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1.
Cell Rep ; 38(2): 110223, 2022 01 11.
Article in English | MEDLINE | ID: mdl-35021072

ABSTRACT

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.


Subject(s)
Lymphocyte Activation/physiology , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Alleles , Animals , B-Lymphocytes/metabolism , Female , Humans , Lymphocyte Activation/genetics , MAP Kinase Kinase 1/physiology , MAP Kinase Kinase 2/genetics , MAP Kinase Kinase 2/physiology , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Male , Mice , Mice, 129 Strain , Mitogen-Activated Protein Kinase 1/metabolism , Phosphorylation , Signal Transduction/physiology , T-Lymphocytes/metabolism
2.
Front Cell Dev Biol ; 9: 639022, 2021.
Article in English | MEDLINE | ID: mdl-34386488

ABSTRACT

Several studies have established the crucial role of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway in hematopoietic cell proliferation and differentiation. MEK1 and MEK2 phosphorylate and activate ERK1 and ERK2. However, whether MEK1 and MEK2 differentially regulate these processes is unknown. To define the function of Mek genes in the activation of the ERK pathway during hematopoiesis, we generated a mutant mouse line carrying a hematopoietic-specific deletion of the Mek1 gene function in a Mek2 null background. Inactivation of both Mek1 and Mek2 genes resulted in death shortly after birth with a severe anemia revealing the essential role of the ERK pathway in erythropoiesis. Mek1 and Mek2 functional ablation also affected lymphopoiesis and myelopoiesis. In contrast, mice that retained one functional Mek1 (1Mek1) or Mek2 (1Mek2) allele in hematopoietic cells were viable and fertile. 1Mek1 and 1Mek2 mutants showed mild signs of anemia and splenomegaly, but the half-life of their red blood cells and the response to erythropoietic stress were not altered, suggesting a certain level of Mek redundancy for sustaining functional erythropoiesis. However, subtle differences in multipotent progenitor distribution in the bone marrow were observed in 1Mek1 mice, suggesting that the two Mek genes might differentially regulate early hematopoiesis.

3.
Development ; 142(17): 2981-95, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26329601

ABSTRACT

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.


Subject(s)
Epithelium/embryology , Epithelium/metabolism , Lung/embryology , Lung/metabolism , Morphogenesis , YY1 Transcription Factor/metabolism , Animals , Apoptosis , Body Patterning , Cartilage/abnormalities , Cartilage/embryology , Cartilage/pathology , Cell Differentiation , Cell Proliferation , DEAD-box RNA Helicases/metabolism , Embryo, Mammalian/abnormalities , Embryo, Mammalian/pathology , Endoderm/embryology , Endoderm/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblast Growth Factor 10/metabolism , Hedgehog Proteins/metabolism , Humans , Lung Diseases/congenital , Lung Diseases/pathology , Mice , Mice, Transgenic , Models, Biological , Myocytes, Smooth Muscle/metabolism , Myofibroblasts/pathology , Phenotype , Pulmonary Blastoma/metabolism , Pulmonary Blastoma/pathology , Ribonuclease III/metabolism , Trachea/abnormalities , Trachea/embryology , Trachea/pathology
4.
Cell Signal ; 27(10): 2068-76, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26208884

