calpastatin system in human hemopoietic cell lines

Archives of Biochemistry and Biophysics 456 (2006) 48–57 www.elsevier.com/locate/yabbi

Characterization of the calpain/calpastatin system in human hemopoietic cell lines Roberto Stifanese a, Monica Averna a, Franca Salamino a, Claudia Cantoni b,c, Maria Cristina Mingari d,e, Carola Prato b, Sandro Pontremoli a, Edon Melloni a,d,¤ a

Department of Experimental Medicine (DIMES), Section of Biochemistry, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy b Department of Experimental Medicine (DIMES), Section of General Pathology and Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy c Giannina Gaslini Institute, Largo G. Gaslini, Genova, Italy d Department of Oncology, Biology and Genetic (DOBIG), University of Genova, Largo R. Benzi 10, 16132 Genova, Italy e National Institute for Cancer research, Largo R. Benzi 10, 16132 Genova, Italy Received 28 July 2006, and in revised form 15 September 2006 Available online 13 October 2006

Abstract As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689–696], a p94-like calpain was now established to be present in six diVerent human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional - and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identiWed by a speciWc mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 M; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells. © 2006 Elsevier Inc. All rights reserved. Keywords: Calcium; Proteolysis; Calpain; Calpastatin; Enzyme regulation; Hemopoietic cell lines

It is generally accepted that calpain, a calcium-requiring cysteine protease, plays an important role in various physiological functions, such as signal transduction, cell growth, diVerentiation and secretion, as well as cytoskeletal reorganization and cell motility [1–9]. In addition to these physiological functions a number of evidences has indicated a relationship between an alteration in the calpain/calpastatin system or mutations in the calpain genes and human

*

Corresponding author. Fax: +39 010 518 343. E-mail address: [email protected] (E. Melloni).

0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.09.022

disease states such as Alzheimer disease, diabetes and muscle dystrophies [10–16]. Among the various calcium-regulated signalling pathways involving calpain activity [for review see 2], it must also be included that concerning the sequential processes leading to the activation of the immunoresponses by competent cells [17–21]. Although 15 gene products of the calpain family are presently known [2], two common calpain forms, - and mcalpain, are constitutively expressed in all mammalian cells diVering at the functional level in the concentration of calcium required for half maximal activity [22–24]. In

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

addition, these two protease isoforms are very similar in terms of speciWcity, mechanism of activation, inhibition by the endogenous protein inhibitor calpastatin, and limited autolysis leading to an increase in Ca2+ sensitivity [25,26]. We have recently demonstrated that peripheral blood mononuclear cells (PBMC)1 contain, in addition to - and m-calpain, a third isoform identiWed as a p94-like calpain, characterized by distinct catalytic and regulatory properties [27]. The Ca2+ requirement of this protease for half maximal activity is in the range of 20–30 M, a concentration in between that required for the two conventional protease forms (- and m-calpain). Activation by Ca2+ does not involve limited autolytic cleavage, and the inhibitory eYciency of calpastatin towards this isoform is signiWcantly lower. At a molecular level, this new calpain form derives from an alternative splicing of CANP 3 gene; a similar splice-variant of the same gene has been shown to occur in rat and human lens [28–30]. The human lymphocyte calpain 3 like (p94-like) protease has lost both the IS1 and the nuclear import sequences contained in IS2. The loss of IS1, an amino acid stretch inserted in the DII catalytic domain, explains the resistance of the protease to the intra-autoproteolytic cleavage [13]. Thus, in spite of an overall structural identity, this calpain isoform displays distinct functional properties with respect to - and m-calpain, being less sensitive to calpastatin inhibition and susceptible to a reversible activation process. These properties could be related to diVerent roles played by this protease in speciWc intracellular sites. Furthermore, PCR analysis revealed that in addition to PBMC, the p94 calpain isoform is expressed in four hemopoietic cell lines being however undetectable in isolated human neutrophils and erythrocytes [27]. On the basis of these observations it seemed interesting to analyse the calpain isoform(s) expressed in these four cell lines and in LCL 721.221 and H9 cells, all resembling the diVerent peripheral blood cell types. The data herewith presented provide the Wrst evidence that in white cells a p94-like isoform is present in signiWcantly larger amounts than that of - and m-calpain. To establish if the function of this protease could be associated to the general context of these cell activities, all cell lines were stimulated with PMA, a well-known activating agent for these cells, and with the Ca2+-ionophore A23187 to directly induce calpain activation through elevation of intracellular Ca2+ concentration [31–33]. In both conditions, the loss of protease activity (consumption), generally attributed to an autolytic process following enzyme activation [1–4] and its subcellular distribution were evaluated [34–36]. The results obtained indicate that stimulation of these cells is accompanied by diVerent patterns of p94-like calpain activation and intracellular redistribution, suggesting a speciWc role for this protease in the biological functions of circulating white cells.

