- Email: [email protected]
Thymic stromal lymphopoietin is regulated by the intracellular calcium
Cytokine 59 (2012) 215–217
Contents lists available at SciVerse ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Short Communication
Thymic stromal lymphopoietin is regulated by the intracellular calcium Na-Ra Han a, Hyung-Min Kim a,⇑,1, Hyun-Ja Jeong b,⇑,1 a b
Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea Biochip Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 December 2011 Received in revised form 12 March 2012 Accepted 11 April 2012 Available online 11 May 2012 Keywords: Calcium Caspase-1 Receptor interacting protein2 Thymic stromal lymphopoietin
a b s t r a c t The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic diseases such as atopic dermatitis. However, it has not been clarified that TSLP would be regulated by intracellular calcium in mast cells yet. To determine it, we blocked intracellular calcium by treatment with calcium chelator, 2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA-AM) in human mast cell line (HMC-1) cells. BAPTA-AM inhibited the production and mRNA expression of TSLP in phorbol myristate acetate plus A23187- stimulated HMC-1 cells. BAPTA-AM also inhibited the nuclear factor-jB activation, IjBa phosphorylation, receptor interacting protein2 (RIP2) expression, and caspase-1 activation in HMC-1 cells. These results provide evidence that calcium regulates the level of TSLP through RIP2/caspase-1/NF-jB/IjBa signal. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as AD and asthma [1]. Moon and Kim reported that TSLP was significantly expressed in stimulated mast cells [2]. Mast cells participate in the inflammatory process in skin leading to atopic dermatitis [3]. When the mast cells are damaged by external stimulation such as inflammatory stimuli, the level of intracellular calcium is raised by an influx of extracellular or a release from an intracellular store [4]. An increase in the intracellular calcium level has been proposed as an essential trigger for mast cell activation [5]. Caspase-1 (previously known as interleukin (IL)-1 converting enzyme) is responsible for activating the inactive precursors of IL-1b that are critical for inflammation [6]. Receptor interacting protein2 (RIP2) is a caspase recruitment domain-containing kinase that interacts with caspase-1 and plays an important role in NF-jB activation [7]. The expression of inflammatory cytokines depends on the activation of NF-jB and phosphorylation of IjBa [8]. Furthermore, TSLP was expressed and produced through caspase-1 and NF-jB in mast cells [2]. 1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA-AM) is a intracellular calcium chelator. In this study, we investigated whether level of TSLP could be regulated through the blockade of intracellular calcium by treatment with BAPTA-AM in human mast cell line (HMC-1) cells.
The materials and methods are described in the supplementary online material (Appendix A).
⇑ Corresponding authors. Tel.: +82 41 540 9681; fax: +82 41 542 9681. 1
E-mail addresses: [email protected] (H.-M. Kim), [email protected] (H.-J. Jeong). These authors equally contributed to this work.
1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.04.015
3. Results and discussion First, we studied the cell viability of BAPTA-AM in HMC-1 cells in accordance with the reports of Jeong et al. [9]. As shown in Fig. S1, we also confirmed that BAPTA-AM (10 lM) did not have the cytotoxicity. In mast cells, an increase of intracellular calcium level produces a large number of cytokines [5]. We stimulated HMC-1 cells with the PKC activator PMA as a substitute for diacylglycerol and A23187 as ionophore (PMACI). We examined whether BAPTA-AM could dose-dependently inhibit intracellular calcium level in PMACI-stimulated HMC-1 cells. The stimulation with PMACI increased calcium release from intracellular stores in HMC-1 cells. However, BAPTA-AM inhibited intracellular calcium level in stimulated HMC-1 cells (Fig. S2). Intriguingly, we hypothesized that the blockade of calcium by treatment with BAPTA-AM would inhibit the production and mRNA expression of TSLP. The TSLP production was significantly inhibited by treatment with BAPTA-AM in PMACI-stimulated cells (10 lM, p < 0.05, Fig. 1a). Next, we studied whether BAPTA-AM can modulate PMACI-induced mRNA expression of TSLP. The mRNA expression of TSLP was up-regulated by PMACI, but the up-regulated TSLP mRNA expression was significantly decreased by treatment with BAPTA-AM (10 lM, p < 0.05, Fig. 1b, c). This is the first study showing an inhibition of TSLP by blockade of calcium in mast cells. In addition, TSLP expression
216
N.-R. Han et al. / Cytokine 59 (2012) 215–217
Fig. 1. Effects of BAPTA-AM on production and mRNA expression of TSLP as well as on activation of NF-jB and phosphorylation of IjBa in HMC-1 cells. (a) HMC-1 cells (4 105) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and then stimulated with PMACI for 7 h. The levels of TSLP in the supernatant were measured with the ELISA method. (b) HMC-1 cells (1 106) were treated with BAPTA-AM for 2 h and then stimulated with PMACI for 5 h. The mRNA was measured with the RT-PCR method. M, marker. (c) The mRNA expression level of TSLP was evaluated by the real time PCR method. (d) HMC-1 cells (5 106) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and stimulated with PMACI for 2 h for activation of NF-jB and phosphorylation of IjBa and measured with Western blot analysis. (e) Each protein level was quantified by densitometry. (f) HMC-1 cells (1 107) were transiently transfected pNF-jB-LUC and pSV40-LUC and treated with BAPTA-AM for 2 h and then stimulated with PMACI for 48 h. The NF-jB activity was assessed with a luciferase assay. NE, nuclear extract; CE, cytoplasmic extract; pIjBa, phosphorylation of IjBa. #p < 0.05; significantly different from the unstimulated cells value. ⁄p < 0.05; significantly different from the PMACI-stimulated cells value.
