Céline Fiorini

Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens.

Macroautophagy (hereafter referred to as autophagy) is the major degradative pathway of long-lived proteins and organelles that fulfils key functions in cell survival, tissue remodeling and tumor suppression. Consistently, alterations in autophagy have been involved in a growing list of pathologies including toxic injury, infections, neurodegeneration, myopathies and cancers. Although critical, the molecular mechanisms that control autophagy remain largely unknown. We have recently exploited the disruption of autophagy by environmental carcinogens as a powerful model to uncover the underlying signaling pathways. Our work published in Cancer Research revealed that the sustained activation of the MAPK ERK pathway by the carcinogen Lindane or the MEK1+ oncogene alters autophagy selectively at the maturation step resulting in the accumulation of large defective autolysosomes. Consistent with our findings, a similar defect is observed with other common xenobiotics such as dichlorodiphenyltrichloroethane and biphenol A that specifically activate ERK. Conversely, Pentachlorophenol that activates both ERK and p38, fails to induce autophagic vacuolation. In addition, evidence is provided that abrogation of p38 by SB203580 is sufficient to interfere with the normal autophagic maturation step. Altogether, these findings underscore the critical role played by MAPK ERK and p38 in the tight control of the autophagy process at the maturation step. Addendum to: Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Activity E. Corcelle, M. Nebout, S. Bekri, N. Gauthier, P. Hofman, P. Poujeol, P. Fénichel and B. Mograbi Cancer Res 2006; 66:6861-70

Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen.

The gap junction protein connexin 43 (Cx43) exhibits dynamic trafficking that is altered in most tumor cells and in response to carcinogen exposure. A number of connexin (Cx)-binding proteins are known to be involved in endocytic internalization of gap junctions. Here, we analyzed the discrete molecular interactions that occur between Src, ZO-1 and Cx43 during Cx43 internalization in response to the non-genomic carcinogen γ-hexachlorocyclohexane (HCH). Internalization of the Cx43 gap junction plaque was significantly accelerated in Cx43-GFP transfected 42GPA9 Sertoli cells that were exposed to the carcinogen. HCH induced the rapid recruitment of Src to the plasma membrane, activation of Src within 3 minutes and the efficient inhibition of gap junctional coupling, but had no effect in the presence of the Src inhibitor PP2. Immunoprecipitation experiments demonstrated that HCH increased Cx43-Src interaction and concomitantly decreased Cx43–ZO-1 association. ZO-1 was detected on both sides of the gap junction plaques in untreated cells, but appeared to be mainly localized on one side during HCH-induced internalization. The dissociation of ZO-1 from Cx43 appears to occur specifically on the side of the plaque to which Src was recruited. These findings provide mechanistic evidence by which internalization of the Cx43 gap junction plaque might be initiated, suggesting that Src-mediated dissociation of ZO-1 from one side of the plaque initiates endocytic internalization of gap junctions and that this process is amplified in response to exposure to HCH.

The large GTPase dynamin2: a new player in connexin 43 gap junction endocytosis, recycling and degradation.

Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis‐specific gap junction protein. We previously reported that Cx33 exerts dominant‐negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild‐type Sertoli cells transfected with Cx33‐DsRed2 and Cx43‐green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi‐channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy‐fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens‐1 (ZO‐1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO‐1 specifically interacted with P1 phosphorylated and P0 unphosphorylated isoforms of Cx43, and the ZO‐1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO‐1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant‐negative effect on gap junction plaque.

Connexin 43 gap junction plaque endocytosis implies molecular remodelling of ZO-1 and c-Src partners.

Gap junctions, through their constitutive proteins, connexins (Cx), are involved in several processes including regulation of cellular proliferation, tissue differentiation, homeostasis and neoplasic transformation. Internalization of the gap junction plaque to form annular gap junction is a dynamic process which present similarities with endocytosis and participates in the control of gap junction coupling. Cx43 exhibits dynamic trafficking that needs sequential implication of a large number of protein partners. We have recently shown that ZO-1 localized in both sides of the gap junction plaque was restricted to one side during internalization. The dissociation between ZO-1 and Cx43 particularly occurred on the face where c-Src specifically associated with Cx43, and was abnormally accelerated in response to a carcinogen. In this addendum we summarize and further discuss these results.

Endothelin‐1 inhibits human osteoblastic cell differentiation: Influence of connexin‐43 expression level

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during developmental and differentiation processes. In bone, the involvement of the gap junctional protein, connexin‐43 (Cx43), and of GJIC in osteoblastic differentiation and mineralization of the extracellular matrix has been previously demonstrated. Former studies have shown that endothelin‐1 (ET‐1) was also implicated in the control of osteoblastic proliferation and differentiation. However, depending on the cellular models, ET‐1 has been shown to decrease or increase osteoblastic differentiation markers. As no data were available on the ET‐1 effect on GJIC and Cx43 expression in osteoblastic cells, we analyzed here the possible crosstalk between Cx43 and ET‐1 in a human cell line (hFOB 1.19) which displays different Cx43 expression levels and phenotypes when cultured at 33.5 or 39°C. The presence of ET‐1 (10−8 M) for 2–12 days of culture did not significantly alter the proliferation rate of hFOB cells whatever their phenotype. In contrast, ET‐1 induced a differential inhibitory effect on the biochemical differentiation markers (alkaline phosphatase activity and osteocalcin expression) with a significant reduction in the differentiated phenotype at 39°C, whereas no effects were measured at 33.5°C. The inhibitory effect was linked to a decrease of GJIC and of Cx43 both at transcriptional and protein levels. Altogether, our results suggest that Cx43 expression level could influence the action of ET‐1 on human osteoblastic cell differentiation. Our data also indicate that the gap junctional protein could play a pivotal role in the response of osteoblasts to mitogenic factors implicated in bone pathologies. J. Cell. Biochem. 103: 110–122, 2008.