FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2045474881> ?p ?o ?g. }

• W2045474881 endingPage "2864" @default.
• W2045474881 startingPage "2857" @default.
• W2045474881 abstract "Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. leucine-rich repeat NAip (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from podospora anserina) and TPI (telomerase-associated protein) pyrin domain p38 mitogen-activated protein kinase Caspases are evolutionary conserved cysteine proteases. They are synthesized as inactive proenzymes and become activated following cleavage at specific cleavage sites (Cohen, 1997Cohen G.M. Caspases: the executioners of apoptosis.Biochem J. 1997; 326: 1-16Crossref PubMed Scopus (3982) Google Scholar). Caspases play important roles not only in the induction of apoptosis but also in inflammatory processes. Caspases can be divided into the apoptotic caspases, including caspase-2, -3, -6, -7, -8, -9, and -10 and the inflammatory caspases, including caspase-1, -4, and -5 (Martinon and Tschopp, 2004Martinon F. Tschopp J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases.Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar). Caspase-14 belongs to another subgroup of caspases and has been implicated in keratinocyte differentiation (Eckhart et al., 2000Eckhart L. Declercq W. Ban J. Rendl M. Lengauer B. Mayer C. et al.Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.J Invest Dermatol. 2000; 115: 1148-1151Crossref PubMed Scopus (171) Google Scholar). Caspase-1 is one of the best characterized caspases and is best known for its role in inflammation and innate immune responses to microbial pathogens (Creagh et al., 2003Creagh E.M. Conroy H. Martin S.J. Caspase-activation pathways in apoptosis and immunity.Immunol Rev. 2003; 193: 10-21Crossref PubMed Scopus (263) Google Scholar). Caspase-1 is constitutively expressed as procaspase-1. Cleavage of procaspase-1 to its mature/active form takes place upon assembly of a complex termed the “inflammasome”. To date, the following two types of inflammasomes have been identified: the NALP1 (NACHT (NAip (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from podospora anserina) and TPI (telomerase-associated protein))-, LRR (leucine-rich repeat)-, and PYD (pyrin domain)-containing) inflammasome, which comprises caspase-1, caspase-5, and Asc (apoptosis-associated speck-like protein containing a caspase recruitment domain) (Martinon et al., 2002Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3567) Google Scholar; Martinon and Tschopp, 2004Martinon F. Tschopp J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases.Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar), and the NALP2 and NALP3 inflammasomes, which comprises the caspase recruitment domain-containing protein cardinal, Asc, and caspase-1 (Petrilli et al., 2005Petrilli V. Papin S. Tschopp J. The inflammasome.Curr Biol. 2005; 15: R581Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Processing of procaspase-1 results in the formation of both active and intermediate cleavage fragments (Yamin et al., 1996Yamin T.T. Ayala J.M. Miller D.K. Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme.J Biol Chem. 1996; 271: 13273-13282Crossref PubMed Scopus (156) Google Scholar; Hentze et al., 2003Hentze H. Lin X.Y. Choi M.S. Porter A.G. Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin.Cell Death Differ. 2003; 10: 956-968Crossref PubMed Scopus (137) Google Scholar). Once activated caspase-1 is responsible for the processing of the proinflammatory cytokines IL-1β and IL-18 to their mature forms (Fantuzzi and Dinarello, 1999Fantuzzi G. Dinarello C.A. Interleukin-18 and interleukin-1 beta: two cytokine substrates for ICE (caspase-1).J Clin Immunol. 1999; 19: 1-11Crossref PubMed Scopus (403) Google Scholar). IL-1β and IL-18 plays an important role in host defense against a variety of pathogens. Thus, caspase-1-deficient mice exhibit altered susceptibility to several pathogens (Sansonetti et al., 2000Sansonetti P.J. Phalipon A. Arondel J. Thirumalai K. Banerjee S. Akira S. et al.Caspase-1 activation of IL-1beta and IL-18 are essential for Shigella flexneri-induced inflammation.Immunity. 2000; 12: 581-590Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar; Tsuji et al., 2004Tsuji N.M. Tsutsui H. Seki E. Kuida K. Okamura H. Nakanishi K. et al.Roles of caspase-1 in Listeria infection in mice.Int Immunol. 2004; 16: 335-343Crossref PubMed Scopus (98) Google Scholar; Mariathasan et al., 2005Mariathasan S. Weiss D.S. Dixit V.M. Monack D.M. Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis.J Exp Med. 2005; 202: 1043-1049Crossref PubMed Scopus (318) Google Scholar), and they are resistant to endotoxic shock (Li et al., 1995Li P. Allen H. Banerjee S. Franklin S. Herzog L. Johnston C. et al.Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock.Cell. 1995; 80: 401-411Abstract Full Text PDF PubMed Scopus (1248) Google Scholar). Given the essential role of caspase-1 in the processing of IL-1β and IL-18, the signaling mechanisms underlying caspase-1 activation have been intensively studied but remain only partially defined. Previously, stress-induced activation of caspase-1 and IL-18 has been shown to be mediated through the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in HaCaT cells (Shimizu et al., 1999Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol. 1999; 112: 769-774Crossref PubMed Scopus (112) Google Scholar), in myocardial tissue from rats (Wang et al., 2005Wang M. Tsai B.M. Turrentine M.W. Mahomed Y. Brown J.W. Meldrum D.R. p38 mitogen activated protein kinase mediates both death signaling and functional depression in the heart.Ann Thorac Surg. 2005; 80: 2235-2241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and in plasma from mice (Sekiyama et al., 2005Sekiyama A. Ueda H. Kashiwamura S. Sekiyama R. Takeda M. Rokutan K. et al.A stress-induced, superoxide-mediated caspase-1 activation pathway causes plasma IL-18 upregulation.Immunity. 