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NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma

Deon G Uffort1, Elizabeth A Grimm, Julie A Ellerhorst

  • 1Department of Experimental Therapeutics, The University of Texas, M.D Anderson Cancer Center, Houston, Texas 77030, USA.

The Journal of Investigative Dermatology
|August 1, 2008
PubMed

Insights

Nuclear factor-kappa B (NF-kappaB) activation, driven by the MAPK pathway, stimulates inducible nitric oxide synthase (iNOS) expression in melanoma. This pathway drives iNOS, contributing to melanoma tumor growth.

Area of Science:

  • Oncology
  • Molecular Biology
  • Biochemistry

Background:

  • Tumor expression of inducible nitric oxide synthase (iNOS) is linked to poor outcomes in melanoma patients.
  • The mitogen-activated protein kinase (MAPK) pathway has been previously identified as a regulator of iNOS in melanoma.

Purpose of the Study:

  • To investigate the role of nuclear factor-kappa B (NF-kappaB) in mediating the regulation of iNOS by the MAPK pathway in melanoma.
  • To elucidate the specific mechanisms of NF-kappaB involvement, including dimer formation and interaction with Bcl-3.

Main Methods:

  • Western blotting to confirm iNOS, activated extracellular signal-regulated kinase (ERK), and NF-kappaB expression.
  • Indirect immunofluorescence to detect NF-kappaB p50 and p65 localization.
  • Electrophoretic mobility shift assay (EMSA) to assess NF-kappaB DNA binding activity.
  • Treatment with MEK inhibitor U0126 and NF-kappaB inhibitors to evaluate pathway regulation.
  • Co-immunoprecipitation to study p50 and Bcl-3 interaction.

Main Results:

  • Constitutive iNOS expression was confirmed in melanoma cells.
  • The MAPK pathway was implicated in NF-kappaB activation, as MEK inhibition reduced NF-kappaB DNA binding.
  • NF-kappaB inhibitors suppressed iNOS expression, confirming NF-kappaB's regulatory role.
  • NF-kappaB p50 homodimers were present and interacted with Bcl-3, suggesting a mechanism for iNOS regulation.

Conclusions:

  • The constitutively active MAPK pathway in melanoma stimulates NF-kappaB activation (both hetero- and homodimers).
  • Activated NF-kappaB drives iNOS expression, contributing to melanoma tumorigenesis.
  • Targeting the MAPK/NF-kappaB/iNOS axis may offer therapeutic strategies for melanoma.

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