The overview of Mitogen-activated extracellular signal-regulated kinase (MEK)-based dual inhibitor in the treatment of cancers.

Mitogen-activated extracellular signal-regulated kinase 1 and 2 (MEK1/2) are the critical components of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) signaling pathway which is one of the well-characterized kinase cascades regulating cell proliferation, differentiation, growth, metabolism, survival and mobility both in normal and cancer cells. The aberrant activation of MAPK/ERK1/2 pathway is a hallmark of numerous human cancers, therefore targeting the components of this pathway to inhibit its dysregulation is a promising strategy for cancer treatment. Enormous efforts have been done in the development of MEK1/2 inhibitors and encouraging advancements have been made, including four inhibitors approved for clinical use. However, due to the multifactorial property of cancer and rapidly arising drug resistance, the clinical efficacy of these MEK1/2 inhibitors as monotherapy are far from ideal. Several alternative strategies have been developed to improve the limited clinical efficacy, including the dual inhibitor which is a single drug molecule able to simultaneously inhibit two targets. In this review, we first introduced the activation and function of the MAPK/ERK1/2 components and discussed the advantages of MEK1/2-based dual inhibitors compared with the single inhibitors and combination therapy in the treatment of cancers. Then, we overviewed the MEK1/2-based dual inhibitors for the treatment of cancers and highlighted the theoretical basis of concurrent inhibition of MEK1/2 and other targets for development of these dual inhibitors. Besides, the status and results of these dual inhibitors in both preclinical and clinical studies were also the focus of this review.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy.

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients' CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.

TRIF Regulates BIC/miR-155 via the ERK Signaling Pathway to Control the ox-LDL-Induced Macrophage Inflammatory Response.

Toll/IL-1R-domain-containing adaptor-inducing IFN-ß (TRIF) is an important adaptor for TLR3- and TLR4-mediated inflammatory signaling pathways. Recent studies have shown that TRIF plays a key role in vessel inflammation and atherosclerosis; however, the precise mechanisms are unclear. We investigated the mechanisms of the TRIF-regulated inflammatory response in RAW264.7 macrophages under oxidized low-density lipoprotein (ox-LDL) stimulation. Our data show that ox-LDL induces TRIF, miR-155, and BIC expression, activates the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways, and elevates the levels of IL-6 and TNF-α in RAW264.7 cells. Knockdown of TRIF using TRIF siRNA suppressed BIC, miR-155, IL-6, and TNF-α expression and inhibited the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways. Inhibition of ERK1/2 signaling also suppressed BIC and miR-155 expression. These findings suggest that TRIF plays an important role in regulating the ox-LDL-induced macrophage inflammatory response and that TRIF modulates the expression of BIC/miR-155 and the downstream SOCS1-STAT3-NF-κB signaling pathway via ERK1/2. Therefore, TRIF might be a novel therapeutic target for atherosclerosis.

Activation of PKB/Akt and p44/42 by mechanical stretch utilizes desmosomal structures and the keratin filament.

BACKGROUND: Mechanical stress is an ubiquitous challenge of human cells with fundamental impact on cell physiology. Previous studies have shown that stretching promotes signalling cascades involved in proliferation and tissue enlargement. OBJECTIVE: The present study is dedicated to learn more about cellular structures contributing to perception and signal transmission of cell stretch. In particular, we hypothesized that desmosmal contacts and the adjacent keratin filament build an intercellular matrix providing information about the mechanical load. METHODS: Epidermal cells with different keratin equipment were seeded on flexible silicon dishes and stretched. As read out parameter the activation of PKB/Akt and p44/42 was monitored by Western blotting. Likewise desomosomal contacts were manipulated by depletion or addition of calcium. Moreover, desmoglein 3 and desmocollin 3 were blocked by either specific antibodies or siRNA. RESULTS: It was found that the omission of calcium from the medium, a necessary cofactor for desmosomal cadherins, inhibited stretch mediated activation of PKB/Akt and p44/42. The relevance of desmosomes in this context was further substantiated by experiments using a desmoglein 3 blocking antibody (AK23) and siRNA against desmocollin 3. Moreover, disruption of the keratin filament by sodium orthovanadate also abrogates PKB/Akt and p44/42 activation in response to stretch. Likewise, KEB-7 keratinocytes harbouring a mutation in the keratin 14 gene and genetically modified keratinocytes devoid of any keratin show an altered signalling after stretch indicating the relevance of the keratin filament in this context. CONCLUSION: Besides their important role in cell architecture our results identify desmosomes and keratins as mechanosensing structures.

