Beneficial Effects of Fingolimod in Alzheimer's Disease: Molecular Mechanisms and Therapeutic Potential.

Alzheimer's disease (AD), the most common cause of dementia remains of unclear etiology with current pharmacological therapies failing to halt disease progression. Several pathophysiological mechanisms have been implicated in AD pathogenesis including amyloid-ß protein (Aß) accumulation, tau hyperphosphorylation, neuroinflammation and alterations in bioactive lipid metabolism. Sphingolipids, such as sphingosine-1-phosphate (S1P) and intracellular ceramide/S1P balance are highly implicated in central nervous system physiology as well as in AD pathogenesis. FTY720/Fingolimod, a structural sphingosine analog and S1P receptor (S1PR) modulator that is currently used in the treatment of relapsing-remitting multiple sclerosis (RRMS) has been shown to exert beneficial effects on AD progression. Recent in vitro and in vivo evidence indicate that fingolimod may suppress Aß secretion and deposition, inhibit apoptosis and enhance brain-derived neurotrophic factor (BDNF) production. Furthermore, it regulates neuroinflammation, protects against N-methyl-D-aspartate (NMDA)-excitotoxicity and modulates receptor for advanced glycation end products signaling axis that is highly implicated in AD pathogenesis. This review discusses the underlying molecular mechanisms of the emerging neuroprotective role of fingolimod in AD and its therapeutic potential, aiming to shed more light on AD pathogenesis as well as direct future treatment strategies.

[Effect of acupuncture combined with hypothermia on MAPK/ERK pathway and apoptosis related factors in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe effect of acupuncture combined with hypothermia therapy on MAPK/ERK pathway and apoptosis related factorsin rats suffered cerebral ischemia reperfusion and to explore underlying mechanisms.
 Methods: Middle cerebral artery ischemia model were established.Ninety SD rats were randomly assigned into a blank group, a control group, a model group, an acupuncture group, a mild hypothermia group, and an acupuncture with hypothermia group. After 72 h treatment, nerve function defect scores were observed, and infarction area percent was detected by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining; expressions of Bcl-2 and Bax were examined by immunohistochemistry; apoptotic cells were detected by TUNEL assay; and expression levels of phospho-mitogen-activated protein kinase(p-MEK2) and phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2) in the rats' hippocampus ischemic side were determined by Western blot.
 Results: In the rats of the model group, the neural function defect scores, the infarction area percent, the expression level of Bax, and apoptotic cells increased, while the level of Bcl-2 decreased significantly. The level of p-MEK2 and p-ERK1/2 increased obviously compared with the blank and control groups (P<0.05 or P<0.01). After treatment with acupuncture and hypothermia, the neural function defect scores, infarction area percent, and the level of Bax, apoptotic cells and the levels of p-MEK2 and p-ERK1/2 were significantly decreased, while the level of Bcl-2 in the treatment group was significantly elevated (P<0.05 or P<0.01) compared with the model group. Compared with the acupuncture group or the hypothermia group, the neural function defect scores and the levels of p-MEK2 and p-ERK1/2 in the acupuncture combined with hypothermia group were significantly reduced (P<0.05 or P<0.01).
 Conclusion: Acupuncture and hypothermia therapy can improve cerebral function, and reduce the cerebral injury through down-regulation of Bax level, and up-regulation of Bcl-2 level, which is related to reducing the levels of p-MEK2 and p-ERK1/2. The therapeutic effects on cerebral ischemia reperfusion injury for combination of acupuncture with hypothermia are better than those with single application of acupuncture or hypothermia.

MAPK/AP-1-Targeted Anti-Inflammatory Activities of Xanthium strumarium.

Xanthium strumarium L. (Asteraceae), a traditional Chinese medicine, is prescribed to treat arthritis, bronchitis, and rhinitis. Although the plant has been used for many years, the mechanism by which it ameliorates various inflammatory diseases is not yet fully understood. To explore the anti-inflammatory mechanism of methanol extracts of X. strumarium (Xs-ME) and its therapeutic potential, we used lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells and human monocyte-like U937 cells as well as a LPS/D-galactosamine (GalN)-induced acute hepatitis mouse model. To find the target inflammatory pathway, we used holistic immunoblotting analysis, reporter gene assays, and mRNA analysis. Xs-ME significantly suppressed the up-regulation of both the activator protein (AP)-1-mediated luciferase activity and the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor (TNF)-[Formula: see text]. Moreover, Xs-ME strongly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW264.7 and U937 cells. Additionally, these results highlighted the hepatoprotective and curative effects of Xs-ME in a mouse model of LPS/D-GalN-induced acute liver injury, as assessed by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and histological damage. Therefore, our results strongly suggest that the ethnopharmacological roles of Xs-ME in hepatitis and other inflammatory diseases might result from its inhibitory activities on the inflammatory signaling of MAPK and AP-1.

