Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells Lillian Zernichow 1 , Knut T. Dalen 1 , Kristian Prydz 2 , Jan-Olof Winberg 3 and Svein O. Kolset 1 1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway 2 Department of Molecular Biosciences, University of Oslo, Norway 3 Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway Studies on proteoglycans (PGs) have, to a large extent, focused on molecules located in the extracellu- lar matrix and on cell surfaces [1–3], and their roles in, for example, the regulation of cell adhesion, cell migration, proliferation and wound healing. However, PGs located in different intracellular locations are receiving increasing attention [4,5]. In particular, PGs in storage and secretory granules in cells of the hae- matopoietic lineage have been the subject of several recent studies, as, for instance, in the mast cell, where heparin PG is stored in secretory granules together with histamine and proteases. Generation of mice with a deleted version of the gene for the heparin- synthesizing enzyme N-deacetylase ⁄ N-sulfotransferase- 2 (NDST-2) resulted in the appearance of mast cells with large changes in secretory granule morphology and in greatly reduced levels of the proteases nor- mally confined to these granules [6,7]. Heparin PG in mast cells, accordingly, seems to be of fundamental importance for the generation of storage granules. Recently, serglycin knockout mice were generated [8]. They developed normally and were fertile, but their mast cells were affected in a manner similar to that of the NDST-2 knockout mice. Keywords matrix metalloproteinase; MDCK; plasminogen activator; proteoglycan; serglycin Correspondence S. O. Kolset, Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Box 1046 Blindern, 0316 Oslo, Norway Fax: +47 22 851 398 Tel: +47 22 851 383 E-mail: s.o.kolset@medisin.uio.no (Received 2 November 2005, accepted 2 December 2005) doi:10.1111/j.1742-4658.2005.05085.x Madin–Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with differ- ent levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35 S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activ- ities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35 S-labelled sergly- cin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthe- sis and secretion of serglycin and potential partner molecules. Abbreviations cABC, chondroitinase ABC; CS, chondroitin sulfate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GAG, glycosaminoglycan; HS, heparan sulfate; His-tag, polyhistidine tag; MDCK, Madin–Darby canine kidney; MMP, matrix metalloproteinase; NDST-2, N-deacetylase ⁄ N-sulfotransferase-2; NGAL, neutrophil gelatinase-associated lipocalin; PA, plasminogen activator; PG, proteoglycan; PMA, phorbol 12-myristate 13-acetate; PVDF, poly(vinylidene difluoride); uPA, urokinase-type plasminogen activator. 536 FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS Haematopoietic cells either harbour storage granules or granules destined for constitutive secretion. In human monocytes and macrophages the main PG is serglycin [9]. In these cells, the PG is secreted and acti- vation results in increased synthesis and secretion of PGs [9,10], suggesting that secretion of PGs is linked to inflammatory response. Secreted serglycin may potentially associate with other secreted products from macrophages in the extracellular environment [11,12], or team up intracellularly with such molecules during constitutive secretion. In a recent study in murine macrophages, abrogation of PG biosynthesis with b-d- xylosides resulted in decreased enzyme secretion [13]. In particular, the secretion of urokinase-type plasmino- gen activator (uPA) and matrix metalloproteinase-9 (MMP-9, also referred to as 92 kDa gelatinase B and type IV collagenase) was lowered after xyloside treat- ment. To further study the interrelationship between ser- glycin and proteases, both with regard to biosynthesis and secretion, serglycin was stably transfected into Madin–Darby canine kidney (MDCK) epithelial cells. A polyhistidine tag (His-tag) was introduced at the C-terminus to facilitate the isolation of serglycin. Transfectants with vector without serglycin insert were generated as negative controls. It has previously been shown that MDCK cells secrete MMP-9 [14] and uPA [15]. Secretion of these endogenous proteases was stud- ied in the transfected cells, in both the absence and presence of the phorbol ester phorbol 12-myristate 13-acetate (PMA), previously reported to enhance the secretion of MMP-9 in MDCK cells [14]. Results pre- sented show that the secretion levels of proteases cor- relate with the levels of serglycin mRNA, HisTrap isolated 35 S-labelled serglycin and serglycin core pro- tein detected by western blotting. The MDCK system with transfected serglycin is potentially a useful model to study PGs in relation to secretion of enzymes important in physiological and pathological condi- tions. Results Transfection of serglycin Clones of MDCK cells with serglycin–His-tag were obtained, and the levels