2005; 22: 669-677Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). p38 MAPK is a kinase known to play a key role in inflammatory processes, cellular growth, differentiation as well as cell death, depending on the cell type and stimulus (Kumar et al., 2003Kumar S. Boehm J. Lee J.C. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases.Nat Rev Drug Discov. 2003; 2: 717-726Crossref PubMed Scopus (1019) Google Scholar; Zarubin and Han, 2005Zarubin T. Han J. Activation and signaling of the p38 MAP kinase pathway.Cell Res. 2005; 15: 11-18Crossref PubMed Scopus (1111) Google Scholar). Furthermore, we have previously demonstrated increased activity of p38 MAPK in lesional psoriatic skin compared with non-lesional psoriatic skin (Johansen et al., 2005Johansen C. Kragballe K. Westergaard M. Henningsen J. Kristiansen K. Iversen L. The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.Br J Dermatol. 2005; 152: 37-42Crossref PubMed Scopus (148) Google Scholar). Psoriasis is an immune-mediated inflammatory skin disorder characterized by skin infiltrating lymphocytes causing hyperproliferation and abnormal differentiation of the keratinocytes (Wrone-Smith and Nickoloff, 1996Wrone-Smith T. Nickoloff B.J. Dermal injection of immunocytes induces psoriasis.J Clin Invest. 1996; 98: 1878-1887Crossref PubMed Scopus (381) Google Scholar; Stern, 1997Stern R.S. Psoriasis.Lancet. 1997; 350: 349-353Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It has been suggested that psoriatic keratinocytes have abnormalities in expression and/or activation of different transcription factors as well as abnormalities in several signaling pathways (McKenzie and Sabin, 2003McKenzie R.C. Sabin E. Aberrant signalling and transcription factor activation as an explanation for the defective growth control and differentiation of keratinocytes in psoriasis: a hypothesis.Exp Dermatol. 2003; 12: 337-345Crossref PubMed Scopus (47) Google Scholar; Johansen et al., 2005aJohansen C. Flindt E. Kragballe K. Henningsen J. Westergaard M. Kristiansen K. et al.Inverse regulation of the nuclear factor-kappaB binding to the p53 and interleukin-8 kappaB response elements in lesional psoriatic skin.J Invest Dermatol. 2005; 124: 1284-1292Crossref PubMed Scopus (52) Google Scholar, Johansen et al., 2005Johansen C. Kragballe K. Westergaard M. Henningsen J. Kristiansen K. Iversen L. The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.Br J Dermatol. 2005; 152: 37-42Crossref PubMed Scopus (148) Google Scholar). IL-18 is a member of the IL-1 cytokine family and was initially isolated as an IFN-γ-inducing factor (Okamura et al., 1995Okamura H. Tsutsi H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. et al.Cloning of a new cytokine that induces IFN-gamma production by T cells.Nature. 1995; 378: 88-91Crossref PubMed Scopus (2317) Google Scholar). IL-18 is a proinflammatory cytokine produced from a biologically inactive precursor, proIL-18 (Dinarello, 1999Dinarello C.A. Interleukin-18.Methods. 1999; 19: 121-132Crossref PubMed Scopus (384) Google Scholar, Dinarello, 2000Dinarello C.A. Interleukin-18, a proinflammatory cytokine.Eur Cytokine Netw. 2000; 11: 483-486PubMed Google Scholar). ProIL-18 is proteolytically activated into an active 18-kDa mature form by caspase-1, although it has been described how anti-bacterial peptides can induce IL-18 secretion by a caspase-1-independent, but p38 MAPK-dependent mechanism in keratinocytes (Niyonsaba et al., 2005Niyonsaba F. Ushio H. Nagaoka I. Okumura K. Ogawa H. The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes.J Immunol. 2005; 175: 1776-1784Crossref PubMed Scopus (192) Google Scholar). IL-18 is expressed in many cell types, including Kupffer cells, macrophages, T cells, B cells, osteoblasts, dendritic cells, astrocytes, and keratinocytes (Nakanishi et al., 2001Nakanishi K. Yoshimoto T. Tsutsui H. Okamura H. Interleukin-18 regulates both Th1 and Th2 responses.Annu Rev Immunol. 2001; 19: 423-474Crossref PubMed Scopus (1043) Google Scholar). Furthermore, human keratinocytes have been demonstrated to constitutively produce and release proIL-18 in vitro (Mee et al., 2000Mee J.B. Alam Y. Groves R.W. Human keratinocytes constitutively produce but do not process interleukin-18.Br J Dermatol. 2000; 143: 330-336Crossref PubMed Scopus (72) Google Scholar). Interestingly, it has been reported that the expression of IL-18 is markedly increased in psoriasis (Naik et al., 1999Naik S.M. Cannon G. Burbach G.J. Singh S.R. Swerlick R.A. Wilcox J.N. et al.Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.J Invest Dermatol. 1999; 113: 766-772Crossref PubMed Scopus (119) Google Scholar; Ohta et al., 2001Ohta Y. Hamada Y. Katsuoka K. Expression of IL-18 in psoriasis.Arch Dermatol Res. 2001; 293: 334-342Crossref PubMed Scopus (79) Google Scholar; Gangemi et al., 2003Gangemi S. Merendino R.A. Guarneri F. Minciullo P.L. DiLorenzo G. Pacor M. et al.Serum levels of interleukin-18 and s-ICAM-1 in patients affected by psoriasis: preliminary considerations.J Eur Acad Dermatol Venereol. 