A role for mitogen kinase kinase 3 in pulmonary inflammation validated from a proteomic approach

https://doi.org/10.1016/j.pupt.2014.01.006Get rights and content

Abstract

Proteomics is a powerful tool to ascertain which proteins are differentially expressed in the context of disease. We have used this approach on inflammatory cells obtained from patients with asthma to ascertain whether novel drugs targets could be illuminated and to investigate the role of any such target in a range of in vitro and in vivo models of inflammation.

A proteomic study was undertaken using peripheral blood mononuclear cells from mild asthmatic subjects compared with healthy subjects. The analysis revealed an increased expression of the intracellular kinase, mitogen activated protein kinase (MKK3), and the function of this protein was investigated further in preclinical models of inflammation using MKK3 knockout mice.

We describe a 3.65 fold increase in the expression of MKK3 in CD8+ T lymphocytes obtained from subjects with asthma compared with healthy subjects using a proteomic approach which we have confirmed in CD8+, but not in CD4+ T lymphocytes or human bronchial epithelial cells from asthmatic patients using a Western blot technique. In wild type mice, bacterial lipopolysaccharide (LPS) caused a significant increase in MKK3 expression and significantly reduced airway neutrophilia in MKK3−/− mice (median, 25, 75% percentile; wild/LPS; 5.3 (0.7–9.9) × 105 cells/mL vs MKK3−/−/LPS; 0 (0–1.9) × 105 cells/mL, P < 0.05). In contrast, eosinophilia in sensitized wild type mice challenged with allergen (0.5 (0.16–0.65) × 105 cells/mL) was significantly increased in MKK3−/− mice (2.2 (0.9–3.5) × 105 cells/mL, P < 0.05).

Our results suggest that asthma is associated with MKK3 over-expression in CD8+ cells. We have also demonstrated that MKK3 may be critical for airway neutrophilia, but not eosinophilia, suggesting that this may be a target worthy of further consideration in the context of diseases associated with neutrophil activation such as severe asthma and COPD.

Introduction

Asthma is an inflammatory disorder of the airways that is associated with an increase in airway hyperresponsiveness, leading to recurrent episodes of airflow obstruction. The incidence of asthma continues to increase in most countries, currently affecting over 300 million people worldwide [1], [2]. Despite the availability of numerous therapies, a significant proportion of asthma sufferers are unable to control their disease through the use of the currently available drugs. These deficiencies in current therapy have led to considerable research effort to identify novel drug targets, particularly to help identify novel anti-inflammatory drugs to replace glucocorticosteroids or to be used in the treatment of patients with severe asthma who may be resistant to glucocorticosteroids [3]. High throughput proteomics has become an important research tool for the assessment of clinical samples in oncology [4], [5], neurology [6], cardiovascular diseases [7], diabetes [8] and in inflammation and asthma [9]. Such studies have uncovered the potential role of numerous genes and proteins in these various conditions, although enormous challenges remain to define the functional relevance of many of the proteins identified in such studies.

Mitogen activated protein kinase kinase 3 (MKK3) is a member of the p38 MAPK signalling pathway, which plays a pivotal role in inflammatory processes [10]. MKK3 is expressed in various cells and has been shown to cause a selective increase in p38 MAP kinase activity [11]. MKK3 is known to be activated following numerous stress stimuli in a wide range of cell types [12], [13] and various studies have demonstrated that MKK3 is an important factor in the development of non-allergic inflammatory processes and Th1 responses, both in vivo and in vitro [14], [15], [16], although less is known about the role of MKK3 in the regulation of lung inflammation characteristic of diseases such as asthma and COPD.

The current study describes the identification of upregulated MKK3 expression in peripheral blood CD8+ lymphocytes isolated from subjects with asthma compared to healthy control subjects and the characterization of this protein in the regulation of non-allergic and allergic lung inflammation.

Section snippets

Study subjects and sample collection

Peripheral blood (200 mL) was obtained by antecubital venepuncture into lithium heparin tubes from 22 healthy non-smoking subjects and 18 non-smoking subjects with well characterized mild asthma (Table 1). The latter were allowed treatment with a beta2-agonist prn, but refrained from taking any medication 24 h prior to venepuncture. This study was approved by the Ethics committee of King's College London. This was conducted according to the declaration of Helsinki principles and informed

Comparison of healthy and asthmatic lymphoid cell proteomes

Peripheral blood samples were obtained from 22 healthy and 18 asthmatics subjects. The demographics of the volunteers are summarized in Table 1. Lysates from purified lymphocytes were separated by SDS-PAGE and the protein inventories of each sample were identified using mass spectrometry. 300 proteins were identified in the study and differences among the two groups were evaluated by the presence or absence of protein detection and by differences in their quantity. Among the proteins identified

Discussion

We have identified the upregulation of MKK3 in CD8+ lymphocytes isolated from patients with well characterized atopic asthma compared to healthy subjects. However, our in vivo study in MKK3 knockout mice has revealed the central importance this protein plays in the development of neutrophilic, but not eosinophilic lung inflammation.

As anticipated, LPS-challenged mice showed a significant neutrophilia in the lung that was associated with an increase in MKK3 expression in lung and spleen tissue.