of serglycin mRNA deter- mined using northern blotting. Cell clones transfected with the vector without the serglycin insert were used as negative controls (mock transfectants). No serglycin mRNA was detected in these clones. The serglycin mRNA expression level in the different clones was related to the housekeeping gene 36B4 mRNA expression level. One clone with a lower ser- glycin mRNA expression level (1–7), one with a higher level (1–10) and one of the mock transfectants were selected for further studies. Figure 1 shows nor- thern blots of the selected clones, using 32 P-labelled cDNA probes of serglycin, MMP-9, uPA and 36B4, cultured in both absence and presence of PMA. The northern blot of the unstimulated cells in Fig. 1A shows that the clone with the highest serglycin mRNA level also has the highest mRNA levels of uPA and MMP-9, although the levels are very low for MMP-9. PMA stimulation (Fig. 1B) was shown to lead to an upregulation of the mRNA levels of serglycin, MMP-9 and uPA. Proteoglycan analyses 35 S-labelled macromolecules According to Svennevig et al. [16] and Erickson & Couchman [17], PGs synthesized by MDCK cells are perlecan, agrin, collagen XVIII, biglycan, bamacan AB Fig. 1. mRNA expression levels of serglycin, MMP-9, uPA and 36B4 in serglycin-transfected MDCK clones. Total RNA was isola- ted from MDCK clones and subjected to northern blotting onto the same membrane and hybridized using 32 P-labelled human serglycin, canine MMP-9 and uPA and murine 36B4 cDNA probes. (A) Un- stimulated clones. (B) PMA-stimulated clones. The experiment was repeated independently three times, and the result shown is typical of the three experiments. The northern blots in (A) and (B) for MMP-9 and uPA have been exposed for different periods, due to large differences in expression levels. L. Zernichow et al. Serglycin and proteases FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS 537 and versican. To investigate the extent to which expression of serglycin influenced total PG biosynthe- sis, cells were labelled with 35 S sulfate for 24 h. The labelled macromolecules were separated from unin- corporated 35 S-labelled sulfate by Sephadex G-50 Fine gel chromatography. It has previously been shown that the major fraction of 35 S-labelled macro- molecules in MDCK cells are of PG nature [16]. The level of 35 S-labelled macromolecules therefore indicates the level of PG synthesis. As can be seen in Fig. 2A, the introduction of serglycin into MDCK cells increased the amount of 35 S-labelled macromole- cules in the medium, but did not significantly increase the total amount of 35 S-labelled macromole- cules. Radiolabelling was also performed in the presence of PMA. Figure 2B shows that in PMA- stimulated cells the distribution of PGs changed, so the majority (78–90%) was secreted into the medium, whereas the corresponding value for unstimulated cells was 30–45%. The total increase of 35 S-labelled macromolecules in clones 1–7 and 1–10, relative to the mock, was approximately the same in unstimu- lated and PMA-stimulated cells. HisTrap isolation of His-tagged serglycin His-tagged serglycin in cell fractions and condi- tioned media from unstimulated clones was isolated by HisTrap affinity chromatography using 35 S-labelled macromolecules obtained by Sephadex G-50 Fine gel chromatography. Figure 3A shows the total amount of 35 S-labelled macromolecules loaded onto the HisTrap column, whereas Fig. 3B shows the total amount of 35 S-labelled macromolecules with affinity for the column. The highest level of 35 S-labelled macromolecules with affinity for the HisTrap column, in both cell fractions and conditioned media, was measured in clone 1–10. This level was 2–3 times higher than for clone 1–7, whereas the level for the mock transfected clone was found to be negligible. In both clone 1–10 and 1–7 the highest level of incorporated 35 S-labelled sulfate was measured in the conditioned media. As can be seen in Fig. 3, serglycin contributes very little to the total incorporation of 35 S-labelled sulfate. When comparing the amount of 35 S-labelled macromolecules before and after HisTrap isolation, it was found that only  1 ⁄ 60 of the radio- activity was associated to serglycin in clone 1–7, whereas the corresponding value for clone 1–10 was  1 ⁄ 20. Superose 6 gel chromatography of HisTrap isolated 35 S-labelled serglycin from conditioned media showed that serglycin from clone 1–10 eluted at a slightly more retarded position compared with clone 1–7 (not shown). Furthermore, analyses of 35 S-labelled glycosaminoglycan (GAG) chains obtained from the same material showed a similar trend, with K av values of 0.53 and 0.57 for clone 1–7 and 1–10, respectively. These findings indicate that the GAG chains of 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 1-7 Cell Medium Total 35 S sulfate (cpmx10 6 ) 35 S sulfate (cpmx10 6 ) A B Cell Medium Total 1,42 1,35 1,0 1,38 1,32 1,0 1-10 Mock 1-7 1-10 Mock Fig. 2. 