The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation Through Modulation of NOTCH Signaling

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling

This information is current as Almudena Ruiz-García, Susana López-López, José Javier of September 28, 2021. García-Ramírez, Victoriano Baladrón, María José Ruiz-Hidalgo, Laura López-Sanz, Ángela Ballesteros, Jorge Laborda, Eva María Monsalve and María José M. Díaz-Guerra

J Immunol published online 29 August 2016 Downloaded from http://www.jimmunol.org/content/early/2016/08/27/jimmun ol.1600421

Supplementary http://www.jimmunol.org/content/suppl/2016/08/27/jimmunol.160042 http://www.jimmunol.org/ Material 1.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 29, 2016, doi:10.4049/jimmunol.1600421 The Journal of Immunology

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling

Almudena Ruiz-Garcıá,1 Susana Lo´pez-Lo´pez,1 Jose´ Javier Garcıá-Ramı´rez, Victoriano Baladroń, Marıá Jose´ Ruiz-Hidalgo, Laura Lo´pez-Sanz, A´ ngela Ballesteros, Jorge Laborda, Eva Marıá Monsalve, and Marıá Jose´ M. Dıáz-Guerra

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, Downloaded from and TLR2 activation, and it is enhanced by IFN-g. In this study, we report that induction of Tspan33 expression by TLR and IFN-g is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modu- http://www.jimmunol.org/ lates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-kB–dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target. The Journal of Immunology, 2016, 197: 000–000.

acrophages are key cells for the defense of the organism scribed that TLR signaling in macrophages increases expression against pathogens. Macrophages detect pathogens of different NOTCH receptors (5–7) and activates NOTCH sig- through molecular pattern recognition receptors, in naling. This process can modulate macrophage activation, favor-

M by guest on September 28, 2021 particular TLR, which recognize several conserved microbial ing polarization toward an inflammatory phenotype, characterized structures. Signaling through these receptors represents a critical by higher IL-6, IL-12, and TNF-a production and enhanced cy- event in regulating macrophage activity, affecting cytokine and totoxicity (8, 9), mediated in part by increased NF-kB activity (10, chemokine production, receptor expression, cytotoxic activity, and 11). Indeed, Notch1 deficiency has been related to decreased in- cell migration (1, 2). Macrophages possess a remarkable plasticity flammation in different pathological models (12–15). and can change their functional phenotype in response to envi- NOTCH receptors are expressed on the cell surface as hetero- ronmental signals, becoming adapted to different situations by dimers of their N- and C-terminal fragments generated after fine-tuning their activity (3, 4). In this regard, it has been de- proteolytic cleavage at the trans-Golgi network (16, 17). These NOTCH receptor heterodimers adopt a protease-resistant confor- mation in their basal state, but after ligand binding to their ex- Facultad de Medicina, Centro Regional de Investigaciones Biome´dicas, Unidad de tracellular region, a hidden region is now exposed to an enzyme Biomedicina, Universidad de Castilla–La Mancha/Consejo Superior de Investiga- with a disintegrin and metalloprotease (ADAM) activity, such as ciones Cientificas, 02006 Albacete, Spain ADAM10, which upon cleaving at a site located at the extracel- 1 A.R.-G. and S.L.-L. contributed equally to this work. lular region of the membrane-bound, C-terminal moiety of the ORCIDs: 0000-0002-8348-3727 (J.J.G.-R.); 0000-0003-2508-8398 (M.J.R.-H.); NOTCH receptors creates a new substrate site that can be pro- 0000-0002-9210-838X (J.L.); 0000-0003-4933-4602 (E.M.M.); 0000-0003-3843- 3912 (M.J.M.D.-G.). cessed by a g-secretase complex. This, in turn, produces the re- Received for publication March 10, 2016. Accepted for publication August 8, 2016. lease of the NOTCH intracellular domains (NICD), which are translocated to the nucleus where they bind to the DNA-binding This work was supported by Instituto Carlos III, Ministerio de Economıá y Com- petitividad, Spain Grants PI12/01546 and PI15/00991. protein RBP-J/CSL/CBF1 and other factors. This causes the as- Address correspondence and reprint requests to Dr. Marıá Jose´ M. Dıáz-Guerra and sembly of an active transcriptional activation complex that drives Dr. Eva Marıá Monsalve, Facultad de Medicina/Centro Regional de Investigaciones the expression of NOTCH target genes, in particular the HES and Biome´dicas, Universidad de Castilla–La Mancha, Avenida da Almansa 14, 02006 HEY families of transcription factors (18, 19). Albacete, Spain. E-mail addresses: [email protected] (M.J.M.D.-G.) and [email protected] (E.M.M.) Tetraspanins constitute a superfamily of 33 membrane proteins The online version of this article contains supplemental material. with four transmembrane regions, three short intracellular domains Abbreviations used in this article: ADAM, a disintegrin and metalloproteinase; COX, (two of them corresponding to the N- and C- terminal regions), and cyclooxygenase; Genot, genotyped; iNOS, inducible NO synthase; LTA, lipoteichoic a specific fold in the largest of the two extracellular regions, which acid; NICD, NOTCH intracellular domain; poly(I:C), polyinosinic-polycytidylic contains four or more cysteines in two or more CCG conserved acid; qPCR, quantitative PCR; shRNA, short hairpin RNA; WT, wild-type. sequences (20, 21). These proteins interact with each other and Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 with other integral membrane proteins to form microdomains on

