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Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing
Mechanisms of Ageing and Development 102 (1998) 15 – 23
Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing Dimitris Kletsas a, Eva Caselgrandi b, Daniela Barbieri c, Dimitri Stathakos a, Claudio Franceschi c,d, Enzo Ottaviani e,* a
Institute of Biology, National Center for Scientific Research ‘Demokritos’, Athens 153 10, Greece b Department of Biomedical Sciences, Hygiene and Microbiology Section, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy c Department of Biomedical Sciences, General Pathology Section, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy d INRCA, 60100 Ancona, Italy e Department of Animal Biology, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy
Received 16 October 1997; received in revised form 15 December 1997; accepted 16 December 1997
Abstract Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1–24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human fibroblasts; NEP; ACTH; Ageing; Fetal; Adult; Centenarian
* Corresponding author. Tel.: +39 59 243566; fax: + 39 59 226769; e-mail: [email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00003-7
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1. Introduction Neutral endopeptidase-24.11 (NEP), also known as enkephalinase (EC 3.4.24.11), is a 93-KDa glycosylated ectoenzyme, i.e. an integral membrane protein whose active site faces the extracellular space (Roques et al., 1993). This Zn metallopeptidase was found to be identical to CD10, a type II integral protein (Letarte et al., 1988) used as a marker of the common acute lymphoblastic leukemia antigen (CALLA; Letarte et al., 1988; LeBien and McCormack, 1989; Shipp et al., 1989). NEP exerts its activity by cleaving peptide bonds at the amino side of hydrophobic amino acid residues of small regulatory peptides (Turner and Tanzawa, 1997). Thus, NEP can exert wide regulatory activity by degrading a variety of bioactive peptides, such as opioid peptides, tachykinins, e.g. substance P, members of the natriuretic peptide family, bombesin-like peptides, chemotactic peptides and adrenocorticotropin hormone (ACTH; DuvauxMiret et al., 1992; Turner and Tanzawa, 1997). Accordingly, NEP is involved in many physiological processes, e.g. cardiovascular regulation, inflammatory phenomena, neuropeptide metabolism, cell migration and proliferation (Shipp et al., 1991; Harrison et al., 1995; Turner and Tanzawa, 1997), and, is therefore an important drug target for the treatment of hypertension, pain or diarrhea (Turner and Tanzawa, 1997). The aim of the present study was to determine whether fetal-to-adult transition, and ageing have any effect on NEP activity in human fibroblasts. Furthermore, we examined the possible regulation of NEP activity by ACTH—a key molecule in the stress cascade, capable also of immune functions (Ottaviani and Franceschi, 1997; Ottaviani et al., 1997)—which can be degraded by this endopeptidase. Recently, it has been proposed that stress may play a major role in extending life span (Masoro and Austad, 1996). In humans, the NEP gene is located on chromosome 3, spans more than 80 kb and contains 24 miniexons (Roques et al., 1993). NEP is a highly conserved protein. There are only six non-conservative changes in the 742 amino acids of the human and rat NEPs (Erdo¨s and Skidgel, 1989). NEP presence has been detected in the central nervous system, where its distribution matches that of mand/or d-opioid receptors, and in peripheral tissue, particularly in membranes of the brush border epithelial cells of intestine and kidney, lung, testis, prostate and synovium cells, in neutrophiles, chondrocytes, thymocytes, and in several epithelial and endocrine cells (Connelly et al., 1985; Pierart et al., 1988; Erdo¨s and Skidgel, 1989; Mapp et al., 1992; Roques et al., 1993). It is also expressed in human lung, skin, synovial or muscle fibroblasts in in vitro cultures (Johnson et al., 1985; Bathon et al., 1992; Bou-Gharios et al., 1995; Harrison et al., 1995; Vaghy et al., 1995). Finally, NEP has also been found in invertebrates, in particular in synaptic membranes from the insect Schistocerca gregaria (Isaac, 1988), and in immunocytes from the molluscs Mytilus edulis, Planorbarius corneus and Vi6iparus ater (Shipp et al., 1990; Ottaviani and Caselgrandi, 1997).
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2. Materials and methods
2.1. Cell lines and culture conditions Various normal human fibroblast cell strains with a limited in vitro life span were used in this study: (i) human lung fibroblasts: one strain from a 20-week fetus (Flow 2002, Flow Laboratories, Rickmanswothr, UK) and one from a 20-year adult donor (CCD 19 Lu, ECACC, Salisbury, UK); (ii) human skin fibroblasts: one strain from a 18-week fetus (Detroit 551, Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia, Brescia, Italy) and four from adult donors, i.e. a 7-year old (DSF7), a 23-year old (1BR.3. ECACC Salisbury, UK), a 75-year old (DSF75), and a centenarian. DSF7, DSF75 and the centenarian cell strains were developed in our laboratories from skin explants from healthy volunteers, after obtaining the informed consent of the donors or of the parents, as previously described (Kletsas et al., submitted). The cells were cultured in Eagle’s Minimal Essential Medium (MEM), supplemented with 10% Fetal Calf Serum (FCS). All media were purchased from Seromed (Berlin, Germany). All cell strains were routinely subcultured every 7 days at 1:2 split ratio until they reached in vitro senescence. For Flow 2002, this was after 609 3 passages, for CCD 19 Lu after 509 2 passages, for Detroit 551 after 509 2 passages, for DSF7 after 4792 passages, for 1BR.3 after 409 3 passages, for DSF75 after 409 2 passages (Kletsas et al., submitted,) and for centenarian cells after 28 93 passages. During initial passages, the cultures reached confluency within 1 week of growth, and cells were considered to be in phase II, as defined by Hayflick and Moorhead (1961) and Hayflick (1965). If confluency was not reached within 1 week, the cells were considered to be in phase III, approaching senescence (Kletsas and Stathakos, 1992). In all experiments presented here, we used cell cultures at the middle of phase II of the their in vitro lifespan, unless otherwise specified.
