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

Almudena Ruiz-García, Susana López-López, José Javier 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 October 15, 2016, 197 (8) 3371-3381; DOI: https://doi.org/10.4049/jimmunol.1600421

Abstract

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, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ 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 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB–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.

Introduction

Macrophages are key cells for the defense of the organism against pathogens. Macrophages detect pathogens through molecular pattern recognition receptors, in particular TLR, which recognize several conserved microbial structures. Signaling through these receptors represents a critical event in regulating macrophage activity, affecting cytokine and chemokine production, receptor expression, cytotoxic activity, and cell migration (1, 2). Macrophages possess a remarkable plasticity and can change their functional phenotype in response to environmental signals, becoming adapted to different situations by fine-tuning their activity (3, 4). In this regard, it has been described that TLR signaling in macrophages increases expression of different NOTCH receptors (5–7) and activates NOTCH signaling. This process can modulate macrophage activation, favoring polarization toward an inflammatory phenotype, characterized by higher IL-6, IL-12, and TNF-α production and enhanced cytotoxicity (8, 9), mediated in part by increased NF-κB activity (10, 11). Indeed, Notch1 deficiency has been related to decreased inflammation in different pathological models (12–15).

NOTCH receptors are expressed on the cell surface as heterodimers of their N- and C-terminal fragments generated after proteolytic cleavage at the trans-Golgi network (16, 17). These NOTCH receptor heterodimers adopt a protease-resistant conformation in their basal state, but after ligand binding to their extracellular region, a hidden region is now exposed to an enzyme with a disintegrin and metalloprotease (ADAM) activity, such as ADAM10, which upon cleaving at a site located at the extracellular region of the membrane-bound, C-terminal moiety of the NOTCH receptors creates a new substrate site that can be processed by a γ-secretase complex. This, in turn, produces the release of the NOTCH intracellular domains (NICD), which are translocated to the nucleus where they bind to the DNA-binding protein RBP-J/CSL/CBF1 and other factors. This causes the assembly of an active transcriptional activation complex that drives the expression of NOTCH target genes, in particular the HES and HEY families of transcription factors (18, 19).

Tetraspanins constitute a superfamily of 33 membrane proteins with four transmembrane regions, three short intracellular domains (two of them corresponding to the N- and C- terminal regions), and a specific fold in the largest of the two extracellular regions, which contains four or more cysteines in two or more CCG conserved sequences (20, 21). These proteins interact with each other and with other integral membrane proteins to form microdomains on the plasma membrane, known as tetraspanin-enriched micro domains (22–24). In these microdomains, tetraspanins have been found to interact with integrins and with Ig domain–containing proteins, among others (25). A subgroup of tetraspanins, known as TSPANC8 owing to the eight cysteine residues present at their major extracellular loop, is composed of TSPAN5, 10, 14, 15, and 17 and TSPAN33/PENUMBRA. TSPANC8 members are differentially expressed in cells and tissues, and most of them interact with ADAM10 and regulate its maturation and trafficking to the cell membrane (26–28). ADAM10 is a ubiquitous metalloprotease that cleaves the extracellular regions of a large number of proteins, including NOTCH receptors, as previously described (29, 30).

We have explored the role of TSPAN33 in NOTCH and TLR signaling during macrophage activation. We show in the present study that Tspan33 is induced after macrophage activation by TLR triggering and IFN-γ treatment and that this process is largely dependent on NOTCH signaling. Using genetic approaches to increase or diminish TSPAN33 expression levels in macrophages, we demonstrate that TSPAN33 modulates TLR-induced macrophage activation, increasing cytokine production and cytotoxic activity, mainly through NF-κB activation by enhancing NOTCH signaling. Overall, these results reveal a new role of TSPAN33 in macrophage biology as a regulator of NOTCH-mediated proinflammatory activation.

Materials and Methods

Mice

We generated mice with a myeloid-specific deletion of Notch1, Notch2, or both by crossing Notch1flox/flox, Notch2flox/flox animals to animals with a lysozyme-driven Cre transgene on C57BL/6 genetic background (The Jackson Laboratory). All procedures were approved by the Ethics in Animal Care Committee of the University of Castilla–La Mancha. Mice were genotyped (Genot) by PCR, using genomic DNA obtained from small tail cuts with a GeneJET genomic purification kit (Fermentas), with the oligonucleotides Genot Cre+, Genot Cre−, Genot Notch1, and Genot Notch2 (Table I).