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600421 2 TSPAN33 CONTROLS NOTCH SIGNALING IN MACROPHAGE ACTIVATION the plasma membrane, known as tetraspanin-enriched micro do- porter plasmids pNF-kB–luc, pRBP-J–luc, inducible NO synthase (iNOS)– mains (22–24). In these microdomains, tetraspanins have been luc, cyclooxygenase (COX)-2–luc, and pIFN-b–luc, used to detect found to interact with integrins and with Ig domain–containing NF-kB–, NOTCH-, iNOS-, COX-2–, and IFN-b–dependent transcription activities, respectively, have been previously described (32). pRLTK proteins, among others (25). A subgroup of tetraspanins, known as Renilla-expressing vector (Promega) was used as a control for transfection TSPANC8 owing to the eight cysteine residues present at their efficiency. pCMV6 (empty vector), pCMV6-Tspan33 (Tspan33 expression major extracellular loop, is composed of TSPAN5, 10, 14, 15, and vector), Sh-control (empty vector), Sh-Tspan33 (eBioscience), pLNCX2 17 and TSPAN33/PENUMBRA. TSPANC8 members are differ- (empty vector), pLNCX2-Notch1IC (intracellular Notch1 expression vector), PCD2 (empty vector), and/or PCD2-Notch1 (full-length Notch1 ex- entially expressed in cells and tissues, and most of them interact pression vector) (33) were used together with the reporters. Cells were with ADAM10 and regulate its maturation and trafficking to the stimulated with 100 ng/ml LPS for 24 h after being transfected. Luciferase cell membrane (26–28). ADAM10 is a ubiquitous metalloprotease and Renilla activities were measured by using the Dual-Luciferase reporter that cleaves the extracellular regions of a large number of proteins, assay system (Promega) in a Monolight 3096 (BD Biosciences) following including NOTCH receptors, as previously described (29, 30). the manufacturers’ recommendations. For stable transfections, 3 3 106 Raw 264.7 cells were seeded on 60-mm We have explored the role of TSPAN33 in NOTCH and TLR plates 24 h before transfection with 6.5 mg of either Sh-control, Sh- signaling during macrophage activation. We show in the present Tspan33, pLNCX2, or pLNCX2-Notch1IC plasmids. Cells were selected study that Tspan33 is induced after macrophage activation by TLR with 300 mg/ml G418 (Sigma-Aldrich) for 2 wk. triggering and IFN-g treatment and that this process is largely de- Protein extracts and Western blot analysis pendent on NOTCH signaling. Using genetic approaches to increase or diminish TSPAN33 expression levels in macrophages, we dem- Cells were washed twice with ice-cold PBS, scraped off from the dishes, and collected by centrifugation. Cell pellets were resuspended in RIPA lysis onstrate that TSPAN33 modulates TLR-induced macrophage acti- Downloaded from buffer (25 mM HEPES [pH 7.5], 1.5 mM MgCl2, 0.2 mM EDTA, 1% vation, increasing cytokine production and cytotoxic activity, mainly Triton X-100, 0.3 M NaCl, 20 mM b-glycerophosphate, 0.1% SDS, 0.5% through NF-kB activation by enhancing NOTCH signaling. Overall, deoxycholic acid) supplemented with 10 ml/ml each protease inhibitor and these results reveal a new role of TSPAN33 in macrophage biology I and II phosphatase inhibitors (Sigma-Aldrich). Finally, pellets were ho- 3 as a regulator of NOTCH-mediated proinflammatory activation. mogenized for 30 min at 4˚C and centrifuged at 8000 g for 15 min. Protein concentrations were determined by the Bradford method (Bio- Rad).

Denatured total protein extracts (30–40 mg) were separated in 10% http://www.jimmunol.org/ Materials and Methods polyacrylamide gels, transferred to Nylon membranes (Hybond-C Extra; Mice Amersham Biosciences) and processed according to the Ab suppliers’ We generated mice with a myeloid-specific deletion of Notch1, Notch2,or recommendations. Proteins were detected with ECL (Amersham Phar- both by crossing Notch1flox/flox, Notch2flox/flox animals to animals with a macia Biotech). b-tubulin expression was used as a loading control. lysozyme-driven Cre transgene on C57BL/6 genetic background (The Anti-TSPAN33 Ab was purchased from Proteintech. Anti–COX-2 (no. Jackson Laboratory). All procedures were approved by the Ethics in An- 4842) was acquired from Cell Signaling Technology. Anti-ADAM10 and imal Care Committee of the University of Castilla–La Mancha. Mice were anti-ADAM17 were purchased from Millipore and anti–b-tubulin (T-3952) genotyped (Genot) by PCR, using genomic DNA obtained from small tail was from Sigma-Aldrich. cuts with a GeneJET genomic purification kit (Fermentas), with the oli- + 2 RNA and cDNA purification gonucleotides Genot Cre , Genot Cre , Genot Notch1, and Genot Notch2 by guest on September 28, 2021 (Table I). Total RNA was obtained by using the RNeasy kit (Qiagen) with DNase (Promega) according to the manufacturers’ instructions and evaluated in a Cells and reagents ND-1000 (NanoDrop) spectrophotometer. cDNA was synthesized from Peritoneal macrophages were isolated as previously described (6) from 1 mg of total RNA by using RevertAidH minus first strand cDNA synthesis 2-mo-old Notch1 and Notch2 wild-type (WT) or knockout male mice, 4 d (Fermentas) following the manufacturer’s recommendations. after i.p. injection of 2 ml of 3% sterile thioglycollate broth (w/v in water; Quantitative PCR Life Technologies). Elicited macrophages were seeded at 1 3 105 cells/ 2 cm in complete RPMI 1640 medium (supplemented with 10% FBS, 2 mM Gene expression analysis by quantitative PCR (qPCR) was performed in L-glutamine, and 1% penicillin-streptomycin; all purchased from Lonza) triplicate according to the Fast SYBR Green protocol with the StepOne real- and incubated overnight in complete RPMI 1640 supplemented with 2% time PCR detection system (Applied Biosystems). Specific oligonucleotides FBS. The cells were activated with 100 ng/ml LPS from Salmonella were designed with PrimerQuest computer program (Integrated DNA typhimurium (Sigma-Aldrich), 200 ng/ml polyinosinic-polycytidylic acid Technologies) and are indicated in Table I. The mRNA levels of mouse [poly(I:C); Amersham Biosciences), 5 mg/ml lipoteichoic acid (LTA) from riboprotein P0 (34) or human GAPDH were used as internal controls. Staphylococcus aureus (InvivoGen), and/or 20 U/ml IFN-g. The activation was verified by determining NO production with the Griess reaction. Statistical analysis Raw 264.7 cells (ATCC TIB-71) were subcultured at 6–8 3 104 cells/ cm2 in DMEM medium (Lonza) with the above supplements and incubated The Student unpaired t test was used for statistical analyses between two , overnight in complete DMEM containing 5% FBS before induction with groups. A p value 0.05 was considered significant. 100 ng/ml LPS. Human monocytes were isolated from the blood of healthy donors by Results centrifugation on Ficoll-Paque Plus (Amersham Biosciences) following TSPAN33 expression is induced in activated macrophages in a reported protocols (31) and cultured in DMEM complete medium supplemented or not with 100 ng/ml LPS for 24 h. Human samples were obtained NOTCH-dependent manner and increases Toll receptor– and processed under the European Union and Spanish regulations. dependent NOTCH signaling Human U937 monocytes (ATCC 1593.2) were maintained in complete DMEM medium supplemented with 10 mM HEPES. For differentiation into To investigate the mechanism by which NOTCH receptors contribute macrophages, 1.5 3 106 cells per well were incubated in six-well plates to macrophage activation, we searched for new genes differentially with 10 mM PMA (Sigma-Aldrich) for 24 h. Once attached to the plate, expressed in LPS- and IFN-g–activated peritoneal macrophages cells were incubated during 48 h in fresh medium without PMA. lacking or not Notch1 and Notch2 expression. By using a set of Cell transfections Affymetrix microarrays, we identified Tspan33, among other genes, as a gene preferentially expressed in WT macrophages activated by 5 For transient transfections, 2.5 3 10 Raw 264.7 cells per well were seeded LPS and IFN-g. We confirmed the microarray data by quantitative in triplicate on 12-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day, according to the manufacturer’s rec- PCR (Table1) and Western blot analyses. As shown in Fig. 1A, ommendations, using Opti-MEM medium (Life Technologies) without macrophages activated with LPS rapidly increased Tspan33 mRNA supplements and 1.25 mg total EndoFree plasmid DNA per well. The re- level, and this process was enhanced by IFN-g. TSPAN33 protein The Journal of Immunology 3