Anti-adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways.

It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of ß-catenin.

Identification and optimization of new dual inhibitors of B-Raf and epidermal growth factor receptor kinases for overcoming resistance against vemurafenib.

Epidermal growth factor receptor (EGFR) amplification has been demonstrated to be critical for the inherent and/or acquired resistance against current B-Raf(V600E) inhibitor therapy for melanoma and colorectal cancer patients. We describe the discovery and structure-activity relationship study of a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxamide analogues as novel dual inhibitors of EGFR and B-Raf(V600E) mutant. One of the most promising compounds, 6a, potently inhibited both of the kinases with IC50 values of 8.0 and 51 nM, respectively. The compound also strongly suppressed the proliferation of a panel of intrinsic and acquired resistant melanoma and/or colorectal cancer cells harboring overexpressed EGFR with submicromolar IC50 values. Further mechanism investigation revealed that 6a could sustainably inhibit the activation of the MAPK path way in the resistant SK-MEL-28 PR30 melanoma cancer cells and WiDr colorectal cancer cells with EGFR amplification. Our results support the hypothesis that the EGFR/B-Raf(V600E) dual inhibition might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma and/or colorectal cancers against the current B-Raf(V600E) inhibitor therapy.

Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

Extracellular signal-regulated kinases (ERK) inhibitors from Aristolochia yunnanensis.

Six new sesquiterpenoids, aristoyunnolins A-F (1-6), an artifact of isolation [7-O-ethyl madolin W (7)], and 12 known analogues were isolated from stems of Aristolochia yunnanensis. The structures were determined by combined chemical and spectral methods, and the absolute configurations of compounds 2, 3, 5-7, 9, 14, and 17 were determined by the modified Mosher's method and CD analysis. Compounds 1-19 were screened using a bioassay system designed to evaluate the effect on mitogen-activated protein kinases (MAPKs) signaling pathways. Among three MAPKs (ERK1/2, JNK, and p38), compounds 1, 4, 10-13, 16, 18, and 19 exhibited selective inhibition of the phosphorylation of ERK1/2. Compounds 16 and 19 were more active than the positive control PD98059, a known inhibitor of the ERK1/2 signaling pathway.

Epidermal growth factor receptor expression and signaling are essential in glutamine's cytoprotective mechanism in heat-stressed intestinal epithelial-6 cells.

Epidermal growth factor receptor (EGFR) expression and signaling can induce cellular protection after intestinal inflammation. L-Glutamine (GLN) is known to prevent apoptosis after intestinal injury by activating MAPK and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. However, the role of EGFR expression and signaling in GLN-mediated cellular protection in intestinal epithelial-6 (IEC-6) cells after heat stress (HS) is unknown. To address the role of EGFR in GLN-mediated protection, IEC-6 cells were treated with GLN in the presence or absence of EGFR small interfering RNA, the EGFR tyrosine kinase inhibitor AG1478, the ERK1/2 inhibitor PD98059, the p38MAPK inhibitor SB203580, or the PI3-K/Akt inhibitor LY294002 under basal and HS conditions. GLN-mediated cell survival was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Phosphorylated and/or total levels of EGFR, cleaved caspase-3, poly(ADP-ribose) polymerase-1, ERK1/2, p38MAPK, and Akt were assessed by Western blotting. We showed that HS induced a decrease in total, cytoplasmic, and nuclear EGFR levels in IEC-6 cells, which was prevented by GLN supplementation, leading to attenuated apoptosis via EGFR small interfering RNA. Furthermore, the protective effect of GLN was lessened by AG1478, PD98059, and LY294002 but was not affected by SB203580. AG1478 attenuated GLN-mediated increases in ERK1/2 and decreases in p38MAPK phosphorylation. However, AG1478 had no effect on GLN-mediated augmentations in Akt phosphorylation. In summary, EGFR expression was important in the protective mechanism of GLN, as well as GLN-mediated activation of EGFR tyrosine kinase activity. GLN-mediated EGFR signaling activated ERK1/2 and decreased p38MAPK signaling. However, GLN-mediated Akt phosphorylation after HS seems to be independent of EGFR signaling.

EGCG inhibits Tat-induced LTR transactivation: role of Nrf2, AKT, AMPK signaling pathway.