35 S-labelled macromolecules in serglycin-transfected MDCK clones. Confluent MDCK clones were labelled with 35 S sulfate for 24 h in both absence and presence of PMA, whereupon the cells fractions and conditioned media were harvested and subjected to Sephadex G-50 Fine gel chromatography to remove unincorporated 35 S sulfate. Each point represents the mean ± SD of measurement on material from three separate wells. The number on top of the black columns, representing the total 35 S sulfate incorporation, is relative to the mock transfectant. (A) Unstimulated clones. (B) PMA-stimulated clones. The experiment was repeated independ- ently three times, and the result shown is typical of the replicate experiments. Serglycin and proteases L. Zernichow et al. 538 FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS serglycin from clone 1–7 are slightly longer than those of clone 1–10. Analysis of the composition of the 35 S-labelled GAGs showed that both clone 1–7 and 1–10 contained  70% chondroitin sulfate (CS) and 30% heparan sulfate (HS) (not shown), indicating that CS is the dominating GAG, in agreement with previ- ous findings [18]. Western blotting of serglycin To further analyse for the presence of serglycin, condi- tioned media of the different clones were subjected to western blotting after chondroitinase ABC (cABC) treatment, using a rabbit polyclonal antibody to human serglycin. As evident in Fig. 4, clones 1–7 and 1–10 contained the serglycin core protein, with the highest amount in the latter. The molecular mass of the serglycin core protein was 35 kDa, in accordance with another study [5]. The same results were observed with a mouse monoclonal antibody to the His-tag (not shown). Enzyme analyses The possible relationship between serglycin and prote- ase secretion was analysed in the different clones using serum-free conditioned media, the chromogenic sub- strates S-2288 and S-2444, and gelatin and plasmino- gen–gelatin zymography. Cell fractions were also analysed. All cell culture experiments were carried out in both the absence and presence of PMA, to ensure 0 100 200 300 400 500 600 700 Cell Medium Total 0 5 10 15 20 25 30 35 Cell Medium Total A B 35 S sulfate (cpm x 10 4 ) 35 S sulfate (cpm x 10 4 ) 1-7 1-10 Mock 1-7 1-10 Mock Fig. 3. HisTrap isolation of 35 S sulfate-labelled His-tagged sergly- cin from serglycin-transfected MDCK clones. Cell fractions and conditioned media from cells exposed to 35 S sulfate for 24 h were buffer exchanged to binding buffer by Sephadex G-50 Fine gel chromatography. The samples were further applied to a 1 mL HisTrap column, pre-equilibrated with binding buffer (20 m M phosphate, 1 M NaCl, 8 M urea and 20 mM imidazole pH 8.0). After a washing step, the samples were eluted with a solution containing 20 m M phosphate, 1 M NaCl, 8 M urea and 500 mM imidazole (pH 8.0). (A) Before HisTrap isolaton. (B) After HisTrap isolation. The experiment was repeated independently more than three times, and the result shown is typical of all the replicate experiments. Fig. 4. Western blot of serglycin core protein in serglycin-trans- fected MDCK clones. Conditioned media from unstimulated MDCK clones were desalted against Milli-Q water by Sephadex G-50 Fine gel chromatography. After freeze-drying, the samples were dis- solved in a small volume of Milli-Q water and treated with cABC as described in Experimental Procedures. Furthermore, the samples were subjected to SDS ⁄ PAGE followed by western blotting using a rabbit polyclonal antibody to human serglycin. The data shown are from a single experiment that was repeated three times with the same results. L. Zernichow et al. Serglycin and proteases FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS 539 comparison of basal and stimulated secretion of pro- teases. Chromogenic substrates Cell fractions and serum-free conditioned media were analysed with respect to a broad spectrum of serine proteases using the chromogenic substrate S-2288 (Fig. 5). Media from clone 1–10 showed the highest enzyme activity in unstimulated cells. When cells were treated with PMA, the enzyme activities in the media were strongly enhanced. Compared with the media, enzyme activities in cell fractions were found to be low. Media from PMA-treated clones con- tained  100-fold more protease activity than medium from unstimulated clones. Also after PMA treatment, medium from clone 1–10 showed the highest activity, but the difference between the clones was much less distinct than observed for the unstim- ulated clones. Further experiments were performed using the uro- kinase substrate S-2444. Cell fractions and serum-free conditioned media from the various clones all con- tained activity towards this substrate, as can be seen in Fig. 6. Again, media from the clone 1–10 had the high- est enzyme activity. Here also PMA treatment resulted in an  100-fold increase in enzyme activity in the clones tested. Clearly, the enzyme activities in serum-free condi- tioned media towards the chromogenic substrates S-2288 and S-2444 were related to the levels of sergly- cin, MMP-9 and uPA mRNA expression in the MDCK-transfected cells. The clone