Table I. Oligonucleotides used for PCR

Gene Forward Primer Reverse Primer Genot Cre+ 59-TTA CAG TCG GCC AGG CTG AC-39 59-CTT GGG CTG CCA GAA TTT CTC-39 Genot Cre2 59-CCC AGA AAT GCC AGA TTA CG-39 59-CTT GGG CTG CCA GAA TTT CTC-39 Genot Notch1 59-TGC CCT TTC CTT AAA AGT GG-39 59-GCC TAC TCC GAC ACC AAT A-39 Genot Notch2 59-TAG GAA GCA GCT CAG CTC ACA G-39 59-ATA ACG CTA AAC GTG CAC TGG AG-39 mP0 59-GAA TCG CTC CTG CAG CAA AG-39 59-CCA GGG TCT CAT CCG CAT T-39 mTspan33 59-GAG CAC AAC AGC AGC ATC CAC ATT-39 59-TGA TGG CAC AAC TCC ACT GAT CCT-39 hTspan33 59-AGG CAA GCA ACA GGA GTA AG-39 59-GTG TCT ACG CTC GGC TAA TG-39 h5NRPD3 59-ATG AAC TGC CAG ATG TCC-39 59-TGT CAG GCA AAA TCA GAA AG-39 mHes1 59-AAG CGC GTC CTG GCA TTG TCT-39 59-CCG CAG GGG CAG CAG TGG T-39 mTspan5 59-AAA GGT GTC GTC CTC TCC ACC-39 59-CAA ATT CTG TGC CAG GCA-39 mTspan10 59-CAC CGG GAC ACC GTT ATT T-39 59-GGC AAG GAG AAG AGG AA-39 mTspan14 59-ACC TGC TCT TCA GCT ACA ATA TC-39 59-CTC TCA CCC AGT CTT GGA ATA AA-39 mTspan15 59-CCG GAG AGG AAT CGA GAA TTA C-39 59-GAG TGG CCA TCA GAC ACT ATA C-39 mTspan17 59-TCG CCC TCC TCC AGA TAT T-39 59-CTC CTA AGC CAC ACA CAG ATA C-39 mADAM10 59-CTG GGA GGT CAG TAT GGA AAT C-39 59-CTC TCT GCT GGC TCA ATG-39 mADAM17 59-GAA GAG ACG AGC TGA ACC TAAC-39 59-CTG CTC TAT CTG CAC TCC ATA C-39 mPsen1 59-GTA TAA CAC ACA AAG AGC GG-39 59-TTT TAC TCC TCT TTC CTC CG-39 mPsen2 59-AAA TGA GCC CAT ATT TCC TG-39 59-GTA GGA GTC TTC TTC CAT CTC-39 Downloaded from was detected about 4 h after LPS and IFN-g treatment (Fig. 1A, previously shown, NOTCH signaling also modulates Tspan33 right panel). To evaluate whether the increased expression of expression in TLR-activated macrophages, thus creating a positive Tspan33 was a general process during TLR macrophage activation, activation regulatory loop that can enhance NOTCH signaling in we evaluated Tspan33 mRNA and protein levels after treating activated macrophages.

