ABSTRACT
The extracellular metalloprotease meprin β is expressed as a homodimer and is primarily membrane bound. Meprin β can be released from the cell surface by its known sheddases ADAM10 and ADAM17. Activation of pro-meprin β at the cell surface prevents its shedding, thereby stabilizing its proteolytic activity at the plasma membrane. We show that a single amino acid exchange variant (G32R) of meprin β, identified in endometrium cancer, is more active against a peptide substrate and the IL-6 receptor than wild-type meprin β. We demonstrate that the change to an arginine residue at position 32 represents an additional activation site used by furin-like proteases in the Golgi, which consequently leads to reduced shedding by ADAM17. We investigated this meprin β G32R variant to assess cell proliferation, invasion through a collagen IV matrix and outgrowth from tumor spheroids. We found that increased meprin β G32R activity at the cell surface reduces cell proliferation, but increases cell invasion.
INTRODUCTION
Meprin β is an extracellular, multi-domain protease that forms disulfide-linked homodimers at the cell surface (Prox et al., 2015). The active site of the protease domain is shielded by an inhibitory pro-peptide and, to gain proteolytic activity, meprin β requires activation by soluble or membrane-bound serine proteases (Ohler et al., 2010; Jäckle et al., 2015). Of note, activation of meprin β is a fate-determining step of the protease. If activated on the cell surface, for example by matriptase-2, meprin β can no longer be proteolytically released (shed) by its known sheddases ADAM10 or ADAM17 (Jefferson et al., 2013; Wichert et al., 2017). When released from the cell surface in its inactive state by ADAM10 or ADAM17, it will be activated by soluble serine proteases, including, for example, kallikrein 4 and kallikrein 5 (Ohler et al., 2010). This difference in localization substantially determines the substrate accessibility, as substrates such as the IL-6 receptor can only be cleaved by membrane-bound meprin β (Arnold et al., 2017). However, not all substrates are accessible for the membrane-bound version of the protease including mucin 2 and pro-collagen I (Wichert et al., 2017; Broder et al., 2013; Schutte et al., 2014). Thus, a well-balanced ratio of shed and membrane-bound meprin β is required for a specific cellular function.
Meprin β displays a unique cleavage preference for negatively charged amino acids around the scissile bond (Becker-Pauly et al., 2011), and has been shown to play important roles for proper collagen I maturation in the skin and for the mucus detachment in the small intestine (Wichert et al., 2017; Broder et al., 2013; Schutte et al., 2014). Increased levels of meprin β have been found in fibrotic conditions of the skin and lung (Becker-Pauly et al., 2007; Biasin et al., 2014). The cellular localization of meprin β has been shown to be apical in epithelial cells of the proximal tubules of the kidney and enterocytes of the small intestine (Wichert et al., 2017; Schutte et al., 2014; Butler et al., 1987; Beynon et al., 1981; Sterchi et al., 1982). It has also been reported that meprin β relocates to the basolateral side during cisplatin-induced acute kidney injury, where it cleaves nidogen-1, a component of the basal membrane. This cleavage of nidogen-1 was not detected in meprin β-deficient animals (Herzog et al., 2015). Additionally, it has been shown in biochemical experiments, that meprin β cleaves the important basal membrane component collagen IV (Kruse et al., 2004).
Previously, we found that the co-expression of the membrane-bound serine protease matriptase-2 together with meprin β increases the proteolytic activity at the cell surface and reduces the amount of shed meprin β (Jäckle et al., 2015; Wichert et al., 2017). Here, we characterize two cancer-associated mutations (G32R and D47A) that are located within the pro-peptide and that likely influence activation and activity of meprin β. Both variants were identified in large-scale screenings of tumor samples and were deposited in the publicly available repositories (e.g. BioMuta-HIVE) (Simonyan and Mazumder, 2014). Previously, we found that the introduction of single amino acid changes can alter the cleavage preference of meprin β, namely, that the D204A variant of meprin β processes the amyloid precursor protein (APP) to a higher extent; however, it is restricted to the secretory pathway and cleaves APP there (Arnold et al., 2015).
Here, we found that both the G32R and D47A variants are transported to the cell surface and exhibit comparable expression with wild-type meprin β. However, the G32R variant showed higher proteolytic activity at the cell surface due to the change to an arginine residue at position 32, which provides an additional activation site, as revealed by N-terminal detection. Tumor cell invasion through a collagen IV matrix was increased for cells expressing the meprin β G32R variant, as was outgrowth length from tumor spheroids compared to that seen for a non-activate able meprin β variant.
