Matriptase-2 inhibits HJV-mediated hepcidin activation

To study the effect of matriptase-2 on hepcidin expression, we transfected Hep3B cells with a hepcidin promoter/firefly luciferase reporter construct (Hep-Luc) and a renilla luciferase vector (Pagani et al., 2008). The expression of matriptase-2^wt or matriptase-2 mutants did not alter hepcidin promoter activity in basal conditions nor after BMP2 stimulation (Fig. 2A). As expected, the presence of HJV enhanced hepcidin activation. Co-expression of HJV and matriptase-2^wt, however, significantly decreased hepcidin reporter gene activation. No decrease in hepcidin expression resulted when HJV was coexpressed with matriptase-2^MASK, while expression of matriptase-2^R774C led to a partial inhibition. A comparable trend was observed when HJV-matriptase-2 cotransfected cells were treated with BMP2 (Fig. 2A), suggesting that matriptase-2 specifically blocks the HJV-mediated effect. To verify the minimal effective concentration of matriptase-2, Hep3B cells were transfected with escalating concentrations of the serine protease. Matriptase-2^wt inhibited hepcidin activation in a dose-dependent manner, matriptase-2^R774C was less active and reached an inhibitory effect comparable to that of the wild type protein only at high concentrations but matriptase-2^MASK was inactive at all concentrations tested (Fig. 2B).

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Figure 2

Matriptase-2 modulation of the hepcidin promoter responses

(A) Hepcidin promoter responses by BMP2 and HJV, in the presence of matriptase-2 (MT2). A firefly luciferase reporter driven by 2.9 kb of the proximal hepcidin promoter was cotransfected into Hep3B cells with pRL-TK, either alone or with HJV and/or MT2 expressing vectors. Relative luciferase activity is calculated as reported in material and method and expressed as a multiple of the activity of cells transfected with the reporter alone. (B) Dose dependent modulation of the hepcidin activity by MT2. Hep3B cells were transfected with increasing concentrations of wild type or mutant MT2 expressing vectors in the presence of fixed amount of HJV expressing vector and treated or not with BMP2 (50 ng/ml). The fold induction is calculated as a ratio between the MT2-mediated and the mock-mediated hepcidin promoter activity, calculated as described in (A). Experiments, made in triplicate, were performed three times. Error bars indicate SD. Exact P-values are shown above bars.

Matriptase-2 cleaves HJV

Based on the observation that matriptase-2 inhibits the HJV-mediated hepcidin activation, we assessed whether it cleaved HJV. Coexpression of matriptase-2^wt and HJV caused a decrease of total (both full length and 33 kDa) HJV. No decrease in HJV occurred upon coexpression of matriptase-2^MASK, suggesting that matriptase-2 cleaves HJV (Fig. 3A). Since matriptase-2 is a transmembrane protease we hypothesized that it cleaves cell surface HJV (m-HJV). To test this hypothesis, HeLa cells were transfected with HJV and increasing concentrations of matriptase-2^wt or the matriptase mutants. Cell surface HJV decreased in a dose dependent manner in cells expressing matriptase-2^wt (Fig. 3B). In contrast, m-HJV was unchanged in cells transfected with matriptase-2^MASK and partially decreased in the presence of matriptase-2^R774C, indicating that the integrity of the protease domain is essential for an efficient cleavage of m-HJV (Fig. 3B). To further assess the residual amount of m-HJV after matriptase-2 cleavage, transfected cells were treated with a phosphatidylinositol-specific phospholipase (PI-PLC) that specifically cuts GPI-anchored proteins and the cleavage products in the supernatant were analyzed. HJV released by PI-PLC migrates as approximately 50 kDa and 33 kDa forms under partially reducing conditions (Fig 3A, lane 2, PI-PLC top panel) or mainly as 33 kDa fragments under strong reducing conditions (Fig. 3A, lane 2, PI-PLC bottom panel). When HJV was coexpressed with matriptase-2^wt, because of matriptase-2 proteolytic activity only traces of residual m-HJV were cleaved by PI-PLC (Fig. 3A, lane 3, PI-PLC panels). As indicated by the amount of m-HJV released by PI-PLC, matriptase-2^MASK was fully inactive (Fig. 3A, lane 5, PI-PLC panels), whereas matriptase-2^R774C (Fig. 3A, lane 4, PI-PLC panels) had partial activity.