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii.

BACKGROUND: Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. RESULTS: Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CONCLUSION: CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.

Instructive role of M-CSF on commitment of bipotent myeloid cells involves ERK-dependent positive and negative signaling.

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.

Inhibition of Protein Kinases AKT and ERK1/2 Reduce the Carotid Body Chemoreceptor Response to Hypoxia in Adult Rats.

The carotid body is the main mammalian oxygen-sensing organ regulating ventilation. Despite the carotid body is subjected of extensive anatomical and functional studies, little is yet known about the molecular pathways signaling the neurotransmission and neuromodulation of the chemoreflex activity. As kinases are molecules widely involved in motioning a broad number of neural processes, here we hypothesized that pathways of protein kinase B (AKT) and extracellular signal-regulated kinases ½ (ERK1/2) are implicated in the carotid body response to hypoxia. This hypothesis was tested using the in-vitro carotid body/carotid sinus nerve preparation ("en bloc") from Sprague Dawley adult rats. Preparations were incubated for 60 min in tyrode perfusion solution (control) or containing 1 µM of LY294002 (AKT inhibitor), or 1 µM of UO-126 (ERK1/2 inhibitor). The carotid sinus nerve chemoreceptor discharge rate was recorded under baseline (perfusion solution bubbled with 5 % CO(2) balanced in O(2)) and hypoxic (perfusion solution bubbled with 5 % CO(2) balanced in N(2)) conditions. Compared to control, both inhibitors significantly decreased the normoxic and hypoxic carotid body chemoreceptor activity. LY294002- reduced carotid sinus nerve discharge rate in hypoxia by about 20 %, while UO-126 reduces the hypoxic response by 45 %. We concluded that both AKT and ERK1/2 pathways are crucial for the carotid body intracellular signaling process in response to hypoxia.

Across-Species Transfer of Protection by Remote Ischemic Preconditioning With Species-Specific Myocardial Signal Transduction by Reperfusion Injury Salvage Kinase and Survival Activating Factor Enhancement Pathways.

RATIONALE: Reduction of myocardial infarct size by remote ischemic preconditioning (RIPC), that is, cycles of ischemia/reperfusion in an organ remote from the heart before sustained myocardial ischemia/reperfusion, was confirmed in all species so far, including humans. OBJECTIVE: To identify myocardial signal transduction of cardioprotection by RIPC. METHODS AND RESULTS: Anesthetized pigs were subjected to RIPC (4×5/5 minutes hindlimb ischemia/reperfusion) or placebo (PLA) before 60/180 minutes coronary occlusion/reperfusion. Phosphorylation of protein kinase B, extracellular signal-regulated kinase 1/2 (reperfusion injury salvage kinase [RISK] pathway), and signal transducer and activator of transcription 3 (survival activating factor enhancement [SAFE] pathway) in the area at risk was determined by Western blot. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Plasma sampled after RIPC or PLA, respectively, was transferred to isolated bioassay rat hearts subjected to 30/120 minutes global ischemia/reperfusion. RIPC reduced infarct size in pigs to 16±11% versus 43±11% in PLA (% area at risk; mean±SD; P<0.05). RIPC increased the phosphorylation of signal transducer and activator of transcription 3 at early reperfusion, and AG490 abolished the protection, whereas RISK blockade did not. Signal transducer and activator of transcription 5 phosphorylation was decreased at early reperfusion in both RIPC and PLA. In isolated rat hearts, pig plasma taken after RIPC reduced infarct size (25±5% of ventricular mass versus 38±5% in PLA; P<0.05) and activated both RISK and SAFE. RISK or SAFE blockade abrogated this protection. CONCLUSIONS: Cardioprotection by RIPC in pigs causally involves activation of signal transducer and activator of transcription 3 but not of RISK. Protection can be transferred with plasma from pigs to isolated rat hearts where activation of both RISK and SAFE is causally involved. The myocardial signal transduction of RIPC is the same as that of ischemic postconditioning.

Involvement of PI3K, MAPK ERK1/2 and p38 in Functional Stimulation of Mesenchymal Progenitor Cells by Alkaloid Songorine.

