JBC Papers in Press. Published on April 4, 2018 as Manuscript RA118.002175
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.002175

Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune
semaphorin SEMA4D
Kou Motani1* and Hidetaka Kosako1
From the 1Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 318-15 Kuramoto-cho, Tokushima 770-8503, Japan*

To whom correspondence should be addressed: Kou Motani: Division of Cell Signaling, Fujii Memorial

Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan;
motani@tokushima-u.ac.jp; Tel. 81-88-634-6411; Fax. 81-88-634-6405

Keywords: ADAM, cell surface protein, inflammation, proteomics, post-translational modification, STING,
SEMA4D

unexplored. Here, we analyzed the RAW264.7
macrophage secretome to comprehensively identify

ABSTRACT

proinflammatory factors released into the extracellular

Stimulator of interferon genes (STING) is anmembranemedium upon STING activation. In total, we identified

protein that mediates cytosolic pathogen DNA–1,299 proteins in macrophage culture supernatants, of

induced innate immunity and inflammatory responses

which 23 were significantly increased after STING

in host defenses. STING is activated by cyclic di-activation. These proteins included IRF3-dependent

nucleotides and is then translocated to the Golgi

cytokines, as well as previously unknown targets ofapparatus, an event that triggers STING assembly with

STING,

the downstream enzyme TANK-binding kinase 1

SEMA4D/CD100, which possesses proinflammatory(TBK1). This assembly leads to the phosphorylation ofcytokine-like activities. Unlike for canonical cytokines,

the transcription factor interferon regulatory factor 3

the expression of the SEMA4D gene was not up-(IRF3), which, in turn, induces expression of type-I

regulated.

interferon (IFN) and chemokine genes. STING also

membrane-bound SEMA4D was cleaved into a soluble

mediates inflammatory responses independently ofform, suggesting the presence of a post-translational

IRF3, but these molecular pathways are largely

shedding machinery. Importantly, the SEMA4D

endoplasmic

reticulum(ER)-resident

1

such

as

Instead,

the

upon

immuneSTING

semaphorin

activation,

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Running title: SEMA4D shedding by STING

shedding was blocked by TMI-1, an inhibitor of the(cGAMP) which is produced as an intracellular second

sheddase

17

messenger when cGAMP synthase (cGAS) recognizes(ADAM17), but not by the TBK1 inhibitor BX795.

cytosolic DNA (12-15). Upon binding to cGAMP,

These results suggest that STING activates ADAM17

STING causes its dimerization and translocation fromandsolublethe ER to the Golgi apparatus (16-18). After relocation,

proinflammatory SEMA4D independently of the

STING recruits a serine/threonine kinase TBK1,

TBK1/IRF3-mediated transcriptional pathway.

leading to the phosphorylation of IRF3 and

ADAM

that

his

metallopeptidase

activation

domainproduces

upregulation of type-I IFN and IFN-stimulated genes
Introduction(ISG) including IFN- and CXCL10 (19,20).
IFN- production via STING-TBK1-IRF3 axis

various pathogens triggers innate immune andis crucial for development of anemia in DNaseII

inflammatory responses, which are essential for hostdeficient mice (21). However, another phenotype

defense (1). In contrast, endogenous self-DNA doespolyarthritis is dependent of STING but not IRF3 (21).

not induce an immune response normally, because it isSimilarly, systemic inflammation characterized by

localized to the nucleus or mitochondria. However,interstitial

inadequate accumulation of self-DNA in cytoplasm

hypercytokinemia

can also activate innate immunity (2). During animal

mutations of STING gene has been shown to be IRF3-

development,

independent using a mouse model (22). These

tissueturnoveranddefinitivelung

disease,insuggestskin

patientsthatlesionswithadditional

andactivating

erythropoiesis, apoptotic cells and pyrenocytes areobservations

signaling

engulfed by macrophages (3,4) and their DNA ispathway(s) is induced downstream of STING.