RESULTS
Localization and cell surface expression of meprin β variants G32R and D47A
At first, we evaluated the cellular localization of the G32R and D47A variants. Both of these amino acid positions are located in the pro-peptide of the protease and might modulate its activity (Fig. 1A). To assess the cell surface level, we transfected Cos-7 cells with each variant and performed cell surface protein biotinylation experiments. We observed no significant difference between the variants and wild-type meprin β; however, a trend to a lower surface expression was found for the G32R variant (Fig. 1B,C). We confirmed this finding by fluorescence microscopy, demonstrating that wild-type and the two meprin β variants were found mainly at the cell surface (Fig. 1D). Thus, the expression pattern and cellular localization of the two amino acid exchange variants did not differ from that of wild-type meprin β.
Expression pattern of G32R, D47A and wild-type meprin β. (A) Molecular model of meprin β on the cell surface with one monomer shown in dark gray and the other in light gray. The insert shows the pro-peptide (orange) and the three active site histidine residues (cyan) that coordinate the zinc ion (gold). The positions of the two amino acid exchange variants [glycine to arginine (G32R), and aspartate to alanine (D47A)] are shown in red. (B) Representative western blot of a surface biotinylation of Cos-7 cells, with the transferrin receptor 1 (transferrin-R 1) used as a loading control for the membrane fraction. Cells were transfected with the meprin β variants indicated and, after biotinylation, the biotin-bound proteins were pulled down and analyzed by western blotting. (C) Quantification (mean±s.d.) of four individual experiments as shown in B. Meprin β amounts in the lysate were divided by the amount in the surface fraction, and all three variants were detected at the surface to the same extent (n=4). ns, not significant (P>0.05, one-way ANOVA with Tukey post-hoc test). (D) Immunofluorescence images of Cos-7 cells expressing wild-type, D47A and G32R (red) meprin β, with the endoplasmic reticulum marker protein disulfide isomerase A6 (PDIA6) (green).
The G32R meprin β variant shows increased activity
As we hypothesized that the exchange of a glycine residue into an arginine residue or an aspartate residue into an alanine residue might change meprin β activation and activity, we transfected HEK cells that were double-negative for ADAM10 and ADAM17 (denoted ADAM10/17−/−) cells with G32R, D47A and wild-type expression plasmids for meprin β or a control plasmid (mock). The ADAM10/17−/− cell line was used to prevent shedding mediated by endogenous ADAM10 or ADAM17, which would change the protease level at the cell surface. By using a peptide cleavage assay, we found that the activity of the G32R variant at the cell surface was significantly higher than the activity detected for D47A and wild-type meprin β (Fig. 2A). In this assay, a meprin β-specific peptide substrate that is linked to a fluorogenic group (MCA) at the N-terminus and a quencher (DPN) at the C-terminus (see Materials and Methods) was added to the cells (Broder and Becker-Pauly, 2013). Upon cleavage by the protease, an increase in fluorescence intensity was observed (Fig. S1A). Western blot analysis was used a control for equal expression of all meprin β variants (Fig. S1B). As previously we identified the serine protease matriptase-2 (MT-2) as a membrane-bound activator of meprin β (Jäckle et al., 2015), we were interested whether the two exchange variants can be further activated by MT-2. Therefore, the respective meprin β variant and MT-2 were co-expressed in ADAM10/17−/− HEK cells. The increase in proteolytic activity was again measured by performing the fluorogenic peptide cleavage assay (Fig. S1C) and western blot analysis of cell lysates was used a control for equal expression (Fig. S1D). For wild-type meprin β and the D47A variant, a significant increase in activity was detected after co-transfection with MT-2. Again, we observed an increased activity of the G32R variant in single-transfected cells, which was increased after co-transfection with MT-2. This increase was not significant; however, the basal activity of the G32R variant is already elevated (Fig. 2B), and thus the relative increase in activity is smaller than for D47A and wild-type meprin β.