Fig. 2. Effect of BAPTA-AM on RIP2/caspase-1 pathway in PMACI-stimulated HMC-1 Cells. (a) HMC-1 cells (5 106) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and stimulated with PMACI for 30 min for RIP2 activation or 1 h for caspase-1 activation and measured with Western blot analysis. (b) All the protein levels were quantified by densitometry. (c) HMC-1 cells (5 106) were treated with BAPTA-AM for 2 h and then stimulated with PMACI for 1 h. CI, treatment with caspase-1 inhibitor in PMACIstimulated cells. #p < 0.05; significantly different from the unstimulated cells value, ⁄p < 0.05; significantly different from the PMACI-stimulated cells value. (d, e) Catalytic activity of recombinant caspase-1 (0.5 units) was measured by WEHD-pNA (substrate), BAPTA-AM, BAPTA, or caspase-1 inhibitor for various times. B, no substrate group; C, substrate-added group; BAPTA-AM, substrate and BAPTA-AM-added group; BAPTA, substrate and BAPTA-added group; CI, substrate and CI (500 nM)-added group. #p < 0.05; significantly different from no substrate group value, ⁄p < 0.05; significantly different from substrate-added group value.
N.-R. Han et al. / Cytokine 59 (2012) 215–217
was significantly elevated in the epidermis of lesional skin from individuals with acute and chronic atopic dermatitis. [10]. Thus, we can assume that blockade of calcium can help us to treat atopic diseases aggravated by increased TSLP. Furthermore, we studied which mechanism is responsible for regulating TSLP level by blockade of calcium. TSLP expression was mediated via NF-jB in human airway smooth muscle cells or mast cells [2,11]. Thus, we studied whether BAPTA-AM could inhibit TSLP level through NF-jB/IjBa pathway. The stimulation with PMACI increased the translocated levels of NF-jB in nucleus and phosphorylation of IjBa in cytosol, but the translocated levels of NF-jB and phosphorylation of IjBa were significantly suppressed by the treatment with BAPTA-AM (p < 0.05, Fig. 1d, e). Several studies have reported that the translocation of NF-jB in nucleus and phosphorylation of IjBa in cytosol were blocked by BAPTA-AM in RBL-2H3 mast cells [12]. Our result also showed BAPTA-AM inhibited NF-jB/IjBa activation in HMC-1 cells. In addition, BAPTA-AM dose-dependently reduced NF-jB luciferase activity increased in PMACI- stimulated HMC-1 cells (p < 0.05, Fig. 1f). From previous reports and our result, we could confirm that blockade of calcium reduced the level of TSLP through NFjB/IjBa signal in mast cells. Upon receipt of proinflammatory stimuli, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation [13]. RIP2 is an upstream activator of NF-jB and caspase-1 induces NF-jB activation [6]. Caspase-1 level was significantly increased in serum from allergic asthmatic patients as compared to normal person [14]. The production of inflammatory cytokines was reduced in macrophages of RIP2/ mice [15]. Thus, we studied whether the activations of RIP2 and caspase-1 would be inhibited through the blockade of intracellular calcium in PMACI-stimulated cells. Treatment with BAPTA-AM significantly inhibited the activations of RIP2 and caspase-1 in PMACI-stimulated cells (p < 0.05; Fig. 2a, b). In addition, BAPTA-AM significantly inhibited the caspase-1 enzymatic activity increased by PMACI (p < 0.05, Fig. 2c). Finally, we examined whether BAPTA-AM and BAPTA could inhibit the cleavage of caspase-specific peptide (tetrapeptide WEHD-pNA) by recombinant caspase-1 by evaluating the binding reaction between recombinant caspase-1 and tetrapeptide WEHDpNA. The binding affinity between recombinant caspase-1 and tetrapeptide WEHD-pNA was significantly inhibited by BAPTA-AM (0.1, 1, and 10 lM) or BAPTA (0.1, 1, and 10 lM) for various times (p < 0.05, Fig. 2d, e). Thus, we could confirm that RIP2/caspase-1 activation is induced through the regulation of calcium level in mast cells.
