Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

Abstract

The present study demonstrates that the oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin α are capable of generating biologically active IL-1β from its precursor pro-IL-1β. Amino-acid sequencing analysis reveals that meprin A and meprin α cleave pro-IL-1β at the His¹¹⁵-Asp¹¹⁶ bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin β site. The biological activity of the pro-IL-1β cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1β product produced by meprin β or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1β, meprin inhibitor actinonin significantly reduces levels of serum IL-1β. Meprin A and meprin α may therefore play a critical role in the production of active IL-1β during inflammation and tissue injury.

INTRODUCTION

Meprins are zinc-dependent metalloproteinases of the ‘astacin’ family that are highly expressed as oligomeric proteins at the brush-border membranes of the kidney and intestine [1–2]. Meprin A, the heterooligomeric form comprised of α and β subunits, is the predominant form expressed abundantly in the apical membranes of renal proximal tubules [2–4]. Meprin A is anchored to the brush-border membranes through the membrane-spanning domain of the β subunit [5]. Thus, meprin A containing both α and β subunits is a membrane-associated protease anchored to the brush-border membranes through the β subunit. Meprins are capable of mediating proteolysis of a wide variety of proteins in vitro. Meprins are able to cleave extracellular matrix (ECM) proteins [4, 6–8], peptide hormones, growth factors [6, 9], and cytokines [10,11].

In addition to the abundant expression of meprin A in the kidney and small intestines, the expression of meprin α and meprin β, most likely in their homooligomeric forms, has been reported in other tissues and cells. Meprin α has been shown to be expressed in the large intestines and colon [12,13] and in cell culture systems [5] including Caco-2 colon carcinoma cells [13] and organ cultures of human fetal gut that constitutively secrete meprin α in the culture supernatants. Both meprin α and meprin β are expressed in the leukocytes of inflamed lamina propria mucosae [12] and mouse mesenteric lymph nodes [14]. In the human epidermis, each meprin subunit is expressed in distinct layers of epidermis [15].

Interleukin-1 beta, a key proinflammatory cytokine produced by multiple cells including monocytes, macrophages, neutrophils, and hepatocytes, plays important roles in many acute and chronic inflammatory diseases ranging from infections to tumor invasiveness [16–19]. Our recent studies demonstrated that meprin β can proteolytically process inactive pro-IL-1β into a biologically active form [11]. It is generally accepted that IL-1β is synthesized as an inactive precursor of 32 kDa (pro-IL-1β), which is processed into a 17-kDa active form (IL-1β) by caspase-1 [20,21]. However, many reports have suggested that caspase-1-independent proteolytic processing of pro-IL-1β can also occur. Caspase-1-independent processing has been reported previously in macrophages treated with zymosan and turpentine [22], Fas-stimulated neutrophils [23], and leptin-induced release of IL-1β. Some extracellular proteases have been shown to be capable of processing pro-IL-1β [11,24]. A recent study showed that LPS-induced pro-IL-1β processing occurred in caspase-1-deficient cells [25] caspase-1-deficient mice [26].

In view of the abundant expression of meprin A and meprin α in various organs and inflammatory sites, the present study was therefore undertaken to study whether meprin A and meprin α are capable of producing biologically active IL-1β in vitro and in vivo. Furthermore, the studies examined the specific site of cleavage in the pro-IL-1β molecule by meprin A and meprin α.

MATERIALS AND METHODS

Reagents

Chemicals for cell culture were purchased from Invitrogen (Carlsbad, CA) and all other chemicals from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Antibodies against human IL-1β and against human meprin β were from Santa Cruz Biologicals (Santa Cruz, CA).

Purification of meprin A from rat kidney cortex

Meprin A was purified from the kidney cortices essentially as we described previously [4].