with the highest levels of serglycin, MMP-9 and uPA mRNA and level of HisTrap isolated 35 S sulfate-labelled serglycin, and amount of serglycin core protein detected by western blotting, in conditioned media, i.e. clone 1–10, also had the highest secretion of the enzymes analysed with chromomogenic substrates, both basal and after PMA treatment. To investigate the nature of the enzyme activities measured, serum-free conditioned media were incuba- ted in both the absence and presence of the enzyme inhibitors amiloride and Pefabloc. The enzyme activity recognizing the substrate S-2288 was inhibited  90% in the presence of 2 mm Pefabloc, demonstrating that this activity is of a serine protease nature (Table 1). Furthermore, an inhibition of  70% of the enzyme activity was observed in the presence of 2 mm amilo- ride, indicating that a major part of the serine protease activity measured with S-2288 is due to plasminogen activators (PAs). To investigate whether the activity recognizing the substrate S-2444 is indeed uPA, we made use of the inhibitor amiloride, considered to be a specific inhibitor of uPA [19]. When serum-free condi- tioned media from the different clones were incubated in the presence of 2 mm amiloride, the activities were inhibited  95%, demonstrating that this activity is of uPA nature (Table 2). A B Fig. 5. Analysis of enzyme activities in serglycin-transfected MDCK clones using the chromogenic substrate S-2288. Confluent MDCK clones were cultured for 24 h under serum-free conditions in both absence and presence of PMA. Cell fractions and conditioned media were harvested and analysed for enzyme activities by using the chromogenic substrate S-2288. Each point represents the mean and standard deviation of measurement on material from three sep- arate wells. (A) Unstimulated clones. (B) PMA-stimulated clones. The S-2288 activity assay was repeated independently more than three times, and the result shown is typical of all the replicate experiments. Note the difference in the scales of the vertical axis for the unstimulated and PMA-stimulated clones. Serglycin and proteases L. Zernichow et al. 540 FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS Gelatin and plasminogen–gelatin zymography To further investigate the relationship between sergly- cin and protease secretion in MDCK clones, the possible presence of gelatinases in the serum-free con- ditioned media of the different clones was investigated by gelatin zymography. The rationale for measuring gelatinase activities is that these enzymes are known to interact with PGs [20,21]. No gelatinolytic bands could be detected in the serum-free conditioned media of any of the clones tes- ted (Fig. 7A). In contrast, when the cells were treated with PMA, several gelatinolytic bands were detected (Fig. 7B). By comparing the gelatinolytic bands with those of MMP-9 and MMP-2 standards, it is likely that the  225 and 92 kDa gelatinolytic bands are dimeric and monomeric MMP-9, respectively [22]. The highest degree of gelatinolytic activity was evident in clone 1–10, although no particular difference in inten- sity of the 92 kDa (proform) band could be demon- strated between the different clones. However, the  78 kDa band, probably an active form, had higher intensity in media from clone 1–10 than from 1–7 and the mock transfected clone. The  127 kDa band observed for clone 1–10 may be a complex of mono- meric MMP-9 with neutrophil gelatinase-associated lipocalin (NGAL) [23]. A B Fig. 6. Analysis of enzyme activities in serglycin-transfected MDCK clones using the chromogenic substrate S-2444. Confluent MDCK clones were cultured for 24 h under serum-free conditions in both absence and presence of PMA. Cell fractions and conditioned media were harvested and analysed for enzyme activities by using the chromogenic substrate S-2444. Each point represents the mean and standard deviation of measurement on material from three sep- arate wells. (A) Unstimulated clones. (B) PMA-stimulated clones. The S-2444 activity assay was repeated independently more than three times, and the result shown is typical of all the replicate experiments. Note the difference in the scales of the vertical axis for the unstimulated and PMA-stimulated clones. Table 1. Effect of amiloride and Pefabloc on serine protease activit- ies in transfected MDCK clones. Serum-free conditioned media from unstimulated cells were harvested and analysed for enzyme activities using the chromogenic substrate S-2288 in both the absence and presence of amiloride and Pefabloc, both at a final concentration of 2 m M. The results are calculated as percentages of controls. Each value represents the mean ± SD of measurement made on three independent wells. The assay was repeated inde- pendently three times, and the result shown is typical of the repli- cate experiments. Inhibitor 1–7 % remaining activity 1–10 % remaining activity Mock % remaining activity Control 100 100 100 Pefabloc 14 ± 5 10 ± 3 12 ± 4 Amiloride 27 ± 6 35 ± 2 29 ± 2 Table 2. Effect of amiloride on PA activities in