Expression Of P38 Mitogen-activated Protein Kinase Research Articles

Protective Effects of FR167653 on Chronic Allograft Nephropathy by Inhibiting p38 MAPK in Rats

Background Chronic allograft nephropathy (CAN) remains a challenge for transplant clinicians. Some novel intervention strategies may shed new light on its treatment. Methods An orthotopic kidney transplant model in Fisher-to-Lewis rats was used with administration of cyclosporine alone or in combination with FR167653 (30 mg/kg/d subcuntaneously) to recipients. We analyzed renal function and urinary protein excretion. Animals were sacrificed at 30 weeks posttransplantation for histological and immunohistochemical studies. Results Renal function among vehicle-treated rats deteriorated progressively with substantial proteinuria compared with FR167653-treated rats. FR167653 administration significantly prevented the morphlogical features of CAN and prolonged rat survivals. p38 mitogen-activated protein kinase (MAPK) expression was markedly reduced by FR167653 treatment. Meanwhile, transforming growth factor-β1 and monocyte chemotactic protein-1 expression were significantly down-regulated among FR167653-treated hosts. Conclusion p38 MAPK phosphorylation correlated with CAN progression and inhibition of p38 MAPK by FR167653 may provide a potent novel therapeutic target for its prevention.

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite ( Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ ( p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .

SB203580, a p38 mitogen-activated protein kinase inhibitor, fails to improve functional outcome following a moderate spinal cord injury in rat

We examined the spatial and temporal expression patterns of active p38 mitogen-activated protein kinase (MAPK), an important regulator of immune cell function, following spinal cord injury (SCI). We further assessed whether administration of SB203580, an inhibitor of p38 MAPK activity, would reduce inflammation, improve tissue sparing, and improve functional outcome after SCI. Adult Wistar rats were subjected to a T9/10 SCI contusion of moderate severity and killed at several time points after injury, whereas sham-injured (control) animals only received a laminectomy. In control animals, active p38 MAPK expression was primarily localized to resting microglia within the spinal cord. Over the first 24 h after SCI, a continuing increase in active p38 MAPK expression was evident in neutrophils and activated microglia (OX42+) surrounding the spinal lesion site. At 15 days post-injury, active p38 MAPK was localized to macrophages (ED1+) that now dominated the lesion site. In addition, active p38 MAPK was localized to macrophages within white matter fiber tracts undergoing degeneration, several segments rostral and caudal to the injury site, which persisted for at least 6 weeks. Overall, our results demonstrate that active p38 MAPK is increased within resident and invading immune cells after SCI contusion injury and, therefore, may be an important target to regulate the inflammatory cascade after SCI. However, intrathecal application of SB203580 failed to improve functional outcome after a moderate SCI contusion.

Protoapigenone, a Novel Flavonoid, Induces Apoptosis in Human Prostate Cancer Cells through Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase 1/2

In this study, we investigated the anticancer effect of protoapigenone on human prostate cancer cells. Protoapigenone inhibited cell growth through arresting cancer cells at S and G(2)/M phases as well as inducing apoptosis. Blockade of cell cycle by protoapigenone was associated with an increase in the levels of inactivated phospho (p)-Cdc25C (Ser216) and a decrease in the levels of activated p-cyclin B1 (Ser147), cyclin B1, and cyclin-dependent kinase (Cdk) 2. Protoapigenone triggered apoptosis by increasing the levels of cleaved poly(ADP-ribose) polymerase and caspase-3. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK)1/2 was a critical mediator in protoapigenone-induced cell death. Inhibition of the expression of p38 MAPK and JNK1/2 by pharmacological inhibitors or specific small interfering RNA reversed the protoapigenone-induced apoptosis through decreasing the level of cleaved caspase-3. In contrast, p38 MAPK, but not JNK1/2, was involved in the protoapigenone-mediated S and G(2)/M arrest by modulating the levels of Cdk2 and p-Cdc25C (Ser216). Moreover, in vivo xenograft study showed that protoapigenone had a significant inhibition of prostate tumor growth without major side effects on the mice we tested. This inhibition was associated with induction of apoptosis and activation of p38 MAPK and JNK1/2 in protoapigenone-treated tumor tissues. In conclusion, our results demonstrated protoapigenone suppressed prostate cancer cell growth through the activation of p38 MAPK and JNK1/2, with the potential to be developed as a chemotherapeutic agent for prostate cancer.