macrophages with poly(I:C), a TLR3 agonist, and with LTA, a In mammals, different C8 tetraspanin family members, including http://www.jimmunol.org/ TLR2 agonist, in the presence or not of IFN-g. We observed that, as TSPAN5, 10, 14, 15, 17, and 33, seem to be important in the control shown in Fig. 1B, both TLR agonists increased Tspan33 expression, of NOTCH receptor signaling (26, 27). For that reason, we eval- although to different extents, and this increase was enhanced by uated the expression of the C8 tetraspanin family members in IFN-g. Thus, it seems that increase in Tspan33 expression is a control and TLR-activated macrophages. Only the expression of common feature in TLR-triggered macrophage activation. Finally, Tspan15 and Tspan33 was clearly increased in TLR-activated we also analyzed Tspan33 expression in human monocytes and in macrophages, whereas the mRNA levels of the other C8 tetra- the human promyelocytic cell line U937 and observed that Tspan33 spanins remained constant or slightly decreased after stimulation expression was also increased soon after treatment with LPS in both (Supplemental Fig. 2). Thus, TSPAN33 seems to be the tetra- types of cells (Fig. 1C). spanin more intensely and rapidly induced in TLR-activated by guest on September 28, 2021 We next evaluated whether Tspan33 induction by LPS was macrophages. dependent on NOTCH signaling. As shown in Fig. 2A, Tspan33 expression levels were lower in macrophages lacking both Maturation of ADAM10 increases in TLR-activated NOTCH1 and NOTCH2 receptors than in WT macrophages. macrophages However, this difference was not observed in macrophages lacking TSPAN33 has been described as a chaperon protein that allows either one of these NOTCH receptors (Supplemental Fig. 1). ADAM10 processing and translocation to the plasma membrane Additionally, Tspan33 mRNA and protein expression were in- (27). ADAM10 and ADAM17 have been related with ligand- creased in activated Raw 264.7 cells overexpressing the intracel- dependent and ligand-independent NOTCH processing (30). We lular domain of NOTCH1 (NICD1; Fig. 2B). These results showed have analyzed ADAM10 and ADAM17 expression in control and that induction of Tspan33 expression after TLR signaling is me- activated macrophages. As shown in Fig. 3A, Adam10 mRNA diated, at least in part, by NOTCH signaling in activated macro- expression diminishes slightly in macrophages activated with phages. LPS, whereas Adam17 mRNA levels increase 6–9 h after activa- Genetic studies in Drosophila have shown that ablation of two tion. As both ADAM10 and ADAM17 are synthesized as inactive TSPANC8 tetraspanin genes mimicked Notch deficiency, indicating precursors, we also analyzed ADAM10 and ADAM17 expression that those genes were essential for NOTCH signaling (26). We by Western blot to evaluate the presence of their active mature evaluated the role of TSPAN33 in NOTCH signaling after TLR forms. As shown in Fig. 3B, in macrophages activated with LPS, activation by cotransfecting Raw 264.7 cells with a Tspan33 ex- with or without IFN-g, an increase in the active processed form of pression vector or with a specific Tspan33 short hairpin RNA ADAM10 (55 kDa) was observed, and this correlated with di- (shRNA) together with an RBP-J reporter plasmid. As shown in minished levels of the unprocessed, inactive 85-kDa form. Fig. 2C, forced expression of Tspan33 increased RBP-J reporter ADAM17 was not basally detected in macrophages, but its ex- activity in control or LPS-activated macrophages, whereas a pression increased after activation (Fig. 3B, right panel), accord- specific shRNA partially diminished this effect in LPS-activated ingly to the mRNA levels (Fig. 3A). Both unprocessed (120 kDa) cells. To confirm these results, we generated stable Raw 264.7 and mature ADAM17 forms (85 kDa) were detected after mac- cells expressing control or a specific Tspan33 shRNA. As shown rophage activation. These results indicate that TLR signaling in Fig. 2D, Tspan33 shRNA clearly diminished the expression of promotes ADAM10 and ADAM17 expression and/or activation in Tspan33 (upper panel). This decrease was associated with a lower macrophages, favoring in this way NOTCH processing. expression of Hes1 (a downstream NOTCH target) after TLR4 We evaluated by Western blot the role of TSPAN33 in ADAM10 activation (Fig. 2D, lower panel). These results confirm that and ADAM17 processing in TLR-activated macrophages by TSPAN33 represents an important element in the control of comparing their expression in control and Raw 264.7 cells over- NOTCH activity in TLR-activated macrophages. Moreover, as expressing TSPAN33. As reported for other cell types (27), 4 TSPAN33 CONTROLS NOTCH SIGNALING IN MACROPHAGE ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 1. Tspan33 is expressed in macrophages activated though TLRs and IFN-g.(A) Left panel, qPCR analysis of Tspan33 expression in peritoneal murine macrophages activated with LPS (100 ng/ml), IFN-g (20 U/ml), or both at different times. Data are referred to unstimulated macrophages set to 1. qPCR was performed in triplicate with riboprotein P0 as the internal control. Means 6 SD of three independent experiments are shown. Right panel, Western blot analysis of TSPAN3333 expression in peritoneal macrophages activated with LPS and IFN-g up to 24 h. b-Tubulin expression was used as a loading reference. Image is representative of three independent experiments. *p , 0.05 with respect to control conditions (ﬁrst column), #p , 0.05 with by guest on September 28, 2021 respect to LPS conditions. (B) Left panel, qPCR analysis of Tspan33 expression in peritoneal macrophages activated with poly(I:C) (100 ng/ml) or LTA (200 ng/ml) in the presence or not of IFN-g for the indicated times. Data are referred to unstimulated macrophages set to 1. Means 6 SD of three independent experiments are shown. Right panel, Western blot analysis of TSPAN33 expression in peritoneal macrophages activated as above with poly(I:C) or LTA in the presence of IFN-g up to 24 h. b-Tubulin expression was used as a reference. Images are representative of three independent experiments. *p , 0.05 with respect to control conditions (ﬁrst column), #p , 0.05 with respect to the TLR agonist conditions. (C) qPCR analysis of Tspan33 expression in human monocytes and U937 cells activated with LPS (100 ng/ml) at different times. qPCR was performed in triplicate with riboprotein P0 as the internal control. Means 6 SD of three independent experiments are shown. *p , 0.05 with respect to control conditions.