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Figure 3

Matriptase-2 cleaves membrane HJV

(A) HeLa cells were transfected with HJV in the presence of the empty vector (mock), matriptase-2^wt (MT2^wt), MT2^R774C and MT2^MASK. Whole cell extracts, concentrated media and PI-PLC supernatants were loaded onto a 10% SDS-PAGE and processed for western blot analysis. Anti-FLAG and anti-HJV were used to detect MT2 and HJV respectively. β-ME: beta-mercaptoethanol. (B) Binding assay was used to measure m-HJV in the presence of increasing concentrations of wild type and mutants MT2 expressing vectors and was performed essentially as described in Fig. 1B. Experiments were made in triplicate and performed three times. Error bars indicate SD. Exact P-values refer to MT2^wt versus MT2^R774C. (C) HeLa cells were transfected with MT2^wt and MT2^MASK, in the presence of HJV^wt, HJV^W191C and HJV^R335Q. Whole cell extracts, concentrated media and PI-PLC supernatants were loaded onto a 10% SDS-PAGE and processed for western blot analyses.

s-HJV: soluble HJV; m-HJV: membrane associated HJV. CL: cellular lysates; CM: conditioned medium; PI-PLC: supernatant after PI-PLC cleavage. The equal loading was verified by α-tubulin.

Examination of the cleavage products in the culture media released by matriptase-2 revealed bands migrating between 25 and 30 kDa, in addition to decreased 42 and 33 kDa s-HJV species (Fig. 3A, lane 3, CM panel). In contrast, the former fragments were not present in the media from cells transfected with matriptase-2^MASK (Fig. 3A, lane 5, CM panel) and only barely present in the media from cells expressing matriptase-2^R774C (Fig. 3A, lane 4, CM panel).

To clarify the origin of the cleavage products, we took advantage of two previously developed HJV variants. W191C, which is incorrectly processed, retained into ER and is unable to reach the cell surface. This mutant form is released into the medium as a 42kDa s-HJV (Silvestri et al., 2007). A second HJV mutant R335Q is efficiently exported to plasma membrane but does not release s-HJV, since the amino acid change affects the furin consensus cleavage site (Silvestri et al., 2008). PI-PLC cleaves m-HJV only in cells transfected with HJV^wt (Fig. 3C, lane 2) and HJV^R335Q (Fig. 3C, lane 4) but not with HJV^W191C (Fig. 3C, lane 3). When matriptase-2 was coexpressed with HJV^wt or HJV^R335Q PI-PLC did not release m-HJV due to the serine protease activity (Fig. 3C, lanes 5 and 7, PI-PLC panel). Moreover, cells cotransfected with matriptase-2 and HJV^R335Q (Fig. 3C, lane 7, CM panel) release in the supernatant the same cleavage products as from cleavage of HJV^wt (Fig. 3C, lane 5, CM panel). In contrast, when matriptase-2^wt was coexpressed with HJV^W191C (Fig. 3C, lane 6, CM panel), the 42 kDa s-HJV form was decreased, but no additional fragments were observed. In the same experiments matriptase-2^MASK was used as an inactive control (Fig. 3C, lanes 8–10). We conclude that the 25–30 kDa fragments produced by the matriptase-2 cleavage originate from m-HJV and hypothesize that the 42 kDa s-HJV decrease follows the reduction of HJV due to matriptase-2 overexpression.

To demonstrate the specificity of the process, we tested matriptase-2^wt cleavage ability on uromodulin, a GPI-anchored protein mainly expressed in the kidney (Bernascone et al., 2006). As shown in Figure S2 total, membrane-associated and soluble uromodulin were not affected by the serine protease.