ABSTRACT

The mitogen activated protein kinases ERK1/2 play an important role in response to toll like receptor (TLR) activation and cytokine production, including IL-10 and IL-12. Here, we examined the role of MEK1 in ERK1/2 activation in response to TLR4 agonist by using bone marrow-derived macrophages (BMDMs) from wild type (WT) and Mek1(d/d)Sox2(Cre) mice. Our data demonstrates that MEK1 is essential for ERK1/2 activation in response to LPS. Furthermore, stimulation of the TLR4 receptor of BMDMs derived from Mek1(d/d)Sox2(Cre) mice showed enhanced STAT4 phosphorylation and increased IL-12 secretion, but exhibited a significantly lower IL-10 production as compared to WT macrophages. Most interestingly, TLR ligation in the presence of recombinant IL-10 (rIL-10) or retinoic acid (RA) led to ERK1/2 activation independent of MEK1 in BMDMs derived from Mek1(d/d)Sox2(Cre) mice and led to inhibition of STAT4 and decreased IL-12 levels. Collectively, these data suggest that MEK1 is required for TLR4 mediated ERK activation and in turn regulates the production of IL-10 and IL-12. It also indicates that ERK1/2 can be activated independent of MEK1 in the presence of IL-10 and RA and this activation negatively regulates IL-12, but positively regulates IL-10 production. These findings may have significant implications for the development of drugs that modulate MEK1 activity in the treatment of inflammatory, autoimmune and proliferative diseases such as cancer.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , MAP Kinase Kinase 1/physiology , Macrophages/enzymology , Animals , Enzyme Activation , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System , Macrophages/immunology , Mice, 129 Strain , Phosphorylation , Protein Processing, Post-Translational , STAT4 Transcription Factor/metabolism , Toll-Like Receptor 4/metabolism , Tretinoin/pharmacology
5.
J Biol Chem ; 288(47): 33966-33977, 2013 Nov 22.
Article in English | MEDLINE | ID: mdl-24126911

ABSTRACT

Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Bone Marrow Cells/enzymology , Dual Specificity Phosphatase 1/biosynthesis , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , MAP Kinase Signaling System/drug effects , Macrophages/enzymology , Proto-Oncogene Proteins c-akt/metabolism , Sirolimus/pharmacology , Activating Transcription Factor 1/genetics , Activating Transcription Factor 1/metabolism , Activating Transcription Factor 2/genetics , Activating Transcription Factor 2/metabolism , Animals , Benzamides , Bone Marrow Cells/cytology , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cell Line , Dose-Response Relationship, Drug , Dual Specificity Phosphatase 1/genetics , Enzyme Activation/drug effects , Enzyme Activation/genetics , Enzyme Induction/drug effects , Enzyme Induction/genetics , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/toxicity , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 2/genetics , MAP Kinase Signaling System/genetics , Macrophages/cytology , Mice , Mice, Knockout , Morpholines/pharmacology , Nitric Oxide/genetics , Nitric Oxide/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , Proto-Oncogene Proteins c-akt/genetics , Pyrimidines , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
7.
Mol Cell Biol ; 30(22): 5348-63, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20855530

ABSTRACT

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.


Subject(s)
Branchial Region/embryology , Branchial Region/metabolism , GATA3 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Genes, myc , Proto-Oncogene Proteins c-myc/metabolism , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Branchial Region/anatomy & histology , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/physiology , GATA3 Transcription Factor/genetics , Humans , In Situ Hybridization , Introns , Limb Buds/anatomy & histology , Limb Buds/embryology , Limb Buds/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/genetics , Sequence Alignment
8.
Cancer Res ; 70(9): 3813-22, 2010 May 01.
Article in English | MEDLINE | ID: mdl-20388797

ABSTRACT

Apoptosis and senescence are cellular failsafe programs that counteract excessive mitogenic signaling observed in cancer cells. Melanoma is known for its notorious resistance to apoptotic processes; therefore, senescence, which remains poorly understood in melanomas, can be viewed as a therapeutic alternative. Microphthalmia-associated transcription factor (MITF), in which its M transcript is specifically expressed in melanocyte cells, plays a critical role in melanoma proliferation, and its specific inhibition is associated with G(0)-G(1) growth arrest. Interestingly, decreased MITF expression has been described in senescent melanocytes, and we have observed an inhibition of MITF expression in melanoma cells exposed to chemotherapeutic drugs that induce their senescence. All these observations thereby question the role of MITF in controlling senescence in melanoma cells. Here, we report that long-term depletion of MITF in melanoma cells triggers a senescence program characterized by typical morphologic and biochemical changes associated with a sustained growth arrest. Further, we show that MITF-silenced cells engage a DNA damage response (DDR) signaling pathway, leading to p53 upregulation, which is critically required for senescence entry. This study uncovers the existence of a lineage-restricted DDR/p53 signaling pathway that is inhibited by MITF to prevent senescence and favor melanoma cell proliferation.