Cells and reagents

Peritoneal macrophages were isolated as previously described (6) from 2-mo-old Notch1 and Notch2 wild-type (WT) or knockout male mice, 4 d after i.p. injection of 2 ml of 3% sterile thioglycollate broth (w/v in water; Life Technologies). Elicited macrophages were seeded at 1 × 105 cells/cm2 in complete RPMI 1640 medium (supplemented with 10% FBS, 2 mM l-glutamine, and 1% penicillin-streptomycin; all purchased from Lonza) and incubated overnight in complete RPMI 1640 supplemented with 2% FBS. The cells were activated with 100 ng/ml LPS from Salmonella typhimurium (Sigma-Aldrich), 200 ng/ml polyinosinic-polycytidylic acid [poly(I:C); Amersham Biosciences), 5 μg/ml lipoteichoic acid (LTA) from Staphylococcus aureus (InvivoGen), and/or 20 U/ml IFN-γ. The activation was verified by determining NO production with the Griess reaction.

Raw 264.7 cells (ATCC TIB-71) were subcultured at 6–8 × 104 cells/cm2 in DMEM medium (Lonza) with the above supplements and incubated overnight in complete DMEM containing 5% FBS before induction with 100 ng/ml LPS.

Human monocytes were isolated from the blood of healthy donors by centrifugation on Ficoll-Paque Plus (Amersham Biosciences) following reported protocols (31) and cultured in DMEM complete medium supplemented or not with 100 ng/ml LPS for 24 h. Human samples were obtained and processed under the European Union and Spanish regulations.

Human U937 monocytes (ATCC 1593.2) were maintained in complete DMEM medium supplemented with 10 mM HEPES. For differentiation into macrophages, 1.5 × 106 cells per well were incubated in six-well plates with 10 μM PMA (Sigma-Aldrich) for 24 h. Once attached to the plate, cells were incubated during 48 h in fresh medium without PMA.

Cell transfections

For transient transfections, 2.5 × 105 Raw 264.7 cells per well were seeded in triplicate on 12-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day, according to the manufacturer’s recommendations, using Opti-MEM medium (Life Technologies) without supplements and 1.25 μg total EndoFree plasmid DNA per well. The reporter plasmids pNF-κB–luc, pRBP-J–luc, inducible NO synthase (iNOS)–luc, cyclooxygenase (COX)-2–luc, and pIFN-β–luc, used to detect NF-κB–, NOTCH-, iNOS-, COX-2–, and IFN-β–dependent transcription activities, respectively, have been previously described (32). pRLTK Renilla-expressing vector (Promega) was used as a control for transfection efficiency. pCMV6 (empty vector), pCMV6-Tspan33 (Tspan33 expression vector), Sh-control (empty vector), Sh-Tspan33 (eBioscience), pLNCX2 (empty vector), pLNCX2-Notch1IC (intracellular Notch1 expression vector), PCD2 (empty vector), and/or PCD2-Notch1 (full-length Notch1 expression vector) (33) were used together with the reporters. Cells were stimulated with 100 ng/ml LPS for 24 h after being transfected. Luciferase and Renilla activities were measured by using the Dual-Luciferase reporter assay system (Promega) in a Monolight 3096 (BD Biosciences) following the manufacturers’ recommendations.

For stable transfections, 3 × 106 Raw 264.7 cells were seeded on 60-mm plates 24 h before transfection with 6.5 μg of either Sh-control, Sh-Tspan33, pLNCX2, or pLNCX2-Notch1IC plasmids. Cells were selected with 300 μg/ml G418 (Sigma-Aldrich) for 2 wk.

