Novel role for the N-terminus domain of the a2 isoform of vacuolar ATPase in interleukin-1β production

doi:10.1016/j.humimm.2007.02.010

Human Immunology

Volume 68, Issue 6, June 2007, Pages 469-477

https://doi.org/10.1016/j.humimm.2007.02.010 Get rights and content

Summary

Interleukin–1β (IL-1β) is a mediator cytokine that is released by macrophages and epithelial cells in pregnancy and tumorigenesis before antigen recognition. a2V-ATPase is a protein expressed during pregnancy and tumorigenesis and has a novel role in immune regulation. It is expressed as a 70 kDa molecule in intracellular vesicles. Upon cell stimulation it migrates to the surface followed by the cleavage of a 20 kDa portion (a2 N-terminus domain, a2NTD). This study aimed to determine whether a2NTD could induce IL-1β production in immune cells. Peripheral blood mononuclear cells (PMBC) were stimulated with a2NTD and analyzed for cytokine gene expression by gene arrays. Supernatants were analyzed for IL-1β by enzyme-linked immunosorbent assay, and cells were analyzed for intracellular expression of IL-1α, IL-1β, and TNF-α by flow cytometry. When PBMC were cultured with a2NTD, there was a 2.5-fold increase in IL1A and IL1B gene expression and no induction of TNF gene expression. There was a 72-fold increase in IL-1β in supernatants of PBMC cultured with a2NTD. Finally, there was a 204-fold increase in intracellular expression of IL-1β in monocytes incubated with a2NTD. These results indicate a regulatory role for a2NTD in IL-1 cytokine production and suggest a unique role for this molecule in inflammation.

Introduction

The interleukin (IL)–1 family is a key mediator of inflammation, and its role in the initiation of inflammation has been well documented. The IL-1 family consists of nine members, and among them are IL-1α and IL-1β [1]. The substance IL-1α is synthesized as an active pro-IL-1α form; its cleavage by calpain in the cytoplasm leads to mature IL-1α, which is bound to the cell membrane. In a similar fashion, IL-1β is synthesized as a precursor and is secreted as a 17 kDa active protein after cleavage by IL-1β converting enzyme (caspase-1) [2]. Therefore regulation of IL-1β secretion is a key function in inflammatory conditions, and a role of the vacuolar-type proton pump or V-ATPase in this regulation is proposed in the current study.

V-ATPase is a multisubunit enzyme that regulates the acidification of intracellular compartments as well as the extracellular environment of eukaryotic cells. V-ATPase is present in intracellular vesicles (microvesicles, lysosomes, endosomes) and is important in a variety of cellular processes including membrane fusion, endocytosis, and intracellular transport [3]. V-ATPase is also expressed in the plasma membrane of specialized cells such as macrophages, renal intercalated cells, osteoclasts, and some tumor cells, and regulates the pH of the extracellular environment [4].

The general structure of V-ATPase comprises of one peripheral V₁ domain (which is responsible for ATP hydrolysis) and one membrane-integrated V₀ domain (which translocates H⁺ across the membrane) [5]. The V₀ domain is composed of five subunits: a, c, c′, c″, and d. Subunit a is a membrane-spanning protein with nine transmembrane regions, an intra-lumen C-terminus domain, and a cytoplasmic N-terminus domain [6]. There are four different isoforms (a1, a2, a3, and a4) of the a subunit that show diverse tissue distribution. The a2 isoform (a2V-ATPase; gene name, ATP6V0A2) [7] is particularly important for the immune system because it is expressed mostly in macrophages, as well as other lymphoid subsets.

We cloned and purified a2V-ATPase (formerly known as Regeneration and Tolerance Factor [RTF]) and found that it is a 70-kDa protein [8]. a2V-ATPase has a role in pregnancy [9] and tumorigenesis [10]. Upon cell activation, a2V-ATPase translocates to the plasma membrane, where it resides as a 50-kDa molecule. The remaining hydrophilic external 20 kDa N-terminus domain is proteolytically cleaved and is found in the extracellular environment [9], [11].