1 Abbreviations used: PBMC, peripheral blood mononuclear cells; PMA, Phorbol 12-myristate 13-acetate.

49

Materials and methods Materials Source 15Q resin, Phenyl-Sepharose resin, CNBr-activated Sepharose 4 fast Xow and HRP-linked antimouse secondary antibody were purchased from GE Healthcare. Ca2+-ionophore A23187, PMA (Phorbol 12myristate 13-acetate) and casein were obtained from Sigma–Aldrich. Human erythrocyte calpain was puriWed as reported in [26]. Monoclonal antibodies Monoclonal anti--calpain antibody (mAb 56.3) was prepared as described in [36]. Monoclonal anti-calpastatin antibody (mAb 35.23) was produced as indicated in [37]. Monoclonal anti-m-calpain (anti Domain III/IV) was purchased from Sigma–Aldrich. Monoclonal anti-p94-calpain antibody NCL-CALP-2C4 was obtained from Novocastra Laboratories Ltd. The mAb anti-p94-antibody was solubilized in 1 ml of 20 mM phosphate buVer, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA. The solution was loaded on a column containing human erythrocyte calpain immobilized on a Sepharose (1 ml resin) [38], previously equilibrated with the same buVered saline solution. The unabsorbed protein was used as a source of anti-p94 antibody. Cell culture The following human cell lines were utilized: 2493 and LCL721.221 (B lymphoblastoid cell lines), Jurkat and H9 (T cell leukaemia), NK-92 (NK cell line) [39], MM6 (promyelocytic leukaemia). All these cell lines were cultured at 37 °C (5% CO2) with RPMI 1640 (Sigma–Aldrich) growth medium containing 10% foetal calf serum, 10 U/ml penicillin (Sigma– Aldrich), 100 g/ml streptomycin (Sigma–Aldrich) and 4 mM L-glutamine. NK-92 cell line was cultured as described above in the presence of 100 U/ ml of Interleukin-2 (IL-2). Animals Normotensive Milan Rat Strain were obtained from Carlo Erba Farmaceutica, Milano (Italy).

Methods Determination of the calpain/calpastatin system in cell lines Cells (50 £ 106) were centrifuged at 300g for 5 min, washed three times with Na/Pi solution and lysed by three cycles of freezing and thawing in 1 ml of 50 mM sodium borate, pH 7.5, containing 1 mM EDTA and 0.5 mM 2-mercaptoethanol. Membranes were discarded by centrifugation at 60,000g for 10 min at 4 °C. In order to separate calpastatin from calpain [27], equal amount of the clear supernatants of each clone (3 mg of protein) were submitted to a hydrophobic chromathography onto a PhenylSepharose column (1.2 £ 4 cm), previously equilibrated in 50 mM sodium borate, pH 7.5, containing 0.1 mM EDTA, 0.5 mM 2-mercaptoethanol and 0.3 M NaCl. The Xow rate was 0.5 ml/min. Total calpain activity was eluted from Phenyl-Sepharose resin in a single step with 50 mM sodium borate, pH 7.5, containing 0.1 mM EDTA and 0.5 mM 2-mercaptoethanol without NaCl. Fractions of 1 ml were collected. Calpain activity was assayed using 50 l of each fraction and denatured human globin as substrate as reported in [40]. Fractions containing calpain activity were pooled and submitted to an ion-exchange chromatography onto a Source 15Q column (0.8 £ 2 cm) previously equilibrated in 50 mM sodium borate, pH 7.5, containing 0.1 mM EDTA and 0.5 mM 2-mercaptoethanol, setting a very reproducible procedure using a FPLC system (GE Healthcare). Proteins were eluted with a linear gradient from 0 to 0.5 M NaCl. The Xow rate was 1 ml/min and fractions of 1 ml were collected. Aliquots (50 l) of each fraction were utilized to assay calpain activity [40]. Levels of activity of each calpain isoform were calculated from the area underlying their activity peaks. One unit of calpain activity is deWned as the amount required to cause the release of 1 mol of free –NH2 groups per minute under the speciWed conditions [40]. Unabsorbed proteins eluted from Phenyl-Sepharose resin were collected in fractions of 1 ml. Aliquots (50 l)