Protein extracts and Western blot analysis

Cells were washed twice with ice-cold PBS, scraped off from the dishes, and collected by centrifugation. Cell pellets were resuspended in RIPA lysis buffer (25 mM HEPES [pH 7.5], 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.3 M NaCl, 20 mM β-glycerophosphate, 0.1% SDS, 0.5% deoxycholic acid) supplemented with 10 μl/ml each protease inhibitor and I and II phosphatase inhibitors (Sigma-Aldrich). Finally, pellets were homogenized 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 μg) were separated in 10% polyacrylamide gels, transferred to Nylon membranes (Hybond-C Extra; Amersham Biosciences) and processed according to the Ab suppliers’ recommendations. Proteins were detected with ECL (Amersham Pharmacia Biotech). β-tubulin expression was used as a loading control.

Anti-TSPAN33 Ab was purchased from Proteintech. Anti–COX-2 (no. 4842) was acquired from Cell Signaling Technology. Anti-ADAM10 and anti-ADAM17 were purchased from Millipore and anti–β-tubulin (T-3952) was from Sigma-Aldrich.

RNA and cDNA purification

Total RNA was obtained by using the RNeasy kit (Qiagen) with DNase (Promega) according to the manufacturers’ instructions and evaluated in a ND-1000 (NanoDrop) spectrophotometer. cDNA was synthesized from 1 μg of total RNA by using RevertAidH minus first strand cDNA synthesis (Fermentas) following the manufacturer’s recommendations.

Quantitative PCR

Gene expression analysis by quantitative PCR (qPCR) was performed in triplicate according to the Fast SYBR Green protocol with the StepOne real-time PCR detection system (Applied Biosystems). Specific oligonucleotides were designed with PrimerQuest computer program (Integrated DNA Technologies) and are indicated in Table I. The mRNA levels of mouse riboprotein P0 (34) or human GAPDH were used as internal controls.

Statistical analysis

The Student unpaired t test was used for statistical analyses between two groups. A p value <0.05 was considered significant.

Results

TSPAN33 expression is induced in activated macrophages in a NOTCH-dependent manner and increases Toll receptor–dependent NOTCH signaling

To investigate the mechanism by which NOTCH receptors contribute to macrophage activation, we searched for new genes differentially expressed in LPS- and IFN-γ–activated peritoneal macrophages lacking or not Notch1 and Notch2 expression. By using a set of Affymetrix microarrays, we identified Tspan33, among other genes, as a gene preferentially expressed in WT macrophages activated by LPS and IFN-γ. We confirmed the microarray data by quantitative PCR (Table1) and Western blot analyses. As shown in Fig. 1A, macrophages activated with LPS rapidly increased Tspan33 mRNA level, and this process was enhanced by IFN-γ. TSPAN33 protein was detected about 4 h after LPS and IFN-γ treatment (Fig. 1A, right panel). To evaluate whether the increased expression of Tspan33 was a general process during TLR macrophage activation, we evaluated Tspan33 mRNA and protein levels after treating macrophages with poly(I:C), a TLR3 agonist, and with LTA, a TLR2 agonist, in the presence or not of IFN-γ. We observed that, as shown in Fig. 1B, both TLR agonists increased Tspan33 expression, although to different extents, and this increase was enhanced by IFN-γ. Thus, it seems that increase in Tspan33 expression is a common feature in TLR-triggered macrophage activation. Finally, we also analyzed Tspan33 expression in human monocytes and in the human promyelocytic cell line U937 and observed that Tspan33 expression was also increased soon after treatment with LPS in both types of cells (Fig. 1C).

View this table:
Table I. Oligonucleotides used for PCR
FIGURE 1.
FIGURE 1.

Tspan33 is expressed in macrophages activated though TLRs and IFN-γ. (A) Left panel, qPCR analysis of Tspan33 expression in peritoneal murine macrophages activated with LPS (100 ng/ml), IFN-γ (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 ± SD of three independent experiments are shown. Right panel, Western blot analysis of TSPAN3333 expression in peritoneal macrophages activated with LPS and IFN-γ up to 24 h. β-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 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-γ for the indicated times. Data are referred to unstimulated macrophages set to 1. Means ± 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-γ up to 24 h. β-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 ± SD of three independent experiments are shown. *p < 0.05 with respect to control conditions.

