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Proliferation of smooth muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs. J. Cell. Biochem. 99: 835–844, 2006. © 2006 Wiley-Liss, Inc.

Bortezomib (PS-341) is an inhibitor of the S26 proteasome. Bortezomib induces mitochondrial damage but the exact mechanism remains unclear. We studied PKC-delta, a kinase that is regulated by proteasome degradation and translocates to mitochondria in apoptosis, and examined whether PKC-delta could be a potential mediator of bortezomib-induced mitochondrial damage. Co-incubation of bortezomib with a PKC-delta inhibitor, rottlerin, suppressed bortezomib-induced apoptosis in U937 cells. Western analysis of U937 cells treated with bortezomib revealed accumulation of full-length PKC-delta in the first 4 h. By 16 h an active catalytic fragment of PKC-delta accumulated in mitochondria. The cleavage of PKC-delta after bortezomib treatment was mediated by caspases, because a pan-caspase inhibitor BAF prevented the appearance of the active fragment of PKC-delta. These findings indicate that accumulation of the active PKC-delta fragment in mitochondria is responsible for bortezomib-induced mitochondrial damage.

Phospholipid scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-δ (PKC-δ) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC-δ activator, to investigate the mechanism of PLS3 phosphorylation by PKC-δ. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-δ, but not the kinase-defective PKC-δ, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr21 of PLS3. Mutation of Thr21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-δ in vitro and AD198-induced PLS3 phosphorylation in vivo. Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-δ induces phosphorylation of mitochondrial PLS3 at Thr21 and that PLS3 is a critical downstream effector of PKC-δ in AD198-induced apoptosis.

Bid, a BH3-only pro-apoptotic member of the Bcl-2 family, is cleaved by caspase 8 in apoptosis induced by death domain receptors. The carboxyl terminus of the cleavage product, tBid, remains associated with the amino terminal fragment (nBid) after cleavage. Dissociation of tBid from nBid occurs during targeting of tBid to mitochondria. We use an in vitro system and demonstrate that cardiolipin is sufficient for the dissociation. Monolysocardiolipin, a metabolite of cardiolipin that increases in mitochondria during apoptosis, has the same affinity to tBid as cardiolipin and is also capable of inducing dissociation of tBid from nBid. In contrast, phosphatidylethanolamine could not induce dissociation of tBid from nBid. To determine the site of tBid that interacts with cardiolipin, we performed mutational analysis by eliminating the positive-charged residues in helices 4-6. None of the single mutations can abolish the ability of tBid to target to mitochondria and to induce cytochrome c release, suggesting that positive-charged residues in helices 4-6 may not be required for mitochondrial targeting of tBid.