Mechanism of attenuation by β-hydroxy-β-methylbutyrate of muscle protein degradation induced by lipopolysaccharide

The mechanism of the effect of β-hydroxy-β-methylbutyrate (HMB) on protein degradation induced by lipopolysaccharide (LPS) has been evaluated in murine myotubes. HMB (50 μM) completely attenuated total protein degradation induced by LPS (1–100 ng/ml), formation of reactive oxygen species (ROS) and activation of caspase-3/-8. Specific inhibitors of caspase-3/-8 completely attenuated ROS production, total protein degradation and the LPS-induced autophosphorylation of dsRNA-dependent protein kinase (PKR). Protein degradation in response to LPS or ROS production was not seen in myotubes transfected with mutant PKRΔ6, suggesting that PKR was involved in ROS production, which was essential for total protein degradation. This was confirmed using the antioxidant butylated hydroxytoluene (BHT) which completely attenuated protein degradation in response to LPS. The link between PKR activation and ROS production was mediated through p38 mitogen-activated protein kinase (MAPK), which was activated by LPS in myotubes transfected with wild-type PKR, but not PKRΔ6. Both ROS production and protein degradation induced by LPS were completely attenuated by SB203580, a specific inhibitor of p38MAPK. This suggests that LPS induces protein degradation through a signalling cascade involving activation of caspase-3/-8, activation of PKR and production of ROS through p38MAPK, and that this process is attenuated by HMB.     

Mechanism of induction of muscle protein loss by hyperglycaemia

Treatment of murine myotubes with high glucose concentrations (10 and 25 mM) stimulated protein degradation through the ubiquitin-proteasome pathway, and also caused activation (autophosphorylation) of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α). Phosphorylation of PKR and eIF2α was also seen in the gastrocnemius muscle of diabetic ob/ob mice. High glucose levels also inhibited protein synthesis. The effect of glucose on protein synthesis and degradation was not seen in myotubes transfected with a catalytically inactive variant (PKRΔ6). High glucose also induced an increased activity of both caspase-3 and -8, which led to activation of PKR, since this was completely attenuated by the specific caspase inhibitors. Activation of PKR also led to activation of p38MAPK (mitogen activated protein kinase), leading to ROS (reactive oxygen species) formation, since this was attenuated by the specific p38MAPK inhibitor SB203580. ROS formation was important in protein degradation, since it was completely attenuated by the antioxidant butylated hydroxytoluene. These results suggest that high glucose induces muscle atrophy through the caspase-3/-8 induced activation of PKR, leading to phosphorylation of eIF2α and depression of protein synthesis, together with PKR-mediated ROS production, through p38MAPK and increased protein degradation.     

Mechanism of activation of dsRNA-dependent protein kinase (PKR) in muscle atrophy

The role of Ca²⁺ in the activation of PKR (double-stranded-RNA-dependent protein kinase), which leads to skeletal muscle atrophy, has been investigated in murine myotubes using the cell-permeable Ca²⁺ chelator BAPTA/AM (1,2-bis (o-aminphenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester). BAPTA/AM effectively attenuated both the increase in total protein degradation, through the ubiquitin–proteasome pathway, and the depression of protein synthesis, induced by both proteolysis-inducing factor (PIF) and angiotensin II (Ang II). Since both protein synthesis and degradation were attenuated this suggests the involvement of PKR. Indeed BAPTA/AM attenuated both the activation (autophosphorylation) of PKR and the subsequent phosphorylation of eIF2α (eukaryotic initiation factor 2α) in the presence of PIF, suggesting the involvement of Ca²⁺ in this process. PIF also induced an increase in the activity of both caspases-3 and -8, which was attenuated by BAPTA/AM. The increase in caspase-3 and -8 activity was shown to be responsible for the activation of PKR, since the latter was completely attenuated by the specific caspase-3 and -8 inhibitors. These results suggest that Ca²⁺ is involved in the increase in protein degradation and decrease in protein synthesis by PIF and Ang II through activation of PKR by caspases-3 and -8.     

Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-kappaB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.     

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.     

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Calpha (PKCalpha), degradation of inhibitor-kappaB (I-kappaB) and nuclear migration of nuclear factor-kappaB (NF-kappaB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the alpha-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCalpha by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 muM), an inhibitor of eIF2alpha dephosphorylation, as was activation of PKCalpha. In addition myotubes transfected with a dominant-negative PKR (PKRDelta6) showed no release of arachidonate in response to Ang II, and no activation of PKCalpha. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA(2)/PKC pathway leading to activation of NF-kappaB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR.     

Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase

Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.     

Mechanism of attenuation of protein loss in murine C2C12 myotubes by d-myo-inositol 1,2,6-triphosphate

d-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumor necrosis factor-α/interferon-γ. At a concentration of 100 μM AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitin-proteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the α-subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn²⁺. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several catabolic conditions.     

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.     

Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells

Carnosic acid (CA), a rosemary phenolic compound, has been shown to display anti-cancer activity. We examined the apoptotic effect of CA in human neuroblastoma IMR-32 cells and elucidated the role of the reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) associated with carcinogenesis. The result indicated that CA decreased the cell viability in a dose-dependent manner. Further investigation in IMR-32 cells revealed that cell apoptosis following CA treatment is the mechanism as confirmed by flow cytometry, hoechst 33258, and caspase-3/-9 and poly(ADP-ribose) polymerase (PARP) activation. Immunoblotting suggested a down-regulation of anti-apoptotic Bcl-2 protein in the CA-treated cells. In flow cytometric analysis, CA caused the generation of reactive oxygen species (ROS); however, pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated the CA-induced generation of ROS and apoptosis. This effect was accompanied by increased activation of p38 and by decreased activation of extracellular signal-regulated kinase (ERK) as well as activation of c-Jun NH₂-terminal kinase (JNK). Moreover, NAC attenuated the CA-induced phosphorylation of p38. Silencing of p38 by siRNA gene knockdown reduced the CA-induced activation of caspase-3. In conclusion, ROS-mediated p38 MAPK activation plays a critical role in CA-induced apoptosis in IMR-32 cells.     

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from <Emphasis Type="Italic">Chlamys farreri</Emphasis> in human HaCaT keratinocytes

Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H₂O₂-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.     

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.     

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 diabetes.     

Cryptotanshinone enhances TNF-??-induced apoptosis in chronic myeloid leukemia KBM-5 cells

Cryptotanshinone is a biologically active compound from the root of Salvia miltiorrhiza. In the present study, we investigated the molecular mechanisms by which cryptotanshinone is in synergy with tumor necrosis factor-alpha (TNF-α) for the induction of apoptosis in human chronic myeloid leukemia (CML) KBM-5 cells. The co-treatment of cryptotanshinone with TNF-α reduced the viability of the cells [combination index (CI) < 1]. Concomitantly, the co-treatment of cryptotanshinone and TNF-α elicited apoptosis, manifested by enhanced the number of terminal deoxynucleotide transferase-mediated dUTP-nick-end labeling (TUNEL)-positive cells, the sub-G1 cell populations, and the activation of caspase-8 and -3, in comparison with the treatment with either drug alone. The treatment with cryptotanshinone further suppressed TNF-α-mediated expression of c-FLIP(L), Bcl-x(L), but the increased level of tBid (a caspase-8 substrate). Furthermore, cryptotanshinone activated p38 but not NF-κB in TNF-α-treated KBM-5 cells. The addition of a specific p38 MAPK inhibitor SB203580 significantly attenuated cryptotanshinone/TNF-α-induced apoptosis. The combination treatment of cryptotanshinone and TNF-α also stimulated the reactive oxygen species (ROS) generation. N-acetyl-L-cysteine (NAC, a ROS scavenger) was not only able to block cryptotanshinone/TNF-α-induced ROS production but also the activation of caspase-8 and p38 MAPK. Overall, our findings suggest that cryptotanshinone can sensitize TNF-α-induced apoptosis in human myeloid leukemia KBM-5 cells, which appears through ROS-dependent activation of caspase-8 and p38.     

Peroxisome proliferator-activated receptor gamma-independent activation of p38 MAPK by thiazolidinediones involves calcium/calmodulin-dependent protein kinase II and protein kinase R: correlation with endoplasmic reticulum stress

The thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands that promote increased insulin sensitivity in type II diabetic patients. In addition to their ability to improve glucose homeostasis, TZDs also exert anti-proliferative effects by a mechanism that is unclear. Our laboratory has shown that two TZDs, ciglitazone and troglitazone, rapidly induce calcium-dependent p38 mitogen-activated protein kinase (MAPK) phosphorylation in liver epithelial cells. Here, we further characterize the mechanism responsible for p38 MAPK activation by PPARgamma ligands and correlate this with the induction of endoplasmic reticulum (ER) stress. Specifically, we show that TZDs rapidly activate the ER stress-responsive pancreatic eukaryotic initiation factor 2alpha (eIF2alpha) kinase or PKR (double-stranded RNA-activated protein kinase)-like endoplasmic reticulum kinase/pancreatic eIF2alpha kinase, and that activation of these kinases is correlated with subsequent eIF2alpha phosphorylation. Interestingly, PPARgamma ligands not only activated calcium/calmodulin-dependent kinase II (CaMKII) 2-fold over control, but the selective CaMKII inhibitor, KN-93, attenuated MKK3/6 and p38 as well as PKR and eIF2alpha phosphorylation. Although CaMKII was not affected by inhibition of PKR with 2-aminopurine, phosphorylation of MKK3/6 and p38 as well as eIF2alpha were significantly reduced. Collectively, these data provide evidence that CaMKII is a regulator of PKR-dependent p38 and eIF2alpha phosphorylation in response to ER calcium depletion by TZDs. Furthermore, using structural derivatives of TZDs that lack PPARgamma ligand-binding activity as well as a PPARgamma antagonist, we show that activation of these kinase signaling pathways is PPARgamma-independent.