2.2. Preparation of the samples For the determination of the NEP activity, cells from each strain were seeded in four dishes in order to obtain subconfluent growing cultures. Samples were then washed twice with PBS, and 2 ml MEM was added to each dish. Subsequently, one sample was used to evaluate the cell NEP activity (Control). Phosphoramidon (10 mM) was added to a second sample as a blank of the reaction, and ACTH 10 − 8 M (1 – 24) (Sigma, St. Louis, IL) was added to a third. Finally, ACTH (1 – 24) 10 − 8 M +10 mM phosphoramidon (Sigma) was used in a fourth sample as a control of the NEP activity on ACTH. After incubation for 1 h in the dark at 37°C, the cells were collected by scraping, and then counted and centrifuged for 15 min at 1200 g. The pellet was washed twice with PBS, centrifuged, resuspended in 1.8 ml HEPES buffer, sonicated for 30 s and immediately used to detect NEP activity.
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2.3. Spectrofluorimetric analysis of NEP acti6ity NEP activity was measured as in a previous study (Ottaviani and Caselgrandi, 1997), following the procedure described by Deshodt-Lankman et al. (1990). It must be underlined that for the correct evaluation of NEP activity, the number of cells must be under 700 · 103 cells/ml, as above this value, enzymic activity falls quickly (Ottaviani and Caselgrandi, 1997). NEP cleaves the substrate (Suc-Ala-AlaPhe-AMC, Sigma) to Phe-AMC, and the subsequent fluorescent product (free AMC) can be measured with a fluorescence spectrometer (Perkin Elmer, mod. 204) at an excitation of 367 nm and an emission of 440 nm. The calibration curve was determined using pig NEP enzyme, a kind gift from Dr A.J. Turner (Department of Biochemistry and Molecular Biology, Leeds, UK). The enzymic activity in our experiments is expressed as pmol/106 cells. As reported in a previous paper (Ottaviani and Caselgrandi, 1997), blanks also show values near to the sensitivity limit of the method. Statistical analysis was performed by ANOVA of logarithmic values, followed by the Student-Newman-Keules multiple comparison test to compare the NEP activity of the different cell lines. The Student’s t-test was used to compare control NEP activity, ACTH cultures and ACTH + phosphoramidon cultures in each cell line.
3. Results We first studied the expression of NEP activity in human fetal fibroblasts from two different tissues, i.e. lung and skin. The two cell strains were from fetuses in the same gestation period (second trimester), and the cell cultures were at the middle of phase II of their in vitro lifespan, i.e. passages 38 and 39 for lung fibroblasts and passages 24 and 28 for skin cells. The levels of NEP activity in fetal skin fibroblasts were higher than in fetal lung fibroblasts (Tables 1 and 2). To detect possible alterations during the fetal-to-adult transition, we assessed NEP enzymic activity in four different adult skin fibroblast strains. As shown in Table 1, all strains expressed significantly higher levels of NEP activity compared to fetal fibroblasts. The same change during fetal-to-adult transition was also observed in human lung fibroblasts (Table 2). As in the case of fetal cells, all adult skin cell strains expressed levels of NEP activity higher than those of the adult lung cell strain (Tables 1 and 2). In adult skin fibroblasts from the 7- and 23-year-old donors we found comparable levels of NEP activity (Table 1). In contrast, fibroblasts from the 75-year old individual expressed considerably higher enzymic activity, while those from the centenarian donor did not follow this trend, but showed NEP activity similar to that of cells from young donors. We also studied the effect of in vitro ageing on the levels of NEP activity. Two lung fibroblast strains — fetal and adult—at different passages were used, i.e. passages 38 and 39 (middle of phase II) and 56 (phase III) for fetal cells, and passages 19 (phase II) and 45 (phase III) for adult fibroblasts. Table 2 shows that
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there was a substantial increase in the levels of NEP activity with in vitro ageing of the fibroblasts, the increase being more pronounced in fetal lung fibroblasts. When we incubated fetal skin and lung fibroblasts with ACTH (1–24), a significant increase of NEP activity was observed (Tables 1 and 2). This was also true for two of the adult skin cell strains from the 7-and 23-year-old donors, respectively. In particular, the latter exhibited the largest increase in NEP activity after ACTH-treatment (Table 1). NEP activity in adult lung fibroblasts and in skin fibroblasts from the old and the centenarian donor (Tables 1 and 2), however, decreased after ACTH-treatment. Finally, ACTH induced an intense increase in NEP activity in phase III Flow 2002 cells (passage 56), while it lowered the levels of the enzymic activity in phase III CCD 19Lu cells (passage 45). As expected, phoshoramidon, a strong inhibitor of NEP, inhibited enzymic activity in all cell strains used, demonstrating the specificity of the reaction. Interestingly, in the cell strains in which ACTH provoked an increase in NEP activity, phoshoramidon restored the control levels. As expected, in the other cell strains, a further lowering of NEP activity with respect to the ACTH-treated samples was induced.
