Mycopathologia (2014) 177:75–79 DOI 10.1007/s11046-013-9725-1
Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells Riyoko Tamai • Yusuke Kiyoura
Received: 31 August 2013 / Accepted: 23 December 2013 / Published online: 17 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Galectin-3 is a b-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heatkilled C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by
R. Tamai Y. Kiyoura (&) Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611, Japan e-mail: [email protected]
other microorganisms via up-regulation of galectin-3 secretion. Keywords Candida albicans Galectin-3 Human gingival epithelial cells Cell motility Candida parapsilosis Human gingival fibroblasts
Rapid Communication Candida species are indigenous fungi that cause opportunistic infection in humans [1]. These fungi first interact with epithelial cells that play a crucial role not only in protecting the body against invasion by pathogenic organisms, but also in regulating the cytokine network. C. albicans is capable of inducing proinflammatory cytokine production in various cell types via Toll-like receptor (TLR) 2, TLR4, dectin-1, dectin-2 and galectin-3 [2–5]. Galectin-3, a b-galactoside-binding lectin, is expressed on various cells, including epithelial cells which serve as barriers to oral candidiasis, and binds to molecules containing galactose-b1,4-N-acetylglucosamine, such as epidermal growth factor receptor, integrin b1 and mucin [6– 8]. Neisseria meningitides, a gram-negative coccus, increases its interaction with phagocytic cells by binding to galectin-3 [9]. Galectin-3 is also released from cells and functions extracellularly to activate cells [10]. Some microorganisms are known to rapidly up-regulate galectin-3 secretion by macrophages and
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gastric epithelial cells [11, 12]. The released galectin-3 binds to and kills C. albicans [13, 14]. Candida species can also penetrate tissues including oral mucosa and come into contact with human gingival fibroblasts, which are the major constituents of periodontal tissue [15]. C. albicans can exert its effects by regulating host cell signaling. For example, C. albicans activates focal adhesion kinase (FAK) and regulates cell motility, which may be important for galectin-3 secretion [16]. Calpain 2, an intracellular calcium-dependent protease, cleaves FAK and regulates adhesion dynamics [17]. Interestingly, calpain small subunit 1 (CAPNS1), a small regulatory subunit of calpain 2, is required for galectin-3 secretion [18]. It was recently reported that CAPNS1 interacts with and activates phosphatidylinositol-3 kinase (PI3K), a mediator of FAK signaling [19– 21]. The aim of this study was to determine whether C. albicans and C. parapsilosis up-regulate galectin-3 secretion by oral gingival epithelial cells and human gingival fibroblasts. C. albicans OH-1 was isolated from the oral cavity of an elderly individual [22]. C. parapsilosis JCM1612 was obtained from the RIKEN Bioresource Center. These fungi were aerobically grown at 37 °C in Sabouraud’s dextrose broth supplemented with 1 % yeast extract. Ca9-22, a human gingival epithelial cell line, was cultured under standard conditions in minimum essential medium (MEM; Sigma) containing 10 % heat-inactivated fetal bovine serum (FBS; Biowest), penicillin (100 U/ml) and streptomycin (100 lg/ml) as previously described [23]. Human gingival fibroblasts were prepared from clinically inflamed gingival tissue, according to a previously published method [15], and maintained under standard conditions in a-MEM (Sigma) containing 10 % FBS, penicillin (100 U/ml) and streptomycin (100 lg/ml). Tissue samples were collected from subjects who provided written informed consent. Levels of secreted human galectin-3 in culture supernatants were measured in triplicate by enzymelinked immunosorbent assay (ELISA; R&D Systems, Inc.). Briefly, C. albicans or C. parapsilosis was washed three times with phosphate-buffered saline (PBS) prior to use. Heat-killed C. albicans (HKCA) was prepared by incubation at 95 °C for 30 min. Confluent Ca9-22 cells (2 9 104 cells/well) or human gingival fibroblasts (1 9 104 cells/well) were grown in 96-well flat-bottomed plates (FalconÒ) for 24 h. Cells were then washed once with serum-free medium and incubated for 18 h with serum-free medium. Ca9-22