Matriptase-2 does not cleave s-HJV

To understand whether the 42 kDa s-HJV is cleaved directly by matriptase-2, we incubated matriptase-2-transfected cells with exogenous s-HJV (Fig. 4A) and observed that matriptase-2 overexpressing cells did not cleave s-HJV in media of HJV-transfected cells. To confirm that the decrease of HJV in cell extracts was due to matriptase-2 overexpression, cells were transfected with increasing amounts of matriptase-2^wt and constant amounts of HJV (Fig. 4B). At low expression levels, matriptase-2^wt reduced only m-HJV, releasing the corresponding soluble fragments in the supernatant. We also observed that the serine-protease domain cleaved from matriptase-2 ectodomain was released in the cell culture supernatant. To understand whether the soluble matriptase-2 domain retained proteolytic activity on HJV, we incubated HJV-transfected cells with conditioned media from matriptase-2^wt transfected cells (Fig. 4C). Neither s-HJV nor m-HJV was cleaved, indicating that the released serine-protease domain is inactive on HJV.

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Figure 4

Matriptase-2 does not cleave soluble HJV

(A) HeLa cells were transfected with empty vector or matriptase-2 (MT2) expressing plasmid, and then incubated with an exogenous source of soluble HJV. Whole cell extracts and concentrated media were then analyzed by western blot. A scheme of the experiment is shown bottom left. (B) HeLa cells were transfected with HJV and increasing concentration of MT2^wt expressing vectors. Whole cell extracts, concentrated media and PI-PLC derived supernatants were then analyzed by western blot. (C) HeLa cells were transfected with HJV cDNA expressing vector and incubated with exogenous soluble MT2. Whole cell extracts, concentrated media and PI-PLC derived proteins were analyzed by western blot. A scheme of the experiment is shown bottom right.

s-HJV and s-MT2 refer to soluble proteins. m-HJV indicates membrane-bound HJV. CL: cellular lysates; CM: conditioned medium; PI-PLC: supernatant after PI-PLC cleavage. The equal loading was verified by α-tubulin.

HJV interacts with matriptase-2

Our results demonstrate that matriptase-2 specifically cleaves m-HJV, generating the 25–30 kDa soluble fragments. If overexpressed, matriptase-2 causes a reduction of all HJV species including the 42 kDa soluble form.

To test whether the two proteins interact, HeLa cells were transiently transfected with matriptase-2^wt, in the presence of HJV or an empty vector. Two days after transfection, cell lysates were immunoprecipitated with the anti-HJV antibody and the immunoprecipitates were analyzed by immunoblotting with anti-FLAG, which recognizes matriptase-2. The anti-HJV immunoprecipitate showed the presence of matriptase-2 (Fig. 5A). To confirm the specificity of the interaction, matriptase-2 was immunoprecipitated by the anti-FLAG, and the precipitates analyzed with anti-HJV. Matriptase-2 forms a complex with HJV. HJV also specifically coimmunoprecipitates with matriptase-2^MASK (Fig. 5B), demonstrating that the matriptase-2 ectodomain is involved in HJV interaction. We observed that the amount of matriptase-2^MASK precipitated with HJV was greater than that of matriptase-2^wt, likely because the proteolytic activity of the overexpressed matriptase-2 cleaved HJV. The matriptase-2^R774C variant also maintains the ability to interact with HJV (data not shown).

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Figure 5

Matriptase-2 interacts with HJV

HeLa cells were cotransfected with matriptase-2^wt (MT2^wt)(A) and MT2^MASK (B) in the presence of HJV or an empty vector. Precleared whole cell extracts were immunoprecipitated with anti-HJV and revealed with the anti-FLAG antibody, which recognizes MT2 (panels A and B) or immunoprecipitated with anti-FLAG and revealed with anti-HJV (panel A). To control for transfection, whole cell extracts were loaded and revealed with anti-HJV and anti-FLAG antibodies.