4. Conclusions We showed that BAPTA-AM inhibits the expression and production of TSLP through the blockade of RIP2/caspase-1/NF-jB/IjBa signal cascade in mast cells. In other words, calcium plays the
217
crucial role in regulating TSLP through RIP2/caspase-1/NF-jB/IjBa signal. Overall, blockade of calcium can help to treat inflammatory and atopic diseases aggravated by TSLP. Conflict of interest The authors declare no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20110027280). References [1] Shan L, Redhu NS, Saleh A, Halayko AJ, Chakir J, Gounni AS. Thymic stromal lymphopoietin receptor-mediated IL-6 and CC/CXC chemokines expression in human airway smooth muscle cells: role of MAPKs (ERK1/2, p38, and JNK) and STAT3 pathways. J Immunol 2010;184:7134–43. [2] Moon PD, Kim HM. Thymic stromal lymphopoietin is expressed and produced by caspase-1/NF-jB pathway in mast cells. Cytokine 2011;54:239–43. [3] Damsgaard TE, Olesen AB, Sørensen FB, Thestrup-Pedersen K, Schiøtz PO. Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin. Arch Dermatol Res 1997;289:256–60. [4] Hayakawa M, Hayakawa H, Matsuyama Y, Tamemoto H, Okazaki H, Tominaga S. Mature interleukin-33 is produced by calpain-mediated cleavage in vivo. Biochem Biophys Res Commun 2009;387:218–22. [5] Ma HT, Beaven MA. Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases? Adv Exp Med Biol 2011;716:62–90. [6] Oh HA, Ryu JG, Cha WS, Kim HM, Jeong HJ. Therapeutic effects of traditional Korean medicine Jeechool-Whan in allergic rhinitis model. TANG 2012;2:e9. [7] Sarkar A, Duncan M, Hart J, Hertlein E, Guttridge DC, Wewers MD. ASC directs NF-kappaB activation by regulating receptor interacting protein-2 (RIP2) caspase-1 interactions. J Immunol 2006;176:4979–86. [8] Brodsky M, Halpert G, Albeck M, Sredni B. The anti-inflammatory effects of the tellurium redox modulating compound, AS101, are associated with regulation of NFkappaB signaling pathway and nitric oxide induction in macrophages. J Inflamm (Lond) 2010;7:3. [9] Jeong HJ, Hong SH, Park RK, An NH, Kim HM. Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway. Life Sci 2005;77:2179–92. [10] Soumelis V, Reche PA, Kanzler H, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 2002;3:673–80. [11] Redhu NS, Saleh A, Halayko AJ, Ali AS, Gounni AS. Essential role of NF-jB and AP-1 transcription factors in TNF-a-induced TSLP expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2011;300:L479–485. [12] Jeong HJ, Hong SH, Lee DJ, Park JH, Kim KS, Kim HM. Role of Ca(2+) on TNFalpha and IL-6 secretion from RBL-2H3 mast cells. Cell Signal 2002;14:633–9. [13] Krieg A, Le Negrate G, Reed JC. RIP2-beta: a novel alternative mRNA splice variant of the receptor interacting protein kinase RIP2. Mol Immunol 2009;46:1163–70. [14] Lamkanfi M, Kalai M, Saelens X, Declercq W, Vandenabeele P. Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity. J Biol Chem 2004;279:24785–93. [15] Kobayashi K, Inohara N, Hernandez LD, et al. RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 2002;416:194–9.