We next evaluated whether Tspan33 induction by LPS was dependent on NOTCH signaling. As shown in Fig. 2A, Tspan33 expression levels were lower in macrophages lacking both NOTCH1 and NOTCH2 receptors than in WT macrophages. However, this difference was not observed in macrophages lacking either one of these NOTCH receptors (Supplemental Fig. 1). Additionally, Tspan33 mRNA and protein expression were increased in activated Raw 264.7 cells overexpressing the intracellular domain of NOTCH1 (NICD1; Fig. 2B). These results showed that induction of Tspan33 expression after TLR signaling is mediated, at least in part, by NOTCH signaling in activated macrophages.

FIGURE 2.
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-γ (20 U/ml) for different times. Means ± 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-γ for 24 h. The dotted black lines indicate where parts of the image were joined. β-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-γ. Means ± SD of three independent qPCR experiments are shown. β-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 ± 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 ± 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.

Genetic studies in Drosophila have shown that ablation of two TSPANC8 tetraspanin genes mimicked Notch deficiency, indicating that those genes were essential for NOTCH signaling (26). We evaluated the role of TSPAN33 in NOTCH signaling after TLR activation by cotransfecting Raw 264.7 cells with a Tspan33 expression vector or with a specific Tspan33 short hairpin RNA (shRNA) together with an RBP-J reporter plasmid. As shown in Fig. 2C, forced expression of Tspan33 increased RBP-J reporter activity in control or LPS-activated macrophages, whereas a specific shRNA partially diminished this effect in LPS-activated cells. To confirm these results, we generated stable Raw 264.7 cells expressing control or a specific Tspan33 shRNA. As shown in Fig. 2D, Tspan33 shRNA clearly diminished the expression of Tspan33 (upper panel). This decrease was associated with a lower expression of Hes1 (a downstream NOTCH target) after TLR4 activation (Fig. 2D, lower panel). These results confirm that TSPAN33 represents an important element in the control of NOTCH activity in TLR-activated macrophages. Moreover, as previously shown, NOTCH signaling also modulates Tspan33 expression in TLR-activated macrophages, thus creating a positive activation regulatory loop that can enhance NOTCH signaling in activated macrophages.

In mammals, different C8 tetraspanin family members, including TSPAN5, 10, 14, 15, 17, and 33, seem to be important in the control of NOTCH receptor signaling (26, 27). For that reason, we evaluated the expression of the C8 tetraspanin family members in control and TLR-activated macrophages. Only the expression of Tspan15 and Tspan33 was clearly increased in TLR-activated macrophages, whereas the mRNA levels of the other C8 tetraspanins remained constant or slightly decreased after stimulation (Supplemental Fig. 2). Thus, TSPAN33 seems to be the tetraspanin more intensely and rapidly induced in TLR-activated macrophages.

Maturation of ADAM10 increases in TLR-activated macrophages

TSPAN33 has been described as a chaperon protein that allows ADAM10 processing and translocation to the plasma membrane (27). ADAM10 and ADAM17 have been related with ligand-dependent and ligand-independent NOTCH processing (30). We have analyzed ADAM10 and ADAM17 expression in control and activated macrophages. As shown in Fig. 3A, Adam10 mRNA expression diminishes slightly in macrophages activated with LPS, whereas Adam17 mRNA levels increase 6–9 h after activation. As both ADAM10 and ADAM17 are synthesized as inactive precursors, we also analyzed ADAM10 and ADAM17 expression by Western blot to evaluate the presence of their active mature forms. As shown in Fig. 3B, in macrophages activated with LPS, with or without IFN-γ, an increase in the active processed form of ADAM10 (55 kDa) was observed, and this correlated with diminished levels of the unprocessed, inactive 85-kDa form. ADAM17 was not basally detected in macrophages, but its expression increased after activation (Fig. 3B, right panel), accordingly to the mRNA levels (Fig. 3A). Both unprocessed (120 kDa) and mature ADAM17 forms (85 kDa) were detected after macrophage activation. These results indicate that TLR signaling promotes ADAM10 and ADAM17 expression and/or activation in macrophages, favoring in this way NOTCH processing.

FIGURE 3.
FIGURE 3.

