MEK-dependent Manner Research Articles

A molecular basis for neurofibroma-associated skeletal manifestations in NF1

PurposePlexiform neurofibromas (pNF) develop in children with neurofibromatosis type 1 (NF1) and can be associated with several skeletal comorbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. MethodsWe used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used dual-energy X-ray absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. ResultsWe detected increased nonmineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. ConclusionResults indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.

To investigate whether ERK/MNK/eIF4E contributes chemoresistance in ovarian cancer. The phosphorylated levels of Erk, Mnk, and eIF4E were systematically analyzed in ovarian cancer patients before and after chemotherapy, and ovarian cancer cells exposed to short- and long-term chemo-agent treatment. The roles of Erk/Mnk/eIF4E were investigated using pharmacological and genetic approaches. Increased phosphorylation levels of ERK, Mnk1, and eIF4E were observed in ovarian cancer cell exposed to chemotherapeutic agents, and paclitaxel-resistant SK-OV-3-r cells, and is a common response of ovarian cancer patients undergoing chemotherapy. MEK inhibitor U0126 inhibits basal and chemodrug-induced phosphorylation of ERK as well as Mnk1 and eIF4E, suggesting that Mnk1/eIF4E are the downstream signaling of ERK pathway and chemotherapy agents activate ERK/MNK/eIF4E in a MEK-dependent manner. eIF4E overexpression promotes ovarian cancer cell growth without affecting migration. In addition, ovarian cancer cells with eIF4E overexpression are more resistant to chemotherapeutic agents in aspect of growth inhibition and apoptosis induction compared to control cells. In contrast, eIF4E depletion augments chemotherapeutic agents' effect in ovarian cancer cells. These demonstrate that eIF4E play roles in growth and chemoresistance in ovarian cancer. MEK inhibitor U0126 also significantly enhances chemotherapeutic agents' inhibitory effects. Our work shows that ERK/Mnk/eIF4E activation is critically involved in ovarian cancer chemoresistance and inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Oncogenic KRAS signalling promotes the Wnt/β-catenin pathway through LRP6 in colorectal cancer.

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in β-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced β-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting β-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/β-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/β-catenin signalings during oncogenesis.

Abstract A05: The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer

Abstract The RAS/RAF/MEK/ERK pathway is suspected to be strongly implicated in malignant transformation of colorectal epithelial cells since KRAS and BRAF genes are mutated in up to 60% of human colorectal cancers (CRCs). Despite intensive efforts made in the understanding of KRAS and BRAF functions in colorectal carcinogenesis, little is known about the oncogenic regulation of ERK activity in CRC cells, especially by the ERK-specific nuclear DUSP phosphatases. We have recently demonstrated in CRC cells that ERK1/2 activity is confined to the cytoplasm because of a pervanadate-dependent phosphatase activity. Accordingly, DUSP4 mRNA was found to be highly expressed, in a MEK-dependent manner, in CRC cells. Thus, DUSP4 and other nuclear DUSPs may function as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during colorectal carcinogenesis. We thus speculate that the modification of nuclear ERK activity might strongly impact on the oncogenic properties of CRC. Methods: Expression of DUSPs was analyzed by PCR in CRC cells and qPCR in human colorectal tumors from different stages. To elucidate the role of nuclear DUSPs, RNA interference was used to specifically silence their expression in human HT29 CRC cell line, a cell line exhibiting BRafV600E mutation. Inducible expression of a DUSP4 inactivated mutant (DUSP4C284S) was done in a model of IEC-6 cells that express an inducible form of BRaf oncogene (BRafV600E:ER). Results: 1- DUSP2, DUSP4 and DUSP5 mRNAs were found to be highly expressed, in a MEK-dependent manner, in CRC cells. 2- DUSP4 and DUSP5 mRNA levels were markedly increased in colorectal tumors in comparison to healthy matched adjacent tissues. 3- Silencing of DUSP 2, 4 or 5 in HT29 cells increased ERK phosphorylation levels and markedly reduced colony formation in soft agar. 4- Furthermore, expression of DUSP4C284S in BRafV600E-expressing IEC-6 cells increased ERK phosphorylation into the nucleus and markedly reduced colony formation in soft agar. Conclusion: We are currently searching by which mechanisms sustained nuclear ERK activity reduces colorectal tumor cell growth. Therefore, targeting nuclear DUSP activity could become a new strategy to treat CRC. Citation Format: Sebastien Cagnol, Nathalie Rivard. The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A05. doi: 10.1158/1557-3125.RASONC14-A05

