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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
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Stress-activated protein kinases are negatively regulated by cell density

D Lallemand1, J Ham, S Garbay

  • 1Unité des Virus Oncogènes, Unité associée 1644 du Centre National de la Recherche Scientifique, Paris Cedex 15 France.

The EMBO Journal
|October 2, 1998
PubMed

Insights

Culture confluency inhibits the JNK/SAPK pathway, reducing c-Jun phosphorylation. This stress response restricts signaling to cells near wounds, impacting cell migration and tissue repair.

Area of Science:

  • Cellular signaling
  • Molecular biology
  • Biochemistry

Background:

  • The JNK/SAPK pathway is activated by UV irradiation, TNFalpha, PDGF, and EGF, leading to c-Jun phosphorylation and increased transactivation.
  • c-Jun phosphorylation at Ser63 is a key event in regulating gene expression and cellular responses.
  • Cell culture confluency is a common experimental condition that can influence cellular signaling pathways.

Purpose of the Study:

  • To investigate the effect of culture confluency on JNK/SAPK pathway activation and c-Jun phosphorylation in mouse fibroblasts.
  • To determine the molecular mechanisms underlying confluency-induced inhibition of JNK/SAPK signaling.
  • To examine the role of c-Jun phosphorylation in wound healing and cell migration in response to mitogenic and chemotactic agents.

Main Methods:

  • Utilized specific antibodies to detect phosphorylated c-Jun (Ser63) in fibroblasts under varying culture conditions.
  • Performed transfection experiments to assess the involvement of small G-proteins (cdc42, Rac1) and the actin cytoskeleton in pathway regulation.
  • Conducted monolayer wounding experiments to observe c-Jun phosphorylation patterns in response to growth factor and UV stimulation.

Main Results:

  • Culture confluency significantly inhibited JNK/SAPK pathway activation and subsequent c-Jun N-terminal phosphorylation.
  • The inhibition of JNK/SAPK signaling by confluency occurred at or upstream of cdc42 and Rac1, and was dependent on the actin microfilament network.
  • In contrast, the classical MAPK pathway remained insensitive to culture confluency.
  • Following stimulation, c-Jun phosphorylation was predominantly observed in cells bordering wounded areas, highlighting a role in directed cell migration.

Conclusions:

  • Culture confluency acts as a modulator of stress-dependent signaling cascades, specifically inhibiting the JNK/SAPK pathway.
  • This confluency-mediated inhibition restricts c-Jun N-terminal phosphorylation to cells at the wound edge, suggesting a mechanism to coordinate cell migration during tissue repair.
  • The findings provide insights into how cellular density influences signaling pathways critical for wound healing and cellular responses to external stimuli.

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