TSPAN33 increased ADAM10 processing, as reflected by the activated with LPS. However, no effect was observed in Raw accumulation of the 55-kDa form in control and activated cells 264.7 cells transfected with the active intracellular domain of (Fig. 3C). However, ADAM17 processing was not affected by NOTCH1 (NICD1). Thus, it seems that TSPAN33 increases TSPAN33 (data not shown). Our data indicate that TSPAN33 NOTCH signaling in TLR- activated macrophages by increasing enhances the processing of ADAM10, which in turn could in- NOTCH processing at the plasma membrane. crease NOTCH1 processing and activation. Although several groups (26, 28), including us (Fig. 3B), have shown that TSPAN33 favors ADAM10 maturation, alternately, the TSPAN33 increases NOTCH signaling in TLR-activated modulation of g-secretase activity by the interaction of tetraspa- macrophages by favoring its processing nins with presenilins has also been described (35, 36). We have To further confirm that the effect of TSPAN33 on NOTCH activity used specific shRNAs to diminish the expression of both was related to an increase in NOTCH receptor processing, we ADAM10 and Presenilin1 (Psen1), the presenilin more highly analyzed NOTCH activity using an RBP-J–LUC reporter in Raw expressed in activated macrophages (Supplemental Fig. 3A, 3B), 264.7 cells transfected with a Tspan33 expression vector in the to evaluate the effect of TSPAN33 on NOTCH activity when the presence of a full-length Notch1 expression vector or the consti- levels of these proteins are low. As shown in Fig. 4C, ADAM10 tutively active intracellular domain of NOTCH1 (NICD1). As and Psen1 shRNAs strongly decreased NOTCH activity in control shown in Fig. 4A, expression of NOTCH1 increased basal and and LPS-activated macrophages; however, increased expression of LPS-induced RBP-J–LUC activity, and this effect was enhanced TSPAN33 augmented NOTCH signaling in both cases. In line in the presence of TSPAN33. In contrast, NICD1 highly in- with these results, lowering TSPAN33 in addition to ADAM10 or creased the RBP-J–dependent reporter activity, but expression of PSEN1 decreased NOTCH activity in both cases (Supplemental TSPAN33 did not modify this effect. Similarly, diminished ex- Fig. 3C). Interestingly, in LPS-activated macrophages, the po- pression of TSPAN33 with a specific shRNA in Raw 264.7 cells tentiation of NOTCH signaling by TSPAN33 is slightly, but sig- (shTspan33; Fig. 4B) correlated with a lower RBP-J–dependent nificantly, more elevated when ADAM10 levels are diminished reporter activity in cells transfected with full-length NOTCH1 and than when PSEN1 levels are low. These results could argue for a The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. TLR-induced Tspan33 expression depends on NOTCH activity and regulates NOTCH signaling in activated macrophages. (A) Left panel, qPCR analysis of Tspan33 expression in WT and Notch1/Notch2 knockout (KO) peritoneal murine macrophages activated with LPS (100 ng/ml) in the presence or not of IFN-g (20 U/ml) for different times. Means 6 SD of three independent experiments are shown. **p , 0.01 between the corresponding conditions in WT and Notch1/Notch2 KO macrophages. Right panel, Western blot analysis of TSPAN33 expression in WT and KO Notch1/Notch2 peritoneal macrophages activated with LPS in the presence or not of IFN-g for 24 h. The dotted black lines indicate where parts of the image were joined. b-Tubulin expression was used as a loading reference. Image is representative of three independent experiments that are quantified in the graph next to it. *p , 0.05 with respect to each WT condition. (B) qPCR (upper panel) and Western blot (lower panel) analysis of Tspan33 mRNA and protein expression in control (Raw/Vector) and Raw 264.7 macrophages overexpressing the active intracellular domain of NOTCH1 (Raw-NICD1) activated with LPS, with or without IFN-g.Means6 SD of three independent qPCR experiments are shown. b-Tubulin expression was used as a loading reference. Data are representative of three independent experiments. (C) Notch transcriptional activity analysis in Raw 264.7 cells transiently transfected with an RBP-J luciferase reporter (RBP-J–LUC), an empty vector, or a Tspan33 expression vector (upper panel) or with control or Tspan33-specific shRNAs (lower panel). One day after transfection, cells were stimulated with LPS for 24 h before analysis. pRLTK was used as an internal control vector for transfection and normalized luciferase/Renilla values are represented. The means 6 SD of three independent experiments are shown. *p , 0.05 with respect to each control condition (left column of each graph), #p , 0.05 with respect to the same condition in cells transfected with the empty vector (upper panel) or with the short hairpin control vector (lower panel). (D) qPCR analysis of Tspan33 (upper panel) and Hes-1 (lower panel) mRNA expression in Raw 264.7 cells stably transfected with control (Raw-Sh control) or Tspan33-specific shRNAs (Raw- ShTspan33) activated with LPS for 4 h. Means 6 SD of three independent experiments are shown. *p , 0.05 with respect to the same condition in Raw 264.7 control cells, #p , 0.05 with respect to the same conditions in cells transfected with the short hairpin control vector. KO, knockout. role of TSPAN33 not only in ADAM10 processing, but also in the NOTCH2 has also been reported in these cells (5, 8, 14). To analyze modulation of g-secretase activity. Nevertheless, we cannot rule the involvement of TSPAN33 in TLR-mediated NOTCH activation, out that the effect of TSPAN33 falls only on ADAM10 processing, we modified the expression of TSPAN33 in macrophages with di- as the product of ADAM10 activity is the substrate of g-secretase. minished expression of NOTCH1 and NOTCH2 receptors with More experiments using truncated mutant NOTCH receptors previously evaluated shRNAs (Supplemental Fig. 3D) and measured lacking the ADAM10 processing site are necessary to exactly RBP-J–LUC activity. As shown in Fig. 4D, this activity diminished delineate whether TSPAN33 modulates Presenilin1/g-secretase in control and LPS-activated macrophages in the presence of specific activity in activated macrophages, as has been shown in Caeno- shRNAs for Notch1 and Notch2, arguing for the important role of rhabditis elegans and human cells (36). these receptors in NOTCH signaling in macrophages. Interestingly, NOTCH1 has been suggested to be one of the most important increased expression of TSPAN33 compensated in part for the de- NOTCH receptors in TLR-activated macrophages, but expression of crease of NOTCH signaling in macrophages with reduced Notch1 6 TSPAN33 CONTROLS NOTCH SIGNALING IN MACROPHAGE ACTIVATION

could modulate proinflammatory macrophage activation. To evaluate the role of TSPAN33 in LPS-activated Raw 264.7 cells, we measured the transcriptional activity of typical proinflammatory genes, such as Cox-2, iNOS, and IFN-b, in the presence of increased or reduced TSPAN33 levels. As shown in Fig. 5A, forced expression of TSPAN33 increased LPS-dependent promoter activity in all cases. In contrast, transfection with a specific Tspan33 shRNA that diminishes Tspan33 mRNA levels (Fig. 2B) correlated with lower promoter activities (Fig. 5B). To further confirm these results, we used stable Raw 264.7 transfectants with increased (Raw-Tspan33) or diminished (Raw- shTspan33) TSPAN33 levels, and evaluated the expression of COX-2. As shown in Fig. 5C, increased levels of Cox-2 mRNA and protein were detected in cells with higher TSPAN33 expression. On the contrary, a decreased level of Cox-2 was observed in Raw 264.7 cells with diminished TSPAN33 expression (Fig. 5D). Therefore, our data show that TSPAN33 modulates the expression of TLR-activated proinflammatory genes. Downloaded from TSPAN33 increases NF-kB activation in TLR-activated macrophages by favoring NOTCH maturation and signaling NF-kB is a key transcription factor in TLR activation whose activity has been described to be enhanced by NOTCH signaling (10, 38–40). We wondered whether the modulation of proin-

