Research ArticleAldosterone up-regulates MMP-9 and MMP-9/NGAL expression in human neutrophils through p38, ERK1/2 and PI3K pathways
Introduction
Aldosterone is now recognized as a hormone implicated not only in homeostasis and blood pressure regulation but also in inflammation under physiopathological conditions. Results obtained from clinical data and animal models have demonstrated direct hypertrophic, thrombotic and fibrotic effects of aldosterone in organs such as heart and kidneys, and mineralocorticoid receptor (MR) antagonists diminish these harmful effects [1], [2], [3], [4]. Other data indicate that aldosterone plays an active role in vascular wall inflammation and its remodelling. This hormone promotes notably smooth muscle cell proliferation, NADPH oxidase activity, dysfunction and adhesion molecule increase in endothelial cells, leukocyte adhesion and infiltration [5], [6], [7], [8], [9]. Moreover, attenuation of atherosclerosis development by MR antagonists or aldosterone synthase inhibitors has been demonstrated [10], [11]. Accumulating data show that MR activation of immune cells, like macrophages, is also implicated in these events. Aldosterone increases monocyte/macrophage NADPH oxidase, NO and ROS production [12], [13]. Kidneys from rats infused with aldosterone present macrophage infiltration and spironolactone, a MR antagonist, counteracts this effect [14]. Specific deletion of MR from macrophages attenuates the hypertrophic and fibrotic effects, as well as the altered blood pressure normally observed in mice treated with deoxycorticosterone or l-NAME/AngII [15], [16], [17]. However, until now, neutrophils have received little attention despite the demonstration that these cells express mineralocorticoid receptors [18]. Moreover, neutrophils are the most abundant blood leukocytes and the first to arrive close to any inflamed tissue and to migrate through it. Activated neutrophils are then able to recruit other leukocytes such as monocytes and lymphocytes by chemokine and leukotriene secretion and help their diapedesis by pro-inflammatory cytokine and matrix metalloprotease (MMP) release [19]. Some chemokines such as CXCL8/IL-8, implicated in neutrophil oxidative bursts and chemotaxis, as well as pro-inflammatory cytokines like proIL-1β are cleaved by MMPs, such as MMP-9, into a biologically active form [20], [21]. Degradation of the extracellular matrix by collagenases, like MMP-2 and MMP-9, favors the entry of immune cells in altered tissue, which constitutes a prerequisite for tissue repair [22]. When activated, neutrophils are able to excrete large amounts of pro and active MMP-9 proteins. Cytokines such as IL-8, IL-1β and TNFα or the chemoattractant peptide formyl-Met-Leu-Phe are known to activate neutrophil MMP-9 release, corresponding to inflammatory or/and infectious conditions. A wide variety of other cells like astrocytes, chondrocytes, epithelial cells, fibroblasts, keratinocytes, mesangial cells, smooth muscle cells, and neurons are able also to secrete pro and active MMP-9 in response to cytokines such as IL-1β, TNFα and TGFβ [23], [24]. However, only neutrophils synthetize a protein complex termed “MMP-9/NGAL”, in which proMMP-9 is linked covalently to neutrophil gelatinase-associated lipocalin (NGAL) [25]. Approximately 20–30% of proMMP-9 is located in this complex where NGAL protects it from protease degradation [26].
Based on the above data, we hypothesized that aldosterone could regulate pro and active MMP-9 neutrophil production as well as that of the MMP-9/NGAL complex.
Herein, we show that aldosterone increases MMP-9 mRNA, proMMP-9, active MMP-9 and MMP-9/NGAL complex expression in granulocyte-differentiated HL-60 cells and in human neutrophils. This up-regulation is mediated by the activation of PI3kinase, p38 and to a lesser extent ERK1/2 pathways. Finally, we show that the MR antagonist, spironolactone, counteracts aldosterone effects on MMP-9 mRNA, proMMP-9, active MMP-9 and MMP-9/NGAL complex.
Section snippets
Cell culture and treatments
The HL-60 human leukemia cell line was cultured at 37 °C under 5% CO2 in RPMI 1640 medium containing 10% heat inactivated fetal calf serum (Eurobio, Courtaboeuf, France), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM Hepes, 2 mM l-glutamine, 1 mM sodium pyruvate (all purchased from Sigma-Aldrich) and 1× non-essential amino-acids (Gibco-BRL, Invitrogen, Baisley, UK). HL-60 cells were differentiated into the neutrophil-lineage by adding 1.3% of DMSO during three days and then 0.65% of DMSO for
Effect of aldosterone on MMP-9 mRNA levels in HL-60 and human PMN
Differentiated HL-60 cells were incubated with aldosterone 10−7, 10−8 or 10−9 M for different time periods. As shown in Fig. 1A, aldosterone upregulated MMP-9 mRNA expression in HL-60 cells in a time- and dose-dependent manner. Indeed, this increase appeared already after 1 h 30 min of incubation and peaked at 6 h. Furthermore, at 6 h, MMP-9 mRNA levels had increased 10-, 6.1- and 3.7-fold with aldosterone at 10−7, 10−8 and 10−9 M, respectively, compared to unstimulated cells. In order to verify that
Discussion
Little information exists about potential regulation of neutrophil functions by aldosterone and MR despite the fact that neutrophils are the most abundant leukocytes and are present in altered tissue from the onset to the end of inflammation/repair. Matrix metalloproteases (MMPs) play an important role during inflammation by degrading extracellular matrix components but also, in the case of MMP-9, by cleaving VEGF-A and some chemokines into biologically active forms useful, respectively, for
Source funding
This work was financially supported by the Fondation de France.
Acknowledgments
We would like thank Dr V. Latger-Cannard and M. Siest (Department of Biological Haematology, Centre Hospitalier Universitaire, Vandoeuvre-les-Nancy, France) for human healthy volunteers recruitment and for blood drawings. We are also grateful to Dr S. Grandemange (Faculty of Sciences, Vandoeuvre les Nancy, France) for performing annexin V studies.
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