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cells were then washed twice with serum-free medium and incubated for the indicated durations with live C. albicans (MOI 1), C. parapsilosis (MOI 1) or HKCA (MOI 100) diluted in culture medium. Culture supernatants were collected and filtered with a 0.45 lm GHP NanosepÒ MF centrifugal device (PALLÒ Life Sciences) at 14,0009g for 2 min. Levels of secreted human galectin-3 in the supernatants were measured by ELISA. For the inhibition assay, cells were pretreated with inhibitors (Sigma) at the indicated concentrations for 30 min, prior to the addition of C. albicans for 15 min. Cytochalasin D (an actin polymerization inhibitor) was used at a concentration of 0.5 or 2.0 lg/ml. ALLN (a calpain inhibitor) and LY294002 (a PI3K inhibitor) were used at either 10 or 50 lM. Data were analyzed using one-way analysis of variance (ANOVA) and the Bonferroni or Dunn method. Results are presented as the mean ± standard error (SE) of triplicate wells. P \ 0.05 was considered significant. In this study, C. albicans and C. parapsilosis rapidly up-regulated galectin-3 secretion by Ca9-22 cells and human gingival fibroblasts. We first investigated whether C. albicans up-regulates galectin-3 secretion by Ca9-22 cells and human gingival fibroblasts. Treatment with live C. albicans at a multiplicity of infection (MOI) of 1 significantly increased galectin-3 secretion by Ca9-22 cells after 5 min, with secretion peaking after 45 min (Fig. 1a). However, galectin-3 secretion began to decrease after 1 h. HKCA (MOI 100) and live C. parapsilosis (MOI 1) up-regulated galectin-3 secretion by Ca9-22 cells to levels similar to that obtained with live C. albicans (Fig. 1a, b). Similar results were observed when human gingival fibroblasts were treated with live C. albicans and C. parapsilosis (Fig. 1c). We next examined whether activation of FAK, PI3K and/or calpain is required for the up-regulation of galectin-3 secretion by C. albicans and C. parapsilosis in Ca9-22 cells (Fig. 2). Treatment of cells with cytochalasin D, an actin polymerization inhibitor, did not inhibit the C. albicans- or C. parapsilosis-mediated up-regulation of galectin-3 secretion. ALLN (a calpain inhibitor) and LY294002 (a PI3K inhibitor) did not significantly reduce galectin-3 secretion either. These results suggest that the C. albicans- or C. parapsilosis-induced up-regulation of galectin-3 secretion by Ca9-22 cells was independent of cell motility. Galectin-3 mediates fungal recognition with dectin1 on the cell surface and regulates keratinocyte motility
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Fig. 2 Galectin-3 secretion is independent of FAK, PI3K and calpain activation. Ca9-22 cells were pre-incubated with DMSO, cytochalasin D (Cyto D), ALLN or LY294002 for 30 min, followed by incubation with or without live C. albicans (MOI 1) or C. parapsilosis (MOI 1) for 15 min. Culture supernatants were collected, and galectin-3 secretion was measured by ELISA. Results are presented as mean (SE) of triplicate cultures from three independent experiments. **P \ 0.01 compared to vehicle
Fig. 1 Rapid galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts co-cultured with C. albicans or C. parapsilosis. a Galectin-3 secretion by Ca9-22 cells incubated with live C. albicans (MOI 1) or heat-killed C. albicans (HKCA; MOI 100). b Galectin-3 secretion by Ca9-22 cells co-cultured with live C. parapsilosis (MOI 1). c Galectin-3 secretion by human gingival fibroblasts co-cultured with live C. albicans or C. parapsilosis (MOI 1). Culture supernatants were collected, and galectin-3 secretion was measured by ELISA. Results are presented as mean (SE) of triplicate cultures from three independent experiments. *P \ 0.05 and **P \ 0.01 compared to vehicle
after being released from the cytoplasm [5, 24]. C. albicans has a cell wall composed of b-glucans, mannans and chitins, and secretes proteinases [4, 25].