Cell surface activity and shedding of the meprin β variants. (A) ADAM10/17-deficient HEK cells were transfected with a control plasmid (Mock) or one of the three meprin β variants [wild-type (WT), D47A or G32R]. The proteolytic activity was determined using a peptide cleavage assay and the meprin β inhibitor actinonin was added as a control (n=4). ***P<0001; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). Representative activity measurements and control western blots are presented in Fig. S1A,B. (B) As in A, but cells were co-transfected with the known meprin β activator matriptase-2 (MT-2). Note, that the activity is not significantly increased for the G32R variant, as the activity is already higher when compared to the other meprin β variants (n=4). **P<0.01; ***P<0.001; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). Representative activity measurements and control western blots are shown in Fig. S1C,D. (C) Representative western blot of the IL-6 receptor (IL-6R) and its soluble form (sIL-6R) generated by cells transfected with the indicated form of meprin β. Cell culture supernatant was collected after 24 h and used for precipitation of the sIL-6R. Note the increased signal for the sIL-6R upon co-expression with the G32R variant. (D) Quantification of four individual experiments as shown in C. The ratio of IL-6R to sIL-6R (relative shed ILR) were determined and analyzed (n=4). *P<0.05; **P<0.01; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). (E) Meprin β variants were co-expressed with ADAM17 in ADAM10/17-deficient HEK cells. To assess the shedding potential of ADAM17 against the different meprin β variants, cell culture supernatant and cell lysate was analyzed by western blotting, and the ratio of shed to membrane-bound meprin β was quantified (n=4). *P<0.05; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). A representative western blot is shown in Fig. S2A. (F) After activation of shed meprin β from experiments as in E with trypsin, activity was determined by performing a peptide cleavage assay (n=4). ***P<0.001; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). Representative activity measurements are shown in Fig. S2B. All graphs show mean±s.d. Activity for all experimental groups was normalized to the median value of the activity measured for wt meprin β.
To analyze the proteolytic activity towards a membrane-bound substrate, we co-transfected ADAM10/17−/− HEK cells with the different meprin β variants and the known protein substrate IL-6 receptor (IL-6R) (Arnold et al., 2017). The soluble proteolytically shed IL-6R (sIL-6R) was precipitated from the cell culture supernatant and analyzed by western blotting (Fig. 2C). The releases of sIL-6R into the supernatant was significantly higher after co-transfection with G32R than for D47A or wild-type meprin β (Fig. 2D). Taken together, these results show that G32R variant of meprin β has a significantly increased proteolytic activity at the cell surface without the need for additional expression of activating serine proteases.
Shedding of meprin β G32R mediated by ADAM17 is impaired
Activation of meprin β and its shedding from the cell surface are closely connected (Wichert et al., 2017). We have previously found that only inactive pro-meprin β can be shed by ADAM10 and ADAM17, but not active membrane-bound meprin β (Wichert et al., 2017). We hypothesized, that increased activation of G32R at the cell surface influences meprin β shedding. Thus, we co-expressed ADAM17 together with the meprin β variants in ADAM10/17−/− HEK cells. Western blot analysis of cell culture supernatants revealed that the G32R variant was shed to a significantly lesser extend than D47A or wild-type meprin β (Fig. 2E; Fig. S2A). To assess the activity of shed meprin β in the cell supernatant, the protease was activated with trypsin, as only inactive pro-meprin β is released by ADAM proteases. As indicated by our western blot analysis, a significantly decreased activity for the G32R variant was seen in the peptide cleavage assay (Fig. 2F; Fig. S2B).
The consensus activation site of meprin β cleaved by different tryptic serine proteases is located between arginine 61 and asparagine 62 (Jäckle et al., 2015). Thus, a mutant that carries a serine at position 61 cannot be activated (Fig. 3A) (Jäckle et al., 2015). We introduced an arginine residue at position 32, to create a G32R/R61S double mutant (Fig. 3A). The peptide cleavage assay revealed a significant increase in the activity of the double mutant (G32R/R61S) compared to the R61S single mutation alone (Fig. 3B). This suggests, that the arginine residue at position 32 can be cleaved by different serine proteases and that this cleavage leads to a partial activation of meprin β. To further investigate the removal of the pro-peptide, we generated meprin β variants that are additionally N-terminally Strep-tagged (Peters et al., 2019). Thus, the activated and non-activated meprin β can be discriminated (Fig. 3C). To further elucidate the activating mechanism, different serine protease inhibitors were used. To inhibit cell surface activation of meprin β, Pefabloc, a non-membrane transmissible inhibitor was added to the cells. To inhibit intracellular furin-like serine proteases in the Golgi network, a proprotein convertase inhibitor (PCI) was applied. Untreated conditions revealed no significant difference between WT and R61S variants (Fig. 3D,E), which might be due to the fast turnover of active meprin β at the cell surface as revealed by co-expression experiments with MT-2 (Jäckle et al., 2015). However, both variants carrying an arginine residue at position 32 (G32R and G32R/R61S) have a significantly lower N-terminal than C-terminal signal (Fig. 3D,E). The addition of Pefabloc did not lead to a marked increase in N-terminal Strep-tag signal for G32R and G32R/R61S, indicating only a minor cleavage at position R32 at the cell surface (Fig. 3F,G). Inhibition of furin-like proprotein convertases by using PCI increased the level of detectable N-terminus (Strep-tag) to wild-type and R61S levels for G32R and G32R/R61S variants, respectively (Fig. 3H,I). Thus, we suggest that the protein can be cleaved at R32 in the Golgi, unlike R61, which is only accessible for cell surface serine proteases (Fig. 3J).