degraded by the lysosomal enzyme DNaseII. If thisImportantly, lack of proinflammatory cytokine genes

degradation machinery is impaired in macrophages,

such as TNF-, IL-6 or IL-1 inhibited arthritis of theaccumulated apoptotic or pyrenocyte DNA leaks from

DNaseII-null mice (8). Although STING activatesthe lysosome into the cytoplasm and triggers a strong

another transcriptional factor NF-κB which induces

inflammatory response, leading to severe anemia andthe proinflammatory cytokine gene expression, thispolyarthritis (5-9). Of note, loss-of-function mutations

activation is rather weak (11,23,24). Thus, it is unclear

in DNaseII have been recently identified in patients

whether NF-κB-mediated gene activation is sufficientwith autoinflammation featuring severe neonatal

for STING-mediated inflammatory response.

(10).

Here, we performed unbiased comprehensivebysecretome analysis and identified that activation ofcytoplasmic DNA is an important issue to resolve

STING promoted the cleavage of plasma membrane

inflammatory diseases.

protein SEMA4D to release the inflammatory soluble

anemiaandUnderstanding

deforming
immunearthropathy

response

triggered

STING is an ER-resident transmembrane

form without its gene induction. We also found that

protein that is essential for DNA-induced innate

TNF- protein was dramatically increased in culture

immune signaling (11). STING is activated by binding

supernatant of STING-activated cells, although its

to cyclic di-nucleotides such as cyclic GMP-AMP

mRNA expression was moderately induced in this2

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The detection of cytosolic DNAs derived fromcondition. The production of soluble SEMA4D as welltargets of STING including SEMA4D, LRPAP1 andas TNF- were completely blocked by an inhibitor of

Fam3C. To confirm these results, we performed

ADAM17 sheddase. On the other hand, the treatment

targeted LC-MS/MS analysis using parallel reactionof cycloheximide (CHX) or TBK1 inhibitor did notmonitoring (PRM) which enables highly specific andabrogate the shedding of SEMA4D. These results

accurate protein quantification of multiple proteinssuggest that STING activates ADAM17-mediatedsimultaneously (25). As expected, the protein levels of

post-translational

SEMA4D,

shedding

machinery.

BecauseLRPAP1

and

Fam3C

in

culturesupernatants were increased about 3 to 6 fold bycytokine production, the STING-ADAM17-SEMA4DDMXAA (Fig. 1C and Table S2). DMXAA did notaxis may be required for optimal inflammatory

induce release of other class 4 semaphorin member

response.

SEMA4B or chemokine CCL9 (Fig. 1C and Table S2),
confirming that specific set of proteins were released

Results

by stimulation with the STING agonist. Among

Identification of SEMA4D as a novel protein released

identified 23 proteins, SEMA4D has been wellby STING agonist

characterized as a proinflammatory protein (26).

To determine the inflammatory factors released

Notably, the number of peptide spectrum matchesfrom STING-activated cells, we prepared culture(PSMs) of SEMA4D was relatively high among themsupernatants from Raw264.7 murine macrophage cells(Table 1), suggesting that SEMA4D is an abundant

treated with or without DMXAA, a cell-permeable

protein in culture supernatant of the STING-activatedsynthetic STING agonist. Like a natural ligand,cells. Thus, the activation of STING causes the release

DMXAA directly binds to murine STING and causesof the inflammatory factor SEMA4D in addition toits conformational activation (15). The DMXAA-

known cytokines.

treated cells were cultured for 4 h under the serum-free
condition to prevent contamination of serum-derived

STING induces SEMA4D shedding independent ofabundant proteins, but no significant cell death was

gene expression

observed at least during the 4-h incubation (data not

To further confirm the STING-mediatedshown). After collection of proteins from therelease of SEMA4D, we performed Western blotting

conditioned medium, we performed an unbiased

analysis of culture supernatant using antibody

proteomic analysis by shotgun LC-MS/MS (Fig. 1A).

