Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2009) 33, 1144–1148 (Printed in Great Britain)
Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases
Xiao‑Jing Yu, Chun‑Yang Li*, Yong‑Hao Xu, La‑Mei Chen and Chun‑Lei Zhou
Department of Dermatology, Qilu Hospital, University of Shandong, Jinan 250012, China


Abstract

Psoriasis is a chronic disease characterized by keratinocyte hyperproliferation and inflammation. It has been demonstrated that the expression of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. CGRP has been previously described to influence proliferation of several cell types, such as Schwann cell, tracheal epithelial cells, and human gingival fibroblasts. In the present study, we determined the effect of CGRP on HaCaT keratinocyte proliferation and the role of mitogen-activated protein kinases (MAPKs) in CGRP induced keratinocyte proliferation. Our data indicate CGRP increased [3H]-thymidine incorporation and MTT activity of HaCaT in a concentration-dependent manner. CGRP also enhanced serum-induced HaCaT cell proliferation. HaCaT cells cultured with CGRP had a significant increase in phosphorylated ERK1/2, p38 and JNK, and CGRP induced DNA synthesis was inhibited by PD 98059 or SB 203580, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK) and p38, respectively. These findings suggest that HaCaT cell proliferate in response to CGRP, which is mediated by phosphorylation of ERK1/2 and p38 MAPK.


Keywords: CGRP, Cell proliferation, ERK1/2, JNK, p38 MAPK, HaCaT keratinocytes, Psoriasis.

*Corresponding author. Tel.: +86 531 82169390; fax: +86 531 86927544.


1 Introduction

Psoriasis is a chronic hyperproliferative skin disease characterized by keratinocyte hyperproliferation and inflammation (Baker and Fry, 1992; Creamer et al., 1997). Although dysfunction of the immune system is known to be an important factor in the pathogenesis of psoriasis, there is also strong evidence that keratinocyte hyperproliferation contribute to the disease. The proliferation and differentiation of keratinocytes are controlled by a complex network of growth factors and cytokines (Luger and Schwarz, 1990). Calcitonin gene-related peptide (CGRP), a 37-amino acid peptide, is one of the most abundant neuropeptides in human and rodent skin. It has been demonstrated that the expression of CGRP is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis (Jiang et al., 1998; Farber and Raychaudhuri, 1999). These findings suggest that CGRP may play a significant role in the pathophysiologic process of psoriasis.

The effects of CGRP on cell proliferation are more complex and context dependent. CGRP has been previously described to positively influence proliferation of Schwann cells (Cheng et al., 1995), tracheal epithelial cells (White et al., 1993), and human Gin-1 gingival fibroblasts (Kawase et al., 1999). On the contrary, it was reported to posses antiproliferative effects on cultured smooth muscle cells (SMC) (Deng et al., 2006; Qin et al., 2004; Chattergoon et al., 2005). The proliferation of several phenotypes of cells is mediated by mitogen-activated protein kinases (MAPKs), a family of serine-threonine protein. There are three well-characterized MAPK subfamilies in mammalian cells: extracellular-signal-regulated protein kinase (ERK), the p38 mitogen-activated protein kinases (p38 MAPKs) and the c-Jun N-terminal kinase (JNK) (Cowan and Storey, 2003). On activation by phosphorylation of both threonine and tyrosine residues, these kinases phosphorylate intracellular enzymes and transcription factors. The activation of MAPKs is a key component in signal transduction associated with cell proliferation.

This study was designed to investigate the effect of CGRP on cell proliferation by the human keratinocyte cell line HaCaT, and the role of MAPKs in CGRP induced HaCaT proliferation. We found that CGRP increased HaCaT proliferation and the proliferative effects were mediated by phosphorylation of ERK1/2 and p38.

2 Materials and methods

2.1 Materials

Human α-calcitonin gene-related peptide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (Saint Louis, MO). PD 98059, SB 203580 and SP 600125 were obtained from Biosource (Camarillo, CA). [3H]-thymidine was purchased from Institute of High Energy Physics, Chinese Academy of Sciences (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA). Mouse antiphospho-ERK1/2 (p-ERK1/2) antibody, rabbit anti-ERK1/2 antibody, mouse antiphospho-p38 (p-p38) antibody, mouse anti-p38 antibody, mouse antiphospho-JNK (p-JNK) antibody, mouse anti-JNK antibody and rabbit anti-actin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