AIMS: Transcription is a crucial step for human immunodeficiency virus 1 (HIV-1) gene expression in infected host cells. The HIV-1 Tat activates the nuclear factor-kappa B (NF-κB) signaling transduction pathway, which is necessary for viral replication. Epigallocatechin-3-gallate (EGCG) has antioxidant, anti-inflammatory, and anti-viral properties. In this study, we investigated the effects of EGCG on Tat-induced HIV-1 transactivation and potential mechanisms by which EGCG inhibited activation of NF-κB pathway. MAIN METHODS: HeLa-CD4-long terminal repeat (LTR)-ß-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The reactive oxygen species (ROS) levels and glutathione (GSH) levels were measured. In addition, the protein expressions were assayed by western blotting. KEY FINDINGS: Tat caused a significant decrease in the intracellular glutathione (GSH) levels, a mild increase in the expression of nuclear levels of NF-E2-related factor-2 (Nrf2), a significant increase in the levels of NF-κB (phosphorylation of p65 and IKK) and a significant increase in ROS production. EGCG supplementation significantly improved the changes associated with Tat-induced oxidative stress by increasing nuclear levels of Nrf2, decreasing levels of NF-κB and ROS production. EGCG reversed Tat-mediated AKT activation and AMPK inhibition in MAGI cells. EGCG inhibited Tat-induced LTR transactivation in a dose-dependent manner. SIGNIFICANCE: The results suggest that Nrf2 signaling pathway may be the primary target for prevention of Tat-induced HIV-1 transactivation by EGCG, and EGCG also reduce NF-κB activation by inhibiting AKT signaling pathway and activating AMPK signaling pathway.

Schisandrin B suppresses TGFß1 signaling by inhibiting Smad2/3 and MAPK pathways.

TGFß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Schisandra chinensis (S. chinensis), which is used as an oriental herbal medicine, is effective in the treatment of vascular injuries that cause aberrant TGFß1 signaling. In this study, we investigated whether S. chinensis extract and its active ingredients inhibit TGFß1 signaling in A7r5 vascular smooth muscle cells. We found that S. chinensis extract suppressed TGFß1 signaling via inhibition of Smad2/3 phosphorylation and nuclear translocation. Among the active ingredients of S. chinensis extract, schisandrin B (SchB) most potently inhibited TGFß1 signaling. SchB inhibited sustained phosphorylation and nuclear translocation of Smad2/3. Moreover, SchB suppressed TGFß1-induced phosphorylation of p38 and JNK, which contributed to Smad2/3 inactivation. The present study is the first to demonstrate that S. chinensis extract and SchB inhibit TGFß1 signaling. Our results may help future investigations to understand vascular fibrosis pathogenesis and to develop novel therapeutic strategies for treatment of vascular fibrotic diseases.

L-3-n-butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695.

Amyloid precursor protein (APP) is cleaved by α-secretase, within the amyloid-ß (Aß) sequence, resulting in the release of a secreted fragment (αAPPs) and precluding Aß production. We investigated the effects of a promising anti-AD new drug, l-3-n-butylphthalide (L-NBP), on APP processing and Aß generation in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. L-NBP significantly increased αAPPs release, and reduced Aß generation. The steady-state full-length APP levels were unaffected by L-NBP. It suggested that L-NBP regulated APP processing towards to the non-amyloidogenic α-secretase pathway. Protein kinase C (PKC) and mitogen activated protein (MAP) kinase might be involved in L-NBP-induced αAPPs secretion. L-NBP significantly increased PKCα and ɛ activations, lowered PKCγ activation and increased the phosphorylation of p44/p42 MAPK. Furthermore, PKC and MAPK inhibitors partially reduced L-NBP-induced αAPPs secretion. The results suggested alternative pharmacological mechanisms of L-NBP regarding the treatment of Alzheimer's disease (AD).

MAPKs are not involved in triptolide-induced cell growth inhibition and apoptosis in prostate cancer cell lines with different p53 status.

Triptolide showed excellent antitumor activity against several solid tumors. However, its mechanism has not been fully understood. To further elucidate it, the effects of mitogen activated protein kinases (MAPKs) on the activity of triptolide towards prostate cancer cell lines were investigated in the present study using both LNCaP (p53 positive and androgen-dependent) and PC-3 (p53 deficient and androgen-independent) cells. Our results showed that triptolide exerted potent growth inhibitory and apoptotic effects on both cell lines, and the effects were independent of the expression of p53. Although upregulation of ERK and JNK phosphorylation was observed after the triptolide treatment, the results with inhibitors showed that these MAPKs were not involved in the mechanism of triptolide activity in human prostate cancer cell lines with different p53 status.

Tanshinone II A induces apoptosis and S phase cell cycle arrest in activated rat hepatic stellate cells.