There are several lines of evidence suggesting a novel role of a2V-ATPase in immune cell activation [12], [13], [14]. Recent studies suggest a role for a2V-ATPase in the inflammatory response. It was shown that a2V-ATPase could modulate IL-1β secretion from a human macrophage cell line [15]. Furthermore, it has been found that the N-terminus domain of the a2V-ATPase caused an increase in IL-10 production and a considerable reduction in IL-2 production from stimulated T cells [9]. In addition, the N-terminus domain of the a2V-ATPase is expressed in cycling and pregnant endometrium and IL-1β increases its mRNA expression [16].

Taken together, these data suggested a role for a2V-ATPase in cytokine production and prompted us to define further its role in inflammation by detecting the effect of the N-terminus domain of the a2V-ATPase upon an array of cytokines generally and IL-1 specifically. The aim of this research was to explain the unique role of the N-terminus domain of the a2V-ATPase in IL-1β secretion from immune cells, which may provide a control point for inflammation.

Section snippets

Confocal microscopy

Peripheral blood mononuclear cells (PBMC) were incubated in a 96-well plate in RPMI-1640 medium supplemented with 10% fetal bovine serum, in the absence or presence of 2 μg/ml phytohemmaglutinin (PHA) (Sigma, St. Louis, MO). After 72 hours, both PHA-stimulated and nonstimulated PBMC were washed twice in phosphate-buffered saline, permeabilized with 70% ice cold ethanol for 15 minutes, and reacted with 7 μl fluorescein isothiocyanate (FITC)–conjugated anti–a2V-ATPase mAb (2C1 clone) for 15

Translocation of a2V-ATPase to the cell membrane upon stimulation

Because the a2V-ATPase is known to be a vesicular protein, we wanted to determine whether, under stimulatory conditions, a2V-ATPase would translocate and come to the cell surface. To show the localization of a2V-ATPase in activated versus nonactivated cells, PBMC were stimulated with PHA, permeabilized and detected for the expression of a2V-ATPase, after staining with FITC-conjugated anti-a2V-ATPase mAb (clone 2C1, which binds to a peptide in the transmembrane domain). As Figure 1A shows,

Discussion

Because of the first identification of a2NTD as a major protein in the maintenance of pregnancy, its early effects were targeted to Th2 cytokines, such as IL-10 [9]. The current research was conducted to investigate the role of a2NTD in cytokine secretion. We found that a2NTD can regulate IL-1α and IL-1β cytokine production in monocytes. There was an increase in IL1A and IL1B gene expression in PBMC treated with a2NTD, whereas there was no induction of the TNF gene. There was also an increase

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Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its ‘a2’ isoform (V-ATPase-V0a2) is a component of functional plasma–membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the ‘a’ subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs.
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Furthermore, a2NTD causes an increase in IL-10, a modest reduction of IFN-γ and a considerable reduction in IL-2 production from stimulated T cells (Lee et al., 2001). In addition, a2NTD regulates the IL-1α and IL-1β cytokine production at the gene and cellular level (Ntrivalas et al., 2007a). There was an increase in IL-1α and IL-1β gene expression in peripheral blood mononuclear cells (PBMC) treated with a2NTD.
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¹: Richard Derks is currently in the Department of Surgery, University of Wisconsin, Madison, WI.

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Novel role for the N-terminus domain of the a2 isoform of vacuolar ATPase in interleukin-1β production

Summary

Introduction

Section snippets

Confocal microscopy

Translocation of a2V-ATPase to the cell membrane upon stimulation

Discussion

Am J Clin Nutr

J Biol Chem

Biochim Biophys Acta

Mol Cell

Hum Immunol

Hum Immunol

J Biol Chem

J Biol Chem

Blood

Immunity

Biol Cell

Targeting IL-1 in inflammatory disease: new opportunities for therapeutic intervention

Nat Rev Drug Discov

The vacuolar (H+)-ATPases—nature’s most versatile proton pumps

Nat Rev Mol Cell Biol

Different roles of proteolipids and 70-kDa subunits of V-ATPase in growth and death of cultured human cells

Genes Cells