Interplay between MEK and PI3 kinase signaling regulates the subcellular localization of protein kinases ERK1/2 and Akt upon oxidative stress

ERK and Akt kinases are key components that participate in numerous regulatory processes, including the response to stress. Using novel tools for quantitative immunofluorescence, we show that oxidant exposure controls the intracellular activation and localization of ERK1/2 and Akt. Oxidative stress alters the nuclear/cytoplasmic levels of the kinases, drastically changing phospho-ERK1/2 and phospho-Akt(Ser473) levels in the nucleus. Moreover, pharmacological inhibition of PI3 kinase modulates the intracellular distribution of phospho-ERK1/2, whereas MEK inhibition affects phospho-Akt(Thr308) and phospho-Akt(Ser473). Our studies identify a new signaling link in the nucleus of stressed cells, where changes in phospho-ERK1/2 levels correlate directly with changes in phospho-Akt(Ser473).     

Dual and opposing roles of ERK in regulating G(1) and S-G(2)/M delays in A549 cells caused by hyperoxia

This study explores the role of ERK activation in regulating G(1) and S-G(2)/M delays during hyperoxia. We demonstrate here that exposing A549 human alveolar type 2 adenocarcinoma cells to hyperoxia (95% O(2)) for 0.5-24 h time-dependently increases phospho-ERK, phospho-p53(Ser15), p53, and p21(CIP1) protein levels. Decreasing phospho-ERK with the pharmacological inhibitors, PD98059 and U0126, markedly suppresses hyperoxia-stimulated phospho-p53(Ser15), p53, and p21(CIP1), and also restores the hyperoxia-reduced kinase activities of cyclin D1/E1-Cdks. Our results suggest that ERK activation during hyperoxia contributes to the p53/p21-mediated G(1) checkpoint. However, inhibition of ERK signaling during hyperoxia further delays S-phase entry and progression. Hyperoxia induces significant expression of cyclin A/B1 and translocation of cyclin A into nuclei while marginally decreasing cyclin A/B1-Cdks kinase activities, which may be related to nuclear association with p21. Interestingly, inhibition of ERK signaling markedly suppresses the elevation of cyclin A/B1 proteins and cyclin A/B1-Cdks kinase activities during hyperoxia. Taken together, the results presented here suggest that hyperoxia-activated ERK acts upstream of p53 and p21 to suppress G(1)-Cdk activities; however, it is also required for induction of cyclin A/B1 and maintenance of cyclin A/B1-Cdk activities that oppose delays in S-phase entry and progression.     

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons

ABSTRACT: BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. Results: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and p-CREB, and reverses cystitis-induced increases in micturition frequency. Conclusion: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Synthesis,characterization and Akt phosphorylation inhibitory activity of cyclopentanecarboxylate-substituted alkylphosphocholines

Akt is activated in most human cancers and contributes to cell growth, proliferation and cellular survival pathway. Accordingly, it is an attractive target for anticancer therapy. A series of novel alkylphosphocholines, incorporating cyclopentanecarboxylate in the phospholipid head group with trans and cis orientations, were synthesized and evaluated for their Akt phosphorylation inhibitory activities and cytotoxicities against human cancer cell lines, A549, MCF-7 and KATO III. Among the synthesized compounds, 5a, 5b and 6c exhibited potent inhibitory Akt phosphorylation effects with IC₅₀ value of 3.1, 2.0 and 3.0 μM, respectively, and their potencies were better than those of three reference compounds miltefosine, perifosine and edelfosine. All the new compounds, except 5d and 6e, displayed more potent growth inhibition against A549 cells than reference compounds. Specifically, compound 5b exhibited most remarkable cytotoxicities on A549 cells as well as MCF-7 and KATO III cells. Importantly, the cytotoxic effects of these compounds correlated with their Akt phosphorylation inhibitory activities.     

Vanadyl bisacetylacetonate induced G1/S cell cycle arrest via high-intensity ERK phosphorylation in HepG2 cells

In recent years the anticancer properties of vanadium compounds have been noticed, but the underlying mechanisms are not well understood. In the present work, we found that vanadyl bisacetylacetonate ([VO(acac)(2)]) blocked cell cycle progression permanently at G1 phase in a dose- and time-dependent manner in HepG2 cells. This was further evidenced by the growth regulatory signals during the G1 stage. After the treatment with [VO(acac)(2)], the level of phosphorylation of retinoblastoma tumor suppressor protein (pRb) and the expressions of cyclin D1, cyclin E and cyclin A were reduced, while the expression of a cyclin-dependent kinase inhibitor p21 was increased dose-dependently. In the meantime, neither O(2)(*-) nor H(2)O(2) level was observed to increase. Interestingly, the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) and Akt were highly activated. After 1-h pretreatment with a lower concentration of MEK inhibitor U0126, the level of phosphorylated pRb was restored, indicating a release of cell cycle arrest. Taken together, we suggested that [VO(acac)(2)]-induced proliferation inhibition was caused by G1/S cell cycle arrest, which resulted from the decreased level of phosphorylated pRb in its active hypophosphorylated form via a highly activated ERK signal in HepG2 cells. The results presented here provided new insight into the development of vanadium compounds as potential anticancer agents.     

Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation. cell density; cyclin D1; hepatocyte growth factor; cell cycle; extracellular signal-regulated kinases     

Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action

Bufalin, a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Chansu showed inhibitory effects against human prostate, hepatocellular, endometrial and ovarian cancer cells, and leukemia cells. However, whether or not bufalin has inhibitory activity against the proliferation of human non–small cell lung cancer (NSCLC) cells is unclear. The aim of this study is to study the effects of bufalin on the proliferation of NSCLC and its molecular mechanisms of action. The cancer cell proliferation was measured by MTT assay. The apoptosis and cell cycle distribution were analyzed by flow cytometry. The protein expressions and phosphorylation in the cancer cells were detected by Western blot analysis. In the present study, we have demonstrated that bufalin suppressed the proliferation of human NSCLC A549 cell line in time- and dose-dependent manners. Bufalin induced the apoptosis and cell cycle arrest by affecting the protein expressions of Bcl-2/Bax, cytochrome c, caspase-3, PARP, p53, p21WAF1, cyclinD1, and COX-2 in A549 cells. In addition, bufalin reduced the protein levels of receptor expressions and/or phosphorylation of VEGFR1, VEGFR2, EGFR and/or c-Met in A549 cells. Furthermore, bufalin inhibited the protein expressions and phosphorylation of Akt, NF-κB, p44/42 MAPK (ERK1/2) and p38 MAPK in A549 cells. Our results suggest that bufalin inhibits the human lung cancer cell proliferation via VEGFR1/VEGFR2/EGFR/c-Met–Akt/p44/42/p38-NF-κB signaling pathways; bufalin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.     

Ginsenoside-Rg2 affects cell growth via regulating ROS-mediated AMPK activation and cell cycle in MCF-7 cells

BackgroundGinsenoside-Rg2 (G-Rg2) is a protopanaxatriol-type ginsenoside isolated from ginseng. It has been found to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, and anticancer effects.PurposeThis study aimed to investigate the anticancer effects of G-Rg2 on estrogen receptor-positive MCF-7 breast cancer (BC) cells, and the underlying mechanisms involving in reactive oxygen species (ROS) production.Study design/MethodsCell viability, cell cycle distribution, apoptosis, and ROS production were measured following exposure to G-Rg2. The protein expression levels of p-ERK1/2, p-Akt, PARP, p-Rb, cyclin D1, CDK6, and p-AMPK were quantified using western blot analysis. The in vivo activity of G-Rg2 was assessed in a xenograft model. Immunohistochemistry staining for p-Rb and p-AMPK was performed in tumor tissues.ResultsG-Rg2 significantly decreased cell viability but increased cell apoptosis. In MCF-7 cells, G-Rg2 increased ROS production by inhibiting ERK1/2 and Akt activation. G-Rg2-induced ROS induced G0/G1 cell cycle arrest and AMPK phosphorylation. In the xenograft model, the 5 mg/kg G-Rg2-treated group showed decreased tumor volume and weight, similar to the 5 mg/kg 4-OHT-treated group, compared to the control group. Immunohistochemistry staining showed that G-Rg2 treatment decreased Rb phosphorylation, while increasing AMPK phosphorylation in tumor tissues.ConclusionG-Rg2 has potential anticancer effects by increasing the ROS-AMPK signaling pathway and inhibiting ERK1/2 and Akt activation-mediated cell proliferation and cell cycle progression in MCF-7 BC cells.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Choline Plasmalogens Isolated from Swine Liver Inhibit Hepatoma Cell Proliferation Associated with Caveolin-1/Akt Signaling

