Protein Kinase C Delta Induces Apoptosis of Vascular Smooth Muscle Cells through Induction of the Tumor Suppressor p53 by Both p38-dependent and p38-independent Mechanisms

Abstract

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. In the present study, we examined the novel PKC isoform protein kinase C delta (PKC␦) and its role in vascular SMC apoptosis. In A10 SMCs, overexpression of PKC␦ was sufficient to induce apoptosis, whereas inhibition of PKC␦ diminished H 2 O 2 -induced apoptosis. Moreover, evidence is provided that the tumor suppressor p53 is an essential mediator of PKC␦-induced apoptosis in SMCs. Activation of PKC␦ led to accumulation as well as phosphorylation of p53 in SMCs; this induction correlated with apoptosis. Furthermore, blocking p53 induction with small interference RNA or targeted gene deletion prevented PKC␦-induced apoptosis, whereas restoring p53 expression rescued the ability of PKC␦ to induce apoptosis in p53 null SMCs. We also establish that PKC␦ regulates p53 at both transcriptional and post-translational levels. Specifically, the transcriptional regulation required p38 MAPK, whereas the post-translational modification, at least for serine 46, did not involve MAPK. Additionally, PKC␦, p38 MAPK, and p53 co-associate in cells under conditions favoring apoptosis. Together, our data suggest that SMC apoptosis proceeds through a pathway that involves PKC␦, the intermediary p38 MAPK, and the downstream target tumor suppressor p53. Apoptosis of vascular smooth muscle cells (SMCs)3 is a well established component of the remodeling that occurs during normal development of the circulatory system (1, 2) as well as during the course of neointimal formation after intervention for atherosclerosis (3-5). Because increased total cellularity is a prominent feature of an occluding neointima, the balance between proliferation and apoptosis during vessel healing appears central to this pathologic process (6, 7). Indeed, accumulating evidence suggests that abnormal SMC apoptosis leads to neointimal hyperplasia (8 -11). However, despite the importance of SMC apoptosis, the precise molecular mechanism underlying the regulation of apoptotic pathways in SMCs remains largely undetermined.Apoptosis is a multistage, genetically controlled process of selective cell deletion. Protein kinases regulate the early stages of apoptosis by phosphorylating key proteins (12, 13), whereas caspases, a family of cysteine proteases, are the main effectors whose activation results in the characteristic morphological changes associated with programmed cell death such as membrane blebbing, chromatin condensation, and DNA fragmentation (14,15).Members of the protein kinase C (PKC) family are activated by diverse stimuli and participate in multiple cellular processes such as growth, differentiation, and apoptosis (16). The novel PKC isoform, protein kinase C delta (PKC␦), has been shown to be associated with the response to DNA damage and other apoptotic stimuli in specific cell types (17)(18)(19)(20). The critical role of PKC␦ in vascular SMC apoptosis and pathogenesis of a neointimal lesion has been recently demonstrated using PKC␦ "knock-out" mice....

Inhibition of PKCδ reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer

“…We hypothesized that PKCδ would phosphorylate p53, leading to p53 activation and consequent renal apoptosis. This hypothesis was mainly based on 2 considerations: (a) a role for p53 has been demonstrated in cisplatin nephrotoxicity (34,(40)(41)(42)(43)(44)(45)(46)(47), and (b) PKCδ has recently been implicated in p53 phosphorylation and activation (56,57). This hypothesis, seemingly logical, is nonetheless not supported by our results.…”

The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

“…We propose the following chain of events underlies the SFHFKSGSL peptide-mediated membrane blebbing. In response to prolonged treatment with PMA or oxidative stress caused by ionizing radiation, activated PKC␦ can be cleaved, presumably by caspase-3, and the released catalytic domain is translocated to the nucleus (21,63), although there is a lag of several hours between treatment and PKC␦ cleavage. Nuclear PKC␦ phosphorylates and thereby inactivates both the DNA damage checkpoint protein hRad9 and DNA-dependent protein kinase; the latter is essential for double strand break repair (64,65).…”

“…The greatest numbers of spectra for phosphorylated peptides were identified for two serine positions in BVR, Ser 21 and Ser 230 . To demonstrate PKC␦ specificity, GST-BVR, without any prior kinase treatment, was also mapped in parallel, using chymotrypsin and trypsin digestion.…”

“…Therefore, its kinase activity plays a determining role in the regulation of cell death (20); for instance, in PMA-stimulated cells, activation of caspase-3 results in cleavage of PKC␦ between the regulatory and catalytic domains of the PKC, leading to translocation of the catalytic domain into the nucleus and hence the onset of apoptosis (21). Conversely, the proliferative effects of PKC␦ likely involve its activation of ERK1/2, which are the upstream kinases for a host of transcriptional factors, including Elk1 and NF-B, that in turn regulate cell growth, proliferation, and survival (22,23).…”

Loss-of-function of the protein kinase C δ (PKCδ) causes a B-cell lymphoproliferative syndrome in humans

“…4,12 Numerous studies have shown that PKCd is involved in proapoptic signaling upon phorbol ester stimulation and after DNA damage. 13 Therefore, we tested PMAinduced cell death in the patient's cells (EBV-transformed B cells and PBMCs).…”