50

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

of these fractions were heated for 1 min at 100 °C and utilized to assay calpastatin activity using denatured human globin as substrate and the single human erythrocyte calpain [2], which has been shown to be the calpain isoform more sensitive to calpastatin inhibition [41]. The calpastatin levels present in each cell line were calculated from the area underlying the calpastatin activity peak. One unit of calpastatin activity is the amount required to inhibit 1 U of calpain. Protein content was determined with the Bradford method [42]. Determination of the calpain isoforms present in human erythrocyte and rat brain Human erythrocyte (50 ml of packed cells) were isolated and lysed as reported in [26]. Freshly collected rat brain (2 g of fresh tissue) was lysed as reported in [41]. Equal amounts of these clear supernatants (3 mg of protein) were then submitted to the same chromatographic steps and in the conditions previously reported in this Section. Calpain activity was assayed as described above and in [40]. Calcium requirement of MM6 calpains following exposure to calcium ions PuriWed calpains (5 U each) isolated from MM6 cell line as previously described were incubated at 25 °C in the presence of 100 M (peak 1 and 2) or 500 M Ca2+ (peak 3) in a Wnal volume of 0.6 ml for 1 min. EDTA was then added to reach a Wnal concentration of 100 M over that of [Ca2+]. Aliquots of 25 l were utilized to assay calpain activity in the presence of increasing [Ca2+] from 0 to 100 M for peak 1 and 2 and from 0 to 1 mM for peak 3 [25]. Calpain levels in stimulated cells Cells (50 £ 106 for each experiment) were diluted in 50 ml of RPMI 1640 growth medium containing 10% foetal calf serum, 10 U/ml penicillin, 100 g/ml streptomycin and 4 mM L-glutamine and incubated for 30 min at 37 °C and 5% CO2 in the presence of 0.1 M Ca2+-ionophore A23187 and 1 mM CaCl2. Alternatively, Ca2+-ionophore A23187 and 1 mM CaCl2 were replaced with 100 ng/ml of PMA. Cells were centrifuged at 300g for 5 min and lysed by freezing and thawing in 1 ml of sodium borate, pH 7.5, containing 1 mM EDTA and 0.5 mM 2-mercaptoethanol. Calpain isozymes were separated as indicated previously in this Section. The levels of calpain isoforms were calculated from the area underlying each activity peak. Calpain detection by Western blot analysis Equal amounts of each calpain isoform (0.5 g), puriWed as previously, described, were subjected to SDS–PAGE carried out as reported in [43]. After electrophoresis, proteins were transferred to nitrocellulose sheets as indicated in [44]. The sheets were then incubated with the appropriate monoclonal antibody as speciWed elsewhere in the manuscript. The immunoreactive material was detected by a HRP-linked secondary antibody [45] and developed with an ECL® detection system (GE Healthcare). Zymogram analysis Zymogram analysis was carried out as described in [46,47] following the modiWcations reported in [48]. BrieXy, equal amounts (1 g) of each calpain isoform isolated as previously reported from MM6 cell line, were diluted in the absence or in the presence of 1 g of puriWed MM6 calpastatin [41] in 60 l (Wnal volume) of 0.1 M Tris/HCl, pH 6.8, containing 20% glycerol, 10 mM 2-mercaptoethanol and 10 mM EDTA and loaded on a 10% polyacrylamide gel, containing 1 mg/ml casein. The electrophoretic run was carried out in 25 mM Tris/HCl, pH 8.0, containing 125 mM glycine, 1 mM EDTA and 10 mM 2-mercaptoethanol for 2 h at 4 °C and 125 V. At the end of the electrophoretic run, the gels were incubated overnight at 25 °C in the presence of 10 mM CaCl2 under gentle shaking. The gels were stained with Coomassie brilliant blue. Calpain immobilization The puriWed p94-like calpain (0.5 g diluted in 50 l of 50 mM sodium borate, pH 7.5, containing 0.1 mM EDTA) was spotted following the procedure reported in [35] on a nitrocellulose sheet (0.5 £ 0.5 cm; Bio-Rad Biosciences) and left for 15 min at 4 °C in a humidiWed chamber. The sheet

was then washed with 1 mM EDTA and saturated with 5% nonfat skimmed milk powder. The nitrocellulose sheet was then incubated in 0.1 ml sodium borate, pH 7.5, containing the speciWc calpain ligands reported elsewhere in the manuscript. The mixtures were incubated at 4 °C for 30 min and mAb 56.3 (0.2 g) was then added. Calpain was detected using a HRP-linked secondary antibody [45] developed with an ECL® detection system (GE Healthcare). Immunoreactive material was detected by subjecting the probed nitrocellulose sheets to autoradiography and quantiWed with a Shimadzu CS9000 densitometer using a Wxed wavelength of 590 nm as reported in [35]. Confocal microscopy Control or stimulated cells (2 £ 10 6 cells for each experiment) were diluted in 10 ml of RPMI 1640 growth medium containing 10% foetal calf serum, 10 U/ml penicillin, 100 g/ml streptomycin and 4 mM L-glutamine. Cells were incubated for 30 min at 37 °C and 5% CO2 in the absence or in the presence of the stimuli indicated elsewhere. Cells were then centrifuged at 300g for 5 min and washed three times with 0.15 M NaCl solution containing 20 mM phosphate buVer, pH 7.5. Cells were Wxed and permeabilized with Triton/paraformaldehyde method [34]. Calpains were detected by confocal microscopy as described in [35,49], using anti-calpain mAb 56.3 or anti-m-calpain mAb as primary antibody and a Xuorescein isothiocyanate-conjugated sheep anti-mouse IgG as secondary antibody (GE Healthcare). The excitation/emission wavelengths for Xuorescein-labeled antibodies were, respectively, of 488/522 nm. Fluorescence was quantiWed with Laser Pix Software (BioRad Bioscience) as reported in [35].