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, improves blood circulation and treats chronic hepatitis and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is the predominant event in liver fibrosis. The therapeutic goal in liver fibrosis is the reversal of fibrosis and selective clearance of activated HSCs. We used rat HSCs transformed by Simian virus 40 (t-HSC/Cl-6) to overcome the limitations inherent in studying subcultures of HSCs. Treatment of t-HSC/Cl-6 cells with tanshinone IIA inhibited cell viability in a dose- and time-dependent manner. Tanshinone IIA induced apoptosis as demonstrated by DNA fragmentation, poly(ADP-ribose) polymerase and caspase-3 cleavage, increased Bax/Bcl-2 protein ratio, and depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol. Furthermore, this compound markedly induced S phase cell cycle arrest, and down-regulated cyclins A and E, and cdk2. Thus, tanshinone IIA induces apoptosis and S phase cell cycle arrest in rat HSCs in vitro.

Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin.

Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2O2-decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process.

The molecular mechanism of gypenosides-induced G1 growth arrest of rat hepatic stellate cells.

AIM OF THE STUDY: Gypenosides, the saponins extract derived from Gynostemma pentaphyllum Makino, have been used for treating hepatitis and cancer in Asia. Our previous study demonstrates that gypenosides inhibit the onset and improve the recovery of liver fibrosis induced by CCl4 in rats. In this study, we used the isolated rat hepatic stellate cells (HSCs) as a model to study the cellular mechanism of gypenosides-inhibited liver fibrosis. MATERIALS AND METHODS: Rat HSCs was treated with PDGF, gypenosides or vehicle. Cell viability was assessed by trypan blue staining. Apoptosis and cell cycle were evaluated by flow cytometry. The activation or inhibition of signal molecules was detected by Western blotting. RESULTS: Our results showed that 500 microg/ml gypenosides decreased PDGF-induced rat HSCs numbers (8750+/-2629 versus 103,000+/-6683, p<0.001, 95% confidence interval) and arrested cells at the G1 phase without the presence of sub-G1 fraction. Analysis of PDGF-induced proliferative molecules including phosphorylation of Akt and p70 S6K, gypenosides inhibited the activation of this signal pathway. Furthermore, gypenosides down-regulated the protein expression of cell cycle G1-specific cyclin D1 and D3. CONCLUSIONS: Gypenosides inhibited PDGF-induced HSCs proliferation by inhibiting the signal pathway of PDGF-Akt-p70 S6K and down-regulation of cyclin D1 and D3 expression.

Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis.

Rat hepatic stellate cells (HSC-T6) were incubated for 24 h with 10-180 microM of t10c12 (98%), c9t11 (96%) and a mixed form (c9,t11:t10,c12; 41%:44%) of conjugated linoleic acid (CLA). The MTS dye reduction was measured to verify cell viability in a dose-dependent manner. Among the three CLAs, c9,t11-CLA exhibited the most intense cytotoxic effect on HSCs, the survival rate of which was reduced to 60% under 80 microM of treatment, while cell survival was slightly affected by the mixed form. Three CLA-induced cell deaths were determined by measuring DNA fragmentation using 4',6-diamidino-2-phenylindole staining. The degrees of DNA fragmentation were the most severe in HSC treated with 80 microM of c9,t11-CLA. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase and mitogen-activated or extracellular signal-regulated protein kinase (MEK) 1 and 2 were not activated in the t10,c12-CLA treatment. This suggests that the MEK-dependent apoptosis signal is crucial in HSC, which is induced by c9,t11 and mixed CLA. In order to evaluate the protective effect of CLA on carbon tetrachloride (CCl4)-induced hepatic fibrosis in vivo, animals were treated with 10% CCl4 to induce hepatic fibrosis during all experimental periods. Rats were divided into two treatment groups: (1) control diet with tap water ad libitum (n=15) and (2) 1% CLA diet with tap water ad libitum (n=15). In the CLA-supplemented rat livers, alpha-smooth muscle actin-positive cells were significantly reduced around the portal vein. In addition, collagen fibers were not detected in the CLA-treated group. These results suggest that 9c,11t-CLA influences cytotoxic effect on HSC in an MEK-dependent manner and preserving liver from fibrosis.

Hibiscus sabdariffa L. water extract inhibits the adipocyte differentiation through the PI3-K and MAPK pathway.