ﬂammatory gene expression by TSPAN33 observed in macro- http://www.jimmunol.org/ phages (Fig. 5) could be mediated, at least in part, by NF-kB. We explored this hypothesis in Raw 264.7 cells cotransfected with an NF-kB–LUC reporter gene together with expression vectors for Tspan33, full-length Notch1, or the constitutively active intracellular domain of NOCH1 (NICD1). In agreement with previous studies (10), elevated expression of Notch1 or NICD1 increased NF-kB activity in control and LPS-activated macrophages (Fig. 6A). In line with our NOTCH signaling data (Fig. 4A), we

observed an increase in TSPAN33-mediated NF-kB activity when by guest on September 28, 2021 NOTCH1 was expressed, but not when the constitutively active FIGURE 3. Analysis of of ADAM10 and ADAM17 expression in activated macrophages. (A) qPCR analysis of Adam10 and Adam17 mRNA NICD1 was expressed (Fig. 6A). Similarly, restricted levels of expression in control and LPS-activated peritoneal macrophages. Data TSPAN33 by shRNAs diminished NF-kB activity in macrophages referred to nonstimulated macrophages are considered as 1. For each gene, activated with LPS and in those overexpressing NOTCH1, but did the mean 6 SD of three independent experiments are shown. *p , 0.05 not affect NF-kB–dependent signaling induced by NICD1 with respect to control conditions. (B) Western blot analysis of ADAM10 (Fig. 6B), arguing for a role of TSPAN33 in NOTCH1 processing. (left panels) and ADAM17 expression (right panel) in peritoneal macro- These results suggest that the effect of TSPAN33 in NF-kB ac- phages activated with LPS (100 ng/ml) in the presence or not of IFN-g tivity is mediated mainly by NOTCH signaling. (20 U/ml). Inactive preforms (high molecular mass) and active forms We also evaluated the effect of TSPAN33 in NF-kB activity of (lower molecular mass) are indicated by arrows. The dotted black lines control and TLR-activated macrophages with diminished Notch1 indicate where parts of the image were joined. b-Tubulin expression was and/or Notch2 expression. As shown in Fig. 6C, NF-kB–LUC used as a loading control. In the case of ADAM10, a graph with the relative proportion of the activated form is shown below the Western blot. activity decreased in control and LPS-activated macrophages in *p , 0.05 with respect to control conditions. (C) Western blot analysis of the presence of a combination of specific shRNAs for Notch1 and ADAM10 expression in Raw 264.7 cells stably transfected with a control Notch2, arguing for the relevant role of these receptors in NF-kB or a Tspan33 expression vector, and activated with LPS for 12 h. Pro- signaling in macrophages, in agreement with previous data (10, ADAM10 and the mature active form are indicated by arrows. The relative 40). However, increased expression of TSPAN33 compensated, in amount of mature ADAM10 is shown in the graph on the right. For (B) and part, for the decrease of NF-kB activity in macrophages with re- # (C), three independent experiments were done. p , 0.05 with respect to duced NOTCH1 and NOTCH2 expression (Fig. 6C). These effects the same conditions in control cells. could be mediated by the elevated NOTCH activity observed previously (Fig. 4D). Thus, TSPAN33 could modulate NF-kB and Notch2 expression. These results suggest that the increase of activity and proinflammatory gene expression by controlling TSPAN33 in the first hours of TLR activation could promote a NOTCH signaling. general increase in NOTCH signaling. Discussion TSPAN33 modulates proinflammatory gene expression in In the last years, multiple studies have uncovered an important role TLR4-activated macrophages of NOTCH signaling in macrophage activation by Toll receptors As NOTCH signaling has been associated with proinflammatory (7–10). However, factors and pathways controlling NOTCH sig- macrophage activation (7–10), and TSPAN33 increases NOTCH naling during this process have not been completely delineated signaling in different cellular types (26, 37), including TLR- yet. In this work, we have characterized the role of TSPAN33, a activated macrophages (Fig. 2C), we wondered whether TSPAN33 tetraspanin implicated in ADAM10 maturation and/or g-secretase The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. TSPAN33 increases NOTCH signaling in TLR-activated macrophages by favoring the liberation of the NOTCH intracellular domain. (A) NOTCH transcriptional activity analysis in Raw 264.7 cells transiently cotransfected with a RBP-J luciferase reporter (RBP-J–LUC), a Tspan33 expression