ERK1/2 Blockade Prevents Epithelial–Mesenchymal Transition in Lung Cancer Cells and Promotes Their Sensitivity to EGFR Inhibition

Overcoming cellular mechanisms of de novo and acquired resistance to drug therapy remains a central challenge in the clinical management of many cancers, including non-small cell lung cancer (NSCLC). Although much work has linked the epithelial-mesenchymal transition (EMT) in cancer cells to the emergence of drug resistance, it is less clear where tractable routes may exist to reverse or inhibit EMT as a strategy for drug sensitization. Here, we demonstrate that extracellular signal-regulated kinase (ERK) 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) signaling plays a key role in directing the mesenchymal character of NSCLC cells and that blocking ERK signaling is sufficient to heighten therapeutic responses to EGF receptor (EGFR) inhibitors. MEK1/2 (MAPKK1/2) inhibition promoted an epithelial phenotype in NSCLC cells, preventing induction of EMT by exogenous TGF-β. Moreover, in cells exhibiting de novo or acquired resistance to the EGFR inhibitor gefitinib, MEK inhibition enhanced the sensitivity to gefitinib and slowed cell migration. These effects only occurred, however, if MEK was inhibited for a period sufficient to trigger changes in EMT marker expression. Consistent with these findings, changes in EMT phenotypes and markers were also induced by the expression of mutant KRAS in a MEK-dependent manner. Our results suggest that prolonged exposure to MEK or ERK inhibitors may not only restrain EMT but also overcome naïve or acquired resistance of NSCLC to EGFR-targeted therapy in the clinic.

PDGF-induced PI3K-mediated signaling enhances the TGF-β-induced osteogenic differentiation of human mesenchymal stem cells in a TGF-β-activated MEK-dependent manner

Transforming growth factor-β (TGF-β) is a critical regulator of osteogenic differentiation and the platelet-derived growth factor (PDGF) is a chemoattractant or mitogen of osteogenic mesenchymal cells. However, the combined effects of these regulators on the osteogenic differentiation of mesenchymal cells remains unknown. In this study, we investigated the effects of TGF-β and/or PDGF on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The TGF-β-induced osteogenic differentiation of UE7T-13 cells, a bone marrow-derived hMSC line, was markedly enhanced by PDGF, although PDGF alone did not induce differentiation. TGF-β induced extracellular signal-regulated kinase (ERK) phosphorylation and PDGF induced Akt phosphorylation. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, suppressed the osteogenic differentiation induced by TGF-β alone. Moreover, U0126 completely suppressed the osteogenic differentiation synergistically induced by TGF-β and PDGF, whereas the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002, only partially suppressed this effect. These results suggest that the enhancement of TGF-β-induced osteogenic differentiation by PDGF-induced PI3K/Akt-mediated signaling depends on TGF-β-induced MEK activity. Thus, PDGF positively modulates the TGF-β-induced osteogenic differentiation of hMSCs through synergistic crosstalk between MEK- and PI3K/Akt-mediated signaling.

Activation of MAPK Pathways due to DUSP4 Loss Promotes Cancer Stem Cell-like Phenotypes in Basal-like Breast Cancer

Basal-like breast cancer (BLBC) is an aggressive disease that lacks a clinically approved targeted therapy. Traditional chemotherapy is effective in BLBC, but it spares the cancer stem cell (CSC)-like population, which is likely to contribute to cancer recurrence after the initial treatment. Dual specificity phosphatase-4 (DUSP4) is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway that is deficient in highly aggressive BLBCs treated with chemotherapy, leading to aberrant MAPK activation and resistance to taxane-induced apoptosis. Herein, we investigated how DUSP4 regulates the MAP-ERK kinase (MEK) and c-jun-NH2-kinase (JNK) pathways in modifying CSC-like behavior. DUSP4 loss increased mammosphere formation and the expression of the CSC-promoting cytokines interleukin (IL)-6 and IL-8. These effects were caused in part by loss of control of the MEK and JNK pathways and involved downstream activation of the ETS-1 and c-JUN transcription factors. Enforced expression of DUSP4 reduced the CD44(+)/CD24(-) population in multiple BLBC cell lines in a MEK-dependent manner, limiting tumor formation of claudin-low SUM159PT cells in mice. Our findings support the evaluation of MEK and JNK pathway inhibitors as therapeutic agents in BLBC to eliminate the CSC population.