Expression and purification of recombinant His-tagged rat meprin α

HEK-293 cells transfected with His₆-tagged meprin α (Clone #2) were kindly provided by Judith S. Bond [27]. The cells were expanded in DMEM supplemented with 10% fetal bovine serum (FBS) and 1x antibiotic-antimycotic (Invitrogen; Carlsbad, CA). For meprin α expression, cells were grown in 150 mm dishes in serum-free DMEM/F12 until they reached 80% confluency. The medium was centrifuged to remove cells and was extensively dialyzed against 10 mM Tris/Cl pH 7.1, 0.1mM PMSF, and 0.1% Nonidet P-40, at 4° C. The dialyzed sample was loaded on an iminodiacetic acid (IDA)-Sepharose column (GE Healthcare; Piscataway, NJ) charged with Cu²⁺ in 10 mM Tris/Cl pH 7.1, 100 mM NaCl. Bound protein was eluted with 20 mM L-histidine in the same buffer. The enzyme activity was monitored by OCK⁺ activity assay [11]. Active fractions were concentrated with a Centriprep YM-30 filtration device (Millipore), and dialyzed against 20 mM Tris/Cl pH 7.5, 100 mM NaCl and stored at 4°C.

Expression, purification, and analysis of human recombinant meprin β

The meprin β expression construct, pMepβΔTM1puro3GW, was prepared and analyzed as described in our previous studies [11].

Analysis of purified meprins α, β and A

Protein concentration was measured with a Bradford dye-binding assay (BioRad; Hercules, CA) and meprin enzyme activity was determined using the fluorogenic peptide substrate H-Abz-Met-Gly-Trp-Met-Asp-Glu-Ile-Asp-Lys(Dnp)-Ser-Gly-OH (OCK⁺; Abz, 2-amino-benzoic acid; Dnp, 2,4-dinitrophenyl) (Diver Drugs, SL; Gava, Spain) as described by Bertenshaw [6]. Briefly, promeprin α or β at 1.32 nM was activated by limited proteolysis with 26.3 nM trypsin at 37° C for 30 min [27]. Trypsin was then inhibited with a two-fold excess of soybean trypsin inhibitor (SBTI) at room temperature for 15 min. The enzyme assay was started by the addition of OCK⁺ at a final concentration of 6.6 μM and the change in fluorescence (excitation at 320 nm/emission at 417 nm) was followed over 20 min at 30° C. All spectroscopic measurements were recorded in 96-well plates (Greiner Bio-One; San Francisco, CA) with a SpectramaxM5 plate reader (Molecular Devices; Sunnyvale, CA). To inhibit meprin activity, actinonin was added to a final concentration of 2.24 μM and incubated at room temperature for 15 min [11]. The protease inhibitor set V without EDTA (Calbiochem; San Diego, CA) was used to inhibit a broad-spectrum of serine and cysteine proteases.

Expression and purification of recombinant proIL-1β

Recombinant proIL-1β was expressed and purified as described in our previous studies [11].

Biological activity assay of processed proIL-1β

Pro-IL-1β (163 nM) was digested with 2.8 nM trypsin-activated meprin α, 2.4 nM meprin β, 1.7 nM meprin A, or 8 nM caspase-1 in a total volume of 100 μl and sterilized using 0.45 μm Millex-GV syringe filters (Millipore). Cell proliferation assays were performed in duplicate in three independent experiments using the helper T-cell line D10S as described [11].

Cecal Ligation Puncture (CLP)

CLP-induced acute kidney injury (AKI) was established as described by Wu et al. [28]. All animals were housed and killed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the Institutional Animal Care and Use Committee. Following the surgery as described previously [28], 1 ml of pre-warmed normal saline was given i.p., animals were placed in individual cages, and set on a warming pad. At 7 h post CLP surgery, mice were anesthetized with isoflurane and blood collected from the vena cava. Serum IL-1β levels were determined using a murine specific sandwich ELISA kit from R&D Systems.

RESULTS

Proteolytic cleavage of proIL-1β with meprin α, meprin β, and meprin A and identification of cleavage sites