Therefore, C. albicans is recognized by many receptors. Although Toll-like receptor (TLR) 2 recognizes C. albicans, Ca9-22 cells express low levels of this receptor and do not respond to its ligand [2, 26]. Integrin b1, an activator of FAK, is an adhesion molecule that mediates the response to fungal cell wall components [27]. However, we previously found that HKCA did not up-regulate integrin b1 expression on Ca9-22 cells, likely because exogenous galectin-3 accelerates the endocytosis of integrin b1 [23, 28]. Thus, the C. albicans-induced up-regulation of galectin-3 secretion is unlikely to be mediated by integrin b1. In the present study, both live C. albicans and HKCA up-regulated galectin-3 secretion by Ca9-22 cells. HKCA lacks proteinase activity and has increased surface expression of 1,3-b-glucan, which is usually found within mannan in the cell wall [3, 29]. Given that, live C. albicans up-regulated galectin-3 secretion to a much greater extent than HKCA, mannan and proteinase activity might be more relevant to galectin-3 secretion than 1,3-b-glucan. C. parapsilosis also rapidly up-regulates galectin-3 secretion to a similar extent as C. albicans, although C. parapsilosis is known to induce much less cytokine production in host cells than C. albicans [30]. Thus, different mannan structures might not influence galectin-3 secretion [31]. We also demonstrated that C. albicans rapidly up-regulates galectin3 secretion by human gingival fibroblasts. Although human gingival fibroblasts express TLR2 [32], C. albicans up-regulated galectin-3 secretion by these cells to almost the same extent as that observed with Ca9-22
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cells. This result suggests that TLR2, which activates PI3K, does not play a role in the C. albicans-mediated up-regulation of galectin-3 secretion. NF-jB is a transcription factor implicated in the production of galectin-3 by UV light and activated by TLR2 signaling [2, 33]. We thus examined whether C. albicans could also activate NF-jB by Ca9-22 cells without serum. However, no NF-jB activation by live C. albicans was observed in the absence of serum (data not shown). C. albicans-mediated galectin-3 secretion was also independent of cell motility, which is known to involve FAK activation. A previous study reported that a mechanotransduction mechanism triggers the rapid secretion of galectin-3 into conditioned medium [34]. Galectin-3 is most likely secreted from membranebound vesicles (exosomes) that accumulate below the plasma membrane and are pinched off before being secreted into the supernatant via the non-classical pathway, i.e., ER/Golgi-independent protein secretion [35]. Thus, galectin-3 secretion is partly independent of specific receptors, such as pattern-recognition receptors for pathogens. However, we found that much less live C. albicans (MOI 1) is required to up-regulate galectin-3 secretion than HKCA (MOI 100). This indicates the potential involvement of specific receptors for mannan and proteinases of C. albicans in galectin-3 secretion, although host cell cytoskeletonassociated proteins are unlikely to be involved. Exogenous galectin-3 promotes the endocytosis of integrin b1, which contributes to invasion by gramnegative bacteria [20, 28, 36]. Galectin-3 also upregulates the production of macrophage-related chemokines, pro-inflammatory cytokines and reactive oxygen species [37, 38]. Our findings suggest that C. albicans and C. parapsilosis rapidly increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion, independently of NF-jB activation and cytoskeleton rearrangements. Acknowledgments This study was supported by a Grant-inAid for Scientific Research (C) (No. 23592709) from the Ministry of Education, Culture, Sports, Science and Technology, and a Grant-in-Aid for Scientific Research from the Ohu University School of Dentistry.
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Mycopathologia (2014) 177:75–79 18. Menon S, Kang CM, Beningo KA. Galectin-3 secretion and tyrosine phosphorylation is dependent on the calpain small subunit, Calpain 4. Biochem Biophys Res Commun. 2011;410:91–6. 19. Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev. 2003;83:731–801. 20. Lagana A, Goetz JG, Cheung P, Raz A, Dennis JW, Nabi IR. Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells. Mol Cell Biol. 2006;26:3181–93. 21. Beltran L, Chaussade C, Vanhaesebroeck B, Cutillas PR. Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity. Proc Natl Acad Sci USA. 2011;108:16217–22. 22. Kamagata-Kiyoura Y, Abe S, Yamaguchi H, Nitta T. Protective effects of human saliva on experimental murine oral candidiasis. J Infect Chemother. 2004;10:253–5. 23. Tamai R, Sugamata M, Kiyoura Y. Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis. Microb Pathog. 2011;51:250–4. 24. Goetz JG, Joshi B, Lajoie P, Strugnell SS, Scudamore T, Kojic LD, et al. Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1. J Cell Biol. 2008;180:1261–75. 25. Schaller M, Schafer W, Korting HC, Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol Microbiol. 1998;29:605–15. 26. Peyret-Lacombe A, Brunel G, Watts M, Charveron M, Duplan H. TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response. Cytokine. 2009;46:201–10. 27. Lavigne LM, O’Brien XM, Kim M, Janowski JW, Albina JE, Reichner JS. Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern. J Immunol. 2007;178: 7276–82. 28. Furtak V, Hatcher F, Ochieng J. Galectin-3 mediates the endocytosis of beta-1 integrins by breast carcinoma cells. Biochem Biophys Res Commun. 2001;289:845–50.