2003; 17: 42-46Crossref PubMed Scopus (41) Google Scholar; Companjen et al., 2004Companjen A. Van der Wel L. Van der Fits L. Laman J. Prens E. Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions.Eur Cytokine Netw. 2004; 15: 210-216PubMed Google Scholar). Given that the expression of IL-18 is elevated in psoriasis and the fact that caspase-1 is the main protein involved in the activation of IL-18, we examined caspase-1 expression in psoriatic skin, and the signaling pathways implicated in the stress-induced activation of IL-18. In this study, we demonstrate for the first time that the activity of caspase-1 is increased in lesional psoriatic skin compared with non-lesional psoriatic skin, whereas no regulation of the expression of procaspase-1 was seen. Moreover, in vitro studies conducted in normal cultured human keratinocytes revealed that the anisomycin-induced activation of preformed proIL-18 was mediated by a p38 MAPK/caspase-1-dependent mechanism, whereas anisomycin-induced new synthesis of proIL-18 mRNA was mediated through p38 MAPK-dependent but caspase-1-independent mechanism. To investigate the protein level and the activity of caspase-1 in psoriatic skin, whole-cell extracts from keratome biopsies obtained from lesional and non-lesional psoriatic skin were isolated. Western blot experiments revealed that the protein level of the intermediate form of caspase-1 was significantly increased (6.3-fold as determined by densitometric analysis, P=0.0034) in cell extracts from lesional psoriatic skin compared with non-lesional psoriatic skin from the same patient. This was seen for all six patients examined (Figure 1a). No changes were seen in the total protein expression level of procaspase-1 in lesional compared with non-lesional psoriatic skin (Figure 1a). In separate sets of biopsies taken from four psoriatic patients (patients 7–10), we further examined caspase-1 using an antibody directed against the p10 subunit of caspase-1. These experiments revealed that also the active p10 subunit of caspase-1 was significantly increased (3.4-fold, P=0.019) in lesional compared with non-lesional psoriatic skin (Figure 1b). In order to determine if the increase in the p10 subunit of caspase-1 seen in psoriatic skin was paralleled by an increased processing of IL-18, we analyzed the IL-18 level in patients 7–10 using an antibody recognizing both proIL-18 and processed IL-18. As seen in Figure 1c, the active/processed form of IL-18 was significantly augmented (3.6-fold, P=0.011) in lesional compared with non-lesional psoriatic skin from the same patient, whereas there were no significant changes in the protein level of proIL-18 between lesional and non-lesional psoriatic skin (Figure 1c). Equal protein loading was assessed by detecting the protein level of β-actin. To further characterize caspase-1, cultured normal human keratinocytes were stimulated with the p38 MAPK activator anisomycin (500 ng/ml) for the indicated time points. By Western blot experiments, anisomycin was shown to increase the protein level of the intermediate form of caspase-1 in a time-dependent manner. After 4 hours of stimulation, there was a significant and maximal increase in the intermediate form of caspase-1 of approximately 6.3-fold (P=0.0087). Not only was the intermediate form of caspase-1 increased in the extracellular medium after stimulation with anisomycin, but also the protein level of procaspase-1 was increased significantly and time dependently, reaching a maximum level after 4 hours of stimulation with anisomycin compared with vehicle-treated cells (3.6-fold, P=0.018) (Figure 2a). No regulation in the protein level of β-actin was found in the extracellular medium after stimulation with anisomycin (Figure 2a). In contrast, the intermediate form of caspase-1 was not changed in the cell lysates after stimulation with anisomycin. Furthermore, no regulation of procaspase-1 was seen in the cell lysates of vehicle-treated or anisomycin-treated keratinocytes (Figure 2a). Stimulation of the keratinocytes with 500 ng/ml anisomycin for 4 hours did not lead to a decrease in cell viability (data not shown). To further characterize the p38 MAPK/caspase-1 signaling pathway, keratinocytes were preincubated with the p38 MAPK inhibitor SB202190 (10 μM) for 45 minutes before stimulated with anisomycin for 4 hours. As shown in Figure 2b, SB202190 clearly and significantly reduced not only the anisomycin-induced increase of the intermediate form of caspase-1 but also the release of procaspase-1 into the extracellular medium (P=0.019 and P=0.0271, respectively), indicating that anisomycin regulates caspase-1 through a p38 MAPK-dependent mechanism (Figure 2b). Also the enzymatic activity of caspase-1 was analyzed in cultured human keratinocytes stimulated with anisomycin. Four hours of stimulation with anisomycin significantly (P=0.0042) increased the enzymatic activity of caspase-1 as determined using the specific substrate, WEHD-pNA (Figure 2c). Furthermore, preincubation with SB202190 for 45 minutes significantly (P=0.01) reduced the anisomycin-induced caspase-1 enzymatic activity (Figure 2c). In contrast, the anisomycin-induced p38 MAPK phosphorylation was not