Plasmalogens play multiple roles in the structures of biological membranes, cell membrane lipid homeostasis and human diseases. We report the isolation and identification of choline plasmalogens (ChoPlas) from swine liver by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC)/MS. The growth and viability of hepatoma cells (CBRH7919, HepG2 and SMMC7721) was determined following ChoPlas treatment comparing with that of human normal immortal cell lines (HL7702). Result indicated that ChoPlas inhibited hepatoma cell proliferation with an optimal concentration and time of 25 μmol/L and 24 h. To better understand the mechanism of the ChoPlas-induced inhibition of hepatoma cell proliferation, Caveolin-1 and PI3K/Akt pathway signals, including total Akt, phospho-Akt(pAkt) and Bcl-2 expression in CBRH7919 cells, were determined by western blot. ChoPlas treatment increased Caveolin-1 expression and reduced the expression of phospho-Akt (pAkt) and Bcl-2, downstream targets of the PI3K/Akt pathway. Further cell cycle analysis showed that ChoPlas treatment induced G₁ and G₁/S phase transition cell cycle arrest. The expression of essential cell cycle regulatory proteins involved in the G₁ and G₁/S phase transitions, cyclin D, CDK4, cyclin E and CDK2, were also analyzed by western blot. ChoPlas reduced CDK4, cyclin E and CDK2 expression. Taken together, the results indicate that swine liver-derived natural ChoPlas inhibits hepatoma cell proliferation associated with Caveolin-1 and PI3K/Akt signals.     

Effect of Tenuifoliside A isolated from Polygala tenuifolia on the ERK and PI3K pathways in C6 glioma cells

Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of BDNF, which were blocked by ERK and PI3K inhibitors, respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkB-ERK/PI3K-CREB signaling pathway in C6 glioma cells.     

Dyrk1A, a Serine/Threonine Kinase, is Involved in ERK and Akt Activation in the Brain of Hyperhomocysteinemic Mice

Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse brain disease. Whereas the biological actions linking hyperhomocysteinemia to the cognitive dysfunction are not well understood, we tried to establish relationships between hyperhomocysteinemia and alterations of signaling pathways. In the brain of CBS-deficient mice, a murine model of hyperhomocysteinemia, we previously found an activation of extracellular signal-regulated kinase (ERK) pathway and an increase of Dyrk1A, a serine/threonine kinase involved in diverse functions ranging from development and growth to apoptosis. We then investigated the relationship between Dyrk1A and the signaling pathways initiated by receptor tyrosine kinases (RTK), the ERK and PI3K/Akt pathways. We found a significant increase of phospho-ERK, phospho-MEK, and phospho-Akt in the brain of CBS-deficient and Dyrk1a-overexpressing mice. This increase was abolished when CBS-deficient and Dyrk1A-transgenic mice were treated with harmine, an inhibitor of Dyrk1A kinase activity, which emphasizes the role of Dyrk1A activity on ERK and Akt activation. Sprouty 2 protein level, a negative feedback loop modulator that limits the intensity and duration of RTK activation, is decreased in the brain of CBS-deficient mice, but not in the brain of Dyrk1A transgenic mice. Furthermore, a reduced Dyrk1A and Grb2 binding on sprouty 2 and an increased interaction of Dyrk1A with Grb2 were found in the brain of Dyrk1A transgenic mice. The consequence of Dyrk1A overexpression on RTK activation seems to be a decreased interaction of sprouty 2/Grb2. These observations demonstrate ERK and Akt activation induced by Dyrk1A in the brain of hyperhomocysteinemic mice and open new perspectives to understand the basis of the cognitive defects in hyperhomocysteinemia.     

An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry

Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G(0)-to-G(1) transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1- and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G(1). Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G(0)-to-G(1) transition of the cell cycle.     

The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-microM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 microM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21(Cip/WAF1) on adding 100 microM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21(Cip/WAF1) induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21(Cip/WAF1) inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.     

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1?2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 μM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 μM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 μM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.     

Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: Stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin D1

Thrombin was found to stimulate astrocytes proliferation. In this study, we want to clarify whether thrombin-activated protease-activated receptor will affect the glucose metabolism signaling pathways to accelerate the proliferation of astrocytes. In addition, we study if thrombin has effects on cell cycle transition to promote astrocytes proliferation. We firstly observed that thrombin activated protease-activated receptor 1 (PAR-1) inducing the increases of intracellular Ca²⁺ and ROS production, which contribute to the astrocytes' proliferation. We further confirmed that ROS stabilized HIF-1α, the latter subsequently accelerated glucose uptake in astrocytes. On the other hand, we demonstrated that thrombin triggered PI3K/Akt/cyclin D1 signal transduction, which may promote the cell cycle transition to enhance astrocytes proliferation. As a result, we discovered three signaling pathways mainly accounting for cell proliferation induced by thrombin: (1) thrombin-stimulated ERK, JNK/ROS/HIF-1α and (2) PI3K/Akt/ROS/HIF-1α pathways to increase expression of hexokinase 2 which mediated glucose metabolism in astrocytes, and (3) thrombin stimulates PAR-1/PI3K/Akt/cyclin D1 to promote the cell cycle transition and finally to increase cell proliferation.