Results Calpain isoforms present in human hemopoietic cell lines In order to investigate the pattern of calpain expression in diVerent hemopoietic cell lines, cells were lysed and calpain isoforms present in cell lysates were separately isolated following the procedure previously described [27]. In all cell lines examined [2493 and LCL721.221 (B lymphoblastoid cell lines), Jurkat and H9 (T cell leukaemia), NK92 (NK cell line), MM6 (promyelocytic leukaemia)], three distinct peaks of calpain activity were identiWed each one being diVerently expressed in each cell line. A typical chromatographic separation of the three peaks, obtained from MM6 cell lysates, is shown in Fig. 1. Comparison of this elution proWle with that obtained with rat brain homogenate or with human erythrocyte lysate, revealed that peak 1 is present only in MM6 cells, peak 2 and 3 are detectable also in brain cells and peak 3 is not detectable in erythrocytes. The characterization of the calpain isoforms was accomplished on the basis of the following parameters: (1) Ca2+ concentration required for half maximal activity; (2) production of a form with a decreased Ca2+ requirement generated by autoproteolysis [2]; (3) sensitivity to calpastatin inhibition; (4) inactivation following exposure to high Ca2+ concentration, presumably due to autodegradation [50]; and (5) cross reactivity with the speciWc antibodies. The data obtained are reported in Table 1. Calpain isoforms under peak 2 and 3 showed a Ca2+ requirement typical of - and m-calpain, both forms also undergoing autolysis and increase in Ca2+ sensitivity following exposure to high [Ca2+]. On the basis of these properties and of a high sensitivity to calpastatin inhibi-

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

51

Table 2 Expression of calpain isoforms in human hemopoietic cell lines Cell linea

Calpain isoform activityb (U/mg) p94-like

MM6 NK-92 2493 LCL721.221 Jurkat H9 a

Fig. 1. IdentiWcation of the calpain isoforms expressed in MM6 cell line. MM6 cell line (50 £ 106) were cultured and lysed as previously described, human erythrocyte were isolated and lysed as reported in [26], rat brain was collected and lysed as described in [41]. Total calpain activity was isolated from the clear supernatants (3 mg of protein), following a hydrophobic chromatography on Phenyl-Sepharose column (1.2 £ 4 cm) as reported in Methods and in [27]. Fractions containing total calpain activity were collected and submitted to an ion-exchange chromatography on a Source 15Q (0.8 £ 2 cm) column as previously described. Proteins were eluted with a linear gradient from 0 to 0.5 M NaCl. The Xow rate was 1 ml/min and fractions of 1 ml were collected. Calpain activity was assayed using 50 l of each fraction and denatured human globin as substrate [40]. MM6 cell line (Wlled circles), human erythrocyte (unWlled squares), rat brain (unWlled circles).

tion it could be concluded that the two calpain isoforms present under peak 2 and 3 corresponded to the conventional - and m-calpain. The calpain isoform recovered under peak 1 showed a Ca2+ requirement for half maximum activity in the range of 30 M, an intermediate value between the concentrations required by the two conventional calpains. In addition, the absence of changes in [Ca2+] requirement and a reduced sensitivity to calpastatin inhibition are properties identical to those of the p94like calpain isolated from human PBMC [27]. Thus, this unusual calpain isoform diVers from the classical - and m-calpains, as well as from the p94 muscle speciWc calpain isoform, including most of the p94 variants [28–30,51]. As shown in Table 1, in spite of these diVerences, the loss of catalytic activity in conditions promoting protease activation was found to be a common property of all three iso-

0.65 § 0.1 0.4 § 0.05 1.72 § 0.22 1.55 § 0.18 0.78 § 0.1 1.65

-Calpain

m-Calpain

0.30 § 0.04

0.2 § 0.03 0.8 § 0.1 0.4 § 0.02

c

0.2 § 0.02 c

c

c

c

c

0.25 § 0.02

6

Cells (50 £ 10 for each clone) were collected and lysed as reported in Methods. Calpain isoforms were separated and identiWed as described in Methods and in Fig. 1. b Total calpain activity was calculated from the area of each activity peak. The values reported represent the arithmetical means § SD of four diVerent experiments. c Refers to calpain activity level below 0.01 U/mg.