changed after preincubation with the caspase-1 inhibitor, Ac-YVAD-CMK (100 μM) (Figure 2d). Equal protein loading was confirmed by assessing the protein level of β-actin. To study the effect of anisomycin on IL-18 protein release, cultured normal human keratinocytes were stimulated with anisomycin (500 ng/ml) for different time periods. IL-18 release was then determined in cell-free supernatants by an IL-18-specific ELISA, recognizing only the cleaved/active form of IL-18. A low baseline production of IL-18 protein was detected in vehicle-treated keratinocytes. However, stimulation of the keratinocytes with anisomycin significantly increased the IL-18 protein release in a time-dependent manner (Figure 3a). The maximum increase in IL-18 protein production was seen after 4 hours of stimulation with anisomycin, where the IL-18 protein level had increased approximately 9.5-fold (P=0.0036) compared with vehicle-treated cells. Twenty-four hours after stimulation with anisomycin, the IL-18 secretion had returned to baseline (Figure 3a). To examine the effect of anisomycin on proIL-18, Western blot experiments were performed using an antibody recognizing both proIL-18 and active IL-18. In the extracellular medium, both proIL-18 and active IL-18 were increased significantly over time with a maximum increase of approximately 1.7-fold (P=0.035) and 5.1-fold (P=0.0093), respectively (Figure 3b), seen after 4 hours of stimulation with anisomycin. Experiments conducted with protein extracts from the cell lysates revealed that neither proIL-18 nor active IL-18 expression intracellularly was regulated by anisomycin (Figure 3b). Equal protein loading was confirmed by assessing the protein level of β-actin. To examine whether the anisomycin-induced IL-18 production in cultured human keratinocytes was dependent on p38 MAPK and caspase-1 activation, keratinocytes were preincubated with SB202190 (10 μM) or Ac-YVAD-CMK (100 μM) for 45 minutes before stimulation for 4 hours with anisomycin. In the extracellular medium, the anisomycin-induced release of active IL-18 was significantly (P=0.0082) reduced to vehicle level by SB202190. In addition, preincubating the keratinocytes with the caspase-1 inhibitor, Ac-YVAD-CMK, significantly (P=0.0026) reduced the amount of active IL-18 induced by anisomycin, as determined by ELISA (Figure 4a). In contrast, when preincubating the keratinocytes with the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (50 μM) no regulation was found on the IL-18 protein release, indicating that anisomycin induces the release of active IL-18 by a p38 MAPK/caspase-1-dependent but extracellular signal-regulated kinase 1/2-independent mechanism in cultured human keratinocytes. The secretion of proIL-18 was also shown to be increased by a p38 MAPK-dependent mechanism, because preincubation with SB202190 significantly (P=0.021) inhibited the anisomycin-induced secretion of proIL-18, as determined by Western blotting (Figure 4b). In contrast, the caspase-1 inhibitor, Ac-YVAD-CMK, had no effect on anisomycin-induced secretion of proIL-18 (Figure 4b). Equal protein loading was confirmed by assessing the protein level of β-actin. To further characterize the p38 MAPK/caspase-1 signaling pathway in the regulation of IL-18 expression in cultured normal human keratinocytes, a time course study was performed. The IL-18 mRNA expression was significantly increased over time by anisomycin, as determined by quantitative reverse transcriptase-PCR. The increase in IL-18 mRNA expression was maximal after 12 hours of stimulation and had increased by approximately 4.5-fold (P=0.012) compared with vehicle-treated keratinocytes (Figure 5a). Twenty-four hours after stimulation with anisomycin, IL-18 mRNA expression had almost returned to baseline. To examine the role of p38 MAPK and caspase-1 in the anisomycin-induced IL-18 mRNA expression, cultured normal human keratinocytes were preincubated with either SB202190 (10 μM) or Ac-YVAD-CMK (100 μM) for 45 minutes before stimulating the cells with anisomycin for 12 hours. In cells preincubated with SB202190, the anisomycin-induced IL-18 mRNA expression was significantly reduced by approximately 72% (P=0.0064) compared with vehicle-treated cells (Figure 5b). In contrast, when preincubating keratinocytes with the caspase-1 inhibitor, Ac-YVAD-CMK (100 μM), no regulation in anisomycin-induced IL-18 mRNA expression was found, demonstrating that IL-18 mRNA expression is mediated by a p38 MAPK-dependent but caspase-1-independent mechanism (Figure 5b). Caspase-1 is the converting enzyme required for the cleavage and maturation of the proinflammatory cytokines IL-18 and IL-1β, two cytokines believed to play an important role in the pathogenesis of inflammatory skin diseases like psoriasis (Cooper et al., 1990Cooper K.D. Hammerberg C. Baadsgaard O. Elder J.T. Chan L.S. Taylor R.S. et al.Interleukin-1 in human skin: dysregulation in psoriasis.J Invest Dermatol. 1990; 95: 24S-26SAbstract Full Text PDF Google Scholar; Le and Abbenante, 2005Le G.T. Abbenante G. Inhibitors of TACE and caspase-1 as anti-inflammatory drugs.Curr Med Chem. 2005; 12: 2963-2977Crossref PubMed Scopus (53) Google Scholar; Tak et al., 2006Tak P.P. Bacchi M. Bertolino M. Pharmacokinetics of IL-18 binding protein in healthy volunteers and subjects with rheumatoid arthritis or plaque psoriasis.Eur J Drug Metab Pharmacokinet. 