Meprin A was purified from kidney cortices and recombinant meprin α and meprin β (Figure 1A) were purified from HEK cells stably transfected with meprin α and meprin β, respectively, as described in “Materials and Methods.” The purified meprins were used to examine their ability to proteolytically process pro-IL-1β. For incubation, 22 nM of meprin A or 37 nM of trypsin-activated meprin α or 31 nM of meprin β in 20 mM Tris-HCl, pH 7.5 containing 100 mM NaCl were incubated with 208 nM proIL-1β at 37° C in a total volume of 15 μl for 90 min. Proteolysis was terminated by heating the samples in the presence of sample loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 5% 2-ME) at 95° C for 5 min. Samples were resolved on 10% NuPAGE gels (Invitrogen), blotted to nitrocellulose membranes (BioRad), and probed with polyclonal rabbit anti-human IL-1β antibody (Figure 1B). Meprin α produced a 17-kDa IL-1β fragment that was slightly larger than the one produced by caspase-1 as shown in the figure. Meprin β cleaved proIL-1β to produce a 20-kDa IL-1β product. Meprin A produced both 17-kDa and 20-kDa products. For N-terminal sequencing, 7 μM proIL-1β was digested with 83 nM trypsin-activated meprin α, 71 nM meprin β, or 50 nM meprin A at 37° C in a total volume of 40 μl for 90 min, loaded onto multiple lanes of a 10% NuPAGE gel (Invitrogen), blotted to PVDF, and visualized with Coomassie Brilliant Blue staining. The bands corresponding to the processed IL-1β products were excised and submitted for amino acid sequencing by Edman degradation at the Harvard Microchemistry Facility. As depicted in Figure 2(A and B), the N-terminal amino acid of the 17-kDa fragment resulting from digestion with either meprin α or meprin A differs from the cleaved product produced by caspase-1 by a single additional amino acid. Meprin α or meprin A cleaved pro IL-1β at the His¹¹⁵-Asp¹¹⁶ site whereas caspase-1 cleaved at position Asp¹¹⁶-Ala¹¹⁷. As reported previously, meprin β cleaved pro-IL-1β at the Asn¹¹⁰-Glu¹¹¹ site.

Figure 1.

Purified meprins process pro-IL-1β to mature IL-1β A. Recombinant meprin α and β, and meprin A purified from kidney cortices were purified as described in Materials and Methods. The purified meprins were resolved by SDS-PAGE and visualized with Coomassie blue. B. Meprin α, meprin β, or meprin A were incubated with purified recombinant pro-IL-1β and the reaction products were analyzed by Western blot using a specific antibody to IL-1 β.

Figure 2.

Identification of cleavage sites of meprin A, meprin α, meprin β, and caspase-1 in the pro-IL-1β sequence. A. N-terminal amino acid sequence of the IL-1β products produced by meprin A, meprin α, meprin β, and caspase-1. N-terminal amino acid sequences of the IL-1β products obtained are shown. B. Cleavage sites of meprin A, meprin α, meprin β, and caspase-1 in the pro-IL-1β sequence as revealed by amino acid sequencing are indicated by arrows.

Biological activity assay IL-1β products produced by meprin A and meprin α

As shown in Figure 3, the biological activity of meprin α was almost 3-fold higher than that of meprin β and 1.6-fold higher for meprin A. These studies suggest that IL-1β products produced by meprin A and meprin α have a higher biological activity than those produced by meprin β or caspase-1. We previously showed that biological activity of IL-1β produced by caspase-1 is same to that produced by meprin β [11].

Figure 3.

Meprin A and meprin α produce biologically active IL-1β products from pro-IL-1β. The ability of indicated treatments with purified recombinant pro-IL-1β to induce cell proliferation of the helper T-cell line D10S was assessed as described in the text. Data are expressed relative to the activity of mature IL-1β produced by meprin β (meprin βΔTM) (100%). Bars represent the mean relative expression, thin lines are SEM (n=6).

Meprin inhibitor actinonin prevents sepsis-induced generation of serum IL-1β levels

In both experimental and clinical sepsis, bacterial compounds induce production and release of cytokines, resulting in a significant inflammatory response [18,29,30]. A sepsis-induced inflammatory response causes multi-organ failure and kidneys are often affected [29,30]. The experimental model of sepsis induced by cecal ligation puncture promotes IL-1β and maximum induction occurs between 4 to 8 hours after infection [18, 31,32]. Actinonin, a naturally occurring hydroxamate, has proved to be a most effective inhibitor of meprin A and inhibits meprin α and meprin A at a nanomolar range (Ki=20 nM) compared to about 5 mM to other metalloproteinases [7]. Actinonin protected renal function in a mouse sepsis model of AKI [33] similar to data reported for other models of AKI injury [34,35]. Thus we examined the effect of the meprin inhibitor actinonin on the formation of mature IL-1β from pro-IL-1β in the sepsis model of AKI. As shown in Figure 4, 7 h after CLP serum IL-1β levels increased 24-fold compared to Sham mice. Treatment with the meprin inhibitor actinonin significantly inhibited the generation of IL-1β (Figure 4), suggesting that meprin A plays a functional role in the cleavage of pro-IL-1β during sepsis.

