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Ras is involved in gap junction closure in proliferating fibroblasts or preadipocytes but not in differentiated
H L Brownell1, R P Narsimhan, M J Corbley
1Department of Microbiology, Queen's University, Kingston, Ontario, Canada.
Abstract:
A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.
Insights
Ras oncogene expression inversely affects gap junctional intercellular communication (GJIC). Decreased Ras signaling enhances GJIC, while increased Ras signaling inhibits it, impacting cell proliferation and differentiation.
Area of Science:
- Cell biology
- Oncology
- Molecular genetics
Background:
- Decreased gap junctional intercellular communication (GJIC) is linked to neoplastic cell transformation.
- The Ras oncogene product's role in regulating GJIC requires further investigation.
Purpose of the Study:
- To elucidate the function of the Ras oncogene product in modulating gap junction communication.
- To investigate the impact of altered Ras gene expression on GJIC in murine fibroblasts.
Main Methods:
- Constructed murine C3H10T1/2 fibroblasts with tunable Ras gene expression (up- and down-regulation).
- Quantified GJIC using a novel in situ electroporation technique on adherent cells.
- Assessed the effect of activated Ras(leu61) introduction and endogenous c-ras gene downregulation on junctional permeability.
Main Results:
- Increased activated Ras(leu61) levels proportionally reduced GJIC in 10T1/2 fibroblasts.
- Downregulation of endogenous c-ras gene expression enhanced junctional permeability.
- Ras appears crucial for junction closure in normal cell proliferation pathways.
Conclusions:
- Ras plays a significant role in the junction closure pathway regulating normal cell proliferation.
- Adipocytic differentiation-induced junction closure is independent of Ras, as demonstrated by GJIC abolition in c-Ras-deficient preadipocytes.