2.2 MTT and [3H]-thymidine incorporation assay for cell proliferation

Human keratinocyte cell lines HaCaT cells were cultured in DMEM supplemented with 10% fetal calf serum (FBS), 100 U/ml penicillin and 100μg/ml streptomycin. Cells were seeded in wells of a 96-well plate at a density of 3×103cells/well and allowed to adhere overnight in the presence of 10% FBS medium. Then the medium was changed for DMEM containing 0.5% FBS to make them quiescent for 24h. HaCaT cells were incubated with CGRP (1, 10, 100nM) in serum-free medium (0.2% FBS/DMEM) for 24h. Then a volume of 200μL of 0.5mg/ml MTT in DMEM was added to each well and incubated for 4h at 37°C. Formazan crystals were dissolved in 150μL of DMSO and the absorbance was measured at a wavelength of 570nm with an enzyme-linked immunosorbent assay (ELISA) reader (1500, Thermo Electron Corp., USA).

For [3H]-thymidine incorporation assay, 5μCi/ml [3H]-thymidine was added for the final 8h and the incorporation was terminated by washing the cells with PBS twice. The cells were lysed with 0.2N NaOH and the radioactivity were counted with a scintillation counter (Wallac Guardian 1414, PerkinElmer Life and Analytical Sciences Inc., Boston, MA, USA). To determine whether CGRP may interact with mitogenic substances in serum and affect HaCaT cells proliferation, growth-arrested HaCaT cells were incubated with CGRP (1, 10, 100nM) in DMEM containing 10% FBS for 24h, and [3H]-thymidine incorporation assay was carried. To confirm the involvement of MAPKs in CGRP induced proliferation of HaCaT cells, the cells were pretreated for 2h with 50μM of PD 98059, 10μM of SB 203580, or 10μM of SP 600125, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK), p38 and JNK, respectively. Then 10nM CGRP was added to the medium and the cells were cultured for 24h, followed by [3H]-thymidine incorporation assay.

2.3 Western blot analysis

Stimulated cells were lysed with ice-cold lysis buffer containing 50mM Tris–HCl (pH 7.4), 150mM NaCl, 1%(octylphenoxy) polyethoxyethanol CA-630, 0.25% sodiumdeoxycholate, 1mM EGTA, 1mM sodium fluoride, 1mM sodium orthovanadate, 1mM PMSF, and mammalian protease inhibitor mixture. Protein determination was performed using the Bradford's method. Equal amounts of protein were boiled for 5min and electrophoresed on a 10% SDS-polyacrylamide gel (PAGE) for 45min at 200V. Proteins were transferred to a 0.22μm nitrocellulose membrane (BioRad, Hercules, CA) at 30V for 1h. After blockade with 5% non-fat milk in Tris-buffered saline, the membranes were incubated overnight at 4°C with following primary antibodies: anti-total or anti-phosphorylated ERK1/2 (which recognizes p42 and p44 MAPK), p38, and JNK (which recognizes p46 and p54 JNK). This was followed by incubating the blot with a HRP-conjugated secondary antibody for 1h at room temperature. The equal loading of proteins was confirmed by immunodetecting the blots with anti-β-actin antibody. The blots were then visualized with enhanced chemiluminescence (ECL) according to the manufacture's instruction.

2.4 Statistical analysis

The statistical significance was calculated by the two-sample independent-groups t-test. p<0.05 was considered significant.

3 Results

3.1 CGRP increased HaCaT proliferation

In order to determine whether CGRP acts as a growth factor, cells were seeded in wells of a 96-well plate at a density of 3×103cells/well and allowed to adhere overnight in the presence of 10% FBS medium. Then the medium was changed for DMEM containing 0.5% FBS to make them quiescent for 24h. HaCaT cells were incubated with CGRP (1, 10, 100nM) in serum-free medium for 24h. All concentrations of CGRP (1, 10 and 100nM) induced significant increase in [3H]-thymidine incorporation by the HaCaT cells (Fig. 1). CGRP also induced a significant concentration-dependent increase in MTT activity of the HaCaT cells (Fig. 1). As a parameter of cell proliferation, [3H]-thymidine incorporation is more sensitive than MTT assay.


Fig. 1

CGRP concentration-dependently increased proliferation of HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were incubated with various concentrations of CGRP in 0.2% FBS/DMEM for 24h either [3H]-thymidine incorporation assay or MTT assay. Significant differences were detected at all concentrations of CGRP treatment compared to the untreated control, p<0.01, n=8.


3.2 CGRP augmented serum-induced proliferation

Serum contains a variety of mitogenic substances. HaCaT cells can respond synergistically to a wide variety of mitogen combinations. CGRP may interact with these substances and affect HaCaT cell proliferation. To determine this, we treated confluent, serum-free HaCaT cells with 10% FBS in the absence or presence of CGRP (10nM) for 24h and measured the changes of thymidine incorporation and MTT activity. DNA synthesis and cell number were significantly increased after treatment with 10% FBS compared to the cells cultured in serum-free medium (Fig. 2). The serum-induced increases in thymidine incorporation and MTT activity were further enhanced by addition of 10nM CGRP (Fig. 2).