79 29. Wellington M, Dolan K, Krysan DJ. Live Candida albicans suppresses production of reactive oxygen species in phagocytes. Infect Immun. 2009;77:405–13. 30. Toth A, Csonka K, Jacobs C, Vagvolgyi C, Nosanchuk JD, Netea MG, et al. Candida albicans and Candida parapsilosis induce different T-Cell Responses in human peripheral blood mononuclear cells. J Infect Dis. 2013;208:690–8. 31. Funayama M, Nishikawa A, Shinoda T, Suzuki M, Fukazawa Y. Antigenic relationship between Candida parapsilosis and Candida albicans serotype B. Microbiol Immunol. 1984;28:1359–71. 32. Hatakeyama J, Tamai R, Sugiyama A, Akashi S, Sugawara S, Takada H. Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system. Oral Microbiol Immunol. 2003;18:14–23. 33. Dumic J, Lauc G, Flogel M. Expression of galectin-3 in cells exposed to stress-roles of jun and NF-kappaB. Cell Physiol Biochem. 2000;10:149–58. 34. Baptiste TA, James A, Saria M, Ochieng J. Mechanotransduction mediated secretion and uptake of galectin-3 in breast carcinoma cells: implications in the extracellular functions of the lectin. Exp Cell Res. 2007;313:652–64. 35. Nickel W. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Eur J Biochem. 2003;270:2109–19. 36. Saravanan C, Liu FT, Gipson IK, Panjwani N. Galectin-3 promotes lamellipodia formation in epithelial cells by interacting with complex N-glycans on alpha3beta1 integrin. J Cell Sci. 2009;122:3684–93. 37. Papaspyridonos M, McNeill E, de Bono JP, Smith A, Burnand KG, Channon KM, et al. Galectin-3 is an amplifier of inflammation in atherosclerotic plaque progression through macrophage activation and monocyte chemoattraction. Arterioscler Thromb Vasc Biol. 2008;28:433–40. 38. Fernandes Bertocchi AP, Campanhole G, Wang PH, Goncalves GM, Damiao MJ, Cenedeze MA, et al. A Role for galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury. Transpl Int. 2008;21:999–1007.
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Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells Riyoko Tamai • Yusuke Kiyoura
Received: 31 August 2013 / Accepted: 23 December 2013 / Published online: 17 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Galectin-3 is a b-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heatkilled C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by
R. Tamai Y. Kiyoura (&) Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611, Japan e-mail: [email protected]
other microorganisms via up-regulation of galectin-3 secretion. Keywords Candida albicans Galectin-3 Human gingival epithelial cells Cell motility Candida parapsilosis Human gingival fibroblasts
Rapid Communication Candida species are indigenous fungi that cause opportunistic infection in humans [1]. These fungi first interact with epithelial cells that play a crucial role not only in protecting the body against invasion by pathogenic organisms, but also in regulating the cytokine network. C. albicans is capable of inducing proinflammatory cytokine production in various cell types via Toll-like receptor (TLR) 2, TLR4, dectin-1, dectin-2 and galectin-3 [2–5]. Galectin-3, a b-galactoside-binding lectin, is expressed on various cells, including epithelial cells which serve as barriers to oral candidiasis, and binds to molecules containing galactose-b1,4-N-acetylglucosamine, such as epidermal growth factor receptor, integrin b1 and mucin [6– 8]. Neisseria meningitides, a gram-negative coccus, increases its interaction with phagocytic cells by binding to galectin-3 [9]. Galectin-3 is also released from cells and functions extracellularly to activate cells [10]. Some microorganisms are known to rapidly up-regulate galectin-3 secretion by macrophages and
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gastric epithelial cells [11, 12]. The released galectin-3 binds to and kills C. albicans [13, 14]. Candida species can also penetrate tissues including oral mucosa and come into contact with human gingival fibroblasts, which are the major constituents of periodontal tissue [15]. C. albicans can exert its effects by regulating host cell signaling. For example, C. albicans activates focal adhesion kinase (FAK) and regulates cell motility, which may be important for galectin-3 secretion [16]. Calpain 2, an intracellular calcium-dependent protease, cleaves FAK and regulates adhesion dynamics [17]. Interestingly, calpain small subunit 1 (CAPNS1), a small regulatory subunit of calpain 2, is required for galectin-3 secretion [18]. It was recently reported that CAPNS1 interacts with and activates phosphatidylinositol-3 kinase (PI3K), a mediator of FAK signaling [19– 21]. The aim of this study was to determine whether C. albicans and C. parapsilosis up-regulate galectin-3 secretion by oral gingival epithelial cells and human gingival fibroblasts. C. albicans OH-1 was isolated from the oral cavity of an elderly individual [22]. C. parapsilosis JCM1612 was obtained from the RIKEN Bioresource Center. These fungi were aerobically grown at 37 °C in Sabouraud’s dextrose broth supplemented with 1 % yeast extract. Ca9-22, a human gingival epithelial cell line, was cultured under