Disclosure statement

The authors have no competing interests to declare.

Authors' contribution

CP, KBB and CR conceived and designed the study. TH, YRV, DS designed and performed the human in vitro and animal in vivo studies. CS performed Western blot experiments presented in Fig. 1f. MM provided the MKK3 breeding colony. BC, FW responsible for recruiting patients for proteomic study. CR and KBB performed the proteomic analysis. TH, YRV, DS and CP drafted the manuscript. All authors contributed to the final draft of the manuscript.

Acknowledgements

We thank Professor Richard Flavell, Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA and Roger Davies Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA, USA, for the kind gift of the MKK3−/− mouse line to Professor Mike Marber, King's College London. We also thank Bayer for funding part of this research.

References (50)

  • M. Masoli et al.

    The global burden of asthma: executive summary of the GINA Dissemination Committee report

    Allergy

    (2004)
  • J. Rees

    Asthma control in adults

    BMJ

    (2006)
  • W.C. Cho et al.

    Oncoproteomics: current trends and future perspectives

    Expert Rev Proteomics

    (2007)
  • Y. Lin et al.

    The current state of proteomics in GI oncology

    Dig Dis Sci

    (2009)
  • M.H. Maurer

    Proteomics of brain extracellular fluid (ECF) and cerebrospinal fluid (CSF)

    Mass Spectrom Rev

    (2010)
  • C. Suss et al.

    Proteomic profiling of beta-cells using a classical approach – two-dimensional gel electrophoresis

    Exp Clin Endocrinol Diabetes

    (2008)
  • L.M. Benson et al.

    Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

    J Sep Sci

    (2009)
  • L.H. Pettus et al.

    Small molecule p38 MAP kinase inhibitors for the treatment of inflammatory diseases: novel structures and developments during 2006-2008

    Curr Top Med Chem

    (2008)
  • J. Raingeaud et al.

    MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway

    Mol Cell Biol

    (1996)
  • A. Cuenda et al.

    Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells

    EMBO J

    (1996)
  • A. Cuenda et al.

    Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38)

    EMBO J

    (1997)
  • T. Inoue et al.

    Regulation of p38 MAPK by MAPK kinases 3 and 6 in fibroblast-like synoviocytes

    J Immunol

    (2005)
  • T. Inoue et al.

    Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis

    Proc Natl Acad Sci U S A

    (2006)
  • H.T. Lu et al.

    Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice

    EMBO J

    (1999)
  • Y.H. Son et al.

    Roles of MAPK and NF-kappaB in interleukin-6 induction by lipopolysaccharide in vascular smooth muscle cells

    J Cardiovasc Pharmacol

    (2008)

    • Cited by (8)

    • Discovery of the first chemical tools to regulate MKK3-mediated MYC activation in cancer

      2021, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      It was originally discovered as the activator of its only known substrate, p38 that regulates inflammation and apoptosis.22 Accordingly, MKK3 functions have been associated solely with the regulation of the p38 signaling.23–28 We have demonstrated a novel role of MKK3 as an adaptor protein for MYC.29–31

    • Pharmacological validation of targets regulating CD14 during macrophage differentiation

      2020, EBioMedicine
      Citation Excerpt :

      Mitogen-activated protein kinase 3 (MAP2K3, MKK3) is a kinase that activates p38 MAPK in response to LPS (34) and a variety of other cell stressors such as cigarette smoke (35), Caerulin (36), TGF-β (37) and TNF (38). Therefore, this pathway regulates inflammatory pathology in models of sepsis (39), lung diseases (40–42), myocardial infarction (43) and diabetic nephropathy (44). Based on our data, either monocyte/macrophage differentiation or regulation of CD14 could account for the effects of MAP2K3 in these conditions, however, it may also have other roles in different cell types.

    • A novel MKK gene (AjMKK3/6) in the sea cucumber Apostichopus japonicus: Identification, characterization and its response to pathogenic challenge

      2017, Fish and Shellfish Immunology
      Citation Excerpt :

      Similar expression profiles were observed in L. vannamei [29]. On the one hand, as the dual specificity kinase in MAPK pathways, MKK3/MKK6 can activate the p38-MAPK cascades via specific amino acid residue phosphorylation [38,39]. As well, many previous studies reported that MAPK pathways could control the response of multi-cellular organisms to environmental stress through regulating many physiological processes, such as cell proliferation, cell differentiation, cell apoptosis, etc. [2,31,40].

    • High expression of MKK3 is associated with worse clinical outcomes in African American breast cancer patients

      2020, Journal of Translational Medicine

    • CD8<sup>+</sup> Tc2 cells: Underappreciated contributors to severe asthma

      2019, European Respiratory Review

    • Spectrum of T-lymphocyte activities regulating allergic lung inflammation

      2017, Immunological Reviews
    View all citing articles on Scopus
    1

    Present address: Axikin Pharmaceuticals, Inc., 10835 Road to the Cure, San Diego, CA 92121, USA.

    2

    Present address: Oxford BioTherapeutics, 94A Milton Park, Abingdon, Oxon OX14 4RY, UK.

    View full text
    Copyright © 2014 Elsevier Ltd. All rights reserved.