Table 1 NEP activity in skin cell lines in presence of ACTH (1 – 24) 10−8 M and ACTH (1 – 24) 10−8 M+NEP inhibitor (10 mM phosphoramidon, Pho) Cell lines and passage
Treatment Control
ACTH
ACTH+Pho
Detroit 551 (fetal skin fibroblasts) p. 24 1.379 0.04 p. 28 1.389 0.10
2.1190.05** 2.20 90.12**
1.28 90.05 1.23 90.09
DSF 7 (skin fibroblasts from a 7-year-old donor) p. 20 1.959 0.13 p. 28 2.059 0.05
2.75 9 0.13* 2.58 90.10**
1.74 90.05 1.89 9 0.12
1BR.3 (skin fibroblasts from a 23-year-old donor) p. 23 2.229 0.09
5.08 90.10**
1.99 9 0.12
DSF 75 (skin fibroblasts from a 75-year-old donor) p. 20 3.089 0.15 p. 29 3.719 0.28
2.79 90.06* 2.7690.30**
1.89 9 0.07** 2.09 9 0.16**
Skin from a centenarian donor fibroblasts p. 11 2.209 0.07 p. 16 2.229 0.08
1.30 90.02** 1.1590.20**
1.17 9 0.02** 1.02 9 0.15**
The mean9 standard deviation of three experiments is shown. Data of NEP activity are expressed as pmol/106 cells Statistical analysis was performed by Student’s t-test. **PB0.001 and *PB0.05 vs. control.
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Table 2 NEP activity in lung cell lines in presence of ACTH (1 – 24) 10−8 M and ACTH (1 – 24) 10−8 M+NEP inhibitor (10 mM phosphoramidon, Pho) Cell lines and passage
Treatment Control
ACTH
ACTH+Pho
0.859 0.03 0.889 0.14 2.249 0.75
1.06 9 0.04* 1.1290.06** 7.27 90.50**
0.98 9 0.23 0.75 9 0.18 3.15 9 0.80
CCD 19 (Lu adult lung fibroblasts) p. 19 1.629 0.11 p. 45 2.549 0.30
1.11 90.05* 1.61 90.25**
0.75 9 0.04** 0.97 9 0.16**
2002 (fetal lung fibroblasts) p. 38 p. 39 p. 56
The mean9 standard deviation of three experiments is shown. Data of NEP activity are expressed as pmol/106 cells. Statistical analysis was performed by Student’s t-test. **PB0.001 and *PB0.05 vs. control.
4. Discussion In this report, we present data on NEP activity at different stages of development and ageing in normal human fibroblasts. Skin and lung fibroblast cell lines at different phases of their lifespan and from donors of different ages were used. NEP activity was monitored in vitro and in vivo. Moreover, the effects on NEP activity of the addition to fibroblast cultures of ACTH, was also studied. ACTH, a bioactive peptide involved in many immune and neuroendocrine responses (Ottaviani et al., 1997), can be effectively cleaved by NEP to a-melanocyte-stimulating hormone (a-MSH; Duvaux-Miret et al., 1992). The following are the major findings: (1). All fibroblast cell strains —skin or lung, fetal or adult—used in this study expressed NEP activity; (2). In lung fibroblasts in phase II of their in vitro life span, there was a considerable increase in NEP activity during fetal-to-adult transition. All adult skin fibroblasts expressed substantially higher levels of enzymic activity compared to fetal cells. The only other paper, to our knowledge, reporting differences in NEP activity between fetal and adult cells is that of Harrison et al. (1995). In this study, one fetal lung cell strain expressed 0.52 pmol/106 cells, while one newborn foreskin cell strain expressed values higher by one order of magnitude (5.2 pmol/106 cells). Our values fall within this range. (3). Cells from young donors (7- and 23-year-old) expressed approximately the same levels of NEP activity, while considerably higher levels were found in fibroblasts from the older donor. This is in agreement with the results of Solmi et al. (1996), who reported that the NEP activity of human skin fibroblasts taken from biopsies of elderly subjects (\ 60 years) is far higher than that in fibroblasts from young (20–30 years) donors;
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(4). Cells from the centenarian donor did not follow this trend and exhibited values similar to those found in fibroblasts from young individuals. This result is not unexpected, given our previous results showing that in many cases centenarian cells behave similarly to those from young donors (Grassilli et al., 1996; Sansoni et al., 1997). If confirmed by further experiments, this correlation between an increase in NEP activity and donor age could help to explain the pathogenesis of several age-related diseases, as NEP is involved in immunological derangements. Elevated synovial tissue concentrations of NEP have been found in chronic arthritis, and fibroblasts and synoviocytes are most likely the major cellular sources of this activity (Sreedharan et al., 1990). (5). When, the effect of in vitro cellular ageing on NEP activity after serial passaging in culture is a significant increase in enzymic activity in phase III cells compared to phase II cells, occurred. Considering that NEP is an integral membrane protein, these results are in accordance with previous data showing considerably higher levels of other integral membrane proteins during the in vitro ageing of human fibroblasts. This finding is probably due to the increased surface area of aged cells (Gerhard et al., 1991; Psarras et al., 1994). (6). ACTH induces an increase in NEP activity in the two fetal