We studied the role of intracellular signaling molecules PI3K, МАРK ERK1/2, and р38 in stimulation of realization of the growth potential of mesenchymal progenitor cells by alkaloid songorine in vitro. Inhibitors of PI3K, ERK1/2 and р38 canceled the increase in proliferative activity of progenitor cells, the blockers of ERK1/2 and р38 reduced the intensity of progenitor cell differentiation.

Brief Report: Autocrine Cytokine-Mediated Deficiency of TRAIL-Induced Monocyte Apoptosis in Rheumatoid Arthritis.

OBJECTIVE: Dysregulated apoptosis of monocytes is a pathogenic feature of rheumatoid arthritis (RA). The aim of this study was to investigate the role of TRAIL and TRAIL-induced apoptosis in patients with RA. METHODS: Cell surface expression and serum concentrations of TRAIL were determined in 63 patients with RA, and TRAIL-induced monocyte apoptosis was quantified. Surface expression of TRAILR-1, TRAILR-2, TRAILR-3, TRAILR-4, CXCR1, and CXCR2 was determined, and intracellular signal transduction was investigated. In 8 patients with RA, clinical and laboratory parameters of disease activity were investigated longitudinally, before and after initiation of treatment with tumor necrosis factor (TNF) inhibitors. RESULTS: Serum concentrations of both TRAIL and interleukin-8 (IL-8) were increased in patients with RA, while cell surface expression of the TRAIL receptors TRAILR-1, TRAILR-2, TRAILR-3, and TRAILR-4 was diminished. TRAIL-induced monocyte apoptosis was significantly decreased in RA due to increased TRAIL-induced IL-8 secretion by RA monocytes. The combined effect of TRAIL and IL-8 on monocytes resulted in activation of antiapoptotic pathways, including p42/44 MAPK and p38. Susceptibility to TRAIL-induced apoptosis was restored in RA monocytes after 3 months of TNF inhibition. CONCLUSION: In RA, circulating monocytes with the potential to produce proinflammatory cytokines appear to have defects in several pathways of apoptosis induction, among which is a deficiency in TRAIL-induced apoptosis. Although this resistance to apoptosis might contribute to perpetuation of the disease, it remains to be determined whether specific induction of apoptosis could be therapeutically beneficial.

Negative regulation of the LKB1/AMPK pathway by ERK in human acute myeloid leukemia cells.

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia.

Compression induces Ephrin-A2 in PDL fibroblasts via c-fos.

Ephrin-A2-EphA2 and ephrin-B2-EphB4 interactions have been implicated in the regulation of bone remodeling. We previously demonstrated a potential role for members of the Eph-ephrin family of receptor tyrosine kinases for bone remodeling during orthodontic tooth movement: compression-dependent upregulation of ephrin-A2 in fibroblasts of the periodontal ligament (PDL) attenuated osteogenesis in osteoblasts of the alveolar bone. However, factors affecting the regulation of ephrin-A2 expression upon the application of compressive forces remained unclear. Here, we report a mechano-dependent pathway of ephrin-A2 induction in PDL fibroblasts (PDLFs) involving extracellular signal-regulated kinases (ERK) 1/2 and c-fos. PDLF subjected to compressive forces (30.3 g/cm(2)) upregulated c-fos and ephrin-A2 mRNA and protein expression and displayed increased ERK1/2 phosphorylation. Inhibition of the MAP kinase kinase (MEK)/ERK1/2 pathway using the specific MEK inhibitor U0126 significantly reduced ephrin-A2 messenger RNA upregulation upon compression. Silencing of c-fos using a small interfering RNA approach led to a significant inhibition of ephrin-A2 induction upon the application of compressive forces. Interestingly, ephrin-A2 stimulation of PDLF induced c-fos expression and led also to the induction of ephrin-A2 expression. Using a reporter gene construct in murine 3T3 cells, we found that ephrin-A2 was able to stimulate serum response element (SRE)-dependent luciferase activity. As the regulation of c-fos is SRE dependent, ephrin-A2 might induce c-fos via SRE activation. Taken together, we provide evidence for an ERK1/2- and c-fos-dependent regulation of ephrin-A2 in compressed PDLF and suggest a novel pathway for ephrin-A2 induction emanating from ephrin-A2 itself. We showed previously that ephrin-A2 at compression sites might contribute to tooth movement by inhibiting osteogenic differentiation. The regulatory pathway of ephrin-A2 induction during tooth movement identified in this study might be accessible for pharmacological interventions.

