Antisense Suppression of Protein Kinase C-α and -δ in Vascular Smooth Muscle

Abstract

Understanding and being able to manipulate intracellular signaling pathways which control VSMC gene expression and proliferation will be important in efforts to control neointimal hyperplastic vascular diseases. Activation of the protein kinase C (PKC) family of enzymes is a central event in growth factor-stimulated cells. PKC activation results in the activation of downstream protein kinases including mitogen activated protein kinase (MAPK). PKC isozymes alpha (α) and delta (δ) predominate in cultured rat aortic VSMC and both isozymes are completely downregulated upon prolonged (16–24 hr) stimulation with the PKC activator, phorbol 12,13 dibutyrate (PDBu). At these low levels of PKC, MAPK activation in response to PDBu is nearly abolished. To assess the role of specific PKC isozymes in regulating MAPK, isozyme-specific antisense oligodeoxynucleotides (ODNs) were used to inhibit reexpression of PKC in downregulated cells. ODNs were phosphorothioated to increase stability and contained C-5 propynyl modified pyrimidines which are reported to have increased binding affinity. ODNs were administered in low concentration (400 nM) with a cationic liposome carrier (Lipofectin; GibcoBRL). Optical imaging of cells treated with FITC-labeled ODNs confirmed that virtually all cells took up the ODNs within 2 hr. With this technique, PKCα-specific antisense ODNs selectively inhibited PKCα recovery compared to cells treated with an equal length nonsense ODN (76 ± 3.9,P< 0.001), with no effect on recovery of PKCδ. However, activation of MAPK by PDBu was not significantly inhibited in these PKCα downregulated cells. This suggests that only a small amount of the total PKCα is required for PDBu induced activation of MAPK and/or that PKCδ can mediate the response. Manipulation of PKC isozymes using this model system should allow assessment of the roles of specific isozymes in controlling diverse downstream effectors and events related to VSMC growth and proliferation.