Figure 4.

Effects of actinonin on IL-1 β levels in serum from mice subjected to CLP-induced sepsis. Serum IL-1 β levels were determined using a murine-specific sandwich ELISA kit from R&D Systems. Samples were obtained from C57BL/6 mice 7h after CLP treatment or Sham surgery. Actinonin was administered 30 min before CLP treatment. Data are mean ± SEM (n = 4–6 per group). *P < 0.05 compared to Sham and CLP 7h + actinonin; **P < 0.05 compared to Sham and CLP 7h.

DISCUSSION

Our studies demonstrate that meprin A purified from rat kidney cortices and purified recombinant meprin α are capable of cleaving pro-IL-1β to produce a biologically active form of IL-1β. Meprin A and meprin α produced a 17-kDa IL-1β fragment that was one amino acid larger than that produced by caspase-1. Both meprin A and meprin α were found to cleave the His¹¹⁵-Asp¹¹⁶ bond whereas caspase-1 cleaves the Asp¹¹⁶-Ala¹¹⁷ site. The amino acid residues C-terminal to the cleavage in the P1′ through P4′ sites are DAPV in the IL-1β fragment produced by Meprin A and meprin α. The Ala and Pro at positions P2′ and P3′, respectively, are in complete accordance with meprin A’s preference for these residues as described previously [6]. Also, meprin A has some preference for the Asp in the P1′ position [6]. We have demonstrated previously that recombinant meprin β cleaved pro-IL-1β at the Asn¹¹⁰-Glu¹¹¹ site to result in a biologically active IL-1β fragment [11]. Thus, the IL-1β fragment produced by meprin A and meprin α is five amino acids shorter than that produced by meprin β. In addition to the formation of the major fragment produced by cleavage at His¹¹⁵-Asp¹¹⁶, meprin A also produced a minor fragment whose size was similar to that produced by meprin β; however, the amount of this product was insufficient to allow its N-terminal sequence determination. IL-1β fragments produced by meprin A and meprin α were biologically active and this activity was significantly higher than the IL-1β produced by meprin β or caspase-1. We have reported previously that IL-1β produced by caspase-1 and meprin β had similar biological activity [11]. These studies demonstrate that homo- and hetero-oligomeric forms of meprins are capable of cleaving pro-IL-1β at different sites and producing biologically active fragments. A recent study has shown that pro-IL-18, another substrate of caspase-1, is also processed by meprin β to form biologically active IL-18 [10]. Meprin β cleaved pro-IL-18 at the Asn⁵¹-Asp⁵² bond, suggesting that meprin β prefers an acidic amino acid in the P1′ position. This specificity of meprin β for acidic residues is consistent to our previous studies on the cleavage specificity at Asn¹¹⁰-Glu¹¹¹ bond for pro-IL-1β [11].

Meprin A has been shown previously to be involved in urinary tract infections [1, 36], the pathogenesis of renal ischemia-reperfusion injury [34], cisplatin nephrotoxicity [35], sepsis-induced AKI [33], bladder inflammation [36], and inflammatory bowl disease [1, 10]. The high expression of meprin α in an inflammatory site suggests that meprin A or meprin α may be involved in activation of IL-1β in inflammatory diseases. Tissue damage may be caused by inflammatory mediators and cytokines, including interleukin-1, released in response to the infection [30]. One of the most important and early pro-inflammatory cytokines released in response to sepsis is IL-1β [18,37], which may play a critical role in host defense and tissue remodeling following injury [38]. IL-1β is known to promote its own production and also induce the expression of many pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α [39]. Sepsis is associated with a systemic inflammatory response thought to be orchestrated by pro-inflammatory cytokines. Our studies show that actinonin, a potent inhibitor of meprin A, significantly reduced sepsis-induced IL-1β production. The results of this study, therefore, suggest that meprins may play a critical role in processing pro-IL-1β to its active form during tissue injury and inflammation.