standard conditions in minimum essential medium (MEM; Sigma) containing 10 % heat-inactivated fetal bovine serum (FBS; Biowest), penicillin (100 U/ml) and streptomycin (100 lg/ml) as previously described [23]. Human gingival fibroblasts were prepared from clinically inflamed gingival tissue, according to a previously published method [15], and maintained under standard conditions in a-MEM (Sigma) containing 10 % FBS, penicillin (100 U/ml) and streptomycin (100 lg/ml). Tissue samples were collected from subjects who provided written informed consent. Levels of secreted human galectin-3 in culture supernatants were measured in triplicate by enzymelinked immunosorbent assay (ELISA; R&D Systems, Inc.). Briefly, C. albicans or C. parapsilosis was washed three times with phosphate-buffered saline (PBS) prior to use. Heat-killed C. albicans (HKCA) was prepared by incubation at 95 °C for 30 min. Confluent Ca9-22 cells (2 9 104 cells/well) or human gingival fibroblasts (1 9 104 cells/well) were grown in 96-well flat-bottomed plates (FalconÒ) for 24 h. Cells were then washed once with serum-free medium and incubated for 18 h with serum-free medium. Ca9-22
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cells were then washed twice with serum-free medium and incubated for the indicated durations with live C. albicans (MOI 1), C. parapsilosis (MOI 1) or HKCA (MOI 100) diluted in culture medium. Culture supernatants were collected and filtered with a 0.45 lm GHP NanosepÒ MF centrifugal device (PALLÒ Life Sciences) at 14,0009g for 2 min. Levels of secreted human galectin-3 in the supernatants were measured by ELISA. For the inhibition assay, cells were pretreated with inhibitors (Sigma) at the indicated concentrations for 30 min, prior to the addition of C. albicans for 15 min. Cytochalasin D (an actin polymerization inhibitor) was used at a concentration of 0.5 or 2.0 lg/ml. ALLN (a calpain inhibitor) and LY294002 (a PI3K inhibitor) were used at either 10 or 50 lM. Data were analyzed using one-way analysis of variance (ANOVA) and the Bonferroni or Dunn method. Results are presented as the mean ± standard error (SE) of triplicate wells. P \ 0.05 was considered significant. In this study, C. albicans and C. parapsilosis rapidly up-regulated galectin-3 secretion by Ca9-22 cells and human gingival fibroblasts. We first investigated whether C. albicans up-regulates galectin-3 secretion by Ca9-22 cells and human gingival fibroblasts. Treatment with live C. albicans at a multiplicity of infection (MOI) of 1 significantly increased galectin-3 secretion by Ca9-22 cells after 5 min, with secretion peaking after 45 min (Fig. 1a). However, galectin-3 secretion began to decrease after 1 h. HKCA (MOI 100) and live C. parapsilosis (MOI 1) up-regulated galectin-3 secretion by Ca9-22 cells to levels similar to that obtained with live C. albicans (Fig. 1a, b). Similar results were observed when human gingival fibroblasts were treated with live C. albicans and C. parapsilosis (Fig. 1c). We next examined whether activation of FAK, PI3K and/or calpain is required for the up-regulation of galectin-3 secretion by C. albicans and C. parapsilosis in Ca9-22 cells (Fig. 2). Treatment of cells with cytochalasin D, an actin polymerization inhibitor, did not inhibit the C. albicans- or C. parapsilosis-mediated up-regulation of galectin-3 secretion. ALLN (a calpain inhibitor) and LY294002 (a PI3K inhibitor) did not significantly reduce galectin-3 secretion either. These results suggest that the C. albicans- or C. parapsilosis-induced up-regulation of galectin-3 secretion by Ca9-22 cells was independent of cell motility. Galectin-3 mediates fungal recognition with dectin1 on the cell surface and regulates keratinocyte motility
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Fig. 2 Galectin-3 secretion is independent of FAK, PI3K and calpain activation. Ca9-22 cells were pre-incubated with DMSO, cytochalasin D (Cyto D), ALLN or LY294002 for 30 min, followed by incubation with or without live C. albicans (MOI 1) or C. parapsilosis (MOI 1) for 15 min. Culture supernatants were collected, and galectin-3 secretion was measured by ELISA. Results are presented as mean (SE) of triplicate cultures from three independent experiments. **P \ 0.01 compared to vehicle
Fig. 1 Rapid galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts co-cultured with C. albicans or C. parapsilosis. a Galectin-3 secretion by Ca9-22 cells incubated with live C. albicans (MOI 1) or heat-killed C. albicans (HKCA; MOI 100). b Galectin-3 secretion by Ca9-22 cells co-cultured with live C. parapsilosis (MOI 1). c Galectin-3 secretion by human gingival fibroblasts co-cultured with live C. albicans or C. parapsilosis (MOI 1). Culture supernatants were collected, and galectin-3 secretion was measured by ELISA. Results are presented as mean (SE) of triplicate cultures from three independent experiments. *P \ 0.05 and **P \ 0.01 compared to vehicle
after being released from the cytoplasm [5, 24]. C. albicans has a cell wall composed of b-glucans, mannans and chitins, and secretes proteinases [4, 25].