Analysis of of ADAM10 and ADAM17 expression in activated macrophages. (A) qPCR analysis of Adam10 and Adam17 mRNA expression in control and LPS-activated peritoneal macrophages. Data referred to nonstimulated macrophages are considered as 1. For each gene, the mean ± SD of three independent experiments are shown. *p < 0.05 with respect to control conditions. (B) Western blot analysis of ADAM10 (left panels) and ADAM17 expression (right panel) in peritoneal macrophages activated with LPS (100 ng/ml) in the presence or not of IFN-γ (20 U/ml). Inactive preforms (high molecular mass) and active forms (lower molecular mass) are indicated by arrows. The dotted black lines indicate where parts of the image were joined. β-Tubulin expression was 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. *p < 0.05 with respect to control conditions. (C) Western blot analysis of ADAM10 expression in Raw 264.7 cells stably transfected with a control or a Tspan33 expression vector, and activated with LPS for 12 h. Pro-ADAM10 and the mature active form are indicated by arrows. The relative amount of mature ADAM10 is shown in the graph on the right. For (B) and (C), three independent experiments were done. #p < 0.05 with respect to the same conditions in control cells.

We evaluated by Western blot the role of TSPAN33 in ADAM10 and ADAM17 processing in TLR-activated macrophages by comparing their expression in control and Raw 264.7 cells overexpressing TSPAN33. As reported for other cell types (27), TSPAN33 increased ADAM10 processing, as reflected by the accumulation of the 55-kDa form in control and activated cells (Fig. 3C). However, ADAM17 processing was not affected by TSPAN33 (data not shown). Our data indicate that TSPAN33 enhances the processing of ADAM10, which in turn could increase NOTCH1 processing and activation.

TSPAN33 increases NOTCH signaling in TLR-activated macrophages by favoring its processing

To further confirm that the effect of TSPAN33 on NOTCH activity was related to an increase in NOTCH receptor processing, we analyzed NOTCH activity using an RBP-J–LUC reporter in Raw 264.7 cells transfected with a Tspan33 expression vector in the presence of a full-length Notch1 expression vector or the constitutively active intracellular domain of NOTCH1 (NICD1). As shown in Fig. 4A, expression of NOTCH1 increased basal and LPS-induced RBP-J–LUC activity, and this effect was enhanced in the presence of TSPAN33. In contrast, NICD1 highly increased the RBP-J–dependent reporter activity, but expression of TSPAN33 did not modify this effect. Similarly, diminished expression of TSPAN33 with a specific shRNA in Raw 264.7 cells (shTspan33; Fig. 4B) correlated with a lower RBP-J–dependent reporter activity in cells transfected with full-length NOTCH1 and activated with LPS. However, no effect was observed in Raw 264.7 cells transfected with the active intracellular domain of NOTCH1 (NICD1). Thus, it seems that TSPAN33 increases NOTCH signaling in TLR- activated macrophages by increasing NOTCH processing at the plasma membrane.

FIGURE 4.
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 of NOTCH1 (NICD1) expression plasmids. One day after transfection, cells were stimulated with LPS for 24 h before analysis. The means ± 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 ± 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 ± 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.

Although several groups (26, 28), including us (Fig. 3B), have shown that TSPAN33 favors ADAM10 maturation, alternately, the modulation of γ-secretase activity by the interaction of tetraspanins with presenilins has also been described (35, 36). We have used specific shRNAs to diminish the expression of both ADAM10 and Presenilin1 (Psen1), the presenilin more highly expressed in activated macrophages (Supplemental Fig. 3A, 3B), to evaluate the effect of TSPAN33 on NOTCH activity when the levels of these proteins are low. As shown in Fig. 4C, ADAM10 and Psen1 shRNAs strongly decreased NOTCH activity in control and LPS-activated macrophages; however, increased expression of TSPAN33 augmented NOTCH signaling in both cases. In line with these results, lowering TSPAN33 in addition to ADAM10 or PSEN1 decreased NOTCH activity in both cases (Supplemental Fig. 3C). Interestingly, in LPS-activated macrophages, the potentiation of NOTCH signaling by TSPAN33 is slightly, but significantly, more elevated when ADAM10 levels are diminished than when PSEN1 levels are low. These results could argue for a role of TSPAN33 not only in ADAM10 processing, but also in the modulation of γ-secretase activity. Nevertheless, we cannot rule out that the effect of TSPAN33 falls only on ADAM10 processing, as the product of ADAM10 activity is the substrate of γ-secretase. More experiments using truncated mutant NOTCH receptors lacking the ADAM10 processing site are necessary to exactly delineate whether TSPAN33 modulates Presenilin1/γ-secretase activity in activated macrophages, as has been shown in Caenorhabditis elegans and human cells (36).