2006; 31: 109-116Crossref PubMed Scopus (63) Google Scholar). In this study, we present three essential and novel findings. First, we demonstrate that the mature/active form of caspase-1 is significantly augmented in lesional psoriatic skin compared with non-lesional psoriatic skin. In contrast, no regulation of procaspase-1 between lesional and non-lesional psoriatic skin was found. Secondly, anisomycin increased the secretion of procaspase-1 and proIL-18 by a p38 MAPK-dependent mechanism in cultured normal human keratinocytes. Finally, the anisomycin-induced IL-18 mRNA expression in human keratinocytes was mediated through a p38 MAPK-dependent but caspase-1-independent mechanism. Previous studies have demonstrated p38 MAPK to be involved in the maturation/activation of caspase-1, leading to activation of IL-18 (Shimizu et al., 1999Shimizu H. Banno Y. Sumi N. Naganawa T. Kitajima Y. Nozawa Y. Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol. 1999; 112: 769-774Crossref PubMed Scopus (112) Google Scholar; Sekiyama et al., 2005Sekiyama A. Ueda H. Kashiwamura S. Sekiyama R. Takeda M. Rokutan K. et al.A stress-induced, superoxide-mediated caspase-1 activation pathway causes plasma IL-18 upregulation.Immunity. 2005; 22: 669-677Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar; Wang et al., 2005Wang M. Tsai B.M. Turrentine M.W. Mahomed Y. Brown J.W. Meldrum D.R. p38 mitogen activated protein kinase mediates both death signaling and functional depression in the heart.Ann Thorac Surg. 2005; 80: 2235-2241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Furthermore, the p38 MAPK is activated in psoriasis as previously demonstrated by our group (Johansen et al., 2005Johansen C. Kragballe K. Westergaard M. Henningsen J. Kristiansen K. Iversen L. The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.Br J Dermatol. 2005; 152: 37-42Crossref PubMed Scopus (148) Google Scholar). Therefore, cultured human keratinocytes were stimulated with anisomycin in order to characterize the 38 MAPK/caspase-1/IL-18 signaling pathway. We demonstrate here that the anisomycin-induced maturation/activation of caspase-1 and IL-18 in cultured normal human keratinocytes is mediated by a p38 MAPK-dependent mechanism. However, our data further showed that not only was the active form of caspase-1 and IL-18 increased after stimulation with anisomycin, but also the levels of procaspase-1 and proIL-18 were increased in the extracellular medium by a mechanism involving the p38 MAPK. Secretion of procaspase-1 and proIL-18 from the keratinocytes is a novel and interesting finding indicating that the extracellular environment in the epidermis may be metabolically active in inflammatory skin diseases. Interestingly, no regulation of caspase-1 and IL-18 was found in the cell lysate between vehicle-treated and anisomycin-treated keratinocytes. By quantitative reverse transcriptase-PCR, we demonstrated a steady basal level of IL-18 mRNA expression in cultured human keratinocytes. This finding is consistent with that of previous studies demonstrating that human keratinocytes are capable of synthesizing IL-18 mRNA even in non-stimulated conditions (Naik et al., 1999Naik S.M. Cannon G. Burbach G.J. Singh S.R. Swerlick R.A. Wilcox J.N. et al.Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.J Invest Dermatol. 1999; 113: 766-772Crossref PubMed Scopus (119) Google Scholar; Mee et al., 2000Mee J.B. Alam Y. Groves R.W. Human keratinocytes constitutively produce but do not process interleukin-18.Br J Dermatol. 2000; 143: 330-336Crossref PubMed Scopus (72) Google Scholar). In contrast to the rapid induction of extracellular proIL-18 and active IL-18 seen after 4 hours of stimulation, the anisomycin-induced mRNA expression of IL-18 in cultured human keratinocytes was seen after 6 hours of stimulation with a maximal induction after 12 hours of stimulation. Furthermore, using an inhibitor directed against p38 MAPK, the anisomycin-induced IL-18 mRNA expression was significantly inhibited, whereas preincubation with a caspase-1 inhibitor had no effect on the anisomycin-induced IL-18 mRNA expression. These data indicate that IL-18 is regulated by two different mechanisms by the p38 MAPK: by a rapid mechanism at the post-transcriptional and post-translational level where proIL-18 is processed to its active form by a p38 MAPK/caspase-1-dependent mechanism, and by a more postponed mechanism where IL-18 is regulated at the transcriptional level by a p38 MAPK-dependent but caspase-1 independent mechanism. We also found that both the intermediate form and the active p10 subunit of caspase-1 were significantly increased in lesional psoriatic skin compared with non-lesional psoriatic skin. This is in agreement with a previous study by Yamanaka et al., 2000Yamanaka K. Tanaka M. Tsutsui H. Kupper T.S. Asahi K. Okamura H. et al.Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18.J Immunol. 2000; 165: 997-1003Crossref PubMed Scopus (112) Google Scholar). They used transgenic mice overexpressing skin-specific caspase-1, and they showed that the epidermis around skin ulcers in these mice revealed psoriasis-like changes including parakeratosis. Furthermore, the skin and sera of these mice showed elevated levels of mature IL-18 and IL-1β (Yamanaka et al., 2000Yamanaka K. Tanaka M. Tsutsui H. Kupper T.S. Asahi K. Okamura H. et al.Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18.J Immunol. 