transfected MDCK clones. Serum-free conditioned media from unstimulated cells were harvested and analysed for enzyme activities using the chromo- genic substrate S-2444 in both the absence and presence of amilo- ride (2 m M final concentration). The results are calculated as percentages of controls. Each value represents the mean ± SD of measurement made on three independent wells. The assay was repeated independently three times, and the result shown is typical of the replicate experiments. Inhibitor Clone 1–7 % remaining activity Clone 1–10 % remaining activity Mock % remaining activity Control 100 ± 6 100 ± 6 100 ± 3 Amiloride 0 ± 3 9 ± 4 7 ± 2 L. Zernichow et al. Serglycin and proteases FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS 541 The ability to degrade gelatin is not a unique prop- erty of MMPs. The presence of MMP activity in gela- tin zymography of serum-free conditioned media from the PMA-treated clones was verified by using 100 nm galardin, an inhibitor of MMPs [24]. After incubation with galardin, the gelatinolytic bands shown in Fig. 7B were abolished (not shown), indicating that these gela- tinolytic bands were MMPs. The possible presence of PA activity was also investi- gated using plasminogen–gelatin zymography. Gelatin gels run without plasminogen were used as controls against gels containing plasminogen and gelatin. Indeed, when plasminogen was incorporated into the gels, all the clones displayed PA activity in the 55 kDa region, the highest activity again being evident in the clone with the highest mRNA levels for serglycin, MMP-9 and uPA, i.e. clone 1–10 (Fig. 7C). Here also PMA treat- ment resulted in elevated enzyme activities, and two additional bands with molecular masses of  67 and  35 kDa appeared in the zymogram (Fig. 7D). Upon dilution of media from PMA-stimulated clones, 1–10 showed highest activity (not shown). The PA activity in serum-free conditioned media from unstimulated clones was shown to be uPA, because the  55 kDa band was abolished when including 2 mm amiloride in all incubation steps after gel electrophoresis. In zymograms of serum-free conditioned media from PMA-treated cells, both the  55 and  35 kDa bands were abolished in the presence of amiloride, whereas the intensity of the  67 kDa band was unaltered. The identity of the  67 kDa band is unknown. Western blotting of MMP-9 In an attempt to identify the nature of the gelatinolytic bands, we performed western blotting using a MMP-9 antibody (Fig. 8). Owing to a lack of canine MMP-9 antibody we used a rabbit polyclonal antibody to human MMP-9. As in gelatin zymography, no bands were visualized by western blotting of serum-free con- ditioned media from unstimulated clones (not shown). From the western blot of media from PMA-treated clones in Fig. 8, the presence of MMP-9 could be dem- onstrated. As for the zymography, there was no partic- ular difference in intensity of the MMP-9 monomer (92 kDa) band between the different clones. The rela- tive differences in intensity of the  67 kDa band in the different lanes in Fig. 8 are similar to those of the  78 kDa band in Fig. 7B. These bands may or may not represent the same protein, as the western blotting was performed under reducing conditions, whereas zymography was not. Discussion Human serglycin has been stably transfected into MDCK cells. The results presented show related levels of serglycin and MMP-9 and uPA, both at mRNA and protein levels. In Figs 1, 3 and 8 it can be seen that clone 1–10 has the highest level of serglycin, whereas clone 1–7 has the lowest level. The levels of 35 S sulfate incorporation were not particularly high in the clone with the highest serglycin mRNA expression, 1-7 1-10 Mock 1-7 1-10 Mock 1-7 1-10 Mock 1-7 1-10 Mock C D A B M r (kDa) ~ 200 ~ 127 ~ 92 ~ 78 ~ 67 ~ 55 ~ 37 Fig. 7. Zymograms of serum-free condi- tioned media from serglycin-transfected MDCK clones. Confluent MDCK clones were cultured for 24 h under serum-free conditions in both absence and presence of PMA. Conditioned media were harvested and analysed by zymography. (A) Gelatin zymography. Media from unstimulated clones. (B) Gelatin zymography. Media from PMA-stimulated clones. (C) Plasminogen– gelatin zymography. Media from unstimu- lated clones. (D) Plasminogen–gelatin zymography. Media from PMA-stimulated clones. The data shown are from single experiments that were repeated more than three times with the same results. Serglycin and proteases L. Zernichow et al. 542 FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS level of HisTrap isolated 35 S sulfate-labelled serglycin, and the amount of serglycin core protein detected by western blotting. Figure 3 clearly illustrate that high serglycin levels do not necessarily translate into high levels of 35 S-labelled macromolecules expressed. This indicates that the higher release of proteases in clone 1–10 is related to the biosynthesis and release of ser- glycin, and not the endogenous PGs. This raises inter- esting questions concerning the functions of serglycin in intracellular compartments. Our