NOTCH1 has been suggested to be one of the most important NOTCH receptors in TLR-activated macrophages, but expression of NOTCH2 has also been reported in these cells (5, 8, 14). To analyze the involvement of TSPAN33 in TLR-mediated NOTCH activation, we modified the expression of TSPAN33 in macrophages with diminished expression of NOTCH1 and NOTCH2 receptors with previously evaluated shRNAs (Supplemental Fig. 3D) and measured RBP-J–LUC activity. As shown in Fig. 4D, this activity diminished in control and LPS-activated macrophages in the presence of specific shRNAs for Notch1 and Notch2, arguing for the important role of these receptors in NOTCH signaling in macrophages. Interestingly, increased expression of TSPAN33 compensated in part for the decrease of NOTCH signaling in macrophages with reduced Notch1 and Notch2 expression. These results suggest that the increase of TSPAN33 in the first hours of TLR activation could promote a general increase in NOTCH signaling.

TSPAN33 modulates proinflammatory gene expression in TLR4-activated macrophages

As NOTCH signaling has been associated with proinflammatory macrophage activation (7–10), and TSPAN33 increases NOTCH signaling in different cellular types (26, 37), including TLR-activated macrophages (Fig. 2C), we wondered whether TSPAN33 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-β, 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).

FIGURE 5.
FIGURE 5.

TSPAN33 increases the expression of proinflammatory genes in TLR-activated macrophages. (A and B) Analysis of Cox-2, iNOS, and IFN-β 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 ± SD of three independent 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 ± 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 ± 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.

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.

TSPAN33 increases NF-κB activation in TLR-activated macrophages by favoring NOTCH maturation and signaling

NF-κB 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 proinflammatory gene expression by TSPAN33 observed in macrophages (Fig. 5) could be mediated, at least in part, by NF-κB. We explored this hypothesis in Raw 264.7 cells cotransfected with an NF-κB–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-κB 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-κB activity when NOTCH1 was expressed, but not when the constitutively active NICD1 was expressed (Fig. 6A). Similarly, restricted levels of TSPAN33 by shRNAs diminished NF-κB activity in macrophages activated with LPS and in those overexpressing NOTCH1, but did not affect NF-κB–dependent signaling induced by NICD1 (Fig. 6B), arguing for a role of TSPAN33 in NOTCH1 processing. These results suggest that the effect of TSPAN33 in NF-κB activity is mediated mainly by NOTCH signaling.

FIGURE 6.
FIGURE 6.

TSPAN33 increases NF-κB activity in stimulated macrophages. (A) NF-κB transcriptional activity analysis in Raw 264.7 cells transiently cotransfected with a NF-κB luciferase reporter (NF-κB–LUC) and a Tspan33 expression vector (Tspan33) or the corresponding empty vector (control), together with a full-length Notch1 expression vector or a constitutively active intracellular domain of NOTCH1 expression vector (NICD1). One day after transfection, cells were stimulated with LPS (100 ng/ml) for 24 h before analysis. The means ± SD of six independent experiments are shown. *p < 0.05 with respect to control conditions (Tspan33 empty vector), #p < 0.05 with respect to control conditions (Notch empty vector, indicated as Vector). (B) Analysis of NF-κB transcriptional activity in Raw 264.7 cells similar to that described in (A), but using control and Tspan33 shRNAs. (C) Evaluation of the relevance of Notch1, Notch2, and Tspan33 in the NF-κB transcriptional activity of TLR-activated macrophages. Raw 264.7 cells were cotransfected with a NF-κB luciferase reporter gene and a Tspan33 expression vector or Tspan33-specific shRNA, together with control or a combination of Notch1 and Notch2 shRNAs. Normalized luciferase/Renilla values are referred to the levels of nonstimulated Raw 264.7 cells transfected with empty vectors, set as 100% (first bar on the left). The means ± SD of five independent experiments are shown. *p < 0.05 with respect to conditions with control vectors activated with LPS.