Development of glutathione S‐transferase‐P‐negative foci accompanying nuclear factor‐erythroid 2‐related factor 2 expression during early stage of rat hepatocarcinogenesis

Glutathione S-transferase P (GST-P), a marker for rat hepatic preneoplastic lesions, is suggested to bind to Jun N-terminal kinase (JNK) to repress stress response, and GST-P gene expression is regulated by a transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2). In this study, we examined by immunohistochemistry whether JNK2, p38 mitogen-activated protein kinase, and Nrf2 were expressed in GST-P-positive foci induced by the Solt-Farber protocol. At 2 weeks after partial hepatectomy, all GST-P-positive foci were negative for p38, and 86.4 +/- 5.6% and 64.7 +/- 6.3% of GST-P-positive foci were negative for JNK2 and Nrf2, respectively. Western blot analysis showed decreased p38 mitogen-activated protein kinase and JNK2 expression in livers treated with the protocol. In immunohistochemistry, besides GST-P-positive foci, GST-P-negative foci were detected as p38-negative foci in the surrounding tissues positive for p38. In contrast to GST-P-positive foci, most GST-P-negative foci showed enhanced Nrf2 expression. The number of GST-P-negative foci was 76 +/- 18/10 mm(2) of liver section at 2 weeks, but was undetectable at 1 week. The area of GST-P-negative foci was 0.09 +/- 0.05 mm(2), smaller than that of GST-P-positive ones (0.29 +/- 0.23). After treatment with carbon tetrachloride, small vacuoles due to liver injury were frequently observed inside GST-P-negative foci but less frequently in GST-P-positive foci. However, this treatment resulted in expression of JNK2, p38, and Nrf2 in both foci. These results showed development of GST-P-negative foci during the early stage of hepatocarcinogenesis and suggested that Nrf2 is not responsible for GST-P expression in rat hepatic preneoplastic foci.

PROTECTIVE EFFECT OF N-ACETYLCYSTEINE ON HYPEROXIA-INDUCED LUNG INJURY AND ITS INTERACTION WITH P38 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING PATHWAY

INTRODUCTION: N-Acetylcysteine (NAC) is an effective oxidation inhibitor, but the protection of NAC in hyperoxia-induced lung injury is unknown. OBJECTIVE: The objective of this study was to explore the protective effect of NAC on hyperoxia-induced lung injury and change of p38 mitogen-activated protein kinase (MAPK) expression caused by NAC treatment. METHODS: Forty Wistar rats were randomly assigned to room air (A), hyperoxia injury (B), hyperoxia + NAC (C), hyperoxia + SB203580 (D), or hyperoxia + NAC + SB203580 (E). The lung wet/dry ratio, pathology, and location and quantity of p38 protein were detected. RESULTS: Although pathologic changes in group B included severe alveolar edema with inflammatory cell aggregation and red blood cell leakage, the lung micrographic pictures in groups C, D, and E were improved significantly compared with group B; p38-positive cells increased in group B compared with that in group A and labeled in many types cells in lung tissue, especially in infiltrative inflammatory cells. In groups C, D, and E, the positive cells remarkably decreased compared with those in group B; the quantity of p38 MAPK was higher in group B than in group A, and p38 expression in groups C, D, and E decreased significantly compared with group B but was higher than that in the control group. There was no significant difference of p38 quantity among the 3 groups. CONCLUSIONS: Reactive oxygen species activated phospho-p38 MAPK signaling pathway, and NAC and SB203580 treatments reduced the extent of hyperoxia-induced lung injury, as evidenced by reduction of the wet/dry ratio and lung pathology. NAC may exert a protective effect on hyperoxia-induced lung injury through attenuation of reactive oxygen species–induced p38 MAPK activation.

Ceramide induces p38 MAPK-dependent apoptosis and Bax translocation via inhibition of Akt in HL-60 cells

Ceramide induces apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells. However, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. In this study, the activation of p38 mitogen-activated protein kinase (MAPK) was found to be critical for the induction of apoptosis and subcellular redistribution of Bax. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a dominant-negative p38 MAPK attenuated DNA fragmentation, caspase-3 activation, and Bax translocation in response to ceramide. Overexpression of Akt also led to suppression of Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. We also provide evidence for cross-talk between p38 MAPK and Akt pathways. Expression of myr-Akt or inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 had no effect on p38 MAPK activation by ceramide as assessed by phosphorylation, while inhibition of p38 MAPK by a pharmacological inhibitor or a dominant-negative p38 inhibited Akt dephosphorylation in response to ceramide, suggesting that ceramide-induced p38 MAPK activation negatively regulates the Akt pathway.