fibroblast strains and two of the adult skin cell strains, while phosphoramidon, a specific inhibitor for NEP, returns NEP levels close to control values. The effect of ACTH is in accordance with previous studies undertaken on osteoblast-like cells in culture (Howell et al., 1992). In these experiments, calcitonin, a protein that can be hydrolyzed by NEP, provoked a 60% increase in NEP activity, suggesting that a control mechanism is exerted by the peptides cleaved by NEP. Intriguingly, in adult lung fibroblasts and in two of the adult skin cells, including those from the centenarian donor, ACTH provoked a decrease in NEP activity. This unexpected finding might be the result of differences in the ability of NEP to cleave ACTH on the surface of these cells as opposed to fetal and young fibroblasts. Controversial data exist in the literature on this topic. Haxhiu-Poskurica et al. (1992) found a significant increase in NEP activity in 2 – 3 week and 10-week-old piglets with respect to newborn animals. On the other hand, Zhang et al. (1995) detected that exsanguination employed to induce non-cholinergic airway construction in guinea pig caused a significant decrease in airway NEP activity in both immature and mature animals. The authors surmise that exsanguination increases the production of free oxygen radicals which inactivate NEP. Finally, ACTH has the same effect on lung human fibroblasts, irrespective of in vitro ageing level. Indeed, this peptide increased NEP activity in both phases II and III of fetal lung cells. On the other hand, it lowered enzymic activity in adult fibroblasts in phases II or III, suggesting that the effect of ACTH on NEP activity persists during in vitro cellular ageing.
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Acknowledgements This work is supported by the MOLGERON EU Concerted Action Programme (D.K., D.S., C.F.), the Greek General Secretary for Research and Technology (PENED 1995 Programme; D.K., D.S.) and the Italian National Research Council, MURST and INRCA (C.F., E.O.).
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Letarte, M., Vera, S., Tran, R., Addis, J.B.L., Onizuka, R.J., Quackenbuch, E.J., Jongeneel, C.V., McInnes, R.R.J., 1988. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. Exp. Med. 168, 1247–1253. Mapp, P.I., Walsh, D.A., Kidd, B.L., Cruwys, S.C., Polak, J.M., Blake, D.R., 1992. Localization of the enzyme neutral endopeptidase to the human synovium. J. Reumatol. 19, 1838 – 1844. Masoro, E.J., Austad, S.N., 1996. The evolution of the antiaging action of dietary restriction: a hypothesis. J. Gerentol. 51A, B387–B391. Ottaviani, E., Caselgrandi, E., 1997. Neutral endopeptidase-24.11 (NEP)-like activity in molluscan hemocytes. Peptides 18, 1107–1110. Ottaviani, E., Franceschi, C., 1997. The invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. Immunol. Today 18, 169 – 174. Ottaviani, E., Franchini, A., Franceschi, C., 1997. Pro-opiomelanocortin-derived peptides, cytokines and nitric oxide in immune responses and stress: an evolutionary approach. Int. Rev. Cytol. 170, 79 – 141. Pierart, M.E., Najdovski, T., Appelboom, T.E., Deschodt-Lanckman, M.M., 1988. Effect of human endopeptidase 24.11 (‘‘enkephalinase’’) on IL-1-induced thymocyte proliferation activity. J. Immunol. 140, 3808–3811. Psarras, S., Kletsas, D., Stathakos, D., 1994. Restoration of down-regulated PDGF receptors by TGF-b in human embryonic fibroblasts. Enhanced response during cellular in vitro aging. FEBS Lett. 339, 84–88. Roques, B.P., Noble, F., Dauge, V., Fournie-Zaluski, M.-C., Beaumont, A., 1993. Neutral endopeptidase 24.11: Structure, inhibition, and experimantal and clinical pharmacology. Pharmacol. Rev. 45, 87–146. Sansoni, P., Fagnoni, F., Vescovini, R., Mazzola, M., Brianti, V., Bologna, G., Nigro, E., Lavagetto, G., Cossarizza, A., Monti, D., Franceschi, C., Passeri, M., 1997. T lymphocyte proliferative capability to defined and costimulatory CD28 pathway is not impaired in healthy centenarians. Mech. Ageing Dev. 96, 127–136. Shipp, M.A., Vijayaraghavan, J., Schmidt, E.V., Masteller, E.L., D’Adamio, L., Hersh, L.B., Reinherz, E.L., 1989. Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 (‘‘enkephalinase’’): Direct evidence by cDNA transfection analysis. Proc. Natl. Acad. Sci. USA 86, 297–301. Shipp, M.A., Stefano, G.B., D’Adamio, L., Switzer, S.N., Howard, F.D., Sinisterra, J., Scharrer, B., Reinherz, E.L., 1990. Downregulation of enkephalin-mediated inflammatory responses by CD10/ neutral endopeptidase 24.11. Nature (London) 347, 394 – 396. Shipp, M.A., Tarr, G.E., Chen, C.-Y., Switzer, S.N., Hersh, L.B., Stein, H., Synday, M.E., Reinherz, E.L., 1991. CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung. Proc. Natl. Acad. Sci. USA 88, 10662 – 10666. Solmi, R., Tietz, C., Zucchini, C., Gualandi, G., Pugnaloni, A., Talassi, O., Castaldini, C., Simonelli, L., Biagnini, G., 1996. In vitro study of gingival fibroblasts from normal and inflamed tissue: age-related responsiveness. Mech. Ageing Dev. 92, 31 – 41. Sreedharan, S.P., Goetzl, E.J., Malfroy, B., 1990. Elevated synovial tissue concentration of the common acute lymphoblastic leukaemia antigen (CALLA)-associated neutral endopeptidase (3.4.24.11) in human chronic arthritis. Immunology 71, 142 – 144. Turner, A.J., Tanzawa, K., 1997. Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. FASEB J. 11, 355–364. Vaghy, P.L., Russell, J.S., Lantry, L.E., Stephens, R.E., Ward, P.E., 1995. Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts. Peptides 16, 1367–1373. Zhang, H.-Q., Tai, H.-H., Lai, Y.-L., 1995. Age-dependent mechanism in guinea pig bronchoconstriction induced by exsanguination. Respir. Physiol. 99, 361 – 369.