Therefore, C. albicans is recognized by many receptors. Although Toll-like receptor (TLR) 2 recognizes C. albicans, Ca9-22 cells express low levels of this receptor and do not respond to its ligand [2, 26]. Integrin b1, an activator of FAK, is an adhesion molecule that mediates the response to fungal cell wall components [27]. However, we previously found that HKCA did not up-regulate integrin b1 expression on Ca9-22 cells, likely because exogenous galectin-3 accelerates the endocytosis of integrin b1 [23, 28]. Thus, the C. albicans-induced up-regulation of galectin-3 secretion is unlikely to be mediated by integrin b1. In the present study, both live C. albicans and HKCA up-regulated galectin-3 secretion by Ca9-22 cells. HKCA lacks proteinase activity and has increased surface expression of 1,3-b-glucan, which is usually found within mannan in the cell wall [3, 29]. Given that, live C. albicans up-regulated galectin-3 secretion to a much greater extent than HKCA, mannan and proteinase activity might be more relevant to galectin-3 secretion than 1,3-b-glucan. C. parapsilosis also rapidly up-regulates galectin-3 secretion to a similar extent as C. albicans, although C. parapsilosis is known to induce much less cytokine production in host cells than C. albicans [30]. Thus, different mannan structures might not influence galectin-3 secretion [31]. We also demonstrated that C. albicans rapidly up-regulates galectin3 secretion by human gingival fibroblasts. Although human gingival fibroblasts express TLR2 [32], C. albicans up-regulated galectin-3 secretion by these cells to almost the same extent as that observed with Ca9-22
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cells. This result suggests that TLR2, which activates PI3K, does not play a role in the C. albicans-mediated up-regulation of galectin-3 secretion. NF-jB is a transcription factor implicated in the production of galectin-3 by UV light and activated by TLR2 signaling [2, 33]. We thus examined whether C. albicans could also activate NF-jB by Ca9-22 cells without serum. However, no NF-jB activation by live C. albicans was observed in the absence of serum (data not shown). C. albicans-mediated galectin-3 secretion was also independent of cell motility, which is known to involve FAK activation. A previous study reported that a mechanotransduction mechanism triggers the rapid secretion of galectin-3 into conditioned medium [34]. Galectin-3 is most likely secreted from membranebound vesicles (exosomes) that accumulate below the plasma membrane and are pinched off before being secreted into the supernatant via the non-classical pathway, i.e., ER/Golgi-independent protein secretion [35]. Thus, galectin-3 secretion is partly independent of specific receptors, such as pattern-recognition receptors for pathogens. However, we found that much less live C. albicans (MOI 1) is required to up-regulate galectin-3 secretion than HKCA (MOI 100). This indicates the potential involvement of specific receptors for mannan and proteinases of C. albicans in galectin-3 secretion, although host cell cytoskeletonassociated proteins are unlikely to be involved. Exogenous galectin-3 promotes the endocytosis of integrin b1, which contributes to invasion by gramnegative bacteria [20, 28, 36]. Galectin-3 also upregulates the production of macrophage-related chemokines, pro-inflammatory cytokines and reactive oxygen species [37, 38]. Our findings suggest that C. albicans and C. parapsilosis rapidly increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion, independently of NF-jB activation and cytoskeleton rearrangements. Acknowledgments This study was supported by a Grant-inAid for Scientific Research (C) (No. 23592709) from the Ministry of Education, Culture, Sports, Science and Technology, and a Grant-in-Aid for Scientific Research from the Ohu University School of Dentistry.
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