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VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing NE differentiation through PKA, ERK1/2, and PI3K.

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The effect of vasoactive intestinal peptide (VIP) on intracellular Ca(2+) levels and its relationship with the expression of c-fos and vascular endothelial growth factor (VEGF) as well as with neuroendocrine (NE) differentiation were investigated in human prostate LNCaP cells. VIP induced the expression of c-fos mRNA as studied by reverse transcription polymerase chain reaction (RT-PCR). It was accompanied by VIP stimulation of c-fos protein synthesis, as measured by Western blot analysis. VIP enhanced intracellular Ca(2+) levels as evaluated using the calcium probe fura-2. VIP regulation of c-fos expression depended on [Ca(2+)](i) concentration since the intracellular calcium chelator BAPTA/AM decreased c-fos expression (both mRNA and protein) to basal levels. As shown by means of real-time RT-PCR, VIP stimulated VEGF mRNA expression: the effect was inhibited by 40% in the presence of curcumin (an inhibitor of AP-1 binding), and it was dependent on Ca(2+) since BAPTA/AM inhibited this VIP action by 43%. Similar observations were made on the effects of BAPTA/AM and curcumin on VIP stimulation of VEGF protein expression. Simultaneous treatment of cells with the protein kinase A inhibitor H89 and BAPTA/AM completely blocked this VIP effect, whereas each agent alone led only to a partial inhibition. In addition, the calcium chelator blocked by 37% the ability of VIP to induce NE cell differentiation as estimated by the observation of neurite development. These features support a VIP signalling pathway that could be mediated through both cAMP and [Ca(2+)](i) increase in prostate LNCaP cancer cells. Moreover, our data suggest the implication of c-Fos on the induction of the main angiogenic factor VEGF since the promoter region of the VEGF gene possesses AP-1 (i.e., c-Fos/c-Jun heterodimer) response elements. This feature represents a link between the nuclear oncogene c-fos, angiogenesis and NE differentiation by means of an initiating signal upon VIP receptors.