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1.
Nat Genet ; 55(8): 1288-1300, 2023 08.
Article in English | MEDLINE | ID: mdl-37500727

ABSTRACT

Genome-wide association studies have identified many loci associated with hair and skin disease, but identification of causal variants requires deciphering of gene-regulatory networks in relevant cell types. We generated matched single-cell chromatin profiles and transcriptomes from scalp tissue from healthy controls and patients with alopecia areata, identifying diverse cell types of the hair follicle niche. By interrogating these datasets at multiple levels of cellular resolution, we infer 50-100% more enhancer-gene links than previous approaches and show that aggregate enhancer accessibility for highly regulated genes predicts expression. We use these gene-regulatory maps to prioritize cell types, genes and causal variants implicated in the pathobiology of androgenetic alopecia (AGA), eczema and other complex traits. AGA genome-wide association studies signals are enriched in dermal papilla regulatory regions, supporting the role of these cells as drivers of AGA pathogenesis. Finally, we train machine learning models to nominate single-nucleotide polymorphisms that affect gene expression through disruption of transcription factor binding, predicting candidate functional single-nucleotide polymorphism for AGA and eczema.


Subject(s)
Alopecia Areata , Eczema , Humans , Scalp/metabolism , Chromatin/genetics , Chromatin/metabolism , Genome-Wide Association Study , Transcriptome/genetics , Alopecia Areata/metabolism , Hair Follicle/metabolism , Eczema/genetics , Eczema/metabolism
3.
Dev Cell ; 43(2): 227-239.e5, 2017 10 23.
Article in English | MEDLINE | ID: mdl-28943242

ABSTRACT

Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.


Subject(s)
Casein Kinase Ialpha/metabolism , Cell Self Renewal/physiology , Epidermal Cells , Keratinocytes/cytology , Protein-Arginine N-Methyltransferases/physiology , Stem Cells/cytology , Animals , Cell Differentiation , Cells, Cultured , Epidermis/metabolism , Humans , Infant, Newborn , Keratinocytes/metabolism , Mice , Mice, Knockout , Phosphorylation , Stem Cells/metabolism
4.
Nat Genet ; 47(9): 1056-60, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26258847

ABSTRACT

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.


Subject(s)
Mycosis Fungoides/genetics , Receptors, Tumor Necrosis Factor, Type II/genetics , Sezary Syndrome/genetics , Skin Neoplasms/genetics , Antineoplastic Agents/pharmacology , Base Sequence , Bortezomib/pharmacology , CD28 Antigens/genetics , CTLA-4 Antigen/genetics , Cell Line, Tumor , DNA Mutational Analysis , Drug Resistance, Neoplasm , Gene Expression , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Genome-Wide Association Study , Genomics , Humans , Oncogene Proteins, Fusion/genetics , Point Mutation , Receptors, Tumor Necrosis Factor, Type II/metabolism , Signal Transduction
5.
Dev Cell ; 32(6): 693-706, 2015 Mar 23.
Article in English | MEDLINE | ID: mdl-25805135

ABSTRACT

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.


Subject(s)
Cell Differentiation/genetics , Epidermal Cells , MafB Transcription Factor/genetics , Proto-Oncogene Proteins c-maf/genetics , RNA, Long Noncoding/genetics , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Transfer Techniques , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Mice , Mice, Inbred NOD , Mice, SCID , Organogenesis/genetics , Positive Regulatory Domain I-Binding Factor 1 , RNA Interference , RNA, Small Interfering , Repressor Proteins/biosynthesis , Transcription Factors/biosynthesis , Tumor Suppressor Proteins
6.
Methods Mol Biol ; 1220: 239-55, 2015.
Article in English | MEDLINE | ID: mdl-25388255

ABSTRACT

Mast cells are key effector and immunoregulatory cells in IgE-associated immune responses, including allergic disorders. IgE antibodies bind to the high-affinity IgE receptor, FcεRI, expressed on the surface of mast cells; antigen-induced cross-linking of FcεRI-bound IgE molecules activates the mast cell to release an array of proinflammatory and immunomodulatory mediators. Because mast cells often respond to very low levels of antigen in vivo, the level of FcεRI expressed on the surface of these cells is an important factor in determining the responsiveness of these cells to antigen. FcεRI surface expression is regulated by a number of processes, including FcεRI stabilization, FcεRI recycling, and antigen-induced internalization. Although members of the Rab family of small GTPases and the ubiquitin ligase, Cbl, have recently emerged as major regulators of many of the membrane trafficking events that govern FcεRI expression levels, the mechanisms and intracellular pathways that regulate FcεRI trafficking remain poorly defined. This chapter outlines a number of flow cytometry-based assays that can be used to investigate cell surface FcεRI expression and dynamics (stabilization, recycling, and internalization) on bone marrow-derived mast cells (BMCMCs), the most commonly used model system for studying mast cells in vitro. Given the importance of FcεRI levels to mast cell responsiveness and function, the characterization of FcεRI expression and dynamics on different mast cell populations is critical when trying to compare IgE-dependent processes between different mast cell populations.