Polarization of the vacuolar adenosine triphosphatase delineates a transition to high-grade pancreatic intraepithelial neoplasm lesions.

OBJECTIVES: A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/ß-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/ß-catenin signaling. METHODS: We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1a(Cre/+); LSL-Kras(G12D) mice. Wnt/ß-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated ß-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin. RESULTS: In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/ß-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand. CONCLUSIONS: The v-ATPase regulates Wnt/ß-catenin and EGF receptor signaling in PanINs.

Stretch-activated signaling is modulated by stretch magnitude and contraction.

INTRODUCTION: Stretch therapy is commonly utilized to prevent shortening maladaptation of skeletal muscle. Stretch in combination with isometric contraction prevents shortening, but the signaling mechanisms are not understood. METHODS: Using a soleus tenotomy + stretch rat model, the phosphorylation-activation of mechanosensitive kinases (Akt, p70(S6K), p38 MAPK, and ERK1/2) were measured for various stretch magnitudes, set relative to optimal soleus length (Lo). RESULTS: The kinases were not activated by passive stretch until it exceeded the normal physiological range. Stretch + isometric contraction resulted in relatively strong phosphorylation, even at short lengths. CONCLUSIONS: Whereas passive stretch results in kinase phosphorylation only during extreme lengthening, isometric contraction generated pronounced phosphorylation of kinases at Lo and Lo + 25%, indicating stimulation of pathways that lead to the preservation or increase of muscle length. Understanding the effects of passive and active stretch with respect to Lo and contraction is essential for predicting therapeutic outcomes and influencing optimal muscle length.

Genistein suppresses the isoproterenol-treated H9c2 cardiomyoblast cell apoptosis associated with P-38, Erk1/2, JNK, and NFκB signaling protein activation.

Heart disease (HD) is associated with estrogen and therefore gender and menopausal status. In addition, clinical evidence shows that increased serum norepinephrine is found in patients with HD. Therefore, this study aimed to investigate the cardio-protective effect of genistein, a selective estrogen receptor modulator (SERM) from soy bean extract, in H9c2 cardiomyoblast cells treated with isoproterenol (ISO), a norepinephrine analog. In this in vitro model, image data and results from western blotting shown that ISO treatment was capable of inducing cellular apoptosis, especially the mitochondrial dependent pathway. Treatment of genistein could suppress the expression of mitochondrial pro-apoptotic proteins including Bad, caspase-8, caspase-9, and caspase-3 in H9c2 treated with ISO. By contrast, several survival proteins were expressed in H9c2 treated with genistein, such as phosphor (p)-Akt, p-Bad, and p-Erk1/2. Furthermore, we confirmed that the protective role of genistein was partially mediated through the expression of Erk1/2, Akt, and NF κ B proteins by adding several pathway inhibitors. These in vitro data suggest that genistein may be a safe and natural SERM alternative to hormone therapy in cardio-protection.

Basic fibroblast growth factor upregulates expression of growth hormone gene through extracellular signal-regulated kinase 1/2 in GH4 cells.

Basic fibroblast growth factor (bFGF) is reported to not only play multifunctions in pituitary differentiation and tumor formation, stimulating cell differentiation or proliferation, also stimulate pituitary to secret prolactin, growth hormone (GH) and thyroid stimulating hormone, although obvious effect on growth hormone only responded to high-dose bFGF. Since it is well documented that both bFGF and GH correlate closely to tumorigenesis, development and metastasis, so it is necessary to reveal the relationship between the cytokines. In the present report we investigated the effect of bFGF on transcription level of GH gene in GH4 rat pituitary cells as well as the regulatory mechanism with real-time reverse transcription polymerase chain reaction and western blotting. We observed a significant expressional increase of GH gene in GH4 cells stimulated by bFGF, meanwhile phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was also found in the cells. Further investigation unveiled that PD98059, a specific inhibitor of ERK1/2 signaling pathway markedly impaired the transcriptional increment of GH gene induced by bFGF in the pituitary cells, which indicates that bFGF upregulates GH gene expression through ERK1/2 signaling pathway in GH4 cells. Results may be helpful to elaborate roles of the two cytokines on tumor (Fig. 3, Ref. 25).