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Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing Dimitris Kletsas a, Eva Caselgrandi b, Daniela Barbieri c, Dimitri Stathakos a, Claudio Franceschi c,d, Enzo Ottaviani e,* a
Institute of Biology, National Center for Scientific Research ‘Demokritos’, Athens 153 10, Greece b Department of Biomedical Sciences, Hygiene and Microbiology Section, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy c Department of Biomedical Sciences, General Pathology Section, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy d INRCA, 60100 Ancona, Italy e Department of Animal Biology, 6ia Berengario, 14, Uni6ersity of Modena, 41100 Modena, Italy
Received 16 October 1997; received in revised form 15 December 1997; accepted 16 December 1997
Abstract Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1–24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human fibroblasts; NEP; ACTH; Ageing; Fetal; Adult; Centenarian
* Corresponding author. Tel.: +39 59 243566; fax: + 39 59 226769; e-mail: [email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00003-7
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1. Introduction Neutral endopeptidase-24.11 (NEP), also known as enkephalinase (EC 3.4.24.11), is a 93-KDa glycosylated ectoenzyme, i.e. an integral membrane protein whose active site faces the extracellular space (Roques et al., 1993). This Zn metallopeptidase was found to be identical to CD10, a type II integral protein (Letarte et al., 1988) used as a marker of the common acute lymphoblastic leukemia antigen (CALLA; Letarte et al., 1988; LeBien and McCormack, 1989; Shipp et al., 1989). NEP exerts its activity by cleaving peptide bonds at the amino side of hydrophobic amino acid residues of small regulatory peptides (Turner and Tanzawa, 1997). Thus, NEP can exert wide regulatory activity by degrading a variety of bioactive peptides, such as opioid peptides, tachykinins, e.g. substance P, members of the natriuretic peptide family, bombesin-like peptides, chemotactic peptides and adrenocorticotropin hormone (ACTH; DuvauxMiret et al., 1992; Turner and Tanzawa, 1997). Accordingly, NEP is involved in many physiological processes, e.g. cardiovascular regulation, inflammatory phenomena, neuropeptide metabolism, cell migration and proliferation (Shipp et al., 1991; Harrison et al., 1995; Turner and Tanzawa, 1997), and, is therefore an important drug target for the treatment of hypertension, pain or diarrhea (Turner and Tanzawa, 1997). The aim of the present study was to determine whether fetal-to-adult transition, and ageing have any effect on NEP activity in human fibroblasts. Furthermore, we examined the possible regulation of NEP activity by ACTH—a key molecule in the stress cascade, capable also of immune functions (Ottaviani and Franceschi, 1997; Ottaviani et al., 1997)—which can be degraded by this endopeptidase. Recently, it has been proposed that stress may play a major role in extending life span (Masoro and Austad, 1996). In humans, the NEP gene is located on chromosome 3, spans more than 80 kb and contains 24 miniexons (Roques et al., 1993). NEP is a highly conserved protein. There are only six non-conservative changes in the 742 amino acids of the human and rat NEPs (Erdo¨s and Skidgel, 1989). NEP presence has been detected in the central nervous system, where its distribution matches that of mand/or d-opioid receptors, and in peripheral tissue, particularly in membranes of the brush border epithelial cells of intestine and kidney, lung, testis, prostate and synovium cells, in neutrophiles, chondrocytes, thymocytes, and in several epithelial and endocrine cells (Connelly et al., 1985; Pierart et al., 1988; Erdo¨s and Skidgel, 1989; Mapp et al., 1992; Roques et al., 1993). It is also expressed in human lung, skin, synovial or muscle fibroblasts in in vitro cultures (Johnson et al., 1985; Bathon et al., 1992; Bou-Gharios et al., 1995; Harrison et al., 1995; Vaghy et al., 1995). Finally, NEP has also been found in invertebrates, in particular in synaptic membranes from the insect Schistocerca gregaria (Isaac, 1988), and in immunocytes from the molluscs Mytilus edulis, Planorbarius corneus and Vi6iparus ater (Shipp et al., 1990; Ottaviani and Caselgrandi, 1997).