Fig. 2

CGRP enhanced serum-induced proliferations of HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were treated with 10% FBS in the absence or presence of CGRP (10nM) for 24h and the changes of [3H]-thymidine incorporation and MTT assay were determined. All values are % of untreated control cultured in 0.2% FBS/DMEM. *p<0.01, significant differences compared to control, n=8.


3.3 Roles of MAPKs in CGRP induced proliferation

Next, we determined if MAPKs play any role in CGRP induced increase in proliferation. HaCaT cells were pretreated for one hour with PD 98059, SB 203580, or SP 600125, followed by 24-h CGRP treatment and [3H]-thymidine incorporation assay. The CGRP induced DNA synthesis of HaCaT cells was attenuated by the addition of PD 98059 or SB 203580, but not SP 600125 (Fig. 3).


Fig. 3

Effects of MAPKs inhibitors on CGRP induced increase of proliferation in HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were pretreated for 1h with PD 98059, SB 203580, or SP 600125, prior to 24-h treatment with 10nM of CGRP (CG). DNA synthesis was measured by [3H]-thymidine incorporation assay. Inhibition of phosphorylated ERK1/2 and p38 reduced CGRP induced DNA synthesis. *p<0.01 compared to untreated control (C), **p<0.01 compared to CG, n=8.


In order to demonstrate in direct evidence of the activation of MAPKs, HaCaT cells were treated with 10nM of CGRP for 5, 30min, 24 and 48h, followed by extraction of the cellular protein. The expressions of total and phosphorylated ERK1/2, p38, and JNK were determined by Western analysis. The CGRP induced increases in phospho-ERK1/2 occurred only after 30min treatment and this was not decreased by 24-h treatment (Fig. 4A). CGRP induced rapid increases in phospho-p38 (Fig. 4B) and phospho-JNK (Fig. 4C), beginning as early as 5min after addition of CGRP. Longer treatment (24h) with CGRP led to a decrease in both phospho-p38 and phospho-JNK. There was no change in the expression of total p38, ERK1/2, and JNK.


Fig. 4

CGRP increased expression of phosphorylated MAPKs in HaCaT keratinocytes. Confluent and growth-arrested HaCaT keratinocytes were incubated with 10nM of CGRP for 5, 30min, 24 or 48h prior to protein extraction and Western analysis for phosphorylated or total ERK1/2 (Panel A), p38 (Panel B), and JNK (Panel C). The blots are representatives of 3 independent experiments. n=4.


Furthermore, total and phosphorylated p38, ERK1/2, and JNK were determined using the cellular protein of HaCaT cells treated with CGRP for 24h in the presence or absence of SB 203580, PD 98059, or SP 600125. Western analysis revealed that CGRP induced phosphorylation of ERK1/2 and p38 were inhibited by PD 98059, SB 203580, respectively (Fig. 5). There were no changes in phosphorylation of JNK between cells of control, CGRP, and SP 600125 plus CGRP treatment (Fig. 5). These data suggest that CGRP induced increase in proliferation may be mediated by the activation of ERK1/2 and p38 MAPK.


Fig. 5

Effects of MAPKs inhibitors on CGRP induced activation of MAPKs. Confluent and growth-arrested HaCaT keratinocytes were pretreated for 1h with PD 98059, SB 203580, or SP 600125, prior to 24-h treatment with 10nM of CGRP (CG), followed by protein extraction and Western analysis for phosphorylated or total ERK1/2 (Panel A), p38 (Panel B), and JNK (Panel C). The blots are representatives of 3 independent experiments. n=4.


4 Discussion

HaCaT cells are the product of an experimental line derived from normal epidermal keratinocytes; they are immoral, maintain differentiation potential and are used as a model of keratinocytes function (Boukamp et al., 1988). Although there are some reports on the different responses between normal primary keratinocytes and HaCaT (Weninger et al., 1998; Kwon et al., 2004; Park et al., 2005), HaCaT exhibits most of the characteristics of the basal keratinocytes (Wraight et al., 1994; Wraight and Werther, 1995).