An arginine residue at position 32 serves as an additional activation site in pro-meprin β. (A) Molecular model of membrane-bound meprin β. The insert shows the amino acid changes made to produce the G32R, R61S and G32R/R61S (G32R_R61S) mutants at position 32 and at the canonical activation site at position 61. Colors are as in Fig. 1A. (B) Cell surface activity measurements for ADAM10/17-deficient HEK cells transfected with a control plasmid (Mock) or one of the four meprin β variants [wild-type (WT), R61S, G32R or G32R/R61S]. Note that the insertion of an arginine at position 32 reconstitutes the loss of activity in the R61S mutant (n=4). *P<0.05; **P<0.01 (one-way ANOVA with Tukey post-hoc test). Representative activity measurements and western blots are shown in Fig. S2C,D. Activity was normalized to the median value of the activity measured for wt meprin β. (C) Cartoon of an N-Strep- and C-Flag-tagged meprin β variant. The Strep tag can only be detected for inactive meprin β, while the Flag tag can be detected for both, active and inactive meprin β. (D) Strep–Flag-tagged meprin β variants analyzed 36 h past transfection by western blotting to discriminate between active and inactive meprin β. Actin was used as a loading control. (E) Quantification of results shown in D reveals a significant reduction in N-terminal (Strep tag) to C-terminal (Flag tag) signal ratio (n=4). **P<0.01; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). (F) Representative western blot for experiments as in D, but after the addition of Pefabloc for 12 h at 24 h post transfection. (G) Quantification of results shown in F (n=4). *P<0.05; ***P<0.001; ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). (H) Representative western blot for experiments as in D, but after the addition of proprotein convertase inhibitor for 12 h 24 h post transfection. (I) Quantification of results shown in H (n=4). ns, not significant (P>0.05) (one-way ANOVA with Tukey post-hoc test). (J) Summarizing cartoon illustrating the activation of G32R meprin β in the Golgi and of WT meprin β at the cell surface. All graphs show mean±s.d.
Taken together, we found that the ratio between membrane-tethered and shed meprin β was altered upon the introduction of an arginine residue at position 32. This correlated with an increased proteolytic activity at the cell surface and decreased proteolytic activity in the supernatant. Additionally, we found that G32R is pre-activated in the Golgi and delivered to the cell surface as an (at least partially) active enzyme.
Cell proliferation and migration
We next elucidated whether the shift in proteolytic activity of the meprin β variants has functional consequences, and evaluated cell proliferation and cell invasion, which are hallmarks of cancer development. Increased cell proliferation and decreased apoptosis promote tumor growth. Cell migration/invasion is needed for the formation of metastases released from the primary tumor. Changes in the proliferative and invasive potential of HeLa cells upon transfection with different meprin β variants were measured by using an xCelligence real-time cell analyzer (ACEA Biotechnologies). In these assays, increased proliferation or invasion leads to increased electrical impedance values in the respective experimental setup, expressed in an arbitrary unit, the cell index (CI). We observed decreased cell proliferation upon expression of all meprin β variants in HeLa cells. The reduction seen for the G32R variant was significantly higher when compared to cells that did not express meprin β (Fig. 4A). Another hallmark of cancer cells is their ability to invade through the basal lamina. Here, we mimicked this basal laminar with a layer of collagen IV (Col IV), one major component of it. We transfected HeLa cells and allowed them to invade through a collagen IV matrix. We observed that meprin β-expressing cells showed a significantly higher invasive potential than control cells. Interestingly, we found an even higher invasion rate for cells expressing the G32R variant (Fig. 4B).