Subject(s)
DNA Damage , Melanoma/genetics , Microphthalmia-Associated Transcription Factor/deficiency , Animals , Cell Line, Tumor , Cell Lineage/physiology , Cellular Senescence/physiology , Humans , Melanoma/metabolism , Melanoma/pathology , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Mitosis/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
9.
J Biol Chem ; 284(28): 18699-706, 2009 Jul 10.
Article in English | MEDLINE | ID: mdl-19389708

ABSTRACT

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP/metabolism , Melanosomes/metabolism , alpha-MSH/metabolism , Animals , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Humans , Hydrogen-Ion Concentration , Isoquinolines/pharmacology , Melanins/chemistry , Melanoma, Experimental , Mice , Models, Biological , Monophenol Monooxygenase/antagonists & inhibitors , Monophenol Monooxygenase/metabolism , Morpholines/pharmacology , Skin Pigmentation , Sulfonamides/pharmacology
10.
J Biol Chem ; 283(18): 12635-42, 2008 May 02.
Article in English | MEDLINE | ID: mdl-18281284

ABSTRACT

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field.


Subject(s)
Gene Expression Regulation, Neoplastic , Melanosomes/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , rab GTP-Binding Proteins/genetics , Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Base Sequence , Biological Transport , Cell Line, Tumor , E-Box Elements/genetics , Gene Silencing , Humans , Mice , Microphthalmia-Associated Transcription Factor/deficiency , Molecular Sequence Data , Myosin Type V/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Transcription, Genetic , rab GTP-Binding Proteins/metabolism , rab27 GTP-Binding Proteins
11.
J Biol Chem ; 282(19): 14140-7, 2007 May 11.
Article in English | MEDLINE | ID: mdl-17371876

ABSTRACT

The MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. In mouse melanoma cells, Met expression is increased by alphaMSH via the activation of the cAMP pathway. However, the mechanism by which cAMP regulates MET and the biological consequences of this increase were not known. In the present report, we show that alphaMSH regulates MET expression in both human melanocytes and mouse melanoma cells through a transcriptional mechanism that requires MITF. Furthermore, the adenovirus driven expression of MITF is sufficient to increase MET in melanoma cells. Functional analysis of the MET promoter allows us to identify an E-box motif conserved in both human and mouse promoter that mediates the effect of MITF. Interestingly, up-regulation of MET expression by cAMP leads to an exacerbated HGF signaling and allows HGF to protect melanocytes and melanoma cells from apoptosis. Thus, physiological stimuli or pathological events that would induce MITF expression may lead to increased MET expression thereby favoring melanoma survival. These observations strengthen the roles of MITF and MET in melanoma development.


Subject(s)
Apoptosis , Hepatocyte Growth Factor/metabolism , Melanocytes/drug effects , Melanoma, Experimental/drug therapy , Microphthalmia-Associated Transcription Factor/pharmacology , Proto-Oncogene Proteins c-met/metabolism , alpha-MSH/pharmacology , Adenoviridae/genetics , Animals , Base Sequence , Blotting, Western , Cell Nucleus/metabolism , Cells, Cultured , Chloramphenicol O-Acetyltransferase/metabolism , Electrophoretic Mobility Shift Assay , Foreskin/cytology , Foreskin/metabolism , Gene Expression Regulation , Humans , Infant, Newborn , Luciferases/metabolism , Male , Melanocytes/metabolism , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Molecular Sequence Data , Proto-Oncogene Mas , Proto-Oncogene Proteins c-met/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Transfection , Up-Regulation
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