2000; 165: 997-1003Crossref PubMed Scopus (112) Google Scholar). We demonstrate here that the IL-18 expression is regulated by the p38 MAPK at both the transcriptional and post-transcriptional level. Furthermore, the activity of caspase-1 is highly increased in lesional psoriatic skin compared with non-lesional psoriatic skin. These data together with previous data from our group showing that the p38 MAPK signaling pathway is dysregulated in psoriatic skin (Johansen et al., 2005Johansen C. Kragballe K. Westergaard M. Henningsen J. Kristiansen K. Iversen L. The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.Br J Dermatol. 2005; 152: 37-42Crossref PubMed Scopus (148) Google Scholar, Johansen et al., 2006Johansen C. Funding A.T. Otkjaer K. Kragballe K. Jensen U.B. Madsen M. et al.Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2.J Immunol. 2006; 176: 1431-1438Crossref PubMed Scopus (115) Google Scholar; Funding et al., 2006Funding A.T. Johansen C. Kragballe K. Otkjaer K. Jensen U.B. Madsen M.W. et al.Mitogen- and stress-activated protein kinase 1 is activated in lesional psoriatic epidermis and regulates the expression of pro-inflammatory cytokines.J Invest Dermatol. 2006; 126: 1784-1791Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) strongly indicate that the p38 MAPK/caspase-1 signaling pathway plays an essential role in the pathogenesis of psoriasis. The control of caspase-1 activation and subsequent processing and maturation of IL-18 and IL-1β is critical for the regulation of inflammatory conditions in vivo. Understanding the mechanisms underlying the activation of caspase-1 and IL-18 by stress stimuli may contribute to new therapies for the treatment of inflammatory diseases. In this study, we demonstrated that both the unprocessed and the processed/active forms of caspase-1 and IL-18 were induced post-transcriptionally in cultured normal human keratinocytes after stimulation with anisomycin by a p38 MAPK-dependent mechanism. Furthermore, in vivo studies showed significantly increased levels of active caspase-1 in lesional compared with non-lesional psoriatic skin. The p38 MAPK/caspase-1 signaling pathway may therefore be a new target in the treatment of psoriasis. Keratome biopsies were obtained from lesional and non-lesional plaque-type psoriatic skin taken from the center of a plaque from patients with moderate to severe chronic stable plaque psoriasis from either the upper or lower extremities. For each patient, biopsies were taken from only one anatomical site. Biopsies were taken as paired samples, and biopsies from non-lesional psoriatic skin were taken from the same body region as biopsies from lesional psoriatic skin in a distance of at least 5 cm from a lesional plaque. In this study, the Zimmer™ Electronic Dermatome (Zimmer Orthopaedic, Dover, OH) was used. The dermatome was adjusted according to the degree of infiltration, as evaluated clinically. When non-lesional biopsies were obtained, the depth of cut was reduced. This study was conducted according to the Declaration of Helsinki Principles. The local ethical committee of Aarhus approved all described studies. Informed consent was obtained from each patient. Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as described previously (Kragballe et al., 1985Kragballe K. Desjarlais L. Marcelo C.L. Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro.Br J Dermatol. 1985; 112: 263-270Crossref PubMed Scopus (51) Google Scholar). Second-passage keratinocytes were grown in K-SFM (Gibco, Invitrogen, Carlsbad, CA). Twenty-four hours before stimulation with anisomycin, the medium was changed to keratinocyte basal medium (the same as K-SFM but without growth factors), in which the cells were stimulated. In some experiments, the keratinocytes were pretreated with the caspase-1 inhibitor, Ac-YVAD-CMK (100 μM), the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (50 μM), or the p38α/β inhibitor SB202190 (10 μM) for 45 minutes before stimulation. Cells were grown at 37°C and 5% CO2 in an incubator. Lactate dehydrogenase released from cells with compromised membranes was assayed using the CytoTox-One™ Homogeneous Membrane Integrity Assay (Promega, Madison, WI). Keratinocytes, 10,000 cells per well in 96-well plates, were stimulated with anisomycin or vehicle for 2, 4, 6, and 24 hours, after which 100 μl of CytoTox-One Reagent was added to each well and the reaction was allowed to proceed for 10 minutes before addition of 50 μl/well stop solution. Fluorescence was recorded at a wavelength of 590 nm using Fluoroskan Ascent Fl (BIE & Berntsen, Rodovre, Denmark). Maximum lactate dehydrogenase release control was 2 μl of lysis solution added to control cells before addition of reagent. Percent cytotoxicity was calculated as a ratio of 100 × experimental/maximum lactate dehydrogenase release. Total cell extracts were prepared from keratome biopsies as described previously (Johansen et al., 2006Johansen C. Funding A.T. Otkjaer K. Kragballe K. Jensen U.B. Madsen M. et al.Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2.J Immunol. 2006; 176: 1431-1438Crossref PubMed Scopus (115) Google Scholar). Briefly, biopsies were homogenized in lysis buffer and left on ice for 30 minutes. Then the samples were centrifuged at 10,000 × g for 10 minutes at 4°C, after which the supernatant constitute the cell lysate. Cultured human keratinocytes were lysed as described previously (Johansen et al., 2003Johansen C. Kragballe K. Henningsen J. Westergaard M. Kristiansen K. Iversen L. 1,25(OH)2D3 stimulates AP-1 DNA binding activity by a PI3-kinase/Ras/MEK/ERK1/2 and JNK1 dependent increase in c-Fos, Fra1 and c-Jun expression in human keratinocytes.J Invest Dermatol. 