Hibiscus sabdariffa L., a tropical beverage material and medical herb, is used commonly as in folk medicines against hypertension, pyrexia, inflammation, liver disorders, and obesity. This report was designed to investigate the inhibitory mechanisms of hibiscus extract on adipocyte differentiation in 3T3-L1 preadipocytes. The possible inhibitory pathways that regulate the adipocyte differentiation contain the adipogenic transcription factors, C/EBPalpha and PPARgamma, PI3-kinase, and MAPK pathway. In this study, we examined whether hibiscus extract affected the adipogenesis via these three pathways. To differentiate preadipocyte in adipocyte, confluent 3T3-L1 preadipocytes were treated with the hormone mixture including isobutylmethylxanthine, dexamethasone, and insulin (MDI). Hibiscus extract inhibited significantly the lipid droplet accumulation by MDI in a dose-dependent manner and attenuated dramatically the protein and mRNA expressions of adipogenic transcriptional factors, C/EBPalpha and PPARgamma, during adipogenesis. The increase of phosphorylation and expression of PI3-K/Akt during adipocytic differentiation was markedly inhibited by treatment with hibiscus extract or PI3-K inhibitors. Furthermore, the phosphorylation and expression of MEK-1/ERK known to regulate the early phase of adipogenesis were clearly decreased with the addition of hibiscus extract. Taken together, this report suggests that hibiscus extract inhibits the adipocyte differentiation through the modulation of PI3-K/Akt and ERK pathway that play pivotal roles during adipogenesis.

Mitogen-activated protein kinases in heart development and diseases.

Mitogen-activated protein (MAP) kinases belong to a highly conserved family of Ser-Thr protein kinases in the human kinome and have diverse roles in broad physiological functions. The 4 best-characterized MAP kinase pathways, ERK1/2, JNK, p38, and ERK5, have been implicated in different aspects of cardiac regulation, from development to pathological remodeling. Recent advancements in the development of kinase-specific inhibitors and genetically engineered animal models have revealed significant new insights about MAP kinase pathways in the heart. However, this explosive body of new information also has yielded many controversies about the functional role of specific MAP kinases as either detrimental promoters or critical protectors of the heart during cardiac pathological processes. These uncertainties have raised questions on whether/how MAP kinases can be targeted to develop effective therapies against heart diseases. In this review, recent studies examining the role of MAP kinase subfamilies in cardiac development, hypertrophy, and survival are summarized.

Flashlamp pulsed-dye laser suppressed TGF-beta1 expression and proliferation in cultured keloid fibroblasts is mediated by MAPK pathway.

BACKGROUND AND OBJECTIVES: Our previous clinical study indicated that transforming growth factor-beta1 (TGF-beta1) and mitogen-activated protein kinases (MAPK) are both involved in keloid regression following flashlamp pulsed-dye laser (PDL). To further characterize of this involvement, this work examined whether PDL suppression of TGF-beta1 expression was mediated through MAPK pathway in cultured keloid fibroblasts (KF). STUDY DESIGN/MATERIALS AND METHODS: Primary culture of KF harvested from keloid patients received various dosages of PDL treatment in 585-nm wavelength. TGF-beta1 expressions in KF following various dosages of PDL were assessed. Additionally, MAPK pathway activities were studied using the PD98059 (an ERK inhibitor), SB203580 (a p38 kinase inhibitor), and SP600125 (a JNK inhibitor), to determine the role in keloid following PDL treatment. Activator protein-1 (AP-1), a transcription factor of TGF-beta, was analyzed by electrophoretic mobility shift assay (EMSA). Phosphorylated c-Jun, one of the components of AP-1, was also detected. RESULTS: The observation results demonstrated that optimal dosages of PDL significantly suppressed KF proliferation and TGF-beta1 expression. EMSA study identified PDL downregulation of super-shift of AP-1. Three subtypes of MAPK cascades were augmented between 30 minutes and 4 hours following PDL treatment, particularly phosphorylation of ERK1/2 and p38. Pre-treatment with PD98059, SB203580, but not SP600125, markedly inhibited the downregulating effects of TGF-beta1 and phosphorylated c-Jun expression following PDL treatment. CONCLUSION: PDL induced keloid regression is mediated by triggering MAPK cascades and blockade of AP-1 transcription and TGF-beta expression. Modulation of TGF-beta and MAPK interaction in keloids may provide specific targets for therapeutic intervention.

Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation.

We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl- -free solution or with the Cl- -channel inhibitors: 5-nitro-2-(3-phenylpropylamino)benzoic acid or niflumic acid. Similar declines occurred with either a high-K+ (20 mM) supplemented solution or the K+ channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl- and K+ channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n)-Erk1 and d/n-p38 cells. Volume-sensitive Cl- and K+ channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl- and K+ channel activation, whereas p38 activation occurs as a consequence of RVD.