forms. Additional experiments (data not shown) have indicated that all the calpain isoforms isolated from the six cell lines display identical properties to those reported in Table 1, which refers to the proteases isolated from MM6 cell line. The complete analysis of the calpain isoforms expressed in the six cell lines examined is reported in Table 2. The p94-like calpain isoform is expressed in all cell types and is also the most widely represented. The - and m-calpains are also expressed in some of the cell lines examined, although their activity is very low or almost undetectable. This characterization is conWrmed by immunoblot analysis of the calpain isoforms under the three peaks isolated as shown in Fig. 1, by means of three speciWc antibodies. As shown in Fig. 2 an anti-p94 antibody directed against the NS-region [2] absent in - and m-calpains, recognizes an 80 kD band present only in peak 1. However, the mAb 56.3 directed against domain 2 of -calpain form [35] was found to interact with very high eYciency with calpain present under peak 1 and 2. Finally, a m-calpain speciWc antibody shows cross reactivity with protein contained only in peak 3. Thus, it is possible to conclude that p94-like calpain is the typical protease isoform largely and ubiquitously expressed in these cell lines of hemopoietic origin.

Table 1 Properties of the calpain isoforms isolated from MM6 cell line Calpain peaka

[Ca2+] required for 1/2 Vmaxb (M)

[Ca2+] required for 1/2 Vmax (M) following exposure to high[Ca2+] c

Calpastatin inhibition (Ki, nM)d

Autoinactivation in the presence of high[Ca2+] (%)e

1 2 3

25–35 4–8 120–150

25–35 0.2–0.5 15–25

35 § 5.52 8.5 § 1.2 10.5 § 2.0

58 § 10 65 § 15 55 § 10

a

Calpain activity was separated in the three peaks as described in Methods and in the legend to Fig. 1. Calpain activities were assayed as described in Methods, in the presence of increasing concentrations of Ca2+, from 0 to 1 mM. The values refer to the [Ca2+] required by the calpain form to express 1/2 Vmax. c Calpains under peak 1, 2 and 3 were exposed to Ca2+ ions in the conditions described in Methods. Calpain activity was then assayed in the presence of increasing [Ca2+] from 0 to 100 M for peaks 1 and 2 and from 0 to 1 mM M for peak 3. d Calpastatin inhibition was assayed as described in Methods. The values refer to the concentration of calpastatin causing 50% inhibition. The values reported are the arithmetical mean § SD of three diVerent experiments. e Calpain isoforms (0.5 U each) were incubated in the presence of saturating Ca2+ as indicated in c. After 5 min the protease activity was assayed as reported in Methods. The values refer to the proteolytic activity lost during the incubation period. b

52

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

Fig. 2. IdentiWcation of the MM6 cell line calpain isoforms by immunoblotting. Equal amounts of each calpain isoforms (0.5 g) puriWed as previously described were subjected to SDS–PAGE [43]. After electrophoresis, proteins were transferred to nitrocellulose sheets as in [44]. The sheets were then incubated with the indicated monoclonal antibody. The immunoreactive material was detected as described in Methods and in [45]. (1) p94-like calpain; (2) -calpain; (3) m-calpain.

lines described above. As shown in Fig. 3, all cell types were found to express calpastatin at signiWcantly diVerent levels ranging from 2 U/mg in MM6 cells to 7.5 U/mg in Jurkat cells. These results reveal an additional diVerence occurring in the calpain/calpastatin system of these cell lines, and suggest that in the context of each cell function calpain activation can be controlled by calpastatin at diVerent extent, especially in cells undergoing activation. However, in all cell lines calpain activity directly assayed in crude extracts accounted for a small fraction (approximately less than 10%) of the total calpain activity (data not shown), indicating that the amount of calpastatin is suYcient to control most of calpain activity. MM6 cell line was the only exception, since approximately 20% of the total calpain activity could be assayed in crude extract of these cells, depending on the lowest level of calpastatin units present in these cells. Response of calpain to cell stimulation

Fig. 3. Levels of calpastatin activity in hemopoietic cell lines. Equal amounts of the clear supernatants (3 mg of protein), obtained from lysis of 50 £ 106 cells for each cell line as reported in Methods, were submitted to a hydrophobic chromatography on a Phenyl-Sepharose column (1.2 £ 4 cm). The Xow rate was 0.5 ml/min and unabsorbed proteins were collected in fractions of 1 ml. Aliquots (50 l) of these fractions were heated for 1 min at 100 °C and utilized to assay calpastatin activity [41]. Total calpastatin levels were calculated from the area underlying the calpastatin activity peak. The values reported are the arithmetical mean of three diVerent experiment §SD.