2003; 120: 561-570Crossref PubMed Scopus (50) Google Scholar). In brief, cells were lysed in cold lysis buffer (50 mM Tris-HCl, pH 6.8, 10 mM dithiothreitol, 10 mM β-glycerophosphate, 10 mM NaF, 0.1 mM sodium orthovanadate, 10% glycerol, 2.5% SDS, 25 mM phenylmethanesulfonylfluoride, and 50 × complete (a protease inhibitor cocktail, (Roche, Mannheim, Germany)). Then the lysate was centrifuged at 13,000 × g for 3 minutes, and the supernatant was collected and assayed for protein concentration as described by Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205570) Google Scholar). The protein from the extracellular medium was precipitated with 20% trichloroacetic acid and redissolved in SDS-PAGE buffer before the protein concentration was determined by Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205570) Google Scholar). Both samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with anti-caspase-1 (cat. no. sc-622), anti-p10 (cat. no. sc-515) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-IL-18 (Chemicon International Inc., Temecula, CA), anti-phospho-p38 (Cell Signalling Technology, Beverly, MA), or anti-β-actin (Sigma Chemical Co., St Louis, MO) and detected with anti-rabbit or anti-mouse IgG-horseradish peroxidase (Dako, Glostrup, Denmark) in a standard ECL (enhanced chemiluminescence) reaction (Amersham/Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer's instructions. As a positive control for active caspase-1, we used human active caspase-1 recombinant protein (cat. no. CC126, Millipore, Billerica, MA). Densitometric analysis of the band intensity was carried out using Kodak 1D Image Analysis Software. IL-18 was measured by a sandwich ELISA using two mAb antibodies against two different epitopes of human IL-18, according to the manufacture's protocol (cat. no. 7620, R&D Systems, Oxon, UK). The final result was determined by an ELISA reader (Laboratory systems iEMS Reader MF, Copenhagen, Denmark) at 450 nm. All measurements were performed in doublets. Caspase-1 enzymatic activity was determined using a caspase-1 colorimetric assay kit, according to the manufacture's protocol (cat. no. BF14100, R&D Systems). Briefly, protein from the extracellular medium was precipitated with 20% trichloroacetic acid and added to cold lysis buffer. A volume of 50 μl of cell lysate was added to 2.5 mM dithiothreitol and 50 μl caspase-1 reaction buffer. Each sample was added to 200 μM caspase-1 substrate, WEHD-pNA, followed by 2 hours of incubation at 37°C. The enzymatic activity of caspase-1 was measured on an ELISA reader using a 405 nm wavelength. RNA was purified with the SV Total RNA Isolation System (Promega). Briefly, 175 μl of SV RNA Lysis Buffer was added to the cultured human keratinocytes and lysates prepared by manually scraping the bottom of the wells. The lysates were transferred to sterile Eppendorf tubes and kept at −80°C until RNA purification. The lysates were carefully thawed while on ice. A 350 μl portion of SV RNA Dilution Buffer was added and tubes inverted 10 times to mix. Samples were then put on a heating block for 3 minutes at 70°C and afterwards centrifuged at 13,000 × g at room temperature for 10 minutes. Then the supernatant was isolated and added to 200 μl of 96% ethanol. The mixture was then transferred to spin baskets attached to a Vac-Man with Mini-Prep Vacuum Adapters (Promega). Total RNA was purified according to the vacuum protocol, as described by the manufacturer. Finally, RNA was dissolved in RNase/DNase-free water and stored at −80 °C until further use. For reverse transcription, Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, CA) were used. Primers and probes were purchased from Applied Biosystems. IL-18 mRNA expression was analyzed using Taqman 20 × Assays-On-Demand expression assay mix (assay ID: Hs99999040_m1). The probe was a FAM-labelled MGB probe with a non-fluorescent quencher. Eucaryotic 18S rRNA was used as housekeeping gene. Eucaryotic 18S rRNA mRNA expression was determined by using Taqman 20 × Assays-On-Demand expression assay mix (assay ID: Hs99999901_s1). The probe was a FAM-labelled MGB probe with a non-fluorescent quencher. PCR mastermix was Taqman 2 × Universal PCR Master Mix, No AmpErase (Applied Biosystems). Each gene was analyzed in triplicates. The real-time PCR machine was a Rotorgene-3000 (Corbett Research, Sydney, Australia). Reactions were run as singleplex. Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin no. 2 (ABI Prism 7700 sequencing detection system, Applied Biosystems). Briefly, a standard curve for each gene was made of 3-fold serial dilutions of total RNA from cultured human keratinocytes. The curve was then used to calculate relative amounts of target mRNA in the samples. For statistical analysis, a Student's t-test was performed. To test for normal distribution, a probability test was made. A probability of P<0.05 was regarded as statistically significant. The authors state no conflict of interest. This work was supported by the Leo Research Foundation, the Novo Nordisk Foundation, the Danish Psoriasis Foundation, and the Danish Research Agency." @default.