Cell culture and reagents

Cell culture media and reagents were from Invitrogen and Sigma-Aldrich (St Louis, MO). HeLa cells, which do not express endogenous HJV, and Hep3B cells were cultured respectively in Dulbecco’s modified Eagle’s medium (DMEM) and in Earl’s Minimal Essential Medium (EMEM), supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 200 mg/ml streptomycin, 1 mM sodium pyruvate, and 10% heat-inactivated Foetal Bovine Serum (FBS) at 37°C in 95% humidifier air and 5% CO2. The rabbit polyclonal antisera directed against HJV was obtained as described (Silvestri et al., 2008). Briefly, the cDNA fragment encoding from amino acid 226 to 402 of human HJV was cloned into a pGEX (Amersham Biosciences Europe GmbH, Freiburg, Germany) vector in fusion with GST. The protein was expressed in Escherichia coli, purified by affinity chromatography, and used to elicit antibodies in rabbit. Anti–tubulin, anti-FLAG and anti-cMYC antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma-Aldrich, respectively.

Luciferase assay

A pGL2-basic reporter vector (Promega, Madison, WI, USA) containing a 2.9 Kb fragment of the human hepcidin promoter (Hep-Luc) (Pagani et al., 2008) was used to monitor the hepcidin promoter activation by luciferase assay.

Hep3B cells, seeded at 70–80% of confluency, were transiently transfected with 0.25 μg hepcidin promoter luciferase reporter construct in combination with pRL-TK Renilla luciferase vector (Promega) to control transfection efficiency, and with 0.1 μg/ml of expressing vectors encoding wild type or mutant HJV. Eighteen hours after transfection the medium was changed, cells were serum starved 24 hours in EMEM supplemented with 2% FBS, and then lyzed. When indicated, 50 ng/ml of BMP2 was added to the cell culture. Luciferase activity was determined according to manufacture’s instructions (Dual Luciferase Reporter Assay, Promega). Relative luciferase activity was calculated as the ratio of firefly (reporter) to Renilla luciferase activity and expressed as a multiple of the activity of cells transfected with the reporter alone. Experiments were performed in triplicate.

Western blot analysis

HeLa cells, seeded in 100-mm-diameter dishes up to 70–80% of confluency, were transiently transfected with 20 μg of plasmid DNA and 50 μl of the liposomal transfection reagent Lipofectamine 2000 (Invitrogen) in 3 ml of OptiMem (Invitrogen) according to the manufacturer’s instructions. After 18 hours the medium was replaced with 4 mL of OptiMem and 24 hours later media were collected and concentrated using 5 kDa molecular weight (MW) cutoff ultrafiltration (Amicon Ultra; Millipore, Billerica, MA), and cells were lyzed in RIPA buffer. Proteins were quantified by using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA); equal amount of total proteins (50 μg) were subjected to 10% SDS-PAGE, and then transferred to Hybond C membrane (Amersham Biosciences Europe GmbH, Freiburg, Germany) by standard western blotting technique. Blots were blocked with 2% ECL Advance Blocking Agent (Amersham Biosciences) in TBS (0.5 M Tris-Hcl pH 7.4 and 0.15 M NaCl) containing 0.1% Tween-20 (TBST), incubated 2 hours with rabbit anti-HJV (1:1000), rabbit anti-FLAG (1:1000) or mouse anti-tubulin (1:500). After washing with TBST, blots were incubated 1 hour with relevant HRP-conjugated secondary antisera and developed using a chemiluminescence detection kit (ECL, Amersham Biosciences).

Immunoprecipitation

Immunoprecipitation was performed as follows. Transfected cells were lyzed in NET/Triton buffer (150 mM NaCl, 5 mM EDTA, and 10 mM Tris (pH 7.4) with 1% Triton X-100). Total lysates were cleared with 20 μl of Protein A/G Plus Agarose (Santa Cruz) and 2 μl of irrelevant antibodies (sera from rabbit or mouse) and 500 μg of precleared lysates were incubated with anti-HJV or anti-FLAG at 4°C. After 1 hour, 20 μl of Protein A/G Plus Agarose were added and incubated at 4°C for 12 hours, followed by three washing with NET/Triton buffer. Samples were then eluted with 25 μl of Laemmli Sample Buffer 2x and subjected to SDS-PAGE on 10% acrylamide gel. Immunodetection was performed as described above, except that rabbit TrueBlot (eBioscience, 1:1000 dilution), which does not recognize denatured IgG was used as the secondary antibody.