In this study we have demonstrated that CGRP increases proliferation in serum-free condition and enhances serum-induced proliferation of confluent HaCaT cells. This observation is consistent with the reports that CGRP stimulate DNA synthesis in cultured keratinocytes and proliferation of a human squamous cell carcinoma cell line in vitro (Takahashi et al., 1993). These findings have important clinical significance, because over expression of CGRP was found in psoriasis (Jiang et al., 1998; Farber and Raychaudhuri, 1999). Most recently, it was found that CGRP contributes to the UVB-induced keratinocytes proliferation and acanthosis in UVB-irradiated murine skin (Seike et al., 2002). Moreover, we found that CGRP enhances serum-induced HaCaT cell proliferation that occurs in epidermis of psoriasis where there is inflammation leading to increase in cytokines mitogen for keratinocytes. The findings suggest that the mitogenic effects of the cytokines would be enhanced by CGRP, and augment the HaCaT cell hyperplasia.

Phosphorylation of ERK1/2 has been reported to mediate mitogen-induced proliferation, while the phosphorylation of p38 and JNK are activated by a variety of non-specific stimuli such as changes in oxidation, osmolarity, and inflammatory cytokines (Johnson and Lapadat, 2002; Kyriakis and Avruch, 1996). The important roles of MAPKs activation in keratinocyte proliferation induced by IL-6, hepatocyte growth factor, EGF, estradiol and so on have been reported (Gallucci et al., 2004; Yano et al., 2003; Delehedde et al., 2002; Kaufmann and Thiel, 2002; Verdier-Sevrain et al., 2004). However, it is not known if MAPKs mediate CGRP induced HaCaT cell proliferation. In this study, for the first time, we have demonstrated that CGRP induced proliferation of HaCaT cells is associated with MAPKs. Since the inhibitors of ERK1/2 and p38 blocked CGRP induced proliferation, our data suggest that the activation of ERK1/2 and p38 is important for the CGRP induced increase in HaCaT cell proliferation. In our study, there are some differences in the time required for activation of MAPKs after CGRP stimulation amongst the MAPKs. P38 and JNK were rapidly activated by CGRP, which was as early as 5min. However, the activation of ERK1/2 required prolonged treatment with CGRP (30min). The activation of JNK lasted only 30min, and the blockade of JNK activation failed to inhibit the HaCaT cell proliferation induced by 24 h of CGRP treatment, indicating that the activation of JNK may not be important in mediating CGRP induced proliferation of HaCaT cells.

In conclusion, our results demonstrate that CGRP increases HaCaT cells proliferation, and also enhances serum-induced HaCaT cells proliferation. In addition, the activation of ERK1/2 and p38 play an important role in mediating the CGRP induced proliferation by HaCaT keratinocytes. These findings suggest that CGRP which is elevated expressed in epidermis of psoriasis may involve in the pathogenesis of psoriasis by enhancing keratinocyte proliferation.

Acknowledgements

This work was supported by Natural Science Grant (Y2005C67) from the Shandong Ministry of Science and Technology.

References

Baker BS, Fry, L. The immunology of psoriasis. Br J Dermatol 1992:126:1:1-9
Crossref   Medline   1st Citation  

Boukamp P, Petrussevska, RT, Breitkreutz, D, Hornung, J, Markham, A, Fusenig, NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988:106:3:761-71
Crossref   Medline   1st Citation  

Chattergoon NN, D'Souza, FM, Deng, W, Chen, H, Hyman, AL, Kadowitz, PJ. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2005:288:1:L202-211
Crossref   Medline   1st Citation  

Cheng L, Khan, M, Mudge, AW. Calcitonin gene-related peptide promotes Schwann cell proliferation. J Cell Biol 1995:129:3:789-96
Crossref   Medline   1st Citation  

Cowan KJ, Storey, KB. Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J Exp Biol 2003:206:Pt7:1107-15
Medline   1st Citation  

Creamer D, Allen, MH, Sousa, A, Poston, R, Barker, JN. Localization of endothelial proliferation and microvascular expansion in active plaque psoriasis. Br J Dermatol 1997:136:6:859-65
Crossref   Medline   1st Citation  

Delehedde M, Lyon, M, Vidyasagar, R, McDonnell, TJ, Fernig, DG. Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes. J Biol Chem 2002:277:14:12456-62
Crossref   Medline   1st Citation  

Deng W, St Hilaire, RC, Chattergoon, NN, Jeter, JR, Kadowitz, PJ. Inhibition of vascular smooth muscle cell proliferation in vitro by genetically engineered marrow stromal cells secreting calcitonin gene-related peptide. Life Sci 2006:78:16:1830-8
Crossref   Medline   1st Citation  

Farber EM, Raychaudhuri, SP. Is psoriasis a neuroimmunologic disease? Int J Dermatol 1999:38:1:12-5
Crossref   Medline   1st Citation   2nd  

