Figure 4.

p53 attenuation in normal ovarian fibroblasts elevates expression of critical secreted paracrine communication chemokines and ECM remodeling factors in an NF-κB p65-dependent fashion. (A) Attenuation of p53 in ovarian fibroblasts increases expression of IL-8, IL-6, GRO-α, both immature and mature secreted forms of IL-1α and IL-1β, TRAF-6, and SOCS1, relative to γ-tubulin total cell levels. Double attenuation of p53 and NF-κB p65 either disrupt this stimulation (nearly all chemokines shown and SOCS1) or enhance the stimulation (TRAF-6), indicating that p53 suppression requires p65 signaling to stimulate chemokine secretion. (B) Cytoplasmic (C) and nuclear (N) fractionation before protein lysate harvest indicates that p53 repression activates nuclear localization of p65 phosphorylated at serine 273 and confirms elevated cytoplasmic expression of chemokines IL-1α, IL-1β, IL-6, and IL-8 following p53 attenuation that is primarily dependent on p65 as a driving mechanism, relative to stable cytoplasmic levels of HSP-90 and nuclear protein Lamin A/C. (C) Observations at the translational level are confirmed at the transcriptional level by reverse transcription-polymerase chain reaction amplification of cDNA from total RNA lysates following p53, or p53 and p65, attenuation, relative to stable β-actin RNA levels. Each blotting result indicates representative results from N = 3 experiments.