Activated protein C, an anticoagulant polypeptide, ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases

Our aim was to investigate the changes of mitogen-activated protein kinases (MAPKs) by activated protein C (APC) treatment in rats with severe acute pancreatitis (SAP), and relate them to changes in SAP severity, thus providing evidence for developing clinical therapies. Sprague-Dawley rats were given an intravenous injection of saline (SAP group), APC (50 microg/kg or 10 microg/kg), or CNI1493 just before SAP induction. One group of rats underwent a sham operation (control group). Experimental samples were harvested 16 h after SAP induction. The gene expression of pancreatic MAPKs was evaluated by cDNA microarrays. The mRNA and protein/phosphorylated protein levels of p38 MAPK, extracellular signal-regulated protein kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) and the protein levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were determined in pancreatic tissue. The severity of disease was evaluated by pancreatic histology, the pancreatic wet/dry weight ratio, and the serum amylase level. In rats treated with APC (50 microg/kg) or CNI1493, the severity of pancreatitis and expression of pancreatic TNF-alpha and IL-1beta proteins were attenuated by the decreased expression and activity of p38 MAPK and JNK (vs. the SAP group, P < 0.01). The expression and activity of ERK1/2 were increased in APC-treated rats, especially in the group treated with APC 50 microg/kg (vs. the SAP or CNI1493-treated group, P < 0.01, respectively). Inhibition of expression of pancreatic p38 MAPK and JNK and upregulation of ERK1/2 expression by APC treatment may protect against pancreatic injury, thus ameliorating severity of the disease.

Apoptosis in the kidneys of patients with type II diabetic nephropathy

The occurrence and extent of apoptosis in the kidneys of patients with diabetic nephropathy is largely unknown. We evaluated apoptosis in renal biopsies obtained from patients with early or advanced type II diabetic nephropathy. Apoptosis was about 6- and 3-fold higher, respectively, in glomeruli and tubules in kidneys of patients with early nephropathy than in the normal kidney and this was not further increased in advanced diabetic nephropathy. Glomerular apoptosis was related directly to hemoglobin A1(c) and systolic blood pressure, whereas tubular cell apoptosis correlated to diabetes duration and low-density lipoprotein-cholesterol. Fas, Fas ligand, and p38 mitogen-activated protein kinase expressions were enhanced in glomeruli and tubules; however, this did not correlate with apoptosis. In patients with proteinuria, apoptosis was associated with the subsequent loss of kidney function. When these parameters were subjected to multivariate analysis, only glomerular apoptosis retained a significant independent predictive value. Our findings suggest that apoptosis might be a clinically relevant mechanism of glomerular and tubular cell loss in proteinuric type II diabetic patients.

Viral Vector-Mediated 12/15-Lipoxygenase Overexpression in Vascular Smooth Muscle Cells Enhances Inflammatory Gene Expression and Migration

Increased expression and activity of 12/15-lipoxygenase (12/15-LO) in vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetes and vascular complications. However, the consequences of 12/15-LO overexpression for VSMC migration and inflammatory gene expression are not known. In this study, 12/15-LO was overexpressed using adeno- and baculoviral vectors in human VSMC (HVSMCs) and proatherogenic responses compared with control enhanced green fluorescent protein (EGFP)-expressing cells. HVSMCs transduced with 12/15-LO viruses expressed high levels of enzymatically active protein and produced increased levels of the LO product, 12(S)-hydroxyeicosatetraenoic acid. 12/15-LO-overexpressing HVSMCs exhibited increased oxidant stress, activation of p38 mitogen-activated protein kinase, migration and inflammatory gene expression relative to HVSMCs expressing EGFP. Furthermore, inflammatory gene expression induced by 12/15-LO overexpression was abolished by anti-oxidants, siRNAs targeting p65 (nuclear factor-ĸB), or new-generation baculoviruses expressing inhibitory IĸBα or IĸBα superrepressor mutant. Thus, we have used novel viral vector delivery systems, including baculoviruses, for the first time to deliver foreign genes into VSMCs and thereby demonstrated that 12/15-LO overexpression increases oxidant stress, mitogen-activated protein kinase activation, migration and inflammatory genes in VSMCs and that NF-ĸB is a key downstream effector. Enhanced proatherogenic responses in VSMCs triggered by increased 12/15-LO levels under pathological conditions may contribute to vascular dysfunction.