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2. Materials and methods
2.1. Cell lines and culture conditions Various normal human fibroblast cell strains with a limited in vitro life span were used in this study: (i) human lung fibroblasts: one strain from a 20-week fetus (Flow 2002, Flow Laboratories, Rickmanswothr, UK) and one from a 20-year adult donor (CCD 19 Lu, ECACC, Salisbury, UK); (ii) human skin fibroblasts: one strain from a 18-week fetus (Detroit 551, Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia, Brescia, Italy) and four from adult donors, i.e. a 7-year old (DSF7), a 23-year old (1BR.3. ECACC Salisbury, UK), a 75-year old (DSF75), and a centenarian. DSF7, DSF75 and the centenarian cell strains were developed in our laboratories from skin explants from healthy volunteers, after obtaining the informed consent of the donors or of the parents, as previously described (Kletsas et al., submitted). The cells were cultured in Eagle’s Minimal Essential Medium (MEM), supplemented with 10% Fetal Calf Serum (FCS). All media were purchased from Seromed (Berlin, Germany). All cell strains were routinely subcultured every 7 days at 1:2 split ratio until they reached in vitro senescence. For Flow 2002, this was after 609 3 passages, for CCD 19 Lu after 509 2 passages, for Detroit 551 after 509 2 passages, for DSF7 after 4792 passages, for 1BR.3 after 409 3 passages, for DSF75 after 409 2 passages (Kletsas et al., submitted,) and for centenarian cells after 28 93 passages. During initial passages, the cultures reached confluency within 1 week of growth, and cells were considered to be in phase II, as defined by Hayflick and Moorhead (1961) and Hayflick (1965). If confluency was not reached within 1 week, the cells were considered to be in phase III, approaching senescence (Kletsas and Stathakos, 1992). In all experiments presented here, we used cell cultures at the middle of phase II of the their in vitro lifespan, unless otherwise specified.
2.2. Preparation of the samples For the determination of the NEP activity, cells from each strain were seeded in four dishes in order to obtain subconfluent growing cultures. Samples were then washed twice with PBS, and 2 ml MEM was added to each dish. Subsequently, one sample was used to evaluate the cell NEP activity (Control). Phosphoramidon (10 mM) was added to a second sample as a blank of the reaction, and ACTH 10 − 8 M (1 – 24) (Sigma, St. Louis, IL) was added to a third. Finally, ACTH (1 – 24) 10 − 8 M +10 mM phosphoramidon (Sigma) was used in a fourth sample as a control of the NEP activity on ACTH. After incubation for 1 h in the dark at 37°C, the cells were collected by scraping, and then counted and centrifuged for 15 min at 1200 g. The pellet was washed twice with PBS, centrifuged, resuspended in 1.8 ml HEPES buffer, sonicated for 30 s and immediately used to detect NEP activity.
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2.3. Spectrofluorimetric analysis of NEP acti6ity NEP activity was measured as in a previous study (Ottaviani and Caselgrandi, 1997), following the procedure described by Deshodt-Lankman et al. (1990). It must be underlined that for the correct evaluation of NEP activity, the number of cells must be under 700 · 103 cells/ml, as above this value, enzymic activity falls quickly (Ottaviani and Caselgrandi, 1997). NEP cleaves the substrate (Suc-Ala-AlaPhe-AMC, Sigma) to Phe-AMC, and the subsequent fluorescent product (free AMC) can be measured with a fluorescence spectrometer (Perkin Elmer, mod. 204) at an excitation of 367 nm and an emission of 440 nm. The calibration curve was determined using pig NEP enzyme, a kind gift from Dr A.J. Turner (Department of Biochemistry and Molecular Biology, Leeds, UK). The enzymic activity in our experiments is expressed as pmol/106 cells. As reported in a previous paper (Ottaviani and Caselgrandi, 1997), blanks also show values near to the sensitivity limit of the method. Statistical analysis was performed by ANOVA of logarithmic values, followed by the Student-Newman-Keules multiple comparison test to compare the NEP activity of the different cell lines. The Student’s t-test was used to compare control NEP activity, ACTH cultures and ACTH + phosphoramidon cultures in each cell line.