Levels of calpastatin activity in human hemopoietic cell lines In addition to calpain, the levels of the endogenous inhibitor protein calpastatin were determined in the six cell

In an attempt to deWne the function of the calciumdependent proteolytic system, we have explored whether calpain isoforms were responsive to cell stimulation by measuring the loss of their activity. The conventional and m-calpains, as well the p94-like calpain form, have been shown to undergo autolytic inactivation after exposure to high [Ca2+] (see Table 2). Since this process requires the formation of active calpain species, its occurrence indicates the activation of the protease [52]. To explore the occurrence of these events, we Wrst evaluated the changes in the level of calpain isoforms following stimulation of the various cell lines with PMA or with Ca2+-ionophore. PMA was selected since it is commonly used to promote activation of these cells; Ca2+-ionophore was chosen because of its ability to promote activation of calpain in response to intracellular [Ca2+] increase [53]. The results obtained are reported in Table 3. In all cell lines p94like calpain isoform as well as - and m-calpains (when present in detectable amounts), undergo a 10–15% inactivation, probably due to autolysis, with the only exception of Jurkat cells in which the disappearance of the activity of the p94-like calpain accounts for approximately 30%. This

Table 3 Loss of catalytic activity of p94-like, - and m-calpain isoforms in stimulated human hemopoietic cell lines Cell linea

MM6 NK-92 2493 LCL721.221 Jurkat H9

Calpain activity recovered following stimulation with Ca2+-ionophoreb (% of original)

Calpain activity recovered following stimulation with PMAb (% of original) p94-like

-Calpain

m-Calpain

p94-like

-Calpain

m-Calpain

85 § 6 86 § 7 88 § 6 85 § 7 64 § 8 89 § 6

95 § 4 ¥ 96 § 5 ¥ ¥ ¥

70 § 8 74 § 8 96 § 5 ¥ ¥ 78 § 6

82 § 5 80 § 6 82 § 5 65 § 6 68 § 8 83 § 5

92 § 3 ¥ nd ¥ ¥ ¥

66 § 9 70 § 8 nd ¥ ¥ 74 § 6

nd: not detectable; ¥ refers to calpain activity level below 0.01 U/mg. The values reported in this table refer to the percentage of calpain activity in respect to calpain activity measured in control cells. The values reported represent the arithmetical mean of three diVerent experiments §SD. a Cells were cultured as indicated in Methods. b Cells (50 £ 106 for each experiment) were stimulated, recovered and lysed as reported in Methods. Calpains were separated and quantiWed as reported in Methods.

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

probably occurs because in these cells this protease is the only calpain isoform present and is expressed in a signiWcant lower level. No appreciable diVerences were observed between stimulation with PMA or with Ca2+-ionophore even though a slightly more pronounced inactivation was observed with the ionophore, probably due to a larger eVect of this stimulus on the elevation of intracellular [Ca2+] [53–55].

Calpain isozyme

A

μ-

p94-like

+ 1

B

Subcellular localization of calpain in stimulated human cell lines

m-

+ 2

3

+ 4

50

Light intensity (arbitrary units)

To better deWne the response of calpain in stimulated cells, we have also examined the dynamic intracellular distribution of calpains, since this process and particularly calpain association to plasma membranes, correlates with the extent of activation as well as with the eYciency of the regulatory system [56]. Being the speciWc anti-p94 antibody poorly eVective in intracellular staining we have used for the subcellular localization of this protease the mAb 56.3 shown to be (see Fig. 2) very active in reacting also with this calpain form. The interference of -calpain was assumed to be scarcely relevant being in most cases almost undetectable. Due to the fact that calpastatin is directly involved in aVecting the subcellular localization of calpain [35,48], we have preliminarily established if p94-like calpain as well as - and mcalpain present in these cell lines were able to interact in their calcium free form with calpastatin to form a protease/inhibitor complex [48,56,57]. Using the zymography technique, that allows the separation of free protease from that associated with calpastatin and the quantiWcation of the protease activity, it was possible to establish that the p94-like calpain as well as - and m-calpain isolated from MM6 cell line associate to homologous calpastatin in the absence or at very low (physiological) Ca2+ concentrations (Fig. 4A). Identical results (data not shown) were obtained using the calpain isoforms isolated from each one of the six cell lines examined. Furthermore, we have established that, as previously demonstrated for rat brain or human erythrocyte -calpain [35], the anti -calpain antibody (mAb 56.3) recognizes with a much higher eYciency the p94-like calpain in the conformation acquired following its association with calpastatin (Fig. 4B). As shown in the dots the amount of the antibody bound to calpain increases seven to 8-fold following exposure of the protease to calpastatin, whereas the addition of Ca2+ to the mixture results almost ineVective (Fig. 4B). On the basis of this information we have studied by confocal microscopy the intracellular distribution of calpain using the anti--calpain mAb 56.3 capable to detect both the p94-like and the -calpain forms (see Figs. 2 and 4). The various patterns recorded after cell stimulation with PMA or Ca2+-ionophore are sequentially reported in Fig. 5. In panels A–F, the changes in intracellular Xuorescence intensity are shown, as revealed by the anti--calpain antibody