vector (Tspan33), or the corresponding empty vector (control) together with a full-length NOTCH1 (Notch1) or a constitutively active intracellular domain by guest on September 28, 2021 of NOTCH1 (NICD1) expression plasmids. One day after transfection, cells were stimulated with LPS for 24 h before analysis. The means 6 SD of six independent experiments are shown. *p , 0.05 with respect to each control condition (Tspan33 empty vector), #p , 0.05 with respect to each control condition (Notch1 empty vector, indicated as Vector). (B) NOTCH transcriptional activity analysis similar to that described in (A), but transfecting with control or Tspan33 shRNAs. (C) Evaluation of the relevance of Adam10 and Psen1 in the NOTCH transcriptional activity of LPS-activated macrophages, with or without increased Tspan33 expression. Activity analyses were similar to those described in (A) and are expressed in percentage relative to the control condition (first bar on the left) considered as 100%. The means 6 SD of three independent experiments are shown. *p , 0.05 between specific shRNAs respect to shRNA control, #p , 0.05 between short hairpin Adam10 and shPsen1 conditions. (D) Evaluation of the relevance of Notch1, Notch2, and Tspan33 in the NOTCH transcriptional activity of LPS-activated macrophages. Raw 264.7 cells were cotransfected with the RBP-J luciferase reporter gene (RBP-J–LUC) and the Tspan33 expression vector (TSPAN33), or Tspan33-specific shRNA (sh Tspan33), together with a control (sh C) or a combination of Notch1 and Notch2 shRNAs (sh N1+N2). Normalized luciferase/Renilla values are referred to the levels of non-stimulated Raw 264.7 cells transfected with empty vectors, set as 100% (first bar on the left). The means 6 SD of five independent experiments are shown. LPS was used at 100 ng/ml. *p , 0.05 with respect to control conditions in cells activated with LPS. activation, in NOTCH signaling during macrophage proin- creating a positive regulatory loop that leads to NOTCH signal flammatory activation. Our data show that Tspan33 expression amplification. Our data also suggest the existence of a novel increases in response to TLR signaling, both in murine and human positive regulatory loop in which NOTCH and TSPAN33 interact monocytes/macrophages, including responses through TLR4, to enhance NOTCH signaling, leading to a proinflammatory TLR3, and TLR2, and that this process is enhanced by IFN-g. macrophage phenotype upon TLR macrophage activation. Tspan33 inductionbyTLR/IFN-g is largely dependent on NOTCH Induction of target genes after TLR stimulation requires the signaling, as its expression is noticeably diminished in macrophages combination of downstream signaling events, including tran- lacking Notch1 and Notch2 expression. Moreover, TSPAN33 in- scriptional activation via the NF-kB pathway (1). Our results show creases NOTCH signaling and TLR-induced proinflammatory gene that increased expression of TSPAN33 leads to increased NF-kB expression, thus acting as a new control element in the develop- activity, whereas diminished levels of TSPAN33 result in a lower ment of inflammation. activity of this transcription factor. These results correlated with The proper control of NOTCH signaling appears to be very proinflammatory gene expression. This is, to the best of our important for a variety of cell processes, and several mechanisms knowledge, the first time that the activity of a C8 tetraspanin are involved in it, both ligand-dependent and ligand-independent family member is related to NF-kB activation and to the induc- (41). Recently, it has been reported that furin, the protease im- tion of proinflammatory genes. In this regard, the implication of plicated in NOTCH1 and ADAM10 maturation, is transcription- ADAM10 in the induction of NF-kB and cytokine expression has ally induced by NOTCH1 (42). In this way, different proteins been described in macrophages activated with meprin-b,a implicated in NOTCH signaling are regulated by the pathway, thus different metalloproteinase (43). Moreover, much evidence has 8 TSPAN33 CONTROLS NOTCH SIGNALING IN MACROPHAGE ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 5. TSPAN33 increases the expression of proinflammatory genes in TLR-activated macrophages. (A and B) Analysis of Cox-2, iNOS, and IFN-b promoter activity in Raw 264.7 cells transiently transfected with a Tspan33 expression vector or the control empty vector (A), or with control or Tspan33- specific shRNAs (B). One day after transfection, cells were stimulated with LPS (100 ng/ml) for 24 h before analysis, and luciferase activity was evaluated. Normalized luciferase/Renilla values are referred to the levels of nonstimulated control Raw 264.7 cells, set as 1. The means 6 SD of three independent by guest on September 28, 2021 experiments are shown. *p , 0.05 with respect to LPS-activated conditions with control vector (A) or shRNA control (B). (C) qPCR (upper panel) and Western blot (lower panels) analysis of COX-2 expression in Raw 264.7 cells stably transfected with a Tspan33 expression vector (Raw-Tspan33) or the control empty vector (Raw-vector) induced with LPS for the indicated times. Quantification of Western blot signals is also presented (lower panel). Means 6 SD of three independent experiments are shown. *p , 0.05 respect to the corresponding control conditions. (D) qPCR (upper panel) and Western blot (lower panels) analysis of COX-2 expression in Raw 264.7 cells stably transfected with a control (sh C) or a Tspan33-specific (sh Tspan33) shRNA, activated with LPS for the indicated times. Quantification of Western blots is also presented (lower panel). Means 6 SD of three independent experiments are shown. For (C)and (D) three independent experiments were done.*p , 0.05 with respect to the corresponding shRNA control conditions. related TSPAN33 and C8 tetraspanins with NOTCH signaling. (46). More recent studies have unveiled that NOTCH1 is a sub- Penumbra/Tspan33 was characterized in erythroid progenitors strate for both proteases. ADAM10 is absolutely required for (44), but its role in NOTCH signaling was evidenced in Dro- ligand-induced NOTCH1 signaling, whereas ADAM17 signaling sophila, C. elegans, and mammalian cells (26). These data are in is ligand–independent (30). Other studies have confirmed a similar support of the evidence presented in this study, suggesting that role for ADAM10 in NOTCH2 and NOTCH3 processing (47); TSPAN33 influences NF-kB activity through the modulation of however, a lower sensibility for ADAM17 has been described for NOTCH signaling by the control of ADAM10 and/or g-secretase human NOTCH2 receptors (48). ADAM10-deficient macrophages activity. display an increased anti-inflammatory phenotype, showing ele- Different authors have established that TSPAN33 and other C8 vated IL-10, but reduced production of TNF-a, IL-12, and NO tetraspanins promote NOTCH activation at a pre–g-secretase step (49). This phenotype is similar to that observed in macrophages by regulating ADAM10 trafficking and maturation (26). At least lacking CBF/RBP-J, a factor required for NOTCH signaling (8). two ADAM proteins, ADAM10 and ADAM17, have been related These observations, together with our results showing an in- with NOTCH1 receptor processing. Biochemical studies in flies creased expression of proinflammatory genes in TSPAN33- have indicated that ADAM10 interacts with and cleaves NOTCH overexpressing macrophages, suggest that the axis TSPAN33– to activate signaling. Genetic manipulations resulting in the loss of ADAM10–NOTCH is important in the development of the mac- ADAM10 protease activity lead to developmental defects similar rophage proinflammatory phenotype. to those described for deficiencies in NOTCH signaling (45). Adam10 and Adam17 expression is increased in macrophages In vitro studies in mammalian cells identified ADAM17 as the during proinflammatory activation. Adam17 mRNA expression relevant protease in NOTCH1 processing, excluding a role for increases after TLR signaling, and both the immature and mature ADAM10 in this process. However, genetic studies demonstrated forms of the protein are detected in macrophages. On the contrary, that ADAM10 knockout mice display a classic Notch loss-of- Adam10 mRNA expression does not change, but an increase in its function phenotype (45), whereas ADAM17 mutant mice do not protein maturation after TLR and INF-g signaling is observed. The Journal of Immunology 9