recognizing extracellular domain of SEMA4D. As

In total, we identified 1,299 proteins, of which 23

shown in Fig. 2A, we could detect SEMA4D release 2

proteins are significantly increased (P < 0.01, fold-

and 4 h after DMXAA stimulation. To determine

increase > 4) by DMXAA stimulation (Fig. 1B, Table

whether STING is essential for DMXAA-induced

1 and Table S1). These successfully included IRF3-

release of SEMA4D, Raw264.7 cells lacking STING

dependent cytokines such as IFN- and CXCL10, andwere generated using the CRISPR/Cas9 system. We

the most increased protein was TNF- (about 150-foldconfirmed again that SEMA4D was released along

increase).

with TBK1 activation in DMXAA-stimulated wild-

We also identified novel downstream

3

Downloaded from http://www.jbc.org/ at TOKUSHIMA UNIV (KIN/ACC) on April 8, 2018soluble SEMA4D is known to activate inflammatory

type (WT) cells (Fig. 2B). In contrast, neither

concomitant with an increase of cleaved form of

SEMA4D release nor TBK1 activation was observed

SEMA4D in the culture supernatants. Although an

in STING-deficient cells and this phenotype wasintracellular cleaved form was observed in the cell

rescued by ectopic expression of STING (Fig. 2B).

lysates, its amount did not change after DMXAA

Because STING activates transcription of type I

stimulation. These results indicate that STING

interferon and inflammatory cytokine genes, we

activates

checked the induction of the SEMA4D gene after

SEMA4D on cell surface to release the soluble

DMXAA stimulation. Real-time PCR analysis showed

ectodomain.

thepost-translational

processingofthat DMXAA did not induce the expression of
Disease-associatedexamined the expression levels of canonical cytokine

SEMA4D shedding

mutantof

STING

induces

genes and the IFN- and CXCL10 mRNAs wereSeveral autosomal dominant mutations ofdramatically increased about 500 to 1,500 fold after

STING have been reported in patients withDMXAA stimulation (Fig. 3A). The expression of

autoinflammatory diseases (27,28). These mutations

TNF- mRNA was also up-regulated but only 8-fold

encode an active form of STING protein that triggers

increase although the protein level in the cell medium

IRF3-dependent gene induction at steady state.

was increased about 150 fold, which is comparable to

However, a recent study uncovered that IRF3 is

IFN- and CXCL10 (Fig. 1C and Fig. 3A). These

dispensable for development of systemic inflammation

observations prompted us to speculate that STING

caused by the disease-associated mutant of STING in

activates not only transcription but also post-

mice (22). Therefore, it is possible that the shedding oftranslational machinery to release SEMA4D and TNF-

SEMA4D might be involved in inflammation caused

. To confirm this possibility, the cells were treatedby this STING mutant. To test whether the STING

with CHX to inhibit de novo protein synthesis, andmutant can induce SEMA4D shedding, we establishedthenexpected,stable cell lines that lack endogenous STING but

production of CXCL10 was blocked by treatment of

inducibly expresses WT or the corresponding disease-

CHX in a dose-dependent manner, whereas SEMA4D

associated mutant of mouse STING (V146L). After

was still released even in the presence of high-dose

treatment with doxycycline, STING V146L mutant but

CHX (Fig. 3B). Because SEMA4D is a plasma

not STING WT was accumulated in the perinuclear

membrane protein and its soluble ectodomain isregion, where most of them were co-localized with agenerated by proteolytic cleavage (26), we tested

cis-Golgi matrix protein GM130 (Fig. 4A). We also

whether STING agonist promotes shedding ofobserved phosphorylation of TBK1 in cells expressing

SEMA4D. The total cell lysates and culture

STING V146L but not STING WT (Fig. 4B),

supernatants were prepared at the same time and theconfirming the constitutive activation of this STING

cleavage was analyzed by Western blotting. As shown

mutant. Then, we collected the culture supernatantsin Fig. 3C, the full length membrane form of SEMA4D

and found that release of soluble SEMA4D was

was reduced by DMXAA treatment in the cell lysates,

provoked by the expression of the STING mutant.

stimulated

with

DMXAA.