Electron microscopy and morphometric analysis

HeLa cells were transiently transfected with Lipofectamine 2000 using pcDNA3.1-matriptase-2 (matriptase-2^wt), pcDNA3.1-matriptase-2^MASK (matriptase-2^MASK) and pcDNA3.1-matriptase-2^R774C (matriptase-2^R774C) constructs. After 18 hours, cells were fixed, labeled with a polyclonal rabbit anti-FLAG using the gold-enhance protocol, embedded in Epon-812, and cut as described. Immunoelectron microscopy (EM) images were acquired from thin sections under a Philips Tecnai-12 electron microscope (Philips, Eindhoven, the Netherlands) using an ULTRA VIEW CCD digital camera (Philips). Thin sections were also used to quantify gold particles residing within different compartments of the secretory pathway.

Analysis of soluble HJV

HeLa cells were transiently transfected for 18 hours, as described in “Western blot analysis”, with HJV^wt, HJV^W191C or HJV^R335Q expressing vectors in the presence of matriptase-2^wt cDNA, matriptase-2^MASK or matriptase-2^R774C variants. Cells were then incubated with serum-free media, which were collected after 24 hours, concentrated using Amicon Ultra, and analyzed by SDS-PAGE. When indicated, HJV or matriptase-2 transfected cells were incubated for 24 hours with matriptase-2 or HJV-conditioned cell culture media (see Supplemental Methods for details). Soluble, membrane and whole cellular proteins were then analyzed by SDS-PAGE.

Cell surface protein quantification by binding assay

Quantification of cell surface expression of HJV and/or matriptase-2 expressing vectors were performed as described, with minor modifications. In brief, 10⁴ HeLa cells were seeded in 48-well plates and transfected with 0.4 μg of plasmid DNA complexed with 1 μL of Lipofectamine 2000, according to the manufacturer’s instructions. After 12 hours, the medium was replaced and 24 hours later cells were fixed with 4% paraformaldehyde for 45 minutes at room temperature. Cells were washed with PBS, blocked with 5% nonfat milk in PBS, incubated with rabbit anti-HJV (1:1000) or rabbit anti-FLAG (1:1000) and then with the relative secondary HRP antibody at 37°C. For total HJV or matriptase-2 expression, cells were permeabilized with 0.1% Triton X-100 in PBS, prior to blocking and incubation with anti-HJV or anti-FLAG. Peroxidase activity was measured with an HSR substrate (o-phenylenediamine dihydrochloride), according to the manufacturer’s instructions. The amount of m-HJV or m-matriptase-2 was calculated as the ratio between the absorbance of unpermeabilized and permeabilized cells. Background absorbance was subtracted for each sample. The Student t test was used for statistical calculation.

PI-PLC cleavage of membrane HJV

A total of 10⁶ HeLa cells, transiently transfected with HJV- and matriptase-2- expressing constructs or the empty vector were incubated in DMEM plus 0.3 U/ml phosphatidylinositol-specific phospholipase C (PI-PLC) at 37°C in a 5% CO₂ incubator. After 2 hours, the supernatants were collected. Proteins were precipitated with cold acetone and resuspended in Laemmli sample buffer, in the presence of 4% v/v β-mercaptoethanol (β-ME) when indicated. Samples were then boiled for 10 minutes and loaded on a 10% SDS-PAGE.

We gratefully acknowledge Carlos Lopez-Otin for the matriptase-2-FLAG expressing vector; Roman Polishchuk from the Telethon Electron Microscopy Core Facility (TeEMCoF; Consorzio Mario Negri Sud-Santa Maria Imbaro) for the electron microscopy and morphometric analysis on matriptase-2; Paolo Arosio for the gift of the anti-HJV antibody; Luca Rampoldi for the uromodulin cDNA expressing vector. The authors thank Barry H. Paw (Children’s Hospital Harvard, Boston, MA) who designed the morpholino oligonucleotides and provided protocols.

This work was supported by EU Contract N° LSHM-CT-2006-037296, Italian Telethon Foundation ONLUS Rome Grant GGP05024 and Grant GGP08089, Ministero Istruzione Università e Ricerca, (PRIN 2006) Rome, Italy to CC and National Institutes of Health grant DK 070947 to JK.