Cell proliferation and migration. (A) A cell proliferation assay (see Materials and Methods) revealed that there was significantly reduced proliferation of HeLa cells transfected with the G32R meprin β variant when compared to mock-transfected cells, or cells transfected with wild-type (WT) or D47A meprin β. CI, cell index (presented relative to the results for Mock). (B) A cell invasion assay through a collagen IV matrix revealed a significant increase for cells transfected with meprin β. This increase is significantly higher when cells express the G32R variant. (C) An example spheroid from mock-transfected HeLa cells is shown at 48 h past evasion start. (D) A spheroid with the evasion area marked. In green, a manual outline is shown, which enabled semi-automated finding of cells. (E) Spheroid marked with two circles to assess the maximum outgrowth distance. Scale bars: 250 µm. (F) Statistical analysis of results of the spheroid assay for HeLa cells transfected as indicated. No significant differences were found for the same time point between the groups (n=5) (two-way ANOVA with Tukey's post-hoc test). (G) Statistical analysis of the outgrowth distance over time for the spheroid assay (n=5). *P<0.05 (two-way ANOVA with Tukey's post-hoc test). All graphs show mean±s.e.m.
To assess cellular properties in a more tumor-like environment, we used a spheroid evasion assay. Therefore, we generated tumor spheroids from HeLa cells (Fig. 4C) and evaluated the outgrowth area (Fig. 4D) and the outgrowth distance (Fig. 4E) after 24 h, 48 h and 72 h. The cellular outgrowth did change over time, but was not significantly altered among cells expressing the different meprin β variants at a given time point. Western blotting was performed to confirm similar levels of expression of meprin β variants (Fig. S2D). When comparing the outgrowth distance, we found it to be significantly longer for cells expressing the G32R compared to the inactive R61S variant at 48 h (Fig. 4G).
Meprin β is expressed in murine and human endometrium
As both meprin β variants are found in endometrial tumors (COSMIC database IDs 987719 and 987721), we investigated general expression of this protease in the endometrium. Therefore, we analyzed tissues from wild-type and meprin β-deficient [Mep1b knockout (ko)] animals by immunohistochemistry. We found that meprin β is expressed at the apical side of cells in the mucosal cell layer (Fig. 5A). In unaffected control areas of human endometrium samples, we could confirm the expression of meprin β and, as in the murine sample, found it at the apical side of endometrial gland cells (Fig. 5B). To evaluate the expression in endometrial tumor samples, these were stained for meprin β, too. We observed that the distribution of meprin β was changed from apical to also showing basolateral localization within the cells (Fig. 5B). As the cellular polarity is lost in tumor tissue (Khursheed and Bashyam, 2014), a redistribution of meprin β would be expected. Some cells within the tumor seem to express meprin β at higher levels than others and additional cells positive for meprin β were detected outside the tumor (Fig. 5B). Taken together, we found meprin β expression at the apical side of cells in control endometrial tissue, which was changed in samples from tumor patients.
Expression of meprin β in the endometrium. (A) Sections of endometrium samples from wild-type (Mep1b wt) and meprin β-knockout (Mep1b ko) mice stained with H&E (left) or with antibody against meprin β (right). Note, in Mep1b wt animals a clear signal is detected for meprin β at the apical side of cells (arrows). This signal is missing in Mep1b ko animals. (B) Sections from human endometrium control and tumor sample tissue stained for meprin β. The control section shows a clear signal at the apical side (arrows). In tumor samples, this strict apical expression is lost and more randomly distributed signals can also be detected at the basolateral side (arrows).
DISCUSSION
In this study, we investigated the molecular and cellular consequences of the cancer-associated meprin β variants G32R and D47A, both identified in endometrium cancers. We found that the G32R single amino acid exchange variant had higher cell surface activity compared to wild-type meprin β through a premature activation in the Golgi network and thus changes the balance between the amount of soluble and membrane tethered protease because of impaired shedding by ADAM17. As a consequence, cleavage of a peptide substrate and the IL-6R by the meprin β variant G32R was increased. Previously, we had shown that matriptase-2, a membrane-bound serine protease, activates meprin β at the cell surface and thereby impairs its shedding by ADAM proteases (Jäckle et al., 2015; Wichert et al., 2017). Here, we could show that the G32R variant has a similar effect, but relies on the endogenously expressed proprotein convertases in the Golgi. This renders the G32R mutation a valuable tool to simulate increased meprin β activity at the cell surface. In a previous study, we found that a premature activation of meprin β in the endoplasmic reticulum impairs its transport to the cell surface (Arnold et al., 2015). The present data suggests now that active meprin β can be transported from the Golgi to the cell surface.