53

40 30 20 10 0 1

2

Control +Ca2+

3

4

+Calpastatin +Ca2+

Fig. 4. p94-like calpain/calpastatin interaction in physiological conditions. (A) Zymogram analysis of calpain isoforms and calpastatin in the absence of Ca2+. Calpain isoforms were isolated from MM6 cell line as reported in Methods and in legend to Fig. 1. Fractions containing each calpain activity peak were separately collected and concentrated by ultraWltration. Equal amounts (1 g) of each calpain isoform were submitted to zymogram analysis [46–48] in the conditions reported in Methods, in the absence (¡) or in the presence (+) of 1 g of MM6 calpastatin isolated as previously reported. At the end of the electrophoretic run, the gels were incubated overnight at 25 °C in the presence of 10 mM CaCl2 under gentle shaking. The gels were then stained with Coomassie brilliant blue. (B) Calpastatin-induced conformational change in p94-like calpain. p94-like calpain (0.5 g for each experiment) puriWed from MM6 cell line was spotted on nitrocellulose sheets (0.5 £ 0.5 cm) and left for 15 min at 4 °C in a humidiWed chamber. The sheets were washed with 1 mM EDTA and saturated with 5% non fat skimmed milk powder. The immobilized p94-like calpain was then incubated in the absence (1) or in the presence of 25 M Ca2+ (2) or of 1 g of homologue calpastatin (3) or of 1 g of homologue calpastatin and 25 M Ca2+ (4) at 4 °C for 30 min. mAb 56.3 (0.2 g) was then added. Calpain was detected as reported in Methods and in [45]. The probed nitrocellulose sheets were exposed to autoradiography, quantiWed with a Shimadzu CS9000 densitometer with a Wxed wavelength of 590 nm [35]. The values reported are the arithmetical mean of Wve diVerent experiment §SD.

and recorded by cell scanning. It can be seen that in almost cell lines, calpain is poorly stained in resting conditions and appears to be almost completely localized and homogenously diVused in the cytosol; on the other hand, in MM6 cell line, the protease is in part also associated to the plasma membranes. Following stimulation, the Xuorescence intensity increases from 6- to 8-fold in almost all cell lines analysed and appears to be now distributed in an amount ranging between 85% and 88% in the cytosol, and between 12% and 15% in a membrane-associated form. In two cell types, H9 and especially MM6, the amount of membrane bound Xuorescence reaches a value of approximately 28%

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Fig. 5. Changes in intracellular p94-like calpain/mediated Xuorescence revealed in stimulated hemopoietic cell lines. Indicated cell lines (2 £ 106 cells for each experiment) were incubated in 10 ml of growth medium (see Methods) for 30 min at 37 °C and 5% CO2 in the absence (control, black line and unWlled circles) or in the presence of 0.1 M Ca2+-ionophore and 1 mM Ca2+ (Ionophore, green line and Wlled circles) or 100 ng/ml PMA (PMA, blue line and unWlled squares). Cells were then centrifuged, washed three times with NaCl/Pi, Wxed and permeabilized with Triton/paraformaldehyde method [34]. p94like calpain was detected by confocal microscopy as described in [35,49], using anti--calpain mAb 56.3 as primary antibody and a Xuorescein isothiocyanate-conjugated sheep anti-mouse IgG as secondary antibody. The excitation/emission wavelengths were 488/522 nm for Xuorescein-labeled antibodies and 488–568/605 nm. Fluorescence emission at each determination during cell scanning was quantiWed with Laser Pix Software as in [35] (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper).

R. Stifanese et al. / Archives of Biochemistry and Biophysics 456 (2006) 48–57

and 48%, respectively. Also on the basis of the results shown in Fig. 4B, the large increase in cytosolic Xuorescence, detected in all stimulated cells, can be attributed to the association of calpain/calpastatin. Thus, all these results provide clear indication of that the calpain/calpastatin system, in which the predominant calpain isoform is represented by the p94-like calpain, is involved in the stimulation of hemopoietic cell lines. When cells stimulated with PMA or with Ca2+-ionophore were stained with anti m-calpain antibody, the results obtained were very similar in terms of intracellular distribution (Fig. 6). However, the Xuorescence intensity resulted less pronounced, since although also m-calpain associates with calpastatin (see Fig. 4), the aYnity for the mAb remains unchanged being and thereby no conformational changes can be detected. Comparison between the pictures reported in Figs. 5 and 6 clearly indicate that p94like calpain behaves very diVerently in terms of redistribution between cytosol and plasma membranes during cell stimulation. In fact, in NK cell line NK-92, m-calpain is predominantly located in the cytosol, whereas in MM6 cell line appears to be more extensively associated to plasma membranes.