3. Results We first studied the expression of NEP activity in human fetal fibroblasts from two different tissues, i.e. lung and skin. The two cell strains were from fetuses in the same gestation period (second trimester), and the cell cultures were at the middle of phase II of their in vitro lifespan, i.e. passages 38 and 39 for lung fibroblasts and passages 24 and 28 for skin cells. The levels of NEP activity in fetal skin fibroblasts were higher than in fetal lung fibroblasts (Tables 1 and 2). To detect possible alterations during the fetal-to-adult transition, we assessed NEP enzymic activity in four different adult skin fibroblast strains. As shown in Table 1, all strains expressed significantly higher levels of NEP activity compared to fetal fibroblasts. The same change during fetal-to-adult transition was also observed in human lung fibroblasts (Table 2). As in the case of fetal cells, all adult skin cell strains expressed levels of NEP activity higher than those of the adult lung cell strain (Tables 1 and 2). In adult skin fibroblasts from the 7- and 23-year-old donors we found comparable levels of NEP activity (Table 1). In contrast, fibroblasts from the 75-year old individual expressed considerably higher enzymic activity, while those from the centenarian donor did not follow this trend, but showed NEP activity similar to that of cells from young donors. We also studied the effect of in vitro ageing on the levels of NEP activity. Two lung fibroblast strains — fetal and adult—at different passages were used, i.e. passages 38 and 39 (middle of phase II) and 56 (phase III) for fetal cells, and passages 19 (phase II) and 45 (phase III) for adult fibroblasts. Table 2 shows that
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there was a substantial increase in the levels of NEP activity with in vitro ageing of the fibroblasts, the increase being more pronounced in fetal lung fibroblasts. When we incubated fetal skin and lung fibroblasts with ACTH (1–24), a significant increase of NEP activity was observed (Tables 1 and 2). This was also true for two of the adult skin cell strains from the 7-and 23-year-old donors, respectively. In particular, the latter exhibited the largest increase in NEP activity after ACTH-treatment (Table 1). NEP activity in adult lung fibroblasts and in skin fibroblasts from the old and the centenarian donor (Tables 1 and 2), however, decreased after ACTH-treatment. Finally, ACTH induced an intense increase in NEP activity in phase III Flow 2002 cells (passage 56), while it lowered the levels of the enzymic activity in phase III CCD 19Lu cells (passage 45). As expected, phoshoramidon, a strong inhibitor of NEP, inhibited enzymic activity in all cell strains used, demonstrating the specificity of the reaction. Interestingly, in the cell strains in which ACTH provoked an increase in NEP activity, phoshoramidon restored the control levels. As expected, in the other cell strains, a further lowering of NEP activity with respect to the ACTH-treated samples was induced.
Table 1 NEP activity in skin cell lines in presence of ACTH (1 – 24) 10−8 M and ACTH (1 – 24) 10−8 M+NEP inhibitor (10 mM phosphoramidon, Pho) Cell lines and passage
Treatment Control
ACTH
ACTH+Pho
Detroit 551 (fetal skin fibroblasts) p. 24 1.379 0.04 p. 28 1.389 0.10
2.1190.05** 2.20 90.12**
1.28 90.05 1.23 90.09
DSF 7 (skin fibroblasts from a 7-year-old donor) p. 20 1.959 0.13 p. 28 2.059 0.05
2.75 9 0.13* 2.58 90.10**
1.74 90.05 1.89 9 0.12
1BR.3 (skin fibroblasts from a 23-year-old donor) p. 23 2.229 0.09
5.08 90.10**
1.99 9 0.12
DSF 75 (skin fibroblasts from a 75-year-old donor) p. 20 3.089 0.15 p. 29 3.719 0.28
2.79 90.06* 2.7690.30**
1.89 9 0.07** 2.09 9 0.16**
Skin from a centenarian donor fibroblasts p. 11 2.209 0.07 p. 16 2.229 0.08
1.30 90.02** 1.1590.20**
1.17 9 0.02** 1.02 9 0.15**
The mean9 standard deviation of three experiments is shown. Data of NEP activity are expressed as pmol/106 cells Statistical analysis was performed by Student’s t-test. **PB0.001 and *PB0.05 vs. control.