We also evaluated the effect of TSPAN33 in NF-κB activity of control and TLR-activated macrophages with diminished Notch1 and/or Notch2 expression. As shown in Fig. 6C, NF-κB–LUC activity decreased in control and LPS-activated macrophages in the presence of a combination of specific shRNAs for Notch1 and Notch2, arguing for the relevant role of these receptors in NF-κB signaling in macrophages, in agreement with previous data (10, 40). However, increased expression of TSPAN33 compensated, in part, for the decrease of NF-κB activity in macrophages with reduced NOTCH1 and NOTCH2 expression (Fig. 6C). These effects could be mediated by the elevated NOTCH activity observed previously (Fig. 4D). Thus, TSPAN33 could modulate NF-κB activity and proinflammatory gene expression by controlling NOTCH signaling.

Discussion

In the last years, multiple studies have uncovered an important role of NOTCH signaling in macrophage activation by Toll receptors (7–10). However, factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. In this work, we have characterized the role of TSPAN33, a tetraspanin implicated in ADAM10 maturation and/or γ-secretase activation, in NOTCH signaling during macrophage proinflammatory activation. Our data show that Tspan33 expression increases in response to TLR signaling, both in murine and human monocytes/macrophages, including responses through TLR4, TLR3, and TLR2, and that this process is enhanced by IFN-γ. Tspan33 induction by TLR/IFN-γ is largely dependent on NOTCH signaling, as its expression is noticeably diminished in macrophages lacking Notch1 and Notch2 expression. Moreover, TSPAN33 increases NOTCH signaling and TLR-induced proinflammatory gene expression, thus acting as a new control element in the development of inflammation.

The proper control of NOTCH signaling appears to be very important for a variety of cell processes, and several mechanisms are involved in it, both ligand-dependent and ligand-independent (41). Recently, it has been reported that furin, the protease implicated in NOTCH1 and ADAM10 maturation, is transcriptionally induced by NOTCH1 (42). In this way, different proteins implicated in NOTCH signaling are regulated by the pathway, thus creating a positive regulatory loop that leads to NOTCH signal amplification. Our data also suggest the existence of a novel positive regulatory loop in which NOTCH and TSPAN33 interact to enhance NOTCH signaling, leading to a proinflammatory macrophage phenotype upon TLR macrophage activation.

Induction of target genes after TLR stimulation requires the combination of downstream signaling events, including transcriptional activation via the NF-κB pathway (1). Our results show that increased expression of TSPAN33 leads to increased NF-κB activity, whereas diminished levels of TSPAN33 result in a lower activity of this transcription factor. These results correlated with proinflammatory gene expression. This is, to the best of our knowledge, the first time that the activity of a C8 tetraspanin family member is related to NF-κB activation and to the induction of proinflammatory genes. In this regard, the implication of ADAM10 in the induction of NF-κB and cytokine expression has been described in macrophages activated with meprin-β, a different metalloproteinase (43). Moreover, much evidence has related TSPAN33 and C8 tetraspanins with NOTCH signaling. Penumbra/Tspan33 was characterized in erythroid progenitors (44), but its role in NOTCH signaling was evidenced in Drosophila, C. elegans, and mammalian cells (26). These data are in support of the evidence presented in this study, suggesting that TSPAN33 influences NF-κB activity through the modulation of NOTCH signaling by the control of ADAM10 and/or γ-secretase activity.

Different authors have established that TSPAN33 and other C8 tetraspanins promote NOTCH activation at a pre–γ-secretase step by regulating ADAM10 trafficking and maturation (26). At least two ADAM proteins, ADAM10 and ADAM17, have been related with NOTCH1 receptor processing. Biochemical studies in flies have indicated that ADAM10 interacts with and cleaves NOTCH to activate signaling. Genetic manipulations resulting in the loss of ADAM10 protease activity lead to developmental defects similar to those described for deficiencies in NOTCH signaling (45). In vitro studies in mammalian cells identified ADAM17 as the relevant protease in NOTCH1 processing, excluding a role for ADAM10 in this process. However, genetic studies demonstrated that ADAM10 knockout mice display a classic Notch loss-of-function phenotype (45), whereas ADAM17 mutant mice do not (46). More recent studies have unveiled that NOTCH1 is a substrate for both proteases. ADAM10 is absolutely required for ligand-induced NOTCH1 signaling, whereas ADAM17 signaling is ligand–independent (30). Other studies have confirmed a similar role for ADAM10 in NOTCH2 and NOTCH3 processing (47); however, a lower sensibility for ADAM17 has been described for human NOTCH2 receptors (48). ADAM10-deficient macrophages display an increased anti-inflammatory phenotype, showing elevated IL-10, but reduced production of TNF-α, IL-12, and NO (49). This phenotype is similar to that observed in macrophages lacking CBF/RBP-J, a factor required for NOTCH signaling (8). These observations, together with our results showing an increased expression of proinflammatory genes in TSPAN33-overexpressing macrophages, suggest that the axis TSPAN33–ADAM10–NOTCH is important in the development of the macrophage proinflammatory phenotype.