Abstract 291: ERK1/2 pathway activation controls epithelial-mesenchymal transition in NSCLC cells.

Abstract Understanding and overcoming the cellular mechanisms that underlie de novo and acquired therapy resistance remains a challenge in treating many cancers including non-small cell lung cancer (NSCLC). Despite the prevalence of epidermal growth factor receptor (EGFR) over-activation in NSCLC and other cancers, most patients are resistant to EGFR inhibitors. A number of studies have linked mesenchymal cellular phenotypes and epithelial-mesenchymal transition (EMT) with resistance to chemotherapeutics and EGFR inhibitors across cancer types. Here, we demonstrate in NSCLC cells that MAPK/ERK signaling plays a key role in determining mesenchymal cellular characteristics and response to the EGFR inhibitor gefitinib. In cultured NSCLC cell lines, pharmacological inhibition of MEK pushed cells toward a more epithelial phenotype and prevented EMT induction by exogenous transforming growth factor beta. In addition, chronic MEK inhibition synergistically enhanced cellular sensitivity to gefitinib in a reversible manner and slowed cell migration in wound healing assays in NSCLC cell lines with both de novo and acquired resistance to gefitinib. Importantly, our results demonstrate that the observed cell phenotypic changes occur on a time scale corresponding to that with which epithelial and mesenchymal marker expression change in response to ERK inhibition. This time scale is much larger than that for changes in ERK activity itself, reflecting the need for downstream ERK-dependent processes to affect phenotypic changes. Changes in the same cellular phenotypes and associated changes in epithelial and mesenchymal markers also occurred as a result of KRAS mutant expression in a MEK-dependent manner. Overall, this study draws a strong connection between ERK pathway activation and mesenchymal characteristics in NSCLC and suggests potential new opportunities for expanding the efficacy of currently available EGFR inhibitors. Citation Format: Janine M. Buonato, Matthew J. Lazzara. ERK1/2 pathway activation controls epithelial-mesenchymal transition in NSCLC cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2013-291

Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100ng/mL) stimulated mitogenesis together with the raise in T202/Y204P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y705P-STAT3) and MEK (T202/Y204P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S473) and GSK-3β (S9) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S9P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y216P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.

Oncogenic KRAS and BRAF activation of the MEK/ERK signaling pathway promotes expression of dual-specificity phosphatase 4 (DUSP4/MKP2) resulting in nuclear ERK1/2 inhibition

Gain-of-function mutations in KRAS and BRAF genes are found in up to 50% of colorectal cancers. These mutations result in the activation of the BRAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. Upon activation, ERK1/2 translocate from the cytoplasm to the nucleus. This process has been shown to be required for the induction of many cellular responses, although the molecular mechanisms regulating ERK nuclear function, especially under oncogenic stimulation, remain to be explored. Herein, we examined the spatiotemporal regulation of ERK1/2 activity upon oncogenic activation of KRAS(G12V) and BRAF(V600E) in normal intestinal epithelial crypt cells (IECs). Results demonstrate that expression of these oncogenes markedly stimulated ERK1/2 activities and morphologically transformed IECs. Importantly however, ERK phosphorylation was not observed in the nucleus, but restricted to the cytoplasm of KRAS(G12V)- and BRAF(V600E)-transformed IECs. The absence of nuclear ERK phosphorylation was due to a vanadate-sensitive phosphatase activity. Nuclear ERK dephosphorylation was found to be tightly correlated with the rapid expression of DUSP4 phosphatase induced in an MEK-dependent manner. In addition, MEK-dependent phosphorylation of T361, T363, S390 and S395 residues highly stabilized DUSP4 protein. Finally, in human colorectal cancer cells, ERK1/2 activities were also confined to the cytoplasm and treatment with pervanadate reactivated ERK1/2 in the nucleus. Accordingly, DUSP4 mRNAs were found to be highly expressed, in an MEK-dependent manner, in all colorectal cancer cells analyzed. These findings indicate that DUSP4 functions as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during intestinal tumorigenesis.