As

4

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SEMA4D mRNA (Fig. 3A). As a control, weThus, SEMA4D shedding is an additional phenotype

which commonly undergo proteolytic cleavage by

induced by the disease-associated mutant of STING.

ADAM17 (30-32). Therefore, we tested the possibility
that ADAM17 is required for STING-mediated

ADAM17 but not TBK1 is required for SEMA4D

SEMA4D release. Inhibition of ADAM17 sheddase

shedding

activity by a pharmacological inhibitor TMI-1
abolished DMXAA-induced release of TNF- andconstitutively localized to the Golgi apparatus was able

SEMA4D but not CXCL10 (Fig. 5A). We also

to induce SEMA4D shedding, STING trafficking may

confirmed that TMI-1 inhibited the SEMA4D

regulate its shedding machinery. As shown in Fig. 5A,

shedding induced by the active mutant of STING

treatment of a pharmacological inhibitor brefeldin A

without affecting TBK1 phosphorylation (Fig. 5B).

(BFA) which inhibits anterograde transport from the

These results suggest that STING activates two

ER to Golgi apparatus completely blocked DMXAA-

independent downstream signaling pathways after

induced release of CXCL10, TNF- and SEMA4D,

trafficking to the Golgi; one is TBK1-mediated genesuggesting that Golgi apparatus acts as a platform forinduction and another is ADAM17-mediated cleavage

the induction of all downstream signaling events ofof plasma membrane proteins (Fig. 5C).

STING. It should be noted that BFA also seemed toblock SEMA4D trafficking to the plasma membrane,

Discussion

as the smear bands of SEMA4D faster migrated in

In this study, we identified novel STING-

BFA-treated cells, which may imply incomplete

dependent signaling in which a cell surface protein

glycosylation or degradation in ER. After translocation

SEMA4D is cleaved to produce its soluble ectodomain.

to the Golgi apparatus, STING associates with TBK1,

The increased concentration of soluble SEMA4D intriggering CXCL10 and TNF- gene induction by

serum or plasma is associated with numerous

phosphorylation of IRF3 and NF-B, respectively (29).

inflammatory conditions in humans and mice (26), andSurprisingly, DMXAA-induced SEMA4D shedding

the soluble SEMA4D has been described as awas not blocked by BX795, a specific TBK1 inhibitor

stimulator of both innate and acquired immunity (26).

although it inhibited the induction of CXCL10 (Fig.

STING-dependent polyarthritis in DNaseII-null mice

5A), indicating that SEMA4D shedding is TBK1-

is triggered by innate immune response producing

independent.

NF-B-dependent

proinflammatory cytokines including TNF-, IL-6 andtranscription by BX795 partially not completely

IL-1 but not acquired immunity (8). In this case,inhibited the release of TNF-, further supporting thesoluble SEMA4D may contribute to such cytokine

idea that STING promotes its production through a

production rather than acquired immunity. Indeed,transcription-independent pathway. In addition towhen CD14+ monocytes from rheumatoid arthritis

SEMA4D, our proteomic analysis revealed that TNF-

(RA) patients were treated with recombinant soluble

, SDC4, TGFBR1 and SORL1 were also released into

SEMA4D protein, the cells produced high levels of

extracellular space by STING activation (Table 1 andTNF- and IL-6 through a poorly understood

Table S1). All of them are plasma membrane proteinsmechanism, although its receptor CD72 has been

Blockingof5

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Because the active mutant STING that wasimplicated (33). Furthermore, administration of anti-

mediated by NF-B-dependent transcription and

SEMA4D antibodies to mice with collagen-induced

caspase-1-dependent proteolytic processing.
Howarthritis significantly reduced arthritis score and serum

does

STING

activate

ADAM17-level of TNF- and IL-6 (33). Thus, the soluble

dependent shedding machinery? Since the STING

SEMA4D released from STING-activated cells may

mutant that constitutively localized to the Golgi

induce production of inflammatory cytokines in

induced ADAM17-mediated shedding of SEMA4D

adjacent cells or themselves in a paracrine or autocrine(Fig. 4 and Fig. 5B), STING trafficking from the ER tomanner.