results suggest that the presence and level of serglycin could be important for the secretion of different types of proteases. Fur- thermore, the introduction of serglycin into a cell type not normally expressing this PG, changes the levels of endogenous protease secretion. We have previously shown increased secretion of PGs, which is mainly due to increased secretion of serglycin, in monocytes and macrophages after PMA stimulation [9,10]. An increase in PG secretion in monocytes and macrophages has also been observed after stimulation with lipopolysaccharide and gamma interferon, suggesting that increased serglycin secretion is linked to inflammatory reactions [10]. The biological functions linked to the release of serglycin from mono- cytes and macrophages have not been outlined in any great detail, but it has been suggested that serglycin might be important for the binding and release of important inflammatory molecules, such as chemokines [11]. It has also recently been shown that abrogation of PG biosynthesis in the murine macrophage cell line J774 resulted in a decrease in MMP-9 and uPA secre- tion [13]. It therefore seems as though further progress in studies on the biological functions of serglycin will depend, to a certain extent, on a more thorough understanding of interactions with partner molecules. It will furthermore be important to study serglycin secretion in different cell types. The processes and regulation leading to the formation of serglycin- containing granules, secretory or storage type, will probably differ to a large extent between different ser- glycin-expressing cells. The transfected MDCK cells generated here can be used as a model system to study possible relations between serglycin and different partner molecules. The coordinated levels of serglycin and proteases are important in relation to those cells already known to express serglycin. These include haematopoietic cells, such as mast cells, monocytes and macrophages, plate- lets and also endothelial cells and pancreatic acinar cells [4]. All these cells have granules which contain a large variety of serglycin-binding molecules, including histamine, chymases, gelatinase, granzymes, platelet factor 4, lactoferrin and procarboxypeptidase. With the established MDCK clones we are now able to address questions concerning regulation of serglycin release and interactions with different partner mole- cules. It is of interest to note that histamine, which is an important partner molecule for heparin PG in the mast cell granules, is also important for the genesis of gran- ules. Inactivation of the gene encoding histidine decarboxylase, the enzyme converting histidine to histamine, resulted in reduced storage of PG and pro- teases in the granules [25]. It therefore seems as though there is cross-talk between the different granule com- ponents during granule formation, and that lack of one important component has serious consequences for this process, and will also affect the amount of partner molecules sorted to such granules. Our study shows that introduction of serglycin to MDCK cells leads to changes in secretion levels of proteases, via as yet undefined mechanisms, regulated in relation to the serglycin level. This relationship between the levels of serglycin and protease levels could, accordingly, be in support of cross-talk regulatory mechanisms. The fate of serglycin released from different types of immune cells has not been studied to any great extent. Fig. 8. Western blot of MMP-9 in serglycin-transfected MDCK clones. Serum-free conditioned media from PMA-stimulated MDCK clones were subjected to SDS ⁄ PAGE followed by western blotting using a rabbit polyclonal antibody to human MMP-9. The data shown is from a single experiment that was repeated three times with the same results. L. Zernichow et al. Serglycin and proteases FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS 543 It has been shown that serglycin may bind to CD44, and thereby participate in cell–cell interactions [26], and that it can participate in the delivery of perforin to target cells [27]. Furthermore, it has been shown that serglycin isolated from macrophages is not degra- ded when it is added back to fresh cultures of macro- phages, suggesting extracellular functions after release [28]. It has also been shown that serglycin may bind covalently to a fraction of the MMP-9 secreted from the monocyte cell line THP-1 [29]. This association was shown to alter the biochemical properties of the enzyme. There are several possibilities for serglycin to inter- act with other secreted components, such as enzymes, growth factors or cytokines, and modulate their activ- ities [4]. Hence, both the generation of secretory com- plexes during biosynthesis and granule formation and interactions between secreted components, are proces- ses in which serglycin is most probably an important component, worthy of more detailed study. Experimental procedures Cell culture and transfection Cell culture reagents were purchased from Sigma (St. Louis, MO), unless