NOX2-derived reactive oxygen species are crucial for CD29-induced pro-survival signalling in cardiomyocytes

The highly expressed cell adhesion receptor CD29 (β(1)-integrin) is essential for cardiomyocyte growth and survival, and its loss of function causes severe heart disease. However, CD29-induced signalling in cardiomyocytes is ill defined and may involve reactive oxygen species (ROS). A decisive source of cardiac ROS is the abundant NADPH oxidase (NOX) isoform NOX2. Because understanding of NOX-derived ROS in the heart is still poor, we sought to test the role of ROS and NOX in CD29-induced survival signalling in cardiomyocytes. In neonatal rat ventricular myocytes, CD29 activation induced intracellular ROS formation (oxidative burst) as assessed by flow cytometry using the redox-sensitive fluorescent dye dichlorodihydrofluorescein diacetate. This burst was inhibited by apocynin and diphenylene iodonium. Further, activation of CD29 enhanced NOX activity (lucigenin-enhanced chemiluminescence) and activated the MEK/ERK and PI3K/Akt survival pathways. CD29 also induced phosphorylation of the inhibitory Ser9 on the pro-apoptotic kinase glycogen synthase kinase-3β in a PI3K/Akt- and MEK-dependent manner, and improved cardiomyocyte viability under conditions of oxidative stress. The ROS scavenger MnTMPyP or adenoviral co-overexpression of the antioxidant enzymes superoxide dismutase and catalase inhibited CD29-induced pro-survival signalling. Further, CD29-induced protective pathways were lost in mouse cardiomyocytes deficient for NOX2 or functional p47(phox), a regulatory subunit of NOX. p47(phox)-dependent, NOX2-derived ROS are mandatory for CD29-induced pro-survival signalling in cardiomyocytes. These findings go in line with a growing body of evidence suggesting that ROS can be beneficial to the cell and support a crucial role for NOX2-derived ROS in cell survival in the heart.

Phospholipase D1, but Not D2, Regulates Protein Secretion Via Rho/ROCK in a Ras/Raf-Independent, MEK-Dependent Manner in Rat Lacrimal Gland

A prior study showed that cholinergic agonists activate phospholipase D (PLD). The purpose of this study was to determine whether cholinergic agonists use the PLD pathway to alter protein secretion and to identify the molecular signaling components of this pathway in rat lacrimal gland acini. Rat lacrimal gland acini were isolated by collagenase digestion. Presence and localization of PLD1 and -2 were determined by immunofluorescence and Western blot experiments. Acini were incubated with adenoviruses overnight or the inhibitors 1-butanol, Y-27632, or C3 exotoxin before stimulation with the cholinergic agonist carbachol (Cch, 10(-4) M) for 5 minutes. Western blot analysis was performed for 20 minutes, and protein secretion was measured spectrophotometrically. Activation of ERK, MEK, Pyk2, Ras, and Raf was determined by Western blot analysis. 1-Butanol increased Cch-stimulated protein secretion and decreased ERK activity. Incubation with catalytically inactive PLD1, but not catalytically inactive mutant PLD2 adenovirus, also increased Cch-stimulated protein secretion and decreased ERK activity. Inhibition of Rho with C3 exotoxin and a dominant negative Rho adenovirus and inhibition of ROCK with Y-27632 inhibited Cch-stimulated PLD1 activity, increased protein secretion, and decreased ERK activity. The association of PLD1 and ROCK increased with Cch stimulation, as determined by immunoprecipitation. PMA-stimulated ERK activity was also inhibited by 1-butanol. 1-Butanol had no effect on Cch-stimulated Pyk2, Ras, and Raf activity, but decreased MEK activity. Cholinergic agonists activate PLD1 through Rho and ROCK, which in turn activate MEK and ERK, which attenuate protein secretion in freshly isolated epithelial cells.