In this study, we show that the metalloprotease meprin β is present at the apical side of epithelial cells of the murine and human endometrium. This is in line with the expression at the apical side of enterocytes in the small intestine (Wichert et al., 2017; Schutte et al., 2014) and of proximal tubule cells in the kidney (Butler et al., 1987). The exact function of meprin β in healthy endometrium is currently unknown. However, as meprin β is important for proper mucus detachment in the small intestine (Wichert et al., 2017; Schutte et al., 2014), it might have a similar function in the endometrium. We found that the subcellular localization of meprin β changes in endometrial tumors from the apical to also being present at the basolateral side, which might be induced by a loss of cell polarity in these tumor cells (Khursheed and Bashyam, 2014).
The partial redistribution of meprin β from the apical to the basolateral side enables direct contact with well-described substrates in the basal lamina, such as collagen IV, laminin and nidogen, and makes them accessible for proteolytic cleavage (Kruse et al., 2004). An increased cleavage of nidogen-1 at the basal lamina has been described for meprin β in a mouse model of cisplatin-induced acute kidney injury. In this model, the cell polarity is lost and meprin β is also distributed to the basolateral side (Herzog et al., 2015). Here, we demonstrate that this accessibility enhances the invasive potential of meprin β-expressing HeLa cells through a collagen IV matrix. This is in line with recent studies, demonstrating that meprin β increases transmigration of Lewis lung carcinoma cells through an endothelial cell layer in vitro (Bedau et al., 2017a,b) and that fewer immune cells enter a peripheral site of inflammation in mice deficient for meprin β (Bedau et al., 2017a,b). This indicates a role for meprin β in the promotion of trans-endothelial migration for different cell types. Our tumor spheroid assays reveal only minor effects for meprin β on outgrowth rate and distance. While meprin β might play an important role for the penetration of the basal lamina, its role during cell migration seems less pronounced and other proteases might take over such as soluble and membrane type 1 matrix metalloproteinases (MT1-MMP; also known as MMP14) (Thakur and Bedogni, 2016).
Cell proliferation and survival can be influenced by extracellular proteolytic action. This was shown for the extracellular metalloprotease ADAM17. It cleaves epidermal growth factor receptor ligands from the cell surface and thereby induces cell proliferation. Here, we found that increased activity of meprin β on the cell surface reduced cell proliferation. Another pathway influenced by ADAM17, emerging in different tumor entities, is interleukin 6 (IL-6) and IL-6R-induced trans-signaling (Bergmann et al., 2017; Schmidt et al., 2018). Here, ADAM17 releases the IL-6R from the cell surface of one cell, and then this soluble receptor forms a signaling complex together with its cytokine and gp130 (also known as IL6ST) on another cell to induce cell survival via STAT3 phosphorylation (Schaper and Rose-John, 2015). Interestingly, the G32R variant of meprin β sheds more IL-6R from the cell and we have also previously shown that wild-type meprin β generates biologically active IL-6R, which is capable of inducing trans-signaling (Arnold et al., 2017).
Taken together, we identify a single amino acid exchange variant of meprin β (G32R) that changes its proteolytic activity at the cell surface and thereby diminishes its ADAM17-mediated shedding. We observed that cellular properties, such as proliferation and trans-migration, were directly linked to the amount of active meprin β on the cell surface. Additionally, we could show that the expression at the apical side of endometrial mucosal cells is lost in tumor tissue and found cells positive for meprin β in tumors. The exact role of meprin β in the tumor environment has to be further investigated in upcoming studies.
MATERIALS AND METHODS
Generation of the meprin β G32R and D47A variants
The two meprin β variants, G32R and D47A, were generated on the basis of a wild-type meprin β pcDNA4/TO-construct that carries a C-terminal FLAG tag. Individual nucleotides were exchanged according to the exchange variants reported (BioMuta database) using appropriate primers (Table S1) and the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, CA). The sequence of the obtained variants was confirmed by sequencing of the complete meprin β cDNA inserted in the expression plasmid (GATC Biotech, Konstanz, Germany).