the Golgi may causes activation of ADAM17. STING
activates TBK1 at the Golgi (20), however, inhibitionis cleaved by several proteinases including ADAM17,

of TBK1 activity by BX795 did not suppress theMT1-MMP and ATAMTS4 (26). Because the potent

release of SEMA4D, suggesting involvement of

ADAM17 inhibitor TMI-1 used in this study also

another downstream signaling. Although BX795

inhibits matrix metalloproteinases (MMPs) (34), we

partially blocked the release of TNF-, this

could not exclude the possibility that certain MMPs are

discrepancy may be explained by suppression of its

involved in the STING-mediated SEMA4D shedding.

transcriptional activation. The enzymatic activity ofHowever, our secretome analysis showed that other

ADAM17 is tightly regulated by iRhom2-mediated its

ADAM17 substrates such as TNF-, SDC4, TGFBR1

maturation, localization and conformation (35-39).

and SORL1 were also released into culture supernatant

Upon binding to iRhom2, pro-ADAM17 is exported

by STING activation, suggesting that STING activatesfrom the ER to the Golgi, where pro-protein

ADAM17 to promote shedding of these plasma

convertases such as Furin cleave auto-inhibitory pro-membrane proteins in a cell-intrinsic manner.

domain of ADAM17 to be mature form. The

Although STING is known to activate NF-B tointeraction with iRhom2 is also required for post-Golgi

induce TNF- gene expression, its activation is very

trafficking of mature ADAM17 to the plasma

weak compared to IRF3-mediated IFN- or CXCL10

membrane, and regulates its conformation andgene activation (11,23,24). This is supported by ourproteolytic activity at the cell surface. Interestingly, agene expression experiment shown in Fig. 2A.

recent report shows that iRhom2 directly interacts with

However, the protein level of TNF- in cell medium

STING and mediates its trafficking from the ER to thewas dramatically increased by DMXAA stimulation

Golgi (40). In other words, activation of STING

compared to the mRNA level (Fig. 1C). From these

induces enrichment of iRhom2 in the Golgi apparatus

observations, we propose the model that STING

that may promote ER-Golgi or post-Golgi trafficking

activates both transcriptional and post-translationalofmachineries for efficient release of TNF-. This two-

regulation might be involved. The activity ofstep mechanism comprising NF-B-mediated TNF-ADAM17 has been proposed to be regulated bymRNA induction followed by ADAM17-mediatedphosphorylation of ADAM17 and iRhom2 by either

ectodomain shedding is analogous to the case of

ERK or p38 (38, 39, 41-43). Importantly, STING

another inflammatory cytokine IL-1 whose release isagonists induce MAPK activation independent of

6

ADAM17.

Alternatively,

MAPK-mediatedDownloaded from http://www.jbc.org/ at TOKUSHIMA UNIV (KIN/ACC) on April 8, 2018

It has been reported that membrane SEMA4DTBK1 whose biological significance has remained

from R&D systems (Minneapolis, MN). Anti--

unclear (29).

Tubulin mAb (DM1A) and anti-Flag polyAb werefrom Sigma-Aldrich (St. Louis, MO). Anti-phospho-

ADAM17, our comprehensive proteomic approach

TBK1 (Ser172) mAb (D52C2) and anti-TNF- mAb

identified Fam3C and LRPAP1 as an intracellular(D2D4) were from Cell Signalling (Danvers MA).

protein secreted from STING-activated cells. Fam3C

Anti-STING polyAb was from Proteintech (Chicago,

was originally identified as the interleukin-like

IL). Anti-GM130 mAb (35/GM130) was from BD

epithelial-to-mesenchymal transition (EMT) inducer

Biosciences (Franklin Lakes, NJ). DMXAA was from(ILEI) that is essential