• W2045474881 created "2016-06-24" @default.
• W2045474881 creator A5019367041 @default.
• W2045474881 creator A5035700990 @default.
• W2045474881 creator A5067975004 @default.
• W2045474881 creator A5068079740 @default.
• W2045474881 date "2007-12-01" @default.
• W2045474881 modified "2023-10-14" @default.
• W2045474881 title "The Activity of Caspase-1 Is Increased in Lesional Psoriatic Epidermis" @default.
• W2045474881 cites W1529674879 @default.
• W2045474881 cites W1529895897 @default.
• W2045474881 cites W1571910978 @default.
• W2045474881 cites W1586925433 @default.
• W2045474881 cites W1902683402 @default.
• W2045474881 cites W1942214720 @default.
• W2045474881 cites W1970508213 @default.
• W2045474881 cites W1975153732 @default.
• W2045474881 cites W1983843966 @default.
• W2045474881 cites W1989029694 @default.
• W2045474881 cites W1991048381 @default.
• W2045474881 cites W1996729081 @default.
• W2045474881 cites W1998081672 @default.
• W2045474881 cites W1999967054 @default.
• W2045474881 cites W2000953362 @default.
• W2045474881 cites W2004685815 @default.
• W2045474881 cites W2008261126 @default.
• W2045474881 cites W2014697045 @default.
• W2045474881 cites W2043902549 @default.
• W2045474881 cites W2048970367 @default.
• W2045474881 cites W2050524172 @default.
• W2045474881 cites W2051399616 @default.
• W2045474881 cites W2058237985 @default.
• W2045474881 cites W2065184397 @default.
• W2045474881 cites W2081097999 @default.
• W2045474881 cites W2081130105 @default.
• W2045474881 cites W2088016221 @default.
• W2045474881 cites W2090306216 @default.
• W2045474881 cites W2100438302 @default.
• W2045474881 cites W2109447778 @default.
• W2045474881 cites W2117139129 @default.
• W2045474881 cites W2121768487 @default.
• W2045474881 cites W2133515094 @default.
• W2045474881 cites W2166037275 @default.
• W2045474881 cites W2171831396 @default.
• W2045474881 cites W4240978790 @default.
• W2045474881 cites W4248878742 @default.
• W2045474881 cites W4250164303 @default.
• W2045474881 cites W4293247451 @default.
• W2045474881 cites W82618136 @default.
• W2045474881 doi "https://doi.org/10.1038/sj.jid.5700922" @default.
• W2045474881 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17597823" @default.
• W2045474881 hasPublicationYear "2007" @default.
• W2045474881 type Work @default.
• W2045474881 sameAs 2045474881 @default.
• W2045474881 citedByCount "81" @default.
• W2045474881 countsByYear W20454748812012 @default.
• W2045474881 countsByYear W20454748812013 @default.
• W2045474881 countsByYear W20454748812014 @default.
• W2045474881 countsByYear W20454748812015 @default.
• W2045474881 countsByYear W20454748812016 @default.
• W2045474881 countsByYear W20454748812017 @default.
• W2045474881 countsByYear W20454748812018 @default.
• W2045474881 countsByYear W20454748812019 @default.
• W2045474881 countsByYear W20454748812020 @default.
• W2045474881 countsByYear W20454748812021 @default.
• W2045474881 countsByYear W20454748812022 @default.
• W2045474881 countsByYear W20454748812023 @default.
• W2045474881 crossrefType "journal-article" @default.
• W2045474881 hasAuthorship W2045474881A5019367041 @default.
• W2045474881 hasAuthorship W2045474881A5035700990 @default.
• W2045474881 hasAuthorship W2045474881A5067975004 @default.
• W2045474881 hasAuthorship W2045474881A5068079740 @default.
• W2045474881 hasBestOaLocation W20454748811 @default.
• W2045474881 hasConcept C105702510 @default.
• W2045474881 hasConcept C142724271 @default.
• W2045474881 hasConcept C16005928 @default.
• W2045474881 hasConcept C2776458125 @default.
• W2045474881 hasConcept C2780564577 @default.
• W2045474881 hasConcept C71924100 @default.
• W2045474881 hasConceptScore W2045474881C105702510 @default.
• W2045474881 hasConceptScore W2045474881C142724271 @default.
• W2045474881 hasConceptScore W2045474881C16005928 @default.
• W2045474881 hasConceptScore W2045474881C2776458125 @default.
• W2045474881 hasConceptScore W2045474881C2780564577 @default.
• W2045474881 hasConceptScore W2045474881C71924100 @default.
• W2045474881 hasIssue "12" @default.
• W2045474881 hasLocation W20454748811 @default.
• W2045474881 hasLocation W20454748812 @default.
• W2045474881 hasOpenAccess W2045474881 @default.
• W2045474881 hasPrimaryLocation W20454748811 @default.
• W2045474881 hasRelatedWork W1986850072 @default.
• W2045474881 hasRelatedWork W1988927864 @default.
• W2045474881 hasRelatedWork W1997461283 @default.
• W2045474881 hasRelatedWork W2066907069 @default.
• W2045474881 hasRelatedWork W2073640158 @default.
• W2045474881 hasRelatedWork W2145526653 @default.
• W2045474881 hasRelatedWork W2357541302 @default.
• W2045474881 hasRelatedWork W2601256796 @default.

• next