otherwise stated. MDCK epithelial cells (ATCC, Manassas, VA) were cultured at 37 °C, in 5% CO 2 , in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mml-glutamine, 50 unitsÆmL )1 penicillin, 50 lgÆmL )1 streptomycin and 5% (v ⁄ v) heat-inactivated (56 °C for 30 min) fetal bovine serum (FBS). The cell cul- tures were checked for Mycoplasma infection with Myco- Alert mycoplasma detection assay (Cambrex, Rockland, ME) in routine. Before each experiment the cells were grown to confluency (4 days). Triplicate cultures were used for all experiments. The pcDNA3.1(–) ⁄ Myc-His A vector (Invitrogen Life Technologies, Carlsbad, CA) was used to generate the ser- glycin–His-tag expression vector. To obtain inframe transla- tion into the His sequence, serglycin cDNA was amplified from human serglycin cDNA [9] by PCR with the following primers: upper primer (XbaI): 5¢-CTCTAGAGTCATG ATGCAGAAGCTACTCA-3¢ and lower primer (EcoRI): 5¢-CGAATTCCTTCTAATCCATGTTGACCCAA-3¢. The obtained PCR product was cloned into a pCRII vector with the use of TA Cloning Kit (Invitrogen Life Technol- ogies), cut out with XbaI and EcoRI, both purchased from Promega (Madison, WI), and ligated into XbaI ⁄ EcoRI restricted pcDNA3.1(–) ⁄ Myc-His vector. Correct inframe cloning of the insert was verified by sequencing. Vectors encoding the serglycin–His-tag [pcDNA3.1(serglycin) ⁄ Myc- His] were stably transfected into MDCK cells with the DNA-calcium phosphate procedure as described previously [30,31]. Two days after transfection, cells were given select- ive medium [geneticin (G-418), 500 lgÆmL )1 ]. After two weeks with selective medium, stably transfected single col- onies were picked with cloning rings to obtain homogenous subcell lines stably expressing the serglycin construct. Transfectants with vector without serglycin insert (pcDNA3.1(–) ⁄ Myc-His) were generated as negative con- trols. Preparation and analysis of RNA For northern blot analyses, total RNA was extracted from confluent cells using Trizol Reagent (Invitrogen Life Tech- nologies). Parallel cell cultures were treated with 50 ngÆmL )1 PMA for 24 h prior to RNA extraction. DNA fragments used for generation of serglycin and 36B4 probes were digested and purified from vectors containing human serglycin [pcDNA3.1(serglycin) ⁄ Myc-His] and murine acidic ribosomal phosphoprotein PO (36B4). Partial cDNAs for canine MMP-9 and uPA were amplified by RT-PCR using total RNA from PMA-stimulated MDCK cells, followed by PCR using PfuUltra (Stratagene, La Jolla, CA) and cloned into a pPCR-Sript Amp SK(+) vector (Stratagene), as described previously [32]. The following primers were used: For the 5¢-human serglycin: (5¢-CTCTAGAGTCAT GATGCAGAAGCTACTCA-3¢) and 3 ¢-human serglycin: (5¢-CGAATTCCTTCTAATCCATGTTGACCCAA-3¢). For the 5¢-canine MMP-9: (5¢-TTAGGGAGCACGGAGATG GGTAT-3¢) and 3¢-canine MMP-9: (5¢-GTTGGGCAGA AGCCGTAGAGTTT-3¢), and for the 5¢-canine uPA: (5¢-GTCAGCGCCACACACTGCTT-3¢) and 3¢-canine uPA: (5¢-GCCTTGGGTAGAGCAGACCA-3¢). Correct amplifi- cation was verified by sequencing of the inserts. Fragments containing the partial MMP-9 and uPA cDNAs were ampli- fied with PCR using the vectors as template to generate DNA used for labelling. Total RNA samples (20 lgÆwell )1 ) were separated by electrophoresis in 1% agarose gels and transferred to nylon membranes. Probes were generated by radiolabelling of cDNAs with 32 P-labelled dCTP[aP] (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA) using Megaprime DNA labelling systems (Amersham Bio- sciences, Little Chalfont, Bucks, UK). After hybridization, the nylon membranes were washed and further exposed to autoradiography films for detection. Proteoglycan analyses Radiolabelling of macromolecules For radiolabelling of macromolecules, confluent cells were changed to sulfate-free medium (RPMI 1640) (GibcoBRL Life Technologies, Paisley, UK), containing 2 mml-gluta- mine, 50 unitsÆmL )1 penicillin, 50 lgÆmL )1 streptomycin and 2% (v ⁄ v) FBS, and exposed to 35 S sulfate (100 lCiÆmL )1 ) (Perkin Elmer Life and Analytical Sciences). Serglycin and proteases L. Zernichow et al. 544 FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS Parallel cell cultures were treated with 50 ngÆmL )1 PMA during the radiolabelling. After 24 h incubation, cell frac- tions and conditioned media were harvested. Cell layers were recovered by solubilization in a solution containing 4 m guanidine hydrochloride and 50 mm sodium acetate (pH 6.0). Loose cells were separated from the conditioned media by centrifugation at 400 g for 3 min. Unincorporated 35 S sulfate was removed from cell fractions and media by Sephadex G-50 Fine gel chromatography. Radioactivity was measured using a liquid scintillation counter. Isolation of His-tagged serglycin Cell fractions and conditioned media from cells exposed to 35 S sulfate for 24 h were buffer exchanged to binding buffer containing 20 mm phosphate, 1 m NaCl, 8 m urea and 20 mm imidazole (pH 8.0) by Sephadex G-50 Fine gel chro- matography. In addition, unincorporated 35 S sulfate was removed during this