Cell culture and transient transfection
Cell culture experiments were performed as described previously (Arnold et al., 2015). Briefly, Cos-7 and human embryonic kidney (HEK) cells deficient for ADAM10/17 (Riethmueller et al., 2017) or wild-type were seeded in Dulbecco's modified Eagle's medium (DMEM) plus GlutaMax (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin (P/S) (Thermo Fisher Scientific). Cells were grown to 70% confluence and were then transfected with the respective DNA plasmids by using polyethylenimine (PEI). After 24 h, the cell culture medium was changed to DMEM plus GlutaMax without further supplements to enhance the proteolytic potential of meprin β. After an additional 24 h, the cell supernatant and cells were harvested. Cells were washed three times with PBS, lysed with lysis buffer (PBS pH 7.4, 1% Triton X-100, supplemented with Complete EDTA free (Roche)], and protein content was determined by using a BCA kit (Thermo Fisher Scientific). Samples were mixed with loading buffer and heated to 98°C for 10 min. Detection of soluble IL-6R was performed as described previously (Arnold et al., 2017). For experiments using inhibitors, cells were transfected as described above and medium was changed to inhibitor-containing medium supplemented with 10% FCS and 1% P/S for 12 h prior to cell harvesting. Pefabloc (Merck, Darmstadt, Germany) was added at 0.5 mg/ml and proprotein convertase inhibitor (Merck, Darmstadt, Germany) at 22.5 µM.
Cell surface protein biotinylation
Cos-7 cells were transiently transfected with the respective meprin β variants and protein expression was allowed to procedure for 48 h. Cells were cooled to 4°C and washed with PBS-CM (PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2), and incubated with biotin solution (1 mg/ml NHS-SS-Biotin from Thermo Fisher Scientific in PBS-CM) at 4°C for 30 min, prior to quenching with quenching buffer (50 mM Tris-HCl pH 8.0 in PBS-CM). Then cells were lysed and a BCA assay was performed as described above, and lysate controls were taken for western blot analysis. From the remaining cell lysate, ∼600 µg protein was taken and incubated with 75 µl of Streptavidin–agarose resin (Thermo Fisher Scientific), washed three times with lysis buffer, mixed with loading buffer and heated for denaturation. Subsequent western blot analysis of the biotinylated and non-biotinylated samples revealed the presence of meprin β variants at the cell surface. As a control, transferrin 1 receptor was detected. Antibody details are given in Table S2.
Peptide cleavage assay
To assess the activity of meprin β on the cell surface, a peptide cleavage assay was used as described previously (Arnold et al., 2015). In brief, a substrate peptide, specifically cleaved by meprin β and coupled to a fluorophore (7-methoxycoumarin-4-acetic acid N-succinimidyl ester; MCA) on one side and a quencher (2,4-dinitrophenyl; DPN) on the other side, was added to the cell supernatant. Upon cleavage, an increase in the fluorescence signal is detected at 320 nm. The increase of this signal was detected in a 96-well plate reader (Infinit F200 Pro, Tecan, Crailsheim, Germany) for the time indicated in the experiments. As a control, actinonin (Sigma Aldrich, Darmstadt, Germany), a meprin β inhibitor (10 μM), was added to the cell supernatant.
Activation of meprin β by trypsin
To activate soluble pro-meprin β that had been released to the supernatant through the action of ADAM17, pancreatic trypsin was used as described previously (Wichert et al., 2017).
Histology and immunofluorescence
Murine uterus was removed from wild-type or meprin β-deficient animals (Yura et al., 2009) after devitalization of the mouse in accordance with the local animal protection law (animal protection law of Schleswig-Holstein, internal animal facility approval number 750). The uterus from 12-week-old female mice was fixed in 4% paraformaldehyde (PFA) overnight and was then post-fixed in 1% PFA. Samples were embedded in paraffin and sectioned into 7 µm slices. H&E staining was performed following routine protocols (Arnold et al., 2016).
We retrieved specimens of non-neoplastic endometrium and endometrial cancer from the archive of the Dep. of Pathology, University Medical Center Schleswig-Holstein; approval was given by the Ethics Committee of the University Medical Center Schleswig-Holstein, Schwanenweg 20, 24105 Kiel. Working with human samples was approved by local authorities (D 417/18). Diagnosis was based on histological assessment by a certified consultant pathologist. Immunohistochemical meprin β staining of human tissue specimens was carried out with the Bondmax automated slide staining system (Leica Biosystems), using the Polymer Refine Detection Kit (Leica Biosystems), the Epitope Retrieval Solution 1 for 30 min and anti-meprin β antibody (Table S2, dilution 1:1000). Immunostaining was visualized with DAB and counterstained with hemalaun.