Subject(s)
Gene Expression Regulation , Mast Cells/metabolism , Receptors, IgE/metabolism , Animals , Bone Marrow Cells/cytology , Flow Cytometry , Immunoglobulin E/metabolism , Mast Cells/cytology , Protein Stability , Protein Transport
7.
Nat Genet ; 46(10): 1060-2, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25194279

ABSTRACT

Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.


Subject(s)
Carcinoma, Squamous Cell/genetics , Cell Cycle Proteins/genetics , Kinetochores/metabolism , Microtubule-Associated Proteins/genetics , Point Mutation , Skin Neoplasms/genetics , Aneuploidy , Animals , Carcinogenesis/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cells, Cultured , Female , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/transplantation , Mice, Inbred NOD , Mice, SCID , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Transfection , Transplantation, Heterologous
8.
J Allergy Clin Immunol ; 132(4): 922-32.e1-16, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23810240

ABSTRACT

BACKGROUND: Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. OBJECTIVES: We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. METHODS: C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. RESULTS: Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. CONCLUSIONS: Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro.


Subject(s)
Anaphylaxis/therapy , Antigens/immunology , Desensitization, Immunologic/methods , Immunoglobulin E/immunology , Mast Cells/immunology , 2,4-Dinitrophenol/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Antibody Specificity , Antigens/administration & dosage , Antigens/metabolism , Humans , Hypersensitivity , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Time Factors
9.
PLoS One ; 7(9): e46303, 2012.
Article in English | MEDLINE | ID: mdl-23029469

ABSTRACT

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH(i)), typically due to activation of Na(+)/H(+) exchange (NHE). In the systemic vasculature, ET-1 increases pH(i), Na(+)/H(+) exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH(i) and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH(i) and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3-5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH(i) and NHE activity induced by increasing concentrations of ET-1 (10(-10) to 10(-8) M). We found that application of exogenous ET-1 increased pH(i) and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH(i) or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH(i).


Subject(s)
Endothelin-1/pharmacology , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism , rho-Associated Kinases/metabolism , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Fluoresceins , Fluorescent Dyes , Hydrogen-Ion Concentration , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/enzymology , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Pulmonary Artery/cytology , Pulmonary Artery/drug effects , Pulmonary Artery/enzymology , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , rho-Associated Kinases/antagonists & inhibitors
10.
Am J Pathol ; 181(3): 875-86, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22901752

ABSTRACT

Mouse mast cell protease 4 (mMCP-4), the mouse counterpart of human mast cell chymase, is thought to have proinflammatory effects in innate or adaptive immune responses associated with mast cell activation. However, human chymase can degrade the proinflammatory cytokine TNF, a mediator that can be produced by mast cells and many other cell types. We found that mMCP-4 can reduce levels of mouse mast cell-derived TNF in vitro through degradation of transmembrane and soluble TNF. We assessed the effects of interactions between mMCP-4 and TNF in vivo by analyzing the features of a classic model of polymicrobial sepsis, cecal ligation and puncture (CLP), in C57BL/6J-mMCP-4-deficient mice versus C57BL/6J wild-type mice, and in C57BL/6J-Kit(W-sh/W-sh) mice containing adoptively transferred mast cells that were either wild type or lacked mMCP-4, TNF, or both mediators. The mMCP-4-deficient mice exhibited increased levels of intraperitoneal TNF, higher numbers of peritoneal neutrophils, and increased acute kidney injury after CLP, and also had significantly higher mortality after this procedure. Our findings support the conclusion that mMCP-4 can enhance survival after CLP at least in part by limiting detrimental effects of TNF, and suggest that mast cell chymase may represent an important negative regulator of TNF in vivo.