Our results show that ADAM10 performs an important role in NOTCH signaling in TLR-activated macrophages, although the mediation of ADAM17 cannot be discarded. Indeed, new proteases have been recently described to cleave NOTCH receptors (42). Moreover, it is not clearly deﬁned whether NOTCH activation after TLR signaling requires ligand interaction. Although the prototypical cleavage of NOTCH by mature ADAM10 seems to require conformational changes triggered by ligand binding, new mechanisms of ADAM10 proteolytic activity modulation have been described (50). Additional studies are necessary to clarify this point. Initial studies on the interaction of TSPAN33 and C8 tetraspanins with the NOTCH signaling pathway focused on the regulation of g-secretase activity (35, 36) through the interaction of tetraspanins with presenilins. Our results show the important role that Presenilin1 plays on NOTCH signaling in TLR-activated macrophages, and they suggest that TSPAN33 enhancing of NOTCH signaling could be also mediated by increased g-secretase

activity. Conﬂicting results about the modulation of g-secretase Downloaded from activity by C8 tetraspanins are found in the literature. Using similar truncated mutant NOTCH receptors lacking ADAM10 processing site, Dornier et al. (26) observed that the effect of the C8 tetraspanin family members TSPAN 5 and 14 occurs in a pre–g-secretase step, in U2OS-N1 cells, whereas Dunn et al. (36) clearly showed that

TSPAN33 facilitates the g-secretase step. We cannot discard other http://www.jimmunol.org/ possibilities, such as different tetraspanins modulating the different NOTCH receptors in different ways, making them more or less susceptible for ADAM10 and/or g-secretase proteolysis. New experiments using truncated mutant forms of NOTCH receptors in activated macrophages could help to resolve this divergence. Moreover, it is possible that TSPAN33 could act both favoring ADAM10 processing and g-secretase activity. Macrophages express different members of the C8 tetraspanin family, but TSPAN33 is the one most intensely induced after by guest on September 28, 2021 macrophage activation. Recent studies have shown that C8 tetraspanins exert different effects on the intracellular localization and maturation of ADAM10 and on its ability to cleave NOTCH and other ADAM10 substrates (51). In that sense, our results show that a change in the tetraspanin environment, with increased levels of TSPAN33 after TLR activation, seems to favor NOTCH processing and signaling, inducing NF-kB activation and proinflammatory gene expression. In this line, it is interesting that the increased expression of TSPAN33 is able to trigger high NOTCH- dependent transcriptional activity even when NOTCH1 and NOTCH2 receptor levels are low. Tetraspanins play important roles in the immune system, in- FIGURE 6. TSPAN33 increases NF-kB activity in stimulated macro- cluding survival, proliferation, adhesion, and migration (52). Most A phages. ( ) NF-kB transcriptional activity analysis in Raw 264.7 cells of the tetraspanins present in the immune system are also found in transiently cotransfected with a NF-kB luciferase reporter (NF-kB–LUC) a variety of other tissues, but TSPAN33 and a few more, including and a Tspan33 expression vector (Tspan33) or the corresponding empty CD37 and CD56, display a preferential hematopoietic expression vector (control), together with a full-length Notch1 expression vector or a constitutively active intracellular domain of NOTCH1 expression vector (44, 53). TSPAN33 has been identified as a new marker of acti- (NICD1). One day after transfection, cells were stimulated with LPS vated and malignant B cells, but its role on B cell activation has (100 ng/ml) for 24 h before analysis. The means 6 SD of six independent not been clearly outlined (54). However, the involvement of experiments are shown. *p , 0.05 with respect to control conditions NOTCH signaling and ADAM10 in B cell activation, germinal (Tspan33 empty vector), #p , 0.05 with respect to control conditions center formation, and Ab production has been clearly established (Notch empty vector, indicated as Vector). (B) Analysis of NF-kB tran- (55). Our data suggest that the role of TSPAN33 in these processes scriptional activity in Raw 264.7 cells similar to that described in (A), but could be probably mediated by increased ADAM10 processing C using control and Tspan33 shRNAs. ( ) Evaluation of the relevance of and/or increased g-secretase activity and NOTCH activation, Notch1, Notch2, and Tspan33 in the NF-kB transcriptional activity of TLR-activated macrophages. Raw 264.7 cells were cotransfected with a NF-kB luciferase reporter gene and a Tspan33 expression vector or Tspan33-specific shRNA, together with control or a combination of Notch1 tors, set as 100% (first bar on the left). The means 6 SD of five independent and Notch2 shRNAs. Normalized luciferase/Renilla values are referred to experiments are shown. *p , 0.05 with respect to conditions with control the levels of nonstimulated Raw 264.7 cells transfected with empty vec- vectors activated with LPS. 10 TSPAN33 CONTROLS NOTCH SIGNALING IN MACROPHAGE ACTIVATION similarly to what we have observed in TLR-activated macro- endothelial growth factor receptor-1 and inflammatory cytokine expression in macrophages. J. Immunol. 185: 4363–4373. phages. In this line, a role for TSPAN5 and TSPAN10 in osteoclast 15.Park,J.-S.,S.-H.Kim,K.Kim,C.-H.Jin,K.Y.Choi,J.Jang,Y.Choi,A.-R.Gwon differentiation, a process depending on NOTCH signaling, has S.-H. Baik, U. J. Yun, et al. 2015. 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