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in cytosolic Xuorescence and the amount of available calpastatin. As expected, cell lines displaying the highest calpastatin levels are also those showing the highest green Xuorescence. An inverse relationship was obtained plotting the amount of membrane associated calpain in stimulated cells against total calpastatin activity. In this case, cell lines expressing the highest calpastatin activity are characterized by a lower fraction of membrane-translocated calpain (Fig. 7B). Finally, the regulatory role of calpastatin is further supported by the linear relationship between the increase in cytosolic Xuorescence and the ratio calpastatin/

Correlation between the level of calpastatin expression and the intracellular redistribution of calpain To analyse if the postulated regulatory function of calpastatin could be extended to all six cell lines studied, we have correlated the total calpastatin activity: (1) to the increase in cytosolic Xuorescence (Fig. 7A) and (2) to the amount of the membrane associated calpain (Fig. 7B). Furthermore, the increase in cytosolic Xuorescence was also plotted as a function of the calpastatin/calpain activity ratio (Fig. 7C). The data reported in Fig. 7A indicate that there is an almost linear relationship between the increase

Fig. 6. Changes in intracellular m-calpain/mediated Xuorescence revealed in stimulated MM6 and NK-92 cell lines. MM6 and NK cell clones (2 £ 106 cells for each experiment) were incubated as described in legend to Fig. 5 and in Methods in the absence (Control) or in the presence of 100 ng/ml PMA (PMA) or of 0.1 M Ca2+-ionophore and 1 mM Ca2+ (Ionophore). Cells were treated as described in the legend to Fig. 5. m-Calpain was detected by confocal microscopy as described in [35,49], using anti-m-calpain mAb as primary antibody and a Xuorescein isothiocyanate-conjugated sheep anti-mouse IgG as secondary antibody. The excitation/emission wavelengths were 488/522 nm for Xuorescein-labeled antibodies and 488–568/605 nm. Fluorescence detected in each cell section was quantiWed with Laser Pix Software as in [35].

Fig. 7. Correlation between calpastatin levels and formation of an enzyme/inhibitor complex in cytosol and calpain translocation to the plasma membranes. (A) Correlation between the increase in cytosolic calpain-mediated Xuorescence and cytosolic calpastatin availability. Fluorescence was calculated from the cell scanning as reported in Fig. 5. The calpastatin levels were taken from Fig. 3. (B) Correlation between the amount of calpain associated to plasma membranes and calpastatin levels. Membrane calpain was calculated from the cell scanning as reported in Fig. 5. The calpastatin levels were taken from Fig. 3. (C) Correlation between the increase in cytosolic calpain-mediated Xuorescence and the calpastatin/calpain ratio. Data were taken from Fig. 5, Table 1 and Fig. 3.

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calpain activities (Fig. 7C). Altogether these data are indicating that the rate and the extent of activation of calpain at the level of the plasma membranes are directly related to the availability of calpastatin which thereby prevents translocation of calpain by recruiting calpain into the form of a cytosolic enzyme/inhibitor complex. Discussion The data presented provide the Wrst evidence that a p94like calpain is the isoform most largely present in several human hemopoietic cell lines. The enzymatic properties of this protease are identical to those of the p94-like isoform identiWed in human peripheral blood mononuclear cells, and are diVerent from those of the conventional - and m-calpains that are absent or expressed in much lower amounts in these cell lines. Based on the calpastatin sensitivity shown by this p94-like isoform, although lower than that of the - and m-calpain, it is conceivable to consider that this isoform also diVers from the typical muscle p94 calpain or from its p94 variant [50]. Thus, the properties of this protease and the fact that it is the more highly expressed calcium dependent protease in all these cell clones, suggest a critical role of this enzyme in the context of hemopoietic cell functions. This conclusion is further supported by the observation that in these cells the natural inhibitor calpastatin is also present in diVerent levels from cell to cell type; this results in a diVerent calpain/calpastatin ratio and consequently a variability in the regulatory eYciency of calpain activity. At the functional level stimulation of these cells with diVerent agonists was accompanied by a limited autolytic inactivation of intracellular calpain and especially of the p94-like calpain isoform, this being an indication that calpain activation and thereby calpain activity participate to the cell response. Also the subcellular distribution of the protease(s) that was observed in the context of cell stimulation, is in support of this conclusion. Activation with both PMA and Ca2+-ionophore induced calpain translocation to the plasma membranes and accumulation in the cytosol in the form of a complex with calpastatin: this has been revealed with an anti--calpain antibody capable of recognizing the conformational changes of calpain occurring during its association with calpastatin [35]. These results were obtained since the p94-like calpain behaves, with respect to calpastatin, as the conventional calpain -form although with slightly lower aYnity [27]. Moreover, the extent of calpain translocation to the plasma membranes and its recruitment in the cytosol in association with calpastatin are inversely correlated to the total availability of the inhibitor [35,48]. In fact in MM6 cell lines, which displays the lowest calpastatin content, calpain is bound to the membrane even in unstimulated conditions and reaches values close to 50% following stimulation. Two general considerations can be proposed. The Wrst one is that, as in many other biological systems, also in these human hemopoietic cell lines, cell response is associated with calpain activation and its intracellular redistribution. The second one is that many evidences are indicating the exis-

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