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Table 2 NEP activity in lung cell lines in presence of ACTH (1 – 24) 10−8 M and ACTH (1 – 24) 10−8 M+NEP inhibitor (10 mM phosphoramidon, Pho) Cell lines and passage
Treatment Control
ACTH
ACTH+Pho
0.859 0.03 0.889 0.14 2.249 0.75
1.06 9 0.04* 1.1290.06** 7.27 90.50**
0.98 9 0.23 0.75 9 0.18 3.15 9 0.80
CCD 19 (Lu adult lung fibroblasts) p. 19 1.629 0.11 p. 45 2.549 0.30
1.11 90.05* 1.61 90.25**
0.75 9 0.04** 0.97 9 0.16**
2002 (fetal lung fibroblasts) p. 38 p. 39 p. 56
The mean9 standard deviation of three experiments is shown. Data of NEP activity are expressed as pmol/106 cells. Statistical analysis was performed by Student’s t-test. **PB0.001 and *PB0.05 vs. control.
4. Discussion In this report, we present data on NEP activity at different stages of development and ageing in normal human fibroblasts. Skin and lung fibroblast cell lines at different phases of their lifespan and from donors of different ages were used. NEP activity was monitored in vitro and in vivo. Moreover, the effects on NEP activity of the addition to fibroblast cultures of ACTH, was also studied. ACTH, a bioactive peptide involved in many immune and neuroendocrine responses (Ottaviani et al., 1997), can be effectively cleaved by NEP to a-melanocyte-stimulating hormone (a-MSH; Duvaux-Miret et al., 1992). The following are the major findings: (1). All fibroblast cell strains —skin or lung, fetal or adult—used in this study expressed NEP activity; (2). In lung fibroblasts in phase II of their in vitro life span, there was a considerable increase in NEP activity during fetal-to-adult transition. All adult skin fibroblasts expressed substantially higher levels of enzymic activity compared to fetal cells. The only other paper, to our knowledge, reporting differences in NEP activity between fetal and adult cells is that of Harrison et al. (1995). In this study, one fetal lung cell strain expressed 0.52 pmol/106 cells, while one newborn foreskin cell strain expressed values higher by one order of magnitude (5.2 pmol/106 cells). Our values fall within this range. (3). Cells from young donors (7- and 23-year-old) expressed approximately the same levels of NEP activity, while considerably higher levels were found in fibroblasts from the older donor. This is in agreement with the results of Solmi et al. (1996), who reported that the NEP activity of human skin fibroblasts taken from biopsies of elderly subjects (\ 60 years) is far higher than that in fibroblasts from young (20–30 years) donors;
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(4). Cells from the centenarian donor did not follow this trend and exhibited values similar to those found in fibroblasts from young individuals. This result is not unexpected, given our previous results showing that in many cases centenarian cells behave similarly to those from young donors (Grassilli et al., 1996; Sansoni et al., 1997). If confirmed by further experiments, this correlation between an increase in NEP activity and donor age could help to explain the pathogenesis of several age-related diseases, as NEP is involved in immunological derangements. Elevated synovial tissue concentrations of NEP have been found in chronic arthritis, and fibroblasts and synoviocytes are most likely the major cellular sources of this activity (Sreedharan et al., 1990). (5). When, the effect of in vitro cellular ageing on NEP activity after serial passaging in culture is a significant increase in enzymic activity in phase III cells compared to phase II cells, occurred. Considering that NEP is an integral membrane protein, these results are in accordance with previous data showing considerably higher levels of other integral membrane proteins during the in vitro ageing of human fibroblasts. This finding is probably due to the increased surface area of aged cells (Gerhard et al., 1991; Psarras et al., 1994). (6). ACTH induces an increase in NEP activity in the two fetal fibroblast strains and two of the adult skin cell strains, while phosphoramidon, a specific inhibitor for NEP, returns NEP levels close to control values. The effect of ACTH is in accordance with previous studies undertaken on osteoblast-like cells in culture (Howell et al., 1992). In these experiments, calcitonin, a protein that can be hydrolyzed by NEP, provoked a 60% increase in NEP activity, suggesting that a control mechanism is exerted by the peptides cleaved by NEP. Intriguingly, in adult lung fibroblasts and in two of the adult skin cells, including those from the centenarian donor, ACTH provoked a decrease in NEP activity. This unexpected finding might be the result of differences in the ability of NEP to cleave ACTH on the surface of these cells as opposed to fetal and young fibroblasts. Controversial data exist in the literature on this topic. Haxhiu-Poskurica et al. (1992) found a significant increase in NEP activity in 2 – 3 week and 10-week-old piglets with respect to newborn animals. On the other hand, Zhang et al. (1995) detected that exsanguination employed to induce non-cholinergic airway construction in guinea pig caused a significant decrease in airway NEP activity in both immature and mature animals. The authors surmise that exsanguination increases the production of free oxygen radicals which inactivate NEP. Finally, ACTH has the same effect on lung human fibroblasts, irrespective of in vitro ageing level. Indeed, this peptide increased NEP activity in both phases II and III of fetal lung cells. On the other hand, it lowered enzymic activity in adult fibroblasts in phases II or III, suggesting that the effect of ACTH on NEP activity persists during in vitro cellular ageing.
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Acknowledgements This work is supported by the MOLGERON EU Concerted Action Programme (D.K., D.S., C.F.), the Greek General Secretary for Research and Technology (PENED 1995 Programme; D.K., D.S.) and the Italian National Research Council, MURST and INRCA (C.F., E.O.).
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