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Antisense suppresssion of protein kinase C-alpha and -delta in vascular smooth muscle
S J Busuttil1, D L Morehouse, J R Youkey
1Department of Vascular Surgery and Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennyslvania 17822, USA.
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 (alpha) and delta (delta) 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, PKCalpha-specific antisense ODNs selectively inhibited PKCalpha recovery compared to cells treated with an equal length nonsense ODN (76 +/- 3.9, P < 0.001), with no effect on recovery of PKCdelta. However, activation of MAPK by PDBu was not significantly inhibited in these PKCalpha downregulated cells. This suggests that only a small amount of the total PKCalpha is required for PDBu induced activation of MAPK and/or that PKCdelta 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.
Insights
Targeting protein kinase C (PKC) pathways is key for vascular disease. This study used antisense oligodeoxynucleotides to investigate PKC isozymes, finding PKCalpha downregulation minimally impacts MAPK activation, suggesting PKCdelta may play a role.
Area of Science:
- Cellular and Molecular Biology
- Vascular Biology
- Signal Transduction
Background:
- Vascular smooth muscle cell (VSMC) proliferation and gene expression are regulated by intracellular signaling pathways.
- Protein kinase C (PKC) activation is central to growth factor signaling, leading to downstream mitogen-activated protein kinase (MAPK) activation.
- PKC isozymes alpha and delta are predominant in rat aortic VSMC and can be downregulated by phorbol 12,13 dibutyrate (PDBu).
Purpose of the Study:
- To investigate the role of specific PKC isozymes in regulating MAPK activation in VSMCs.
- To assess the efficacy of isozyme-specific antisense oligodeoxynucleotides (ODNs) in manipulating PKC levels.
- To understand the contribution of PKCalpha and PKCdelta to PDBu-induced MAPK activation.
Main Methods:
- Downregulation of PKC isozymes in cultured rat aortic VSMC using prolonged PDBu stimulation.
- Administration of isozyme-specific antisense ODNs (phosphorothioated, C-5 propynyl modified) complexed with Lipofectin.
- Assessment of ODN uptake via FITC-labeling and optical imaging.
- Measurement of PKC isozyme reexpression and MAPK activation in response to PDBu.
Main Results:
- PKCalpha-specific antisense ODNs selectively inhibited PKCalpha recovery compared to nonsense ODNs (P < 0.001) without affecting PKCdelta recovery.
- Despite significant PKCalpha downregulation, PDBu-induced MAPK activation was not significantly inhibited.
- These findings suggest a minimal requirement of total PKCalpha for MAPK activation or a potential role for PKCdelta.
Conclusions:
- Specific PKC isozymes can be selectively manipulated using antisense ODN technology in VSMCs.
- PKCalpha may not be the sole mediator of PDBu-induced MAPK activation, with PKCdelta potentially playing a compensatory role.
- This model system allows for the assessment of individual PKC isozyme roles in VSMC signaling, proliferation, and disease.