Contents lists available at SciVerse ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Short Communication
Thymic stromal lymphopoietin is regulated by the intracellular calcium Na-Ra Han a, Hyung-Min Kim a,⇑,1, Hyun-Ja Jeong b,⇑,1 a b
Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea Biochip Research Center, Hoseo University, 165, Sechul-ri, Baebang-myun, Asan, Chungnam 336-795, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 December 2011 Received in revised form 12 March 2012 Accepted 11 April 2012 Available online 11 May 2012 Keywords: Calcium Caspase-1 Receptor interacting protein2 Thymic stromal lymphopoietin
a b s t r a c t The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic diseases such as atopic dermatitis. However, it has not been clarified that TSLP would be regulated by intracellular calcium in mast cells yet. To determine it, we blocked intracellular calcium by treatment with calcium chelator, 2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA-AM) in human mast cell line (HMC-1) cells. BAPTA-AM inhibited the production and mRNA expression of TSLP in phorbol myristate acetate plus A23187- stimulated HMC-1 cells. BAPTA-AM also inhibited the nuclear factor-jB activation, IjBa phosphorylation, receptor interacting protein2 (RIP2) expression, and caspase-1 activation in HMC-1 cells. These results provide evidence that calcium regulates the level of TSLP through RIP2/caspase-1/NF-jB/IjBa signal. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as AD and asthma [1]. Moon and Kim reported that TSLP was significantly expressed in stimulated mast cells [2]. Mast cells participate in the inflammatory process in skin leading to atopic dermatitis [3]. When the mast cells are damaged by external stimulation such as inflammatory stimuli, the level of intracellular calcium is raised by an influx of extracellular or a release from an intracellular store [4]. An increase in the intracellular calcium level has been proposed as an essential trigger for mast cell activation [5]. Caspase-1 (previously known as interleukin (IL)-1 converting enzyme) is responsible for activating the inactive precursors of IL-1b that are critical for inflammation [6]. Receptor interacting protein2 (RIP2) is a caspase recruitment domain-containing kinase that interacts with caspase-1 and plays an important role in NF-jB activation [7]. The expression of inflammatory cytokines depends on the activation of NF-jB and phosphorylation of IjBa [8]. Furthermore, TSLP was expressed and produced through caspase-1 and NF-jB in mast cells [2]. 1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA-AM) is a intracellular calcium chelator. In this study, we investigated whether level of TSLP could be regulated through the blockade of intracellular calcium by treatment with BAPTA-AM in human mast cell line (HMC-1) cells.
The materials and methods are described in the supplementary online material (Appendix A).
⇑ Corresponding authors. Tel.: +82 41 540 9681; fax: +82 41 542 9681. 1
E-mail addresses: [email protected] (H.-M. Kim), [email protected] (H.-J. Jeong). These authors equally contributed to this work.
1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.04.015
3. Results and discussion First, we studied the cell viability of BAPTA-AM in HMC-1 cells in accordance with the reports of Jeong et al. [9]. As shown in Fig. S1, we also confirmed that BAPTA-AM (10 lM) did not have the cytotoxicity. In mast cells, an increase of intracellular calcium level produces a large number of cytokines [5]. We stimulated HMC-1 cells with the PKC activator PMA as a substitute for diacylglycerol and A23187 as ionophore (PMACI). We examined whether BAPTA-AM could dose-dependently inhibit intracellular calcium level in PMACI-stimulated HMC-1 cells. The stimulation with PMACI increased calcium release from intracellular stores in HMC-1 cells. However, BAPTA-AM inhibited intracellular calcium level in stimulated HMC-1 cells (Fig. S2). Intriguingly, we hypothesized that the blockade of calcium by treatment with BAPTA-AM would inhibit the production and mRNA expression of TSLP. The TSLP production was significantly inhibited by treatment with BAPTA-AM in PMACI-stimulated cells (10 lM, p < 0.05, Fig. 1a). Next, we studied whether BAPTA-AM can modulate PMACI-induced mRNA expression of TSLP. The mRNA expression of TSLP was up-regulated by PMACI, but the up-regulated TSLP mRNA expression was significantly decreased by treatment with BAPTA-AM (10 lM, p < 0.05, Fig. 1b, c). This is the first study showing an inhibition of TSLP by blockade of calcium in mast cells. In addition, TSLP expression
216
N.-R. Han et al. / Cytokine 59 (2012) 215–217
Fig. 1. Effects of BAPTA-AM on production and mRNA expression of TSLP as well as on activation of NF-jB and phosphorylation of IjBa in HMC-1 cells. (a) HMC-1 cells (4 105) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and then stimulated with PMACI for 7 h. The levels of TSLP in the supernatant were measured with the ELISA method. (b) HMC-1 cells (1 106) were treated with BAPTA-AM for 2 h and then stimulated with PMACI for 5 h. The mRNA was measured with the RT-PCR method. M, marker. (c) The mRNA expression level of TSLP was evaluated by the real time PCR method. (d) HMC-1 cells (5 106) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and stimulated with PMACI for 2 h for activation of NF-jB and phosphorylation of IjBa and measured with Western blot analysis. (e) Each protein level was quantified by densitometry. (f) HMC-1 cells (1 107) were transiently transfected pNF-jB-LUC and pSV40-LUC and treated with BAPTA-AM for 2 h and then stimulated with PMACI for 48 h. The NF-jB activity was assessed with a luciferase assay. NE, nuclear extract; CE, cytoplasmic extract; pIjBa, phosphorylation of IjBa. #p < 0.05; significantly different from the unstimulated cells value. ⁄p < 0.05; significantly different from the PMACI-stimulated cells value.
