Saturated Free Fatty Acid Sodium Palmitate-Induced Lipoapoptosis by Targeting Glycogen Synthase Kinase-3β Activation in Human Liver Cells

Background

Elevated serum saturated fatty acid levels and hepatocyte lipoapoptosis are features of nonalcoholic fatty liver disease (NAFLD).

Aim

The purpose of this study was to investigate saturated fatty acid induction of lipoapoptosis in human liver cells and the underlying mechanisms.

Methods

Human liver L02 and HepG2 cells were treated with sodium palmitate, a saturated fatty acid, for up to 48 h with or without lithium chloride, a glycogen synthase kinase-3β (GSK-3β) inhibitor, or GSK-3β shRNA transfection. Transmission electron microscopy was used to detect morphological changes, flow cytometry was used to detect apoptosis, a colorimetric assay was used to detect caspase-3 activity, and western blot analysis was used to detect protein expression.

Results

The data showed that sodium palmitate was able to induce lipoapoptosis in L02 and HepG2 cells. Western blot analysis showed that sodium palmitate activated GSK-3β protein, which was indicated by dephosphorylation of GSK-3β at Ser-9. However, inhibition of GSK-3β activity with lithium chloride treatment or knockdown of GSK-3β expression with shRNA suppressed sodium palmitate-induced lipoapoptosis in L02 and HepG2 cells. On a molecular level, inhibition of GSK-3β expression or activity suppressed sodium palmitate-induced c-Jun-N-terminal kinase (JNK) phosphorylation and Bax upregulation, whereas GSK-3β inhibition did not affect endoplasmic reticulum stress-induced activation of unfolded protein response.

Conclusions

The present data demonstrated that saturated fatty acid sodium palmitate-induced lipoapoptosis in human liver L02 and HepG2 cells was regulated by GSK-3β activation, which led to JNK activation and Bax upregulation. This finding indicates that GSK-3β inhibition may be a potential therapeutic target to control NAFLD.     

Down-regulation of Cbl-b by bufalin results in up-regulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells

Purpose

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic agent that preferentially induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. Bufalin is a major active ingredient of the traditional Chinese medicine ChanSu. The present study aimed to assess the synergistic effect of bufalin and TRAIL and elucidate the underlying mechanisms in breast cancer cells.

Methods

Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. The expression of proteins was assayed by flow cytometry and/or Western blotting. Transfection studies were used to determine the involvement of DR4, DR5 and Cbl-b in the synergistic effect of bufalin and TRAIL.

Results

MCF-7 and MDA-MB-231 cells were resistant to TRAIL. Both cell lines were dramatically sensitized to TRAIL-induced apoptosis by bufalin. Further experiments indicated that bufalin up-regulated DR4 and DR5, activated ERK, JNK and p38 MAPK and down-regulated Cbl-b. Blocking the up-regulation of DR4 and DR5 by siRNA rendered cells less sensitive to apoptosis induced by the combination of bufalin and TRAIL. Inhibition of the activation of ERK, JNK and p38 MAPK by specific inhibitors attenuated DR4 and DR5 up-regulation. Moreover, down-regulation of Cbl-b by shRNA led to stronger activation of ERK, JNK and p38 MAPK, more up-regulation of DR4 and DR5, and a stronger synergistic effect of bufalin and TRAIL.

Conclusions

Bufalin enhanced TRAIL-induced apoptosis by up-regulating the expression of DR4 and DR5. Bufalin-induced down-regulation of Cbl-b contributed to the up-regulation of DR4 and DR5, which might be partially mediated by the activation of ERK, JNK and p38 MAPK.     

Expression of the MAPK kinases MKK‐4 and MKK‐7 in rheumatoid arthritis and their role as key regulators of JNK

Objective

The mitogen‐activated protein (MAP) kinase JNK is a key regulator of interleukin‐1 (IL‐1)–induced collagenase gene expression and joint destruction in arthritis. Two upstream kinases, MKK‐4 and MKK‐7, have been identified as potential activators of JNK. However, the role of MAP kinase kinases (MAPKKs) and their functional organization within fibroblast‐like synoviocytes (FLS) have not been defined. We therefore evaluated the interactions between the various MAP kinase components and determined their subcellular localization.