Subject(s)
Chymases/metabolism , Inflammation/enzymology , Proteolysis , Sepsis/enzymology , Sepsis/pathology , Serine Endopeptidases/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cell Membrane/metabolism , Disease Models, Animal , Down-Regulation , Inflammation/blood , Inflammation/complications , Inflammation/pathology , Leukocyte Count , Ligation , Mast Cells/enzymology , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , Neutrophils/pathology , Sepsis/blood , Sepsis/complications , Serine Endopeptidases/deficiency , Solubility , Survival Analysis
11.
Blood ; 112(10): 4148-57, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18698003

ABSTRACT

Rab5 is a small GTPase that regulates early endocytic events and is activated by RabGEF1/Rabex-5. Rabaptin-5, a Rab5 interacting protein, was identified as a protein critical for potentiating RabGEF1/Rabex-5's activation of Rab5. Using Rabaptin-5 shRNA knockdown, we show that Rabaptin-5 is dispensable for Rab5-dependent processes in intact mast cells, including high affinity IgE receptor (FcepsilonRI) internalization and endosome fusion. However, Rabaptin-5 deficiency markedly diminished expression of FcepsilonRI and beta1 integrin on the mast cell surface by diminishing receptor surface stability. This in turn reduced the ability of mast cells to bind IgE and significantly diminished both mast cell sensitivity to antigen (Ag)-induced mediator release and Ag-induced mast cell adhesion and migration. These findings show that, although dispensable for canonical Rab5 processes in mast cells, Rabaptin-5 importantly contributes to mast cell IgE-dependent immunologic function by enhancing mast cell receptor surface stability.


Subject(s)
Gene Expression Regulation/physiology , Mast Cells/metabolism , Receptors, IgE/biosynthesis , Vesicular Transport Proteins/metabolism , Animals , Antigens/immunology , Antigens/metabolism , Endocytosis/genetics , Endocytosis/immunology , Endosomes/genetics , Endosomes/immunology , Endosomes/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/immunology , Guanine Nucleotide Exchange Factors/metabolism , Integrin beta1/genetics , Integrin beta1/immunology , Integrin beta1/metabolism , Mast Cells/cytology , Mast Cells/immunology , Mice , RNA, Small Interfering/genetics , Receptors, IgE/genetics , Receptors, IgE/immunology , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/immunology , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/immunology , rab5 GTP-Binding Proteins/metabolism
12.
Nat Med ; 14(4): 392-8, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18376408

ABSTRACT

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.


Subject(s)
Mast Cells/metabolism , Neurotensin/metabolism , Sepsis/metabolism , Animals , Cell Degranulation , Disease Models, Animal , Female , Humans , Hypotension/metabolism , Hypotension/prevention & control , Male , Mast Cells/physiology , Metalloendopeptidases/metabolism , Mice , Mice, Congenic , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Neurotensin/antagonists & inhibitors , Neurotensin/blood , Neurotensin/deficiency , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Neurotensin/metabolism , Sepsis/blood
13.
Blood ; 109(12): 5308-17, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17341663

ABSTRACT

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.


Subject(s)
Gene Expression Regulation , Guanine Nucleotide Exchange Factors/physiology , Mast Cells/physiology , Receptors, IgE/genetics , Bone Marrow Cells , Cell Degranulation , Cells, Cultured , Endocytosis , Guanine Nucleotide Exchange Factors/chemistry , Humans , Interleukin-6/biosynthesis , Mast Cells/cytology , Receptors, IgE/deficiency
14.
Proc Natl Acad Sci U S A ; 103(8): 2659-64, 2006 Feb 21.
Article in English | MEDLINE | ID: mdl-16533754

ABSTRACT

We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.


Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Mast Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Factor/metabolism , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Adhesion/genetics , Cell Movement/genetics , Cell Proliferation , Extracellular Signal-Regulated MAP Kinases/metabolism , Genetic Vectors , Guanine Nucleotide Exchange Factors/genetics , Interleukin-6/metabolism , Lentivirus/genetics , MAP Kinase Kinase 4/metabolism , Mast Cells/drug effects , Mice , Mice, Knockout , Phosphorylation , Signal Transduction , Stem Cell Factor/pharmacology , Transfection , ras Proteins/metabolism
15.
Am J Physiol Lung Cell Mol Physiol ; 289(5): L867-74, 2005 Nov.
Article in English | MEDLINE | ID: mdl-15964895

ABSTRACT

Chronic hypoxia (CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pHi) mediated by activation of Na+/H+ exchange (NHE); however, the effect of CH on PASMC pHi homeostasis is unknown. Thus we measured basal pHi and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na+-dependent recovery from NH4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pHi and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pHi, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation.


Subject(s)
Hypoxia/metabolism , Muscle, Smooth, Vascular/metabolism , Pulmonary Artery/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Base Sequence , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Chronic Disease , Gene Expression , Hydrogen-Ion Concentration , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Hypoxia/genetics , Hypoxia/pathology , In Vitro Techniques , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/pathology , Pulmonary Artery/pathology , Sodium-Hydrogen Exchanger 1 , Sodium-Hydrogen Exchangers/genetics
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