For ATP release experiments, the protocol used was the same as for Lucifer Yellow uptake assays, except Lucifer Yellow was omitted. During the stimulation phase, cells were incubated with DCF solution together with a hemichannel inhibitor, Flufenamic Acid (FFA (Sigma)) used at 0.5 mg/mL. After 2 washes in ECS, extracellular medium was collected on ice and sequentially centrifuged at 300 and 14,000× g to remove cells and cellular debris, respectively. Extracellular ATP was quantified by ‘ATP determination kit’ according to the manufacturer’s instruction. This bioluminescence assay is based on ATP-dependent conversion of D-luciferin into oxyluciferin by luciferase leading to light emission. Luminescence was measured with luminometer microplate reader set at 560 nm. For each experimental condition, the amount of ATP (picomole) was calculated with a standard curve obtained from measurements of samples with known and increasing ATP concentrations. Those values were normalized by respective total protein amount determined by typical colorimetric assay (DC protein assay, Bio-Rad Laboratories) after cell lysis.
4.13. Immunoprecipitation Assay
To test interaction between Cx43 and cortactin, LNCaP cells stimulated with or without ObCM for 17 h were lysed with RIPA buffer. After protein concentration determination, 50 μg of the cell lysate were kept for total quantification of Cx43 and cortactin expression by immunoblot. In parallel, 500 μg of proteins were incubated 15 min at 4 °C with 1 μg of polyclonal rabbit anti-Cterm Cx43 or with irrelevant polyclonal rabbit antibody prepared in ice-cold RIPA buffer. Those samples were then incubated together with 50 μL of protein A/G-sepharose beads for 2 h at 4°C with gentle rocking to capture immune complexes. After washes in RIPA buffer, proteins were eluted from the beads by addition of 2× Laemmli loading buffer. The immunoprecipitates and proteins from whole cell lysates were analyzed by Western blot as described above.
4.14. GTP-Bound Rac1 Pull-Down Assay
The Glutathione S-transferase (GST)-tagged Rac-binding domain (RBD) of p21-activated kinase (PAK) [GST-PAK-RBD domain] was obtained as pGEX-2 T fusion genes (gift of JG Collard, Netherlands Cancer Institute, Amsterdam, NL, USA) and produced as described. Recombinant proteins were prepared as glutathione S-transferase fusion proteins in Escherichia coli (BL21 strain), purified using glutathione-sepharose beads (Amersham Pharmacia Biotech, Little Chalfont, UK), and used as GST-fusion proteins. To measure Rac1 activity, LNCaP cells were cultured and stimulated under the same conditions as for migration, except that they were seeded in 10 cm-diameter culture dishes. After 17 h of stimulation with or without ObCM, cells were lysed using a cell lysis buffer containing 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 2 mM MgCl2
, 1% NP-40 10% glycerol, and antiproteases cocktail. Protein concentration of each sample was determined by DC Protein Assay kit (Bio-Rad laboratories, Hercules, CA, USA) at 750 nm. Twenty micrograms of the cell lysate were kept for total quantification of Rac1 and actin expression by immunoblot. In parallel, 300 μg of proteins from the cell lysate were mixed with 30 μL of GST-PAK-RBD fusion protein coupled with glutathione-sepharose beads at 4 °C overnight with gentle rocking. Complexes containing GTP-bound Rac1 were then washed thrice with lysis buffer and proteins eluted with 2× Laemmli loading buffer. The pull-down complexes and proteins from whole cell lysates were fractionated by a 13% SDS–PAGE, followed by Western blotting with anti-Rac1 and anti-actin as described above. This approach was also used to detect Cx43 pulled-down with GST-PAK-RBD using Cx43 antibody instead of Rac1 antibody, as described in Reference [72
4.15. Statistical Analyses
Results were expressed as the mean of n experiments ± the standard error of the mean (SEM). Statistical analyzes were carried out using GraphPad Prism software (version 5.0, GraphPad Software, San Diego, CA, USA) using data from at least 3 independent experiments. Analysis of variance (ANOVA, ANalysis of VAriance) followed by a Bonferroni post-test was used for the analysis of at least three experimental groups. Unpaired t-test was used to compare two experimental groups. Data were taken statistically significant for a value of p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001).