Elsevier

Life Sciences

Volume 92, Issues 17–19, 20 May 2013, Pages 890-895
Life Sciences

PI3K and Notch signal pathways coordinately regulate the activation and proliferation of T lymphocytes in asthma

https://doi.org/10.1016/j.lfs.2013.03.005Get rights and content

Abstract

Aims

In the present study, we determined whether Phosphoinositide 3-kinase (PI3K) and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 which were key molecules in controlling cell cycling from CD4+ T lymphocyte in animal model of asthma.

Main methods

Ovalbumin (OVA) sensitized murine model of asthma was used to investigate the expression of cyclin D1, cyclin A, and p27kip1 by splenic CD4+ T lymphocytes. We further observed the effect of specific inhibitor of PI3K(LY294002) and specific inhibitor of Notch(DAPT) on the proliferation of such CD4+ T lymphocytes.

Key findings

We found that the expression of cyclinD1 and cyclinA was upregulated at both protein and mRNA levels in asthma group while p27kip1 was down-regulated. Both LY294002 and DAPT inhibit the proliferation of CD4+ T lymphocytes in a time- and dose-dependent manner. Furthermore, LY294002 and DAPT have additive effect in down-regulation of cyclinD1 and upregulation of p27kip1. An upregulation of cyclinA, although not statistically significant, was also observed.

Significance

These data suggested that PI3K signal pathway and Notch signal pathway may coordinately regulate the cell proliferation and differentiation processes through up-regulating cyclinD1 and down-regulating p27kip1 of CD4+ T lymphocytes.

Introduction

Asthma is a chronic airway inflammatory disease increasing in prevalence. T lymphocytes play an important role in the pathogenesis of asthma such as airway inflammation and remodeling through the release of cytokines (Choy et al., 2011, Lloyd and Hessel, 2010, Levine and Wenzel, 2010). Phosphoinositide 3-kinase (PI3K) is one of the key enzymes in cell signaling and is widely distributed in the lung tissue. PI3K is crucial in T cell signaling (Alcazar et al., 2007, Okkenhaug et al., 2006, Deane et al., 2007). Notch signal pathway is also involved in T cell differentiation and participates in T cell activation (Laky and Fowlkes, 2008, Osborne and Minter, 2007, Dallman and Smith, 2005). However, those cell signal pathways are not completely independent of each other, i.e. cross talks take place between different signaling pathways. We hypothesize that signaling pathways between PI3K and Notch coordinate in regulating the proliferation and activation of T lymphocytes in asthma.

Cyclins, cyclin-dependent kinase (CDKs), and cyclin-dependent kinase inhibitor (CKIs) are the main players in the regulation of cell cycling to ensure the normal progress of cell cycling from G1 to G2 (CDKs) and from S phase to M phase (CKIs) (Sherr, 1996, Foijer and Riele, 2006). Cyclin D1 is a key regulator of cells from G1 to S phase, while cyclinA activity is essential for entry from G2 to M phase (Foijer and Riele, 2006, Blagosklonny and Pardee, 2002, Wang et al., 2009). In addition, the CDKs inhibitor p27kip1 also regulates the progression of the cells from G1 to S phase. Overexpression of p27kip1 results in inhibition of the progression of cells from G1 to S phase that halts the proliferation of cells (Boudová et al., 2003). In the present study, we determined whether PI3K and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 of CD4+ T lymphocyte in an animal model of asthma.

Section snippets

Mice

Male BALB/c mice, 4 to 6 weeks old, weighing 18–22 g at the beginning of the experiments, were purchased from Shanghai Slac Laboratory Animal Center (Shanghai, China). Throughout the entire study, they were kept in a pathogen-free environment in the animal center of Wenzhou Medical College. All animals were acclimated for at least 1 week prior to the experiments. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the

Purity and viability of CD4+ T cells

After magnetic bead isolation, the purity of CD4+ T cells was determined by flow cytometry using FITC conjugated anti-CD4 as (90.04 ± 5.18) % (Fig. 1). Cell viability, as determined by trypan blue exclusion, was (94.82 ± 3.21) %.

The expression of cyclinA, cyclinD1 and p27kip1

Surface expression of cyclinA, cyclinD1 and p27kip1 from splenic CD4+ T cells was determined by flow cytometry. As shown in Fig. 2A and B, cyclinA and cyclinD1 levels were significantly higher in the asthma group [(28.0 ± 3.5)% and (14.9 ± 3.4)%] than that in the control group

Discussion

The present study discovered previously unrecognized roles of Notch and PI3K signaling in the proliferation of CD4+ T lymphocytes. First, we provided evidence that in an OVA-induced murine model of asthma, the expression of cyclinA and cyclinD1 is increased in CD4+ T lymphocytes, and p27kip1 expression is decreased. Second, we demonstrated that the proliferation of such CD4+ T lymphocytes is regulated by both Notch and PI3K signaling pathways.

Based on the structure and substrate specificity,

Author contributions

CCL and WXZ conceived and designed the experiments. YN, WXZ and LC performed the experiments. WXZ and YN analyzed the data. XHC, HZ, BBL and YFL participated in the statistical analyses. WXZ wrote the paper. All authors read and approved the final manuscript.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by National Natural Science Foundation of China (no. 81100015) and Zhejiang Provincial Natural Science Foundation (no. Y2090327). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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