Gallucci RM, Sloan, DK, Heck, JM, Murray, AR, O'Dell, SJ. Interleukin 6 indirectly induces keratinocyte migration. J Invest Dermatol 2004:122:3:764-72
Crossref   Medline   1st Citation  

Jiang WY, Raychaudhuri, SP, Farber, EM. Double-labeled immunofluorescence study of cutaneous nerves in psoriasis. Int J Dermatol 1998:37:8:572-4
Crossref   Medline   1st Citation   2nd  

Johnson GL, Lapadat, R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002:298:5600:1911-2
Crossref   Medline   1st Citation  

Kaufmann K, Thiel, G. Epidermal growth factor and thrombin induced proliferation of immortalized human keratinocytes is coupled to the synthesis of Egr-1, a zinc finger transcriptional regulator. J Cell Biochem 2002:85:2:381-91
Crossref   Medline   1st Citation  

Kawase T, Okuda, K, Wu, CH, Yoshie, H, Hara, K, Burns, DM. Calcitonin gene-related peptide acts as a mitogen for human Gin-1 gingival fibroblasts by activating the MAP kinase signalling pathway. J Periodont Res 1999:34:3:160-8
Crossref   Medline   1st Citation  

Kwon YW, Kwon, KS, Moon, HE, Choi, KS, Kim, YS, Jang, HS. Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT. J Invest Dermatol 2004:123:1:152-8
Crossref   Medline   1st Citation  

Kyriakis JM, Avruch, J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 1996:18:7:567-77
Crossref   Medline   1st Citation  

Luger TA, Schwarz, T. Evidence for an epidermal cytokine network. J Invest Dermatol 1990:95:Suppl. 6:100S-4S
Medline   1st Citation  

Park HJ, Kim, HJ, Lee, JH, Lee, JY, Cho, BK, Kang, JS. Corticotropin-releasing hormone (CRH) downregulates interleukin-18 expression in human HaCaT keratinocytes by activation of p38 mitogen-activated protein kinase (MAPK) pathway. J Invest Dermatol 2005:124:4:751-5
Crossref   Medline   1st Citation  

Qin XP, Ye, F, Hu, CP, Liao, DF, Deng, HW, Li, YJ. Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells. Eur J Pharmacol 2004:488:1–3:45-9
Crossref   Medline   1st Citation  

Seike M, Ikeda, M, Morimoto, A, Matsumoto, M, Kodama, H. Increased synthesis of calcitonin gene-related peptide stimulates keratinocyte proliferation in murine UVB-irradiated skin. J Dermatol Sci 2002:28:2:135-43
Crossref   Medline   1st Citation  

Takahashi K, Nakanishi, S, Imamura, S. Direct effects of cutaneous neuropeptides on adenylyl cyclase activity and proliferation in a keratinocyte cell line: stimulation of cyclic AMP formation by CGRP and VIP/PHM, and inhibition by NPY through G protein-coupled receptors. J Invest Dermatol 1993:101:5:646-51
Crossref   Medline   1st Citation  

Verdier-Sevrain S, Yaar, M, Cantatore, J, Traish, A, Gilchrest, BA. Estradiol induces proliferation of keratinocytes via a receptor mediated mechanism. FASEB J 2004:18:11:1252-4
Medline   1st Citation  

Weninger W, Rendl, M, Mildner, M, Tschachler, E. Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes. J Invest Dermatol 1998:111:5:907-11
Crossref   Medline   1st Citation  

White SR, Hershenson, MB, Sigrist, KS, Zimmermann, A, Solway, J. Proliferation of guinea pig tracheal epithelial cells induced by calcitonin gene-related peptide. Am J Respir Cell Mol Biol 1993:8:6:592-6
Medline   1st Citation  

Wraight CJ, Werther, GA. Insulin-like growth factor-I and epidermal growth factor regulate insulin-like growth factor binding protein-3 (IGFBP-3) in the human keratinocyte cell line HaCaT. J Invest Dermatol 1995:105:4:602-7
Crossref   Medline   1st Citation  

Wraight CJ, Murashita, MM, Russo, VC, Werther, GA. A keratinocyte cell line synthesizes a predominant insulin-like growth actor-binding protein (IGFBP-3) that modulates insulin-like growth factor-I action. J Invest Dermatol 1994:103:5:627-31
Crossref   Medline   1st Citation  

Yano S, Komine, M, Fujimoto, M, Okochi, H, Tamaki, K. Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line, HaCaT. Biochem Biophys Res Commun 2003:301:4:841-7
Crossref   Medline   1st Citation  


Received 27 July 2009; accepted 28 July 2009

doi:10.1016/j.cellbi.2009.07.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)