Down-regulation of iNOS and TNF-α expression by kaempferol via NF-κB inactivation in aged rat gingival tissues

The primary objective of this study was to evaluate the ability and mechanism of action of kaempferol, which is contained in extracts from Nelumbo nucifera, a well-known Oriental herb used in traditional medicine, with regard to the inhibition of iNOS and TNF-alpha expression in aged rat gingival tissues. We conducted an investigation into the age-related effects of kaempferol on reactive oxygen species (ROS) and GSH oxidative status in samples of aged gingival tissues. Western blotting was conducted in order to determine the expression of iNOS, TNF-alpha, p38 MAPK, NIK/IKK, p65 and IkappaBalpha in the sample tissues. Electrophoretic mobility shift assays (EMSA) were conducted in an effort to characterize the binding activities of NF-kappaB transcription factors in the aged rat gingival nuclear extracts. Our results indicate that kaempferol reduced ROS levels and augmented GSH levels in a dose-dependent manner in the aged gingival tissues. Kaempferol was shown to effect a significant reduction in iNOS and TNF-alpha protein levels, as compared to control gingival tissue samples. The results of Western blot analysis revealed that kaempferol treatment effected the reduction of iNOS and TNF-alpha expression, decreased nuclear p65 and increased cytosolic p65, down-regulation of Erk, p38, JNK and NIK/IKK expression. The EMSA results also indicated that kaempferol, when administered to the rat tissues, attenuated the NF-kappaB nuclear binding activity. Kaempferol may inhibit ROS generation via the inhibition of iNOS and TNF-alpha expression in aged gingival tissues, via the modulation of the NF-kappaB and mitogen-activated protein kinase (MAPK) pathways.

Rosiglitazone, a PPARγ ligand, modulates signal transduction pathways during the development of acute TNBS-induced colitis in rats

Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARγ), a highly nuclear receptor expressed in the colon, may participate in the control of inflammation, especially in regulating the production of immunomodulatory and inflammatory mediators, cellular proliferation and apoptosis. In order to delve into the anti-inflammatory mechanisms and signalling pathways of PPARγ agonists, we have studied the effects of rosiglitazone, a PPARγ agonist on the extent and severity of acute ulcerative colitis caused by intracolonic administration of 2,4,6-trinitribenzene sulfonic acid (TNBS) in rats. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) activity, tumour necrosis factor α (TNF-α) levels and a histological study of the lesions. We determined prostaglandin E2 production as well as the cyclooxygenases (COX)-1 and -2 expressions by immunohistochemistry and Western blotting. The nuclear factor kappa (NF-κB) p65 and p38 mitogen-activated protein kinase (MAPK) expression levels were also measured by Western blotting. Finally, since PPARγ agonists modulate apoptosis, we tried to clarify its effects under early acute inflammatory conditions. Inflammation following TNBS induction was characterized by increased colonic wall thickness, edema, diffuse inflammatory cells infiltration, necrosis reaching an ulcer index (UI) of 9.66 ± 0.66 cm 2 and increased MPO activity and TNF-α colonic levels. Rosiglitazone treatment significantly reduced the morphological alteration associated with TNBS administration and the UI with the highest dose. In addition, the degree of neutrophil infiltration and the cytokine levels were significantly ameliorated. Rosiglitazone significantly reduced the rise in the prostaglandin (PG) E 2 generation compared with TNBS group. The COX-1 levels remained stable throughout the treatment in all groups. The COX-2 expression was elevated in TNBS group; however rosiglitazone administration reduced the COX-2 overexpression. A high expression of NF-κB p65 and p38 MAPK proteins appeared in colon mucosa from control TNBS-treated rats; nevertheless, PPARγ agonist treatment drastically decreased them. There were no significant changes in apoptosis after rosiglitazone treatment when compared to TNBS group. In conclusion, rosiglitazone seems to modulate the acute colitis through NF-κB p65 and p38 MAPK signalling pathways.

The Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases MK2 and MK3 Cooperate in Stimulation of Tumor Necrosis Factor Biosynthesis and Stabilization of p38 MAPK

MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.

Anandamide-induced Ca 2+ elevation leading to p38 MAPK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

The effect of anandamide on human osteoblasts is unclear. This study examined the effect of anandamide on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca 2+ levels in MG63 osteosarcoma cells. Anandamide at 50–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that anandamide induced expression of ERK, JNK and p38 MAPK. Anandamide-induced cell death and apoptosis were reversed by SB203580, but not by PD98059 and SP600125, suggesting that anandamide's action was via p38 MAPK, but not via ERK and JNK. Anandamide at 1–100 μM induced [Ca 2+] i increases. Removal of extracellular Ca 2+ decreased the anandamide response, indicating that anandamide induced Ca 2+ influx and Ca 2+ release. Chelation of intracellular Ca 2+ with BAPTA reversed anandamide-induced cell death and p38 MAPK phosphorylation. Collectively, in MG63 cells, anandamide induced [Ca 2+] i increases which evoked p38 MAPK phosphorylation. This p38 MAPK phosphorylation subsequently activated caspase-3 leading to apoptosis.

Isoform-Specific Regulation of the Actin-Organizing Protein Palladin during TGF-β1-Induced Myofibroblast Differentiation

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-beta1 treatment preceding alpha-SMA upregulation. TGF-beta1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with alpha-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression.

Effects of sodium ferulate on amyloid-beta-induced MKK3/MKK6-p38 MAPK-Hsp27 signal pathway and apoptosis in rat hippocampus

To observe the effects of sodium ferulate (SF) on amyloid beta (Abeta)1-40-induced p38 mitogen-activated protein kinase (MAPK) signal transduction pathway and the neuroprotective effects of SF. Rats were injected intracerebroventricularly with Abeta1-40. Six hours after injection, Western blotting was used to determine the expressions of phosphorylated mitogen-activated protein kinase kinase (MKK) 3/MKK6, phospho-p38 MAPK, interleukin (IL)-1beta, phospho-MAPK activating protein kinase 2 (MAPKAPK-2), the 27 kDa heat shock protein (Hsp27), procaspase-9, -3, and -7 cleavage, and poly (ADP-ribose) polymerase (PARP) cleavage. Seven days after injection, Nissl staining was used to observe the morphological change in hippocampal CA1 regions. Intracerebroventricular injection of Abeta1-40 induced an increase in phosphorylated MKK3/MKK6 and p38 MAPK expressions in hippocampal tissue. These increases, in combination with enhanced interleukin (IL)-1beta protein expression and reduced phospho-MAPKAPK2 and phospho-Hsp27 expression, mediate the Abeta-induced activation of cell death events as assessed by cleavage of procaspase-9, -3, and -7 and caspase-3 substrate PARP cleavage. Pretreatment with SF (100 mg/kg and 200 mg/kg daily, 3 weeks) significantly prevented Abeta1-40-induced increases in phosphorylated MKK3/MKK6 and p38 MAPK expression. The Abeta1-40-induced increase in IL-1beta protein level was attenuated by pretreatment with SF. In addition, Abeta1-40-induced decreases in phosphorylated MAPKAPK2 and Hsp27 expression were abrogated by administration of SF. In parallel with these findings, Abeta1-40-induced changes in activation of caspase-9, caspase-7, and caspase-3 were inhibited by pretreatment with SF. SF prevents Abeta1-40-induced neurotoxicity through suppression of MKK3/MKK6-p38 MAPK activity and IL-1beta expression and upregulation of phospho-Hsp27 expression.

Low-Density Lipoprotein Modified by Macrophage-Derived Lysosomal Hydrolases Induces Expression and Secretion of IL-8 Via p38 MAPK and NF-κB by Human Monocyte-Derived Macrophages

Modified lipoproteins induce inflammatory reactions in the atherosclerotic arterial wall. We have previously found that macrophages in atherosclerotic lesions secrete lysosomal hydrolases that can modify low-density-lipoprotein (LDL) in vitro to generate "hydrolase-modified LDL" (H-LDL). Here, we studied whether H-LDL exerts inflammatory effects on cultured human macrophages. Using cytokine cDNA arrays, we found that H-LDL induced expression of IL-8, but not of the anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-beta, in human monocyte-derived macrophages. H-LDL induced rapid phosphorylation of the p38 mitogen-activated protein kinase (MAPK), nuclear translocation of 2 transcription factors, nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), and time-dependent secretion of IL-8 from the macrophages. Inhibition of MAPKs and of transcription factors showed that p38 MAPK and NF-kappaB, but not ERK1/2, JNK, or AP-1, were crucial for the H-LDL-induced IL-8 secretion from the macrophages. The results show that by activating p38 MAPK and NF-kappaB, macrophage hydrolases modify LDL into biologically active particles capable of triggering the secretion of IL-8 in macrophages. Thus, activated hydrolase-secreting macrophages in atherosclerotic lesions may sustain a proatherogenic extracellular environment by hydrolyzing LDL and triggering it to act in an autocrine or paracrine fashion to induce IL-8 secretion by the plaque macrophages.

Diabetes alters vascular mechanotransduction: pressure-induced regulation of mitogen activated protein kinases in the rat inferior vena cava

BackgroundDiabetes mellitus is an important risk factor for increased vein graft failure after bypass surgery. However, the cellular and molecular mechanism(s) underlying vessel attrition in this population remain largely unexplored. Recent reports have suggested that the pathological remodeling of vein grafts may be mediated by mechanically-induced activation of the mitogen activated protein kinase (MAPK) signaling pathways and the MAPK-related induction of caspase-3 activity. On the basis of these findings, we hypothesized that diabetes may be associated with alterations in how veins "sense" and "respond" to altered mechanical loading.MethodsInferior venae cavae (IVC) from the non-diabetic lean (LNZ) and the diabetic obese (OSXZ) Zucker rats were isolated and incubated ex vivo under basal or pressurized conditions (120 mmHg). Protein expression, basal activation and the ability of increased pressure to activate MAPK pathways and apoptosis-related signaling was evaluated by immunoblot analysis.ResultsImmunoblot analyses revealed differential expression and activation of extracellular signal-regulated kinase (ERK1/2), p38 and c-Jun NH2-terminal kinase (JNK) MAPKs in the IVCs of diabetic rats as compared to non-diabetic rats. In particular, the expression and basal phosphorylation of p38β- (52.3 ± 11.8%; 45.8 ± 18.2%), JNK 1- (21.5 ± 9.3%; 19.4 ± 11.6%) and JNK3-MAPK (16.8 ± 3.3%; 29.5 ± 17.6%) were significantly higher (P < 0.05) in the diabetic vena cava. An acute increase in IVC intraluminal pressure failed to increase the phosphorylation of ERK1-, JNK-2, or any of the p38-MAPKs in the diabetic obese Zucker rats. Also, IVC loading in the LNZ led to a 276.0 ± 36.0% and 85.8 ± 25.1% (P < 0.05) increase in the cleavage of caspase-3 and caspase-9, respectively, with no effect on these molecules in the OSXZ. No differences were found in the regulation of Bax and Bcl-2 between groups. However, basal expression levels of Akt, phospho-Akt, PTEN, phospho-PTEN and phospho-Bad were higher in the diabetic venae cavae (P < 0.05).ConclusionThese data suggest that diabetes is associated with significant alteration in the ability of the vena cava to activate MAPK- and apoptosis-related signaling. Whether these changes are associated with the increased vein graft attrition seen in the diabetic population will require further investigation.

P38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein.

High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL)-B-cells are quiescent differentiated cells that produce interleukin (IL)-10 and accumulate due to resistance to apoptosis. The mechanisms underlying such resistance are poorly understood. Herein we show that all CLL B-cells tested (30/30) display high mRNA and protein expression of the tumor suppressor Mda-7/IL-24, an IL-10 family member, in comparison to normal B cells. A downstream Mda-7 signaling target, p38 mitogen-activated protein kinase (MAPK) was highly phosphorylated in all CLL cells but not in normal B-cells. Mda-7 expression and p38 MAPK phosphorylation diminished in culture and the latter could be reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfection. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK phosphorylation in CLL without affecting p38 MAPK, bcl2, or Lyn expression, further demonstrating the direct role of Mda-7/IL-24 in p38 MAPK activation. Both pharmacological inhibition of p38 MAPK and Mda-7 silencing augmented spontaneous apoptosis by three-fold in CLL cells cultured in autologous serum, which was reversed by LPS and r-IL-24. We established the role of p38 MAPK in CLL cell survival and demonstrated a paradoxical effect, whereby Mda-7 and IL-24, inducers of apoptosis in diverse cancer cells, promote the survival of CLL B-cells through p38 MAPK activation.