Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms

Abstract

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-α, -β2, -δ, and -μ isoforms was most pronounced. Immunofluorescent staining showed PKC-α and -μ expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-δ was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-α, -δ, and -μ from cytosol to membrane. Activation of PKC-α, -δ, and -μ was further confirmed by kinase assays. Addition of PKC-α inhibitor Gö-6976 at a nanomolar concentration, other PKC inhibitors Gö-6983 and GF-109203X, or PKC-δ-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-α or -δ increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-α, -δ, and -μ. Furthermore, inhibition of PKC-α and -δ blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.

The Role of Protein Kinase D in Neurotensin Secretion Mediated by Protein Kinase C-α/-δ and Rho/Rho Kinase

“…The addition of PMA (100 nM) induced a rapid translocation of PKD WT from the cytosol to the plasma membrane within 1 min after stimulation. These results are consistent with our report that endogenous PKD translocated to the membrane from the cytosol after PMA stimulation in fixed cells (14). In quiescent BON cells transfected with PKD ⌬PH , the fluorescence was present in the cytosol with intense fluorescence in the nuclei (middle panel).…”

“…siRNA Directed Against PKD Inhibits NT Secretion and PKD Phosphorylation in BON Cells-Based on findings that both PMA and bryostatin1 induce the activation of endogenous PKD in BON cells (14), we determined whether PKD is involved in PMA-mediated NT secretion. We took advantage of the RNA interference (RNAi) to selectively reduce PKD expression (Fig.…”

“…We have shown that protein kinase C (PKC), particularly isoforms PKC-␣ and -␦, plays a role in the stimulated release of NT (13)(14)(15). The PKCs comprise a family of intracellular serine/threonine-specific kinases that are, depending on the isoform, typically activated by Ca 2ϩ , lipid second messengers or protein activators and mediate the effects of a wide range of physiological stimuli, including growth factors, hormones, and neurotransmitters (16,17).…”

“…Protein Preparation and Western Blotting-Protein preparation and Western blotting were performed as described previously (14). In brief, equal amounts of protein were resolved on Novex Tris-glycine or Nu-PAGE Bis-Tris gels (Invitrogen) and electrophoretically transferred to polyvinylidene difluoride membranes; the membranes were incubated with primary antibodies overnight at 4°C followed by secondary antibodies conjugated with horseradish peroxidase.…”

“…Immunoprecipitation and in Vitro Kinase Assays-Immunoprecipitation and in vitro kinase assays were performed as described previously (14). In brief, proteins (50 g) were incubated with PKD or GFP antibodies (1:50) on a shaker for 2 h at 4°C followed by another 2 h incubation with 30 l of protein A-Sepharose beads at 4°C.…”