Methods

MKKs were identified by immunohistochemistry of rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Western blotting was used to determine the expression of FLS. Immunoprecipitation experiments using antibodies specific for MKK‐4, MKK‐7, and JNK were performed. Phosphospecific antibodies and immunohistochemistry were used to evaluate the activation state of synovial MKK‐4 and MKK‐7. Confocal microscopy was used to determine the subcellular location of the kinases.

Results

Immunohistochemistry studies demonstrated abundant MKK‐4 and MKK‐7 in RA and OA synovium, but the levels of phosphorylated kinases were significantly higher in RA synovium. MKK‐4 and MKK‐7 were constitutively expressed by cultured RA and OA FLS, and IL‐1 stimulation resulted in rapid phosphorylation of both kinases. JNK was detected in MKK‐4 and MKK‐7 immunoprecipitates. Furthermore, MKK‐4 coprecipitated with MKK‐7 and vice versa, indicating that the 3 kinases form a stable complex in FLS. Confocal microscopy confirmed that JNK, MKK‐4, and MKK‐7 colocalized in the cytoplasm, with JNK migrating to the nucleus after IL‐1 stimulation. The signal complex containing MKK‐4, MKK‐7, and JNK was functionally active and able to phosphorylate c‐Jun after IL‐1 stimulation of FLS.

Conclusion

These studies demonstrate that JNK, MKK‐4, and MKK‐7 form an active signaling complex in FLS. This novel JNK signalsome is activated in response to IL‐1 and migrates to the nucleus. The JNK signalsome represents a new target for therapeutic interventions designed to prevent joint destruction.

     

Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland

In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both alpha- and beta-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4beta-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca(2+)/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a beta-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca(2+) signaling pathways are involved in NE regulation of p38MAPK.     

Serine/threonine protein phosphatase 5 regulates glucose homeostasis in vivo and apoptosis signalling in mouse pancreatic islets and clonal MIN6 cells

Aims/hypothesis

During the development of type 2 diabetes mellitus, beta cells are often exposed to a high glucose/hyperlipidaemic environment, in which the levels of reactive oxygen species (ROS) are elevated. In turn, ROS can trigger an apoptotic response leading to beta cell death, by activating mitogen-activated protein kinase (MAPK) signalling cascades. Here we test the hypothesis that serine/threonine protein phosphatase 5 (PP5) acts to suppress proapoptotic c-Jun N-terminal kinase (JNK) signalling in beta cells.

Methods

Ppp5c ^?/? and Ppp5c ^+/+ mice were subjected to intraperitoneal glucose (IPGTT) or insulin tolerance tests. Pancreatic islets from Ppp5c ^?/? and Ppp5c ^+/+ mice or MIN6 cells treated with short-interfering RNA targeting PP5 were exposed to palmitate or H₂O₂ to activate MAPK signalling. Changes in protein phosphorylation, mRNA expression, apoptosis and insulin secretion were detected by western blot analysis, quantitative RT-PCR or ELISA.

Results

Ppp5c ^?/? mice weighed less and exhibited reduced fasting glycaemia and improved glucose tolerance during IPGTT, but retained normal insulin sensitivity and islet volume. Comparison of MAPK signalling in islets from Ppp5c ^?/? mice and MIN6 cells revealed that the lack of PP5 was associated with enhanced H₂O₂-induced phosphorylation of JNK and c-Jun. Cells with reduced PP5 also showed enhanced JNK phosphorylation and apoptosis after palmitate treatment. PP5 suppression in MIN6 cells correlated with hypersecretion of insulin in response to glucose.

Conclusions/interpretation

PP5 deficiency in mice is associated with reduced weight gain, lower fasting glycaemia, and improved glucose tolerance during IPGTT. At a molecular level, PP5 helps suppress apoptosis in beta cells by a mechanism that involves regulation of JNK phosphorylation.     

The RhoA/ROCK pathway mediates high glucose‐induced cardiomyocyte apoptosis via oxidative stress,JNK, and p38MAPK pathways

Aims

To understand the roles of the RhoA/ROCK and mitogen‐activated protein kinase (MAPK) pathways in high glucose (HG)‐induced apoptosis and oxidative stress in cardiomyocytes.

Materials and methods

Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30 mmol/L D‐glucose, in the presence or absence of fasudil (50 or 100 μM), SB203580, SP600125, or PD98059 (10 μM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl‐2 mRNA levels were determined by quantitative real‐time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl‐2, and cleaved caspase‐3 was analysed by Western blot.

Results

Fasudil, SB203580, and SP600125 effectively inhibited the HG‐induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl‐2 protein expression ratio, and cleaved caspase‐3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl‐2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG‐induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK.

Conclusions

The RhoA/ROCK pathway mediates HG‐induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG‐induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways.     

Fibroblast growth factor-21 protects human skeletal muscle myotubes from palmitate-induced insulin resistance by inhibiting stress kinase and NF-κB

We investigated the effects of fibroblast growth factor-21 (FGF-21) on palmitate-induced insulin resistance in skeletal muscle myotubes. First, to determine the effect of FGF-21 on palmitate-induced insulin resistance, we measured 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose uptake and levels of proteins involved in insulin signaling pathways (IRS-1 and Akt) in human skeletal muscle myotubes (HSMMs) exposed to palmitate for 24h, and compared HSMMs exposed to palmitate and different doses of recombinant FGF-21. Second, to determine the mechanisms underlying the contribution of FGF-21 to palmitate-induced insulin resistance, we compared levels of proteins linked to palmitate-induced insulin resistance (PKC-θ, IKKα/β, JNK, p38, IκBα, and NF-κB) in HSMMs exposed to palmitate and different doses of recombinant FGF-21 for 24h. Palmitate-reduced glucose uptake was restored by FGF-21. Palmitate inhibited phosphorylation of Akt and thereby impaired insulin signaling in HSMMs. FGF-21 prevented palmitate from inhibiting the phosphorylation of Akt. These results indicate that FGF-21 prevented palmitate-induced insulin resistance in HSMMs. Palmitate activated NF-κB in HSMMs, thereby impairing the action of insulin and initiating chronic inflammation. FGF-21 inhibited palmitate-induced NF-κB activation in HSMMs. The results of the present study suggest that FGF-21 prevents palmitate-induced insulin resistance in HSMMs by inhibiting the activation of stress kinase and NF-κB.     

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.     

Exendin-4 and exercise improve hepatic glucose homeostasis by promoting insulin signaling in diabetic rats

Recently, it has been reported that a long-acting glucagon-like peptide-1 (exendin-4) and physical exercise improve hepatic insulin action in diabetic rats. However, this phenomenon remains poorly understood. We investigated the long-term effect that exendin-4 and exercise had on hepatic insulin resistance through the modulation of hepatic and/or hypothalamic insulin signaling in 90% pancreatectomized diabetic rats fed 40% energy fat diets. The rats were divided into 4 groups: exendin-4 only, exendin-4 plus exercise training, saline (control), or exercise training only. Rats in the exendin-4 groups were administered with 150 pmol/kg exendin-4 twice a day for 8 weeks, whereas those in the exercise groups ran on an uphill treadmill with a 15° incline at 20 m/min for 30 minutes 5 days a week. Exendin-4 reduced serum glucagon levels in overnight-fasted rats. Exendin-4 treatment by itself decreased hepatic glucose output at hyperinsulinemic states, and exercise without exendin-4 treatment also had the same effect. Exendin-4 promoted hepatic insulin signaling by potentiating tyrosine phosphorylation of the insulin receptor substrate-2 without changing hypothalamic insulin signaling. Exendin-4 also enhanced hypothalamic glucose sensing. However, exercise improved both hepatic and hypothalamic insulin signaling by activating the phosphorylation of cyclic adenosine monophosphate-responding element binding proteins to induce insulin receptor substrate-2 expression. Exendin-4 and exercise decreased the expression of phosphoenolpyruvate carboxykinase, which in turn reduced hepatic glucose output. Exendin-4 in combination with exercise had no additive effects. In conclusion, exendin-4 and exercise improve hepatic glucose homeostasis by promoting hepatic insulin signaling in diabetic rats.     

Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism

Aims/hypothesis

Although the substitution of saturated fatty acids with oleate has been recommended in the management of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance in skeletal muscle cells are not completely known. Here, we examined whether oleate, through activation of AMP-activated protein kinase (AMPK), prevented palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance.

Methods

Studies were conducted in mouse C2C12 myotubes and in the human myogenic cell line LHCN-M2. To analyse the involvement of AMPK, activators and inhibitors of this kinase and overexpression of a dominant negative AMPK construct (K45R) were used.

Results

Palmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. The induction of ER stress markers by palmitate was prevented by the presence of the AMPK activators AICAR and A-769662. Moreover, the ability of oleate to prevent palmitate-induced ER stress and inflammation (nuclear factor-kappa B [NF-κB] DNA-binding activity and expression and secretion of IL6) as well as insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake was reversed in the presence of the AMPK inhibitor compound C or by overexpression of a dominant negative AMPK construct. Finally, palmitate reduced phospho-AMPK levels, whereas this was not observed in oleate-exposed cells or in palmitate-exposed cells supplemented with oleate.

Conclusions/interpretation

Overall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK.     

p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-kappaB degradation in human coronary artery endothelial cells

Plasma free fatty acids are elevated in patients with type 2 diabetes and contribute to the pathogenesis of insulin resistance and endothelial dysfunction. The p38 MAPK mediates stress, inflammation, and apoptosis. Whether free fatty acids induce apoptosis and/or activate nuclear factor-kappaB inflammatory pathway in human coronary artery endothelial cells (hCAECs) and, if so, whether this involves the p38 MAPK pathway is unknown. hCAECs (passages 4-6) were grown to 70% confluence and then incubated with palmitate at concentrations of 0-300 microm for 6-48 h. Palmitate at 100, 200, or 300 microm markedly increased apoptosis after 12 h of incubation. This apoptotic effect was time (P=0.008) and dose (P=0.006) dependent. Palmitate (100 microm for 24 h) induced a greater than 2-fold increase in apoptosis, which was accompanied with a 4-fold increase in p38 MAPK activity (P<0.001). Palmitate did not affect the phosphorylation of Akt1 or ERK1/2. SB203580 (a specific inhibitor of p38 MAPK) alone did not affect cellular apoptosis; however, it abolished palmitate-induced apoptosis and p38 MAPK activation. Palmitate significantly reduced the level of inhibitor of nuclear factor-kappaB (IkappaB). However, treatment of cells with SB203580 did not restore IkappaB to baseline. We conclude that palmitate induces hCAEC apoptosis via a p38 MAPK-dependent mechanism and may participate in coronary endothelial injury in diabetes. However, palmitate-mediated IkappaB degradation in hCAECs is independent of p38 MAPK activity.     

Antiinflammatory functions of p38 in mouse models of rheumatoid arthritis: Advantages of targeting upstream kinases MKK‐3 or MKK‐6

Objective

Inhibitors of p38 demonstrate limited benefit in rheumatoid arthritis (RA), perhaps due to the antiinflammatory functions of p38α. This study was performed to determine if selective deletion of p38α in macrophages affects the severity of arthritis and whether blocking upstream kinases in the p38 pathway, such as MKK‐3 or MKK‐6, avoids some of the limitations of p38 blockade.

Methods

Wild‐type (WT) mice and mice with selective deletion of p38α in macrophages (p38α^ΔLysM) were injected with K/BxN sera. Antigen‐induced arthritis was also induced in p38α^ΔLysM mice. Mouse joint extracts were evaluated by enzyme‐linked immunosorbent assay, quantitative polymerase chain reaction (qPCR), and Western blot analysis. Bone marrow–derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) and were evaluated by qPCR and Western blotting. Bone marrow chimeras were generated using MKK‐3^−/− and MKK‐6^−/− mice, and K/BxN serum was administered to induce arthritis.

Results

Compared to WT mice, p38α^ΔLysM mice had increased disease severity and delayed resolution of arthritis, which correlated with higher synovial inflammatory mediator expression and ERK phosphorylation. In contrast to WT BMMs cultured in the presence of a p38α/β inhibitor, LPS‐stimulated MKK‐6– and MKK‐3–deficient BMMs had suppressed LPS‐mediated interleukin‐6 (IL‐6) expression but had normal IL‐10 production, dual‐specificity phosphatase 1 expression, and MAPK phosphorylation. WT chimeric mice with MKK‐6– and MKK‐3–deficient bone marrow had markedly decreased passive K/BxN arthritis severity.

Conclusion

Inhibiting p38α in a disease that is dominated by macrophage cytokines, such as RA, could paradoxically suppress antiinflammatory functions and interfere with clinical efficacy. Targeting an upstream kinase that regulates p38 could be more effective by suppressing proinflammatory cytokines while preventing decreased IL‐10 expression and increased MAPK activation.

     

Biochemical and histological effects of exendin-4 (exenatide) on the rat pancreas

Aims/hypothesis  

Exendin-4 is a 39 amino acid agonist of the glucagon-like peptide receptor and has been approved for treatment of type 2 diabetes. Many reports describe an increased incidence of acute pancreatitis in humans treated with exendin-4 (exenatide). Previous studies have evaluated the effect of exendin-4 on beta cells and beta cell function. We evaluated the histological and biochemical effects of exendin-4 on the pancreas in rats.     

Butein inhibits ethanol-induced activation of liver stellate cells through TGF-β, NFκB, p38, and JNK signaling pathways and inhibition of oxidative stress

Background

Butein has been reported to prevent and partly reverse liver fibrosis in vivo; however, the mechanisms of its action are poorly understood. We, therefore, aimed to determine the antifibrotic potential of butein.

Methods

We assessed the influence of the incubation of hepatic stellate cells (HSCs) and hepatoma cells (HepG2) with butein on sensitivity to ethanol- or acetaldehyde-induced toxicity; the production of reactive oxygen species (ROS); the expression of markers of HSC activation, including smooth muscle α-actin (α-SMA) and procollagen I; and the production of transforming growth factor-β1 (TGF-β1), metalloproteinases-2 and -13 (MMP-2and MMP-13), and tissue inhibitors of metalloproteinases (TIMPs). The influence of butein on intracellular signals in HSCs; i.e., nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol was estimated.

Results

Butein protected HSCs and HepG2 cells against ethanol toxicity by the inhibition of ethanol- or acetaldehyde-induced production of ROS when cells were incubated separately or in co-cultures; butein also inhibited HSC activation measured as the production of α-SMA and procollagen I. As well, butein downregulated ethanol- or acetaldehyde-induced HSC migration and the production of TGF-β, TIMP-1, and TIMP-2; decreased the activity of MMP-2; and increased the activity of MMP-13. In ethanol-induced HSCs, butein inhibited the activation of the p38 MAPK and JNK transduction pathways as well as significantly inhibiting the phosphorylation of NF κB inhibitor (IκB) and Smad3.

Conclusions

The results indicated that butein inhibited ethanol- and acetaldehyde-induced activation of HSCs at different levels, acting as an antioxidant and inhibitor of ethanol-induced MAPK, TGF-β, and NFκB/IκB transduction signaling; this result makes butein a promising agent for antifibrotic therapies.     

Cytoprotection by Jun kinase during nitric oxide-induced cardiac myocyte apoptosis

Nitric oxide (NO) induces apoptosis in cardiac myocytes through an oxidant-sensitive mechanism. However, additional factors appear to modulate the exact timing and rate of NO-dependent apoptosis. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase [ERK] 1/2, c-Jun N-terminal kinase [JNK] 1/2, and p38MAPK) in NO-mediated apoptotic signaling. The NO donor S:-nitrosoglutathione (GSNO) induced caspase-dependent apoptosis in neonatal rat cardiac myocytes, preceded by a rapid (<10-minute) and significant (approximately 50-fold) activation of JNK1/2. Activation of JNK was cGMP dependent and was inversely related to NO concentration; it was maximal at the lowest dose of GSNO (10 micromol/L) and negligible at 1 mmol/L. NO slightly increased ERK1/2 beginning at 2 hours but did not affect p38MAPK activity. Inhibitors of ERK and p38MAPK activation did not affect cell death rates. In contrast, expression of dominant-negative JNK1 or MKK4 mutants significantly increased NO-induced apoptosis at 5 hours (56.77% and 57.37%, respectively, versus control, 40.5%), whereas MEKK1, an upstream activator of JNK, sharply reduced apoptosis in a JNK-dependent manner. Adenovirus-mediated expression of dominant-negative JNK1 both eliminated the rapid activation of JNK by NO and accelerated NO-mediated apoptosis by approximately 2 hours. These data indicate that NO activates JNK as part of a cytoprotective response, concurrent with initiation of apoptotic signaling. Early, transient activation of JNK serves both to delay and to reduce the total extent of apoptosis in cardiac myocytes.