Fig. 2. Effect of BAPTA-AM on RIP2/caspase-1 pathway in PMACI-stimulated HMC-1 Cells. (a) HMC-1 cells (5 106) were treated with BAPTA-AM (0.1, 1, and 10 lM) for 2 h and stimulated with PMACI for 30 min for RIP2 activation or 1 h for caspase-1 activation and measured with Western blot analysis. (b) All the protein levels were quantified by densitometry. (c) HMC-1 cells (5 106) were treated with BAPTA-AM for 2 h and then stimulated with PMACI for 1 h. CI, treatment with caspase-1 inhibitor in PMACIstimulated cells. #p < 0.05; significantly different from the unstimulated cells value, ⁄p < 0.05; significantly different from the PMACI-stimulated cells value. (d, e) Catalytic activity of recombinant caspase-1 (0.5 units) was measured by WEHD-pNA (substrate), BAPTA-AM, BAPTA, or caspase-1 inhibitor for various times. B, no substrate group; C, substrate-added group; BAPTA-AM, substrate and BAPTA-AM-added group; BAPTA, substrate and BAPTA-added group; CI, substrate and CI (500 nM)-added group. #p < 0.05; significantly different from no substrate group value, ⁄p < 0.05; significantly different from substrate-added group value.
N.-R. Han et al. / Cytokine 59 (2012) 215–217
was significantly elevated in the epidermis of lesional skin from individuals with acute and chronic atopic dermatitis. [10]. Thus, we can assume that blockade of calcium can help us to treat atopic diseases aggravated by increased TSLP. Furthermore, we studied which mechanism is responsible for regulating TSLP level by blockade of calcium. TSLP expression was mediated via NF-jB in human airway smooth muscle cells or mast cells [2,11]. Thus, we studied whether BAPTA-AM could inhibit TSLP level through NF-jB/IjBa pathway. The stimulation with PMACI increased the translocated levels of NF-jB in nucleus and phosphorylation of IjBa in cytosol, but the translocated levels of NF-jB and phosphorylation of IjBa were significantly suppressed by the treatment with BAPTA-AM (p < 0.05, Fig. 1d, e). Several studies have reported that the translocation of NF-jB in nucleus and phosphorylation of IjBa in cytosol were blocked by BAPTA-AM in RBL-2H3 mast cells [12]. Our result also showed BAPTA-AM inhibited NF-jB/IjBa activation in HMC-1 cells. In addition, BAPTA-AM dose-dependently reduced NF-jB luciferase activity increased in PMACI- stimulated HMC-1 cells (p < 0.05, Fig. 1f). From previous reports and our result, we could confirm that blockade of calcium reduced the level of TSLP through NFjB/IjBa signal in mast cells. Upon receipt of proinflammatory stimuli, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation [13]. RIP2 is an upstream activator of NF-jB and caspase-1 induces NF-jB activation [6]. Caspase-1 level was significantly increased in serum from allergic asthmatic patients as compared to normal person [14]. The production of inflammatory cytokines was reduced in macrophages of RIP2/ mice [15]. Thus, we studied whether the activations of RIP2 and caspase-1 would be inhibited through the blockade of intracellular calcium in PMACI-stimulated cells. Treatment with BAPTA-AM significantly inhibited the activations of RIP2 and caspase-1 in PMACI-stimulated cells (p < 0.05; Fig. 2a, b). In addition, BAPTA-AM significantly inhibited the caspase-1 enzymatic activity increased by PMACI (p < 0.05, Fig. 2c). Finally, we examined whether BAPTA-AM and BAPTA could inhibit the cleavage of caspase-specific peptide (tetrapeptide WEHD-pNA) by recombinant caspase-1 by evaluating the binding reaction between recombinant caspase-1 and tetrapeptide WEHDpNA. The binding affinity between recombinant caspase-1 and tetrapeptide WEHD-pNA was significantly inhibited by BAPTA-AM (0.1, 1, and 10 lM) or BAPTA (0.1, 1, and 10 lM) for various times (p < 0.05, Fig. 2d, e). Thus, we could confirm that RIP2/caspase-1 activation is induced through the regulation of calcium level in mast cells.
