Vasoactive intestinal peptide (VIP) induces c-fos expression in LNCaP prostate cancer cells through a mechanism that involves Ca2+ signalling. Implications in angiogenesis and neuroendocrine differentiation.
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.
Collado
Beatriz
B
Department of Biochemistry and Molecular Biology, Alcalá University, Alcalá de Henares, Spain.
Sánchez
María G
MG
Díaz-Laviada
Inés
I
Prieto
Juan C
JC
Carmena
María J
MJ
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D009479:Neuropeptides / Q000378:metabolism; D011471:Prostatic Neoplasms; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects* / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*
D002614:Chelating Agents; D007546:Isoquinolines; D009479:Neuropeptides; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D013449:Sulfonamides; D042461:Vascular Endothelial Growth Factor A; D014660:Vasoactive Intestinal Peptide; D004533:Egtazic Acid; D000242:Cyclic AMP; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium