Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages

The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of α-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O₂⁻ generated via the oxidase system activated with TS.     

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A₂ (PLA₂) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H₂O₂). Exposure of the smooth muscle cells to H₂O₂ dose-dependently stimulates [¹⁴C] AA release, and enhances the cell membrane associated PLA₂ activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H₇ and sphingosine prevent the cell membrane associated PLA₂ activity, and AA release caused by H₂O₂. Treatment of the smooth muscle cells with H₂O₂ stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H₂O₂ caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA₂ and PKC activities correlate linearly. These results suggest that H₂O₂ caused stimulation of the smooth muscle cell membrane associated PLA₂ activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)Abbreviations AA Arachidonic Acid - PLA₂ Phospholipase A₂ - PKC Protein Kinase C - PBS Phosphate Buffered Saline - HBPS Hank's Buffered Physiological Saline - HEPES 4-(2-Hydroxyethyl)-1-Piperazine N-2-Ethanesulfonate - FCS Fetal Calf Serum - ATP Adenosine Triphosphate - H₇ 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine - DMEM Dulbecco's Modified Eagles Medium - TCA Trichloroacetic Acid     

Protein kinase Cδ mediates MCP-1 mRNA stabilization in vascular smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF β and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct “proximal” signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.     

Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²⁺-dependent induction of oxidative stress

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.     

Protein kinase C δ regulates the release of collagen type I from vascular smooth muscle cells via regulation of Cdc42

Collagen type I is the most abundant component of extracellular matrix in the arterial wall. Mice knocked out for the protein kinase C δ gene (PKCδ KO) show a marked reduction of collagen I in the arterial wall. The lack of PKCδ diminished the ability of arterial smooth muscle cells (SMCs) to secrete collagen I without significantly altering the intracellular collagen content. Moreover, the unsecreted collagen I molecules accumulate in large perinuclear puncta. These perinuclear structures colocalize with the trans-Golgi network (TGN) marker TGN38 and to a lesser degree with cis-Golgi marker (GM130) but not with early endosomal marker (EEA1). Associated with diminished collagen I secretion, PKCδ KO SMCs exhibit a significant reduction in levels of cell division cycle 42 (Cdc42) protein and mRNA. Restoring PKCδ expression partially rescues Cdc42 expression and collagen I secretion in PKCδ KO SMCs. Inhibition of Cdc42 expression or activity with small interfering RNA or secramine A in PKCδ WT SMCs eliminates collagen I secretion. Conversely, restoring Cdc42 expression in PKCδ KO SMCs enables collagen I secretion. Taken together, our data demonstrate that PKCδ mediates collagen I secretion from SMCs, likely through a Cdc42-dependent mechanism.     

Myristic acid specifically stabilizes diacylglycerol kinase δ protein in C2C12 skeletal muscle cells

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.     

Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.     

Involvement of protein kinase Cδ in induction of apoptosis by cationic liposomes in macrophage-like RAW264.7 cells

We have recently demonstrated that reactive oxygen species (ROS) play an important role in RAW264.7 cell apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). In this study, we investigated whether protein kinase Cδ PKCδ) is involved in apoptosis induced by cationic liposomes. Tyrosine phosphorylation, nuclear localization, and cleavage of PKCδ were observed following the treatment of cells with SA-liposomes, suggesting that SA-liposomes activate PKCδ. Rottlerin, a specific inhibitor of PKCδ, inhibited ROS generation and also suppressed apoptosis. Cell surface proteoglycans may contribute to PKCδ activation by SA-liposomes. These findings suggest that PKCδ is strongly associated with apoptosis induced by SA-liposomes.     

Effects and underlying mechanisms of curcumin on the proliferation of vascular smooth muscle cells induced by Chol:MβCD

Proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of various cardiovascular diseases. Curcumin, extracted from Curcumae longae, has been shown a variety of beneficial effects on human health, including anti-atherosclerosis by mechanisms poorly understood. In the present study, we attempted to investigate whether curcumin has any effect on VSMCs proliferation and the potential mechanisms involved. Our data showed curcumin concentration-dependently abrogated the proliferation of primary rat VSMCs induced by Chol:MβCD. To explore the underlying cellular and molecular mechanisms, we found that curcumin was capable of restoring caveolin-1 expression which was reduced by Chol:MβCD treatment. Moreover, curcumin abrogated the increment of phospho-ERK1/2 and nuclear accumulation of ERK1/2 in primary rat VSMCs induced by Chol:MβCD, which led to a suppression of AP-1 promoter activity stimulated by Chol:MβCD. In addition, curcumin was able to reverse cell cycle progression induced by Chol:MβCD, which was further supported by its down-regulation of cyclinD1 and E2F promoter activities in the presence of Chol:MβCD. Taking together, our data suggest curcumin inhibits Chol:MβCD-induced VSMCs proliferation via restoring caveolin-1 expression that leads to the suppression of over-activated ERK signaling and causes cell cycle arrest at G1/S phase. These novel findings support the beneficial potential of curcumin in cardiovascular disease.     

ω-3 Polyunsaturated fatty acids inhibit migration of human vascular smooth muscle cells in vitro

Arterial smooth muscle cell migration from the media to the intima is a crucial process in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) has been proposed to play a key role in the development of advanced atherosclerotic lesions by stimulating the migration and proliferation of vascular smooth muscle cells. Polyunsaturated fatty acids (PUFA) of the ω-3 series, extracted from fish oil has been shown to have beneficial effects on atherosclerosis. In this study, we evaluated the effects of ω-3 PUFA on the migration of human aortic smooth muscle cell (hASMC) in vitro. The migration assay was performed according to the Capsoni's method using transwell culture plates. PDGF, fibrinogen or 10%FCS significantly stimulated hASMC migration, however, ω-3 PUFA significantly inhibited PDGF-induced migration of hASMC. These results suggest that the inhibitory effect of ω-3 PUFA on cell migration may be an important aspect by which ω-3 PUFA exerts its antiatherosclerotic influence.     

Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.     

Involvement of subtypes γ and ε of protein kinase C in colon pain induced by formalin injection

Protein kinase C (PKC) has been widely reported to participate in somatic pain; however, its role in visceral pain remains largely unclear. Using a colon inflammatory pain model by intracolonic injection of formalin in rats, the present study was to examine the role of PKC in visceral pain and determine which subtypes may be involved. The colon pain behavior induced by formalin injection could be enhanced by intrathecal pretreatment with a PKC activator (PMA), and alleviated by a PKC inhibitor (H-7). Wide dynamic range (WDR) neurons in the L6-S1 spinal dorsal horn that were responsive to colorectal distension were recorded extracellularly. It was found that neuronal activity was greatly increased following formalin injection. Microdialysis of PMA near the recorded neuron in the spinal dorsal horn facilitated the enhanced responsive activity induced by formalin injection, while H-7 inhibited significantly the enhanced response induced by formalin injection. Western blot analysis revealed that membrane translocation of PKC-γ and PKC-ε, but not other subtypes, in the spinal cord was obviously increased following formalin injection. Therefore, our findings suggest that PKC is actively involved in the colon pain induced by intracolonic injection of formalin. PKC-γ and PKC-ε subtypes seem to significantly contribute to this process.     

Bcl－2 over－expression and activation of protein kinase C suppresss the Trail－induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.     

Basal protein kinase Cδ activity is required for membrane localization and activity of TRPM4 channels in cerebral artery smooth muscle cells

The melastatin (M) transient receptor potential channel (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. We recently reported that PKCδ activity influences smooth muscle cell excitability by promoting translocation of TRPM4 channel protein to the plasma membrane. Here we further investigate the relationship between membrane localization of TRPM4 protein and channel activity in native cerebral arterial myocytes. We find that TRPM4 immunolabeling is primarily located at or near the plasma membrane of freshly isolated cerebral artery smooth muscle cells. However, siRNA mediated downregulation of PKCδ or brief (15 min) inhibition of PKCδ activity with rottlerin causes TRPM4 protein to move away from the plasma membrane and into the cytosol. In addition, we find that PKCδ inhibition diminishes TRPM4-dependent currents in smooth muscle cells patch clamped in the amphotericin B perforated patch configuration. We conclude that TRPM4 channels are mobile in native cerebral myocytes and that basal PKCδ activity supports excitability of these cells by maintaining localization TRPM4 protein at the plasma membrane.