Tyr Phosphatase-Mediated P-ERK Inhibition Suppresses Senescence in EIA + v-raf Transformed Cells, Which, Paradoxically, Are Apoptosis-Protected in a MEK-Dependent Manner

Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H(2)O(2)-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

MTOR Activation, Lymphangiogenesis, and Estrogen-Mediated Cell Survival: The “Perfect Storm” of Pro-Metastatic Factors in LAM Pathogenesis

Research interest in lymphangioleiomyomatosis (LAM) has grown dramatically in the past decade, particularly among cancer biologists. There are at least two reasons for this: first, the discovery in the year 2000 that LAM cells carry TSC2 gene mutations, linking LAM with cellular pathways including the PI3K/Akt/mTOR axis, and allowing the Tuberous Sclerosis Complex (TSC)-regulated pathways that are believed to underlie LAM pathogenesis to be studied in cells, yeast, Drosophila, and mice. A second reason for the rising interest in LAM is the discovery that LAM cells can travel to the lung, including repopulating a donor lung after lung transplantation, despite the fact that LAM cells are histologically benign. This "benign metastasis" underpinning suggests that elucidating LAM pathogenesis will unlock a set of fundamental mechanisms that underlie metastatic potential in the context of a cell that has not yet undergone malignant transformation. Here, we will outline the data supporting the metastatic model of LAM, consider the biochemical and cellular mechanisms that may enable LAM cells to metastasize, including both cell autonomous and non-cell autonomous factors, and highlight a mouse model in which estrogen promotes the metastasis and survival of TSC2-deficient cells in a MEK-dependent manner. We propose a multistep model of LAM cell metastasis that highlights multiple opportunities for therapeutic intervention. Taken together, the metastatic behavior of LAM cells and the involvement of tumor-related signaling pathways lead to optimism that cancer-related paradigms for diagnosis, staging, and therapy will lead to therapeutic breakthroughs for women living with LAM.

AlphaB-crystallin is mutant B-RAF regulated and contributes to cyclin D1 turnover in melanocytic cells.

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

TGF-Beta Auto-Induction and Connective Tissue Growth Factor Expression in Human Renal Tubule Epithelial Cells Requires N-Ras

Background/Aims: Transforming growth factor (TGF) β is strongly implicated in the progression of renal fibrosis. TGFβ1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFβ production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFβ1 inhibition of proliferation, TGFβ auto-induction and TGFβ1-induced CTGF expression in HKC human renal tubule epithelial cells. Methods and Results: TGFβ1 (0–25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5–10 min of incubation, TGFβ1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFβ3 caused an increase in extracellular TGFβ1, which was significantly reduced in the presence of PD 98059. TGFβ1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFβ1 secretion and TGFβ1-induced cellular and secreted CTGF. Conclusion: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFβ1 auto-induction and TGFβ1-induced CTGF expression in human renal tubule epithelial cells.

Cytoskeletal reorganisation, 1α,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation

Bone tissue is especially receptive to physical stimulation and agents with the capacity to mimic the signalling incurred via mechanical loading on osteoblasts may find an application in a bone regenerative setting. Recently this laboratory revealed that the major serum lipid, lysophosphatidic acid (LPA), co-operated with 1α,25-dihydroxy vitamin D3 (D3) in stimulating human osteoblast maturation. Actin stress fiber accrual in LPA treated osteoblasts would have generated peripheral tension which in turn may have heightened the maturation response of these cells to D3. To test this hypothesis we examined if other agents known to trigger stress fiber accumulation co-operated with D3 in stimulating human osteoblast maturation. Colchicine, nocodazole and LPA all co-operated with D3 to promote MG63 maturation in a MEK dependent manner. In contrast, calpeptin, a direct activator of Rho kinase and stress fiber accumulation did not act with D3 to secure MG63 differentiation. Herein we describe how the signalling elicited via microtubule disruption cooperates with D3 in the development of mature osteoblasts.

Negative and Positive Regulation of MAPK Phosphatase 3 Controls Platelet-derived Growth Factor-induced Erk Activation

MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.