4. Conclusions We showed that BAPTA-AM inhibits the expression and production of TSLP through the blockade of RIP2/caspase-1/NF-jB/IjBa signal cascade in mast cells. In other words, calcium plays the
217
crucial role in regulating TSLP through RIP2/caspase-1/NF-jB/IjBa signal. Overall, blockade of calcium can help to treat inflammatory and atopic diseases aggravated by TSLP. Conflict of interest The authors declare no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20110027280). References [1] Shan L, Redhu NS, Saleh A, Halayko AJ, Chakir J, Gounni AS. Thymic stromal lymphopoietin receptor-mediated IL-6 and CC/CXC chemokines expression in human airway smooth muscle cells: role of MAPKs (ERK1/2, p38, and JNK) and STAT3 pathways. J Immunol 2010;184:7134–43. [2] Moon PD, Kim HM. Thymic stromal lymphopoietin is expressed and produced by caspase-1/NF-jB pathway in mast cells. Cytokine 2011;54:239–43. [3] Damsgaard TE, Olesen AB, Sørensen FB, Thestrup-Pedersen K, Schiøtz PO. Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin. Arch Dermatol Res 1997;289:256–60. [4] Hayakawa M, Hayakawa H, Matsuyama Y, Tamemoto H, Okazaki H, Tominaga S. Mature interleukin-33 is produced by calpain-mediated cleavage in vivo. Biochem Biophys Res Commun 2009;387:218–22. [5] Ma HT, Beaven MA. Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases? Adv Exp Med Biol 2011;716:62–90. [6] Oh HA, Ryu JG, Cha WS, Kim HM, Jeong HJ. Therapeutic effects of traditional Korean medicine Jeechool-Whan in allergic rhinitis model. TANG 2012;2:e9. [7] Sarkar A, Duncan M, Hart J, Hertlein E, Guttridge DC, Wewers MD. ASC directs NF-kappaB activation by regulating receptor interacting protein-2 (RIP2) caspase-1 interactions. J Immunol 2006;176:4979–86. [8] Brodsky M, Halpert G, Albeck M, Sredni B. The anti-inflammatory effects of the tellurium redox modulating compound, AS101, are associated with regulation of NFkappaB signaling pathway and nitric oxide induction in macrophages. J Inflamm (Lond) 2010;7:3. [9] Jeong HJ, Hong SH, Park RK, An NH, Kim HM. Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway. Life Sci 2005;77:2179–92. [10] Soumelis V, Reche PA, Kanzler H, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 2002;3:673–80. [11] Redhu NS, Saleh A, Halayko AJ, Ali AS, Gounni AS. Essential role of NF-jB and AP-1 transcription factors in TNF-a-induced TSLP expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2011;300:L479–485. [12] Jeong HJ, Hong SH, Lee DJ, Park JH, Kim KS, Kim HM. Role of Ca(2+) on TNFalpha and IL-6 secretion from RBL-2H3 mast cells. Cell Signal 2002;14:633–9. [13] Krieg A, Le Negrate G, Reed JC. RIP2-beta: a novel alternative mRNA splice variant of the receptor interacting protein kinase RIP2. Mol Immunol 2009;46:1163–70. [14] Lamkanfi M, Kalai M, Saelens X, Declercq W, Vandenabeele P. Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity. J Biol Chem 2004;279:24785–93. [15] Kobayashi K, Inohara N, Hernandez LD, et al. RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 2002;416:194–9.