Serotonin and cancer: what is the link?

Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic monoamine that acts as a neurotransmitter in the central nervous system, local mediator in the gut and vasoactive agent in the blood. Serotonin exerts its multiple, sometimes opposing actions through interaction with a multiplicity of receptors coupled to various signalling pathways. In addition to its well-known functions, serotonin has been shown to be a mitogenic factor for a wide range of normal and tumoral cells. Serotonin exhibits a growth stimulatory effect in aggressive cancers and carcinoids more often through 5- HT1 and 5-HT2 receptors. In contrast, low doses of serotonin can inhibit tumour growth via the decrease of blood supply to the tumour, suggesting that the role of serotonin on tumour growth is concentration-dependent. Data are also available on serotonin involvement in cancer cell migration, metastatic processes and as a mediator of angiogenesis. Moreover, the progression of some tumours is accompanied by a dysregulation of the pattern of serotonin receptor expressions. Serum serotonin level was found to be suitable for prognosis evaluation of urothelial carcinoma in the urinary bladder, adenocarcinoma of the prostate and renal cell carcinoma. In some cases, antagonists of serotonin receptors, inhibitors of selective serotonin transporter and of serotonin synthesis have been successfully used to prevent cancer cell growth. This review revaluates serotonin involvement in several types of cancer and at different stages of their progression.

A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway.

Gap junctions are intercellular channels organized in plaque that directly link adjacent cells. Connexins (Cx), the constitutive proteins of gap junctions are associated with several partner proteins (cytoskeletal, anchoring) which could participate in plaque formation and degradation. Coimmunoprecipitation and indirect immunofluorescence analyses showed that ZO-1, a tight junction-associated protein, was linked to Cx43 in the testis. By using gamma-hexachlorocyclohexane (HCH), known to induce gap junction endocytosis, we demonstrated that endocytosis increased Cx43/ZO-1 association within the cytoplasm of treated Sertoli cells. In control cells, the two proteins were present, as expected, at the plasma membrane level, but poorly colocalized. The increased intracytoplasmic Cx43/ZO-1 complex was associated with a shift towards increased levels of Cx43 P1 and P2 isoforms. The HCH induced Cx43 hyperphosphorylation was abolished by the ERK inhibitor PD98059 suggesting that this effect could be mediated through activation of the ERK pathway. These data strongly support a novel role for ZO-1 in the turnover of Cx43 during gap junction plaque endocytosis.

Connexin expression and gap junctional intercellular communication in human first trimester trophoblast.

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes, and recently mutation of connexin genes has been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cells coexist and lead to a fusion phenotype (villous trophoblast) and a proliferative/invasive phenotype (extravillous trophoblast). Here we characterized in situ and in vitro the expression of Cx transcripts and proteins in the villous and extravillous trophoblast of first trimester placenta. In addition, the GJIC functionality was investigated using the gap-fluorescence recovery after photobleaching (gap-FRAP) method. We demonstrated in the villous trophoblast the presence of Cx43 mRNA and of Cx43 protein localized between cytotrophoblastic cells and between cytotrophoblastic cells and syncytiotrophoblast. In vitro, a transient functional gap junctional intertrophoblastic communication was demonstrated during the trophoblast fusion leading to the multinucleated syncytiotrophoblast. During the proliferative process of the extravillous trophoblast, Cx40 is expressed in the proximal part of the cell columns. When cytotrophoblastic cells were cultured on Matrigel for 2 days, alpha5beta1 integrin expression was observed concomitant with the presence of Cx40 mRNA and of Cx40 protein between the cells. No evidence for a GJIC was detected in this induced extravillous phenotype. In addition, Cx32 was detected between some aggregated cells after 72 h of culture. Our data show that the presence of Cx43 allows an inter-trophoblastic GJIC and is associated with the fusion process leading to the villous syncytiotrophoblast and that the presence of Cx40 does not allow GJIC and is associated with the extravillous phenotype.

Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells.

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Involvement of gap junctional communication and connexin expression in trophoblast differentiation of the human placenta.

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutation of connexin genes have been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cell coexist leading to a double model: fusion phenotype (villous trophoblast) and proliferative/invasive phenotype (extravillous trophoblast). This review focuses on current knowledge on the connexin expression and the implication of GJIC in trophoblastic differentiation. Experimental evidence obtained in human placenta demonstrates the involvement of connexin 43-gap junctions in the trophoblastic fusion process and of a connexin switch during the spatially and temporally controlled proliferation/invasion process.

Modified expression of testicular gap-junction connexin 43 during normal spermatogenic cycle and in altered spermatogenesis.

In order to better understand the implications of gap junction proteins in spermatogenesis, connexin 43 (Cx43), the most abundant connexin in the testis, was evaluated in testes of wild-type mice and of two mutants with impaired spermatogenesis (ebo/ebo, and jun-d-/-mice). Reverse transcription-polymerase chain reaction (RT-PCR) amplification revealed a constitutive expression of mRNA for Cx43 in both wild-type mice and infertile mutants. In the seminiferous tubules of wild-type mice, indirect immunofluorescence revealed that Cx43 expression was stage-dependent and that the signal was mainly located in the region of Sertoli cell occluding junctions. Colocalization of Cx43 and of the tight-junction-associated protein zonula occludens 1 (ZO-1) was demonstrated in seminiferous tubules by using dual-label immunofluorescence in conjunction with confocal microscopy. The Cx43 staining analyzed by high-resolution confocal microscopy appeared as continuous, anastomozed ribbons and thin dots. The level of Cx43 immunoreactivity was reduced in seminiferous tubules of ebo/ebo and jun-d-/- mutants as compared to the respective wild-type mice. No staining for Cx43 was detected in Sertoli cell-only seminiferous tubules observed sometimes in jun-d-/- mice. The present study represents one of the first in vivo examples of alteration of seminiferous tubule Cx43 in testes with impaired spermatogenesis.

Regulation of tissue-type plasminogen activator and its inhibitor (PAI-1) by lipopolysaccharide-induced phagocytosis in a Sertoli cell line.

The plasminogen activator (PA) system is thought to play a major role in the proteolytic events associated with spermatogenesis. The mechanisms controlling the expression of PA and of its major physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1), in the seminiferous epithelium are still unknown. In the present study we analyzed the expression of PA and PAI-1 in a murine Sertoli cell line (42GPA9) in response to stimulation by lipopolysaccharides (LPS) used to activate the phagocytic activity of these cells. Immortalized Sertoli cells cultured under basal conditions secreted predominantly tissue-type PA (tPA) as demonstrated by zymographic analysis and the presence of tPA transcripts. In zymographic experiments a larger molecular weight proteolytic band corresponding to the formation of PA-PAI-1 complex was also observed. The stimulation of immortalized Sertoli cells by LPS resulted in both alteration of the apparent tPA molecular weight to a higher form and transient increase in PAI-1 biosynthesis. The phorbol ester TPA stimulates similarly PAI-1 synthesis in the Sertoli cell line, while 8-bromo-cAMP has no effect. These results suggest for the first time the existence of a direct linkage between molecular events triggered by phagocytosis and regulation of tPA and PAI-1 in Sertoli cells.

Ultrastructural and biochemical evidence for gap junction and connexin 43 expression in a clonal Sertoli cell line: a potential model in the study of junctional complex formation.

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.