Adam10 and Adam17 expression is increased in macrophages during proinflammatory activation. Adam17 mRNA expression increases after TLR signaling, and both the immature and mature forms of the protein are detected in macrophages. On the contrary, Adam10 mRNA expression does not change, but an increase in its protein maturation after TLR and INF-γ signaling is observed. 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 γ-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 γ-secretase activity. Conflicting results about the modulation of γ-secretase 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–γ-secretase step, in U2OS-N1 cells, whereas Dunn et al. (36) clearly showed that TSPAN33 facilitates the γ-secretase step. We cannot discard other possibilities, such as different tetraspanins modulating the different NOTCH receptors in different ways, making them more or less susceptible for ADAM10 and/or γ-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 γ-secretase activity.

Macrophages express different members of the C8 tetraspanin family, but TSPAN33 is the one most intensely induced after 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-κB 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, including survival, proliferation, adhesion, and migration (52). Most of the tetraspanins present in the immune system are also found in a variety of other tissues, but TSPAN33 and a few more, including CD37 and CD56, display a preferential hematopoietic expression (44, 53). TSPAN33 has been identified as a new marker of activated and malignant B cells, but its role on B cell activation has not been clearly outlined (54). However, the involvement of NOTCH signaling and ADAM10 in B cell activation, germinal center formation, and Ab production has been clearly established (55). Our data suggest that the role of TSPAN33 in these processes could be probably mediated by increased ADAM10 processing and/or increased γ-secretase activity and NOTCH activation, similarly to what we have observed in TLR-activated macrophages. In this line, a role for TSPAN5 and TSPAN10 in osteoclast differentiation, a process depending on NOTCH signaling, has been also described (37). TSPAN33 maps to a hotspot for deletions in acute myeloid leukemia and myelodysplastic syndrome (56), and it has also been described as a marker of malignant B cells (54). It is then tempting to speculate that the modulation of NOTCH signaling could be behind all those processes. In that sense, TSPAN33 seems to be implicated in the activation of different immune cells.

In summary, our results show that TSPAN33 is induced in macrophages by TLR signaling in a NOTCH-dependent manner, and it increases NOTCH signaling, which favors proinflammatory macrophage activation in response to TLR through NF-κB signaling. Thus, our data suggest that the modulation exerted by TSPAN33 on NOTCH signaling constitutes a new control point for the inflammatory response that could represent a target of therapeutic interest in the future. Indeed, the activity of γ-secretase has been successfully manipulated in culture cells by using mAbs against specific tetraspanins (35). In this sense, the hematopoietic preferential expression of TSPAN33 could be interesting to focalize the potential therapeutic effects on those cells, in contrast with the indiscriminate effects of chemical inhibitors.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by funds from Fondo Europeo de Desarrollo Regional Grants PI12/01546 and PI15/00991 (Instituto Carlos III, Ministerio de Economía y Competitividad, Spain).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ADAM
    a disintegrin and metalloproteinase
    COX
    cyclooxygenase
    Genot
    genotyped
    iNOS
    inducible NO synthase
    LTA
    lipoteichoic acid
    NICD
    NOTCH intracellular domain
    poly(I:C)
    polyinosinic-polycytidylic acid
    qPCR
    quantitative PCR
    shRNA
    short hairpin RNA
    WT
    wild-type.

  • Received March 10, 2016.
  • Accepted August 8, 2016.

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