Activated Src requires Cadherin-11, Rac, and gp130 for Stat3 activation and survival of mouse Balb/c3T3 fibroblasts.

We previously demonstrated that engagement of cadherins, cell to cell adhesion molecules, triggers a dramatic increase in levels and activity of the Rac/Cdc42 small GTPases, which is followed by secretion of IL6 family cytokines and activation of their common receptor, gp130, in an autocrine manner. This results in phosphorylation of the Signal Transducer and Activator of Transcription-3 (Stat3) on tyrosine-705, which then dimerizes, migrates to the nucleus, and activates transcription of genes involved in cell division and survival. In the present report we demonstrate that, in mouse Balb/c3T3 fibroblasts, mutationally activated Src527F also increases Rac levels, leading to secretion of IL6 family cytokines and gp130 activation, which triggers the Stat3-ptyr705 increase. Interestingly, our results also demonstrate that cadherin-11 is required to preserve gp130 levels for IL6 family signaling. At the same time, however, activated Src527F downregulates cadherin-11, in a quantitative manner. As a result, Src527F expression to intermediate levels allows sufficient cadherin-11, hence gp130 levels for Stat3 activation, as expected. However, expressed to high levels, Src527F eliminates cadherin-11, hence gp130 signaling, thus abolishing Stat3-ptyr705 stimulation. Taken together, these data establish for the first time a loop between Src, cadherin-11, gp130, and Stat3 activation. This fine balance between Src527F and cadherin-11 levels which is required for Stat3 activation and cellular survival could have significant therapeutic implications.

Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication.

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation. To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication.

A functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner. Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

Stat3 and gap junctions in normal and lung cancer cells.

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as Src suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High Src activity was found to correlate with high levels of the Src effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, i.e., transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high Src activity. In the contrary, Stat3 inhibition in normal cells or in lines with low Src activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability.

Effects of SRC and STAT3 upon gap junctional, intercellular communication in lung cancer lines.

BACKGROUND: We have previously demonstrated a positive correlation between SRC and its effector signal transducer and activator of transcription-3 (STAT3), and a reverse relation between SRC and gap junctional communication (GJIC) in seven non-small cell lung cancer (NSCLC) lines. Since a number of oncogenes besides SRC can affect GJIC, here we examined the actual contribution of the SRC/STAT3 axis to GJIC suppression. MATERIALS AND METHODS: SRC and STAT3 activity levels were examined in SK-LuCi-6, LC-T, QU-DB, SW-1573, BH-E, Calu-6, FR-E, SK-MES, H1299, BEN, WT-E, A549 and SHP-77 cells by western blott analysis, probing with antibodies specific for SRC-ptyr418 or STAT3-ptyr705. GJIC was examined by in situ electroporation. RESULTS: Confluence of all cultured NSCLC cells tested induces a dramatic increase in STAT3 activity, which is independent of SRC action. In addition, the LC-T line had high STAT3-705, despite the fact that SRC-418 expression was low, indicating that other, SRC-independent factors must be responsible for STAT3 activation and GJIC suppression in these cells; however, BH-E and SHP-77 cells with low GJIC, both SRC-418 and STAT3-705 expression were low, indicating that GJIC suppression can be independent of the SRC/STAT3 axis altogether. Our results also show that STAT3 inhibition does not restore GJIC in any of the examined lines, while in the non-transformed rat F111 fibroblast line which has extensive GJIC, STAT3 inhibition actually eliminated junctional permeability. CONCLUSION: Our results indicate a further level of complexity in the relationship between SRC, STAT3 and GJIC in NSCLC than what has been previously demonstrated. In addition, STAT3 is actually required for, rather than suppressing GJIC.

Engaged for survival: From cadherin ligation to STAT3 activation.

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors. This state is mimicked by dense cell cultures. In this review, we integrate some recent findings on a key signal transducer, STAT3 (signal transducer and activator of transcription-3), whose activity is dramatically increased following cadherin-mediated cell to cell adhesion. Cadherin engagement, favored in dense cell cultures, causes a dramatic increase in total Rac/Cdc42 protein levels through inhibition of proteasomal degradation, which is followed by activation of IL-6 and STAT3. The cadherin/Rac/IL-6/STAT3 axis offers a potent survival signal that is a prerequisite for neoplastic transformation, as well as normal tissue function.