step. The samples were further applied to a 1 mL HisTrap HP column (Amersham Biosciences), pre-equilibrated with binding buffer. After a washing step, samples were eluted with a solution containing 20 mm phosphate, 1 m NaCl, 8 m urea and 500 mm imidazole (pH 8.0). Fractions containing radioactivity were pooled, and desalted on PD-10 desalting columns (Amersham Bio- sciences). The samples were further treated with NaOH to release GAGs from the serglycin core protein, as described elsewhere [33]. After desalting, the samples were subjected to HNO 2 and cABC treatment, as described previously [16,32]. cABC (from Proteus vulgaris) was purchased from Seikagaku Corporation (Tokyo, Japan). The amounts of HS and CS, degraded by HNO 2 and cABC treatment, respectively, were calculated from the proportions of degra- dation products by gel chromatography on Superose 6 (Amersham Biosciences), using 1 m NaCl as the mobile phase. The elution profiles were monitored by liquid scintil- lation counting. Western blotting of serglycin Conditioned media from the clones were desalted against Milli-Q water by Sephadex G-50 Fine gel chromatogra- phy. After freeze-drying, the samples were dissolved in a small volume of Milli-Q water and treated with cABC at 37 °C for 2 h to release the serglycin core protein. The cABC treatment was performed in the presence of the serine protease inhibitor Pefabloc SC (Fluka, Buchs, Switzerland) and the cysteine protease inhibitor N-ethyl- maleimide (Sigma), both at a 2 mm final concentration. SDS ⁄ PAGE and western blotting were performed accord- ing to standard procedures. Briefly, samples were treated with 2-mercaptoethanol and separated on 15% polyacryl- amide gels. The proteins in the gels were transferred to nitrocellulose membranes. After blocking with 5% skim- med milk, the membranes were incubated with an affinity-purified rabbit polyclonal antibody to human serglycin, (kindly provided by C. U. Niemann and N. Borregaard, Rigshospitalet, Department of Haemato- logy, Copenhagen, Denmark). A mouse monoclonal anti- body to the His-tag (Roche Diagnostics, Mannheim, Germany) was also used to detect the serglycin core pro- tein. Bound antibodies were detected using peroxidase- linked secondary antibodies, followed by chemilumines- cence detection Molecular masses were estimated using prestained SDS⁄ PAGE standards (Amersham Biosciences). Enzyme analyses For enzyme analyses, confluent cells were changed to serum-free medium (DMEM), containing 2 mml-gluta- mine, 50 unitsÆmL )1 penicillin and 50 lgÆmL )1 streptomy- cin. Parallel cell cultures were treated with 50 ngÆmL )1 PMA. After 24 h incubation, cell fractions and serum-free conditioned media were harvested. Cell layers were recov- ered by solubilization in a solution containing 0.25% (v ⁄ v) Triton X-100 and 10 mm CaCl 2 . Loose cells were separated from the serum-free conditioned media by centrifugation at 400 g for 3 min. Chromogenic substrates Aliquots (100 lL) of the cell fractions and serum-free con- ditioned media were analysed for enzyme activity using the chromogenic substrates H-d-Ile-Pro-Arg-pNA (S-2288) and pyro-Glu-Gly-Arg-pNA (S-2444), essentially as suggested by the manufacturer (Chromogenix, Milan, Italy). Experi- ments were performed at room temperature in both the absence and presence of the enzyme inhibitors amiloride (Sigma), a selective inhibitor of uPA, and Pefabloc SC, an inhibitor of serine proteases, both used at 2 mm final con- centration in analyses of serum-free conditioned media from unstimulated cells, as previously described [13]. Absorbance was read at 405 nm. In the analyses performed, we observed linearity up to absorbance values of  1.5. To assure that the detected activities were within the linearity of the assay, absorption measurements were performed regularly up to 1 h for material from PMA-stimulated cells and up to 24 h for material from unstimulated cells. The activities, measured as changes in absorption with time, were calculated from the initial linear parts of the different curves. As the same amount of either cell fraction or condi- tioned medium was used for the different clones, the result of the activity measurements is presented as the change in absorption at 405 nm h -1 (DA 405nm ⁄ h ). Gelatin and plasminogen–gelatin zymography MMP and PA activities were determined by gelatin and plasminogen–gelatin zymography, respectively. Briefly, L. Zernichow et al. Serglycin and proteases FEBS Journal 273 (2006) 536–547 ª 2006 The Authors Journal compilation ª 2006 FEBS 545 [...]... 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Secretion of proteases in serglycin transfected Madin–Darby canine kidney cells Lillian Zernichow 1 , Knut T. Dalen 1 , Kristian Prydz 2 , Jan-Olof Winberg 3 and Svein O. Kolset 1 1. sergly- cin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in. condi- tions. Results Transfection of serglycin Clones of MDCK cells with serglycin His-tag were obtained, and the levels of serglycin mRNA deter- mined using northern blotting. Cell clones transfected with the