PI3K/Akt-Dependent Transcriptional Regulation and Activation of BMP-2-Smad Signaling by NF-κB in Metastatic Prostate Cancer Cells

BACKGROUND. Bone morphogenetic proteins (BMPs) exert osteoinductive effects in prostate cancer (PC) via uncharacterized mechanisms. In this study, we investigated whether the nuclear transcription factor NF-kappa B, implicated in PC metastasis, is involved in transcriptional regulation and activation of BMP-2 or BMP-4/Smad signaling in PC cells. METHODS. NF-kappa B inhibition was achieved by I kappa B alpha super-repressor adenoviral vector and activation was monitored by EMSA and reporter assays. BMP expression and activation was measured by PCR and reporter assays. Promoter binding assay was performed by chromatin immunoprecipitation (ChIP) assay. Smad1/5/8 phosphorylation was measured by Western blot analysis. RESULTS. PCR and chimeric BMP-2 and BMP-4 luciferase assays demonstrate that NF-kappa B confers robust and selective activation of BMP-2 in p65 overexpressing or rh TNF-alpha-stimulated PC cells. Inhibition of NF-kappa B significantly reduced transcript levels and autocrine production of BMP-2 by rhTNF-alpha! stimulated C4-2B cells and to a lesser extent by the parental LNCaP cells. Selective inhibition of PI3K/Akt suppressed the NF-kappa B-induced BMP-2 promoter activity. Furthermore, suppression of NF-kappa B activation decreased the transcript levels and BMP-2-induced phosphorylation of Smad1/5/8, critical downstream targets of BMP-2 signaling in PC cells. Notably, the activation of BMPRII by BMP-2 is required for modulation of Smad activation by NF-kappa B in PC cells. Based on ChIP analysis, the transcriptional regulation of BMP-2 gene by NF-kappa B may be partially attributed to binding to kappa b site on the BMP-2 promoter. CONCLUSIONS. The data suggest that PI3K/Akt-NF-kappa B axis may promote PC bone metastasis in part by regulating transcription and activation of the BMP-2-Smad signaling cascade in osteotropic PC cells. Prostate 69: 168-180, 2009. (C) 2008 Wiley-Liss. Inc.