Immunofluorescence (IF) staining was performed as described previously (Arnold et al., 2015). Briefly, Cos-7 cells were seeded on glass slides and left overnight. Cells were then washed in PBS three times and fixed in 4% PFA for 30 min. After permeabilization with saponin, cells were incubated with the primary antibody (see Table S2) for 1 h, washed and then incubated with the respective secondary antibody coupled to Alexa Fluor 488 or Alexa Fluor 594. Images were acquired on an Olympus FV 1000 confocal laser scanning microscope (Olympus, Hamburg, Germany).
Cell proliferation and invasion
Transfected HeLa cells were analyzed for their proliferative and invasive potential using the xCELLigence DP real-time cell analyzer (ACEA, Biosciences) according to manufacturer's instructions. Briefly, for proliferation assays, cells were harvested, resuspended in DMEM/GlutaMax with 10% FCS and seeded at 104 cells per well in an E-plate in triplicates. Proliferation was tracked by measuring the impedance on the bottom well electrode, representing the area covered by cells, as relative CI units every 30 min for 72 h. Data are presented as the area under the curve of the resulting graph, normalized to control-transfected HeLa cells. For invasion assays, transfected HeLa cells were harvested, resuspended in DMEM/GlutaMax with 1% FCS and seeded at 5×104 cells per well in a CIM-plate in triplicates. Each well was previously coated with 12 µg of human placenta-derived collagen IV (Sigma Aldrich) and allowed to dry, providing a matrix that cells had to invade through, to be measured on the flip side of an 8 µm pore membrane. CI measurements were performed at 30 min intervals for 24 h. Data are presented as end point CI values, normalized to those of mock transfected HeLa cells.
Production and analysis of tumor spheroids
Tumor spheroids were generated from 10,000 HeLa cells following a previously described protocol (Vinci et al., 2015). HeLa cells were transfected 24 h before spheroid formation was started. Images of tumor spheroids were acquired (5× magnification, Leica) at 24 h, 48 h and 72 h after matrix (Geltrex® Matrix, Gibco, Life Technologies, catalog no. A1413202) was supplemented.
To analyze the maximum evasion length, spheroids were marked with circles and the distance from the center of the spheroid circle to the furthest cell that evaded out was measured. Afterwards, the radius of the spheroid was subtracted. Data were analyzed using ImageJ and GraphPad Prism 6.
To analyze the evasion area, cells that evaded from the spheroid were selected automatically in Image-Pro 10 (Media Cybernetics) and noise signals were deleted manually. The evasion area was calculated by the sum of pixels of the selected area. Statistical analysis was performed using GraphPad Prism 6.
Molecular imaging
Molecular graphics were produced using UCSF Chimera (Pettersen et al., 2004). The basis for imaging was the crystal structure of pro-meprin β (PDB ID 4GWM) and a molecular model of the membrane-bound form, containing the EGF-like domains, the transmembrane helix and the C-terminal tail (Arolas et al., 2012). Single amino acid exchanges were produced with the UCSF Chimera command line tool swapaa (https://www.cgl.ucsf.edu/chimera/).
Statistical analysis
All statistical analysis was performed using GraphPad Prism 6, and the test indicated in the figure legend was used for the individual experiments. The density of western blot signals was detected using ImageJ. For soluble protein signals, the corresponding lysate form was used for quantification.
Acknowledgements
We acknowledge Inez Götting, Bettina Facompré and Katrin Neblung-Masuhr for excellent technical help.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: P.A., C.B.-P.; Methodology: W.L., O.H., S.S.; S.K., C.B. Investigation: H.S., W.L., S.K., C.B., P.A.; Resources: F.P., C.R.; R.L., C.B.-P. Data curation: H.S., W.L., P.A.; Writing - original draft: P.A.; Writing - review & editing: all authors; Visualization: H.S., W.L.; P.A.; Supervision: C.B.-P., P.A.; Project administration: P.A.; Funding acquisition: C.B.-P., P.A.
Funding
HS was supported by a stipend from the medical faculty of the Christian Albrechts University Kiel. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – project number 125440785 [SFB 877 Projects A9, A15 (to C.B.-P.) and A13 (to P.A.)].
Supplementary information
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.220665.supplemental
- Received May 23, 2018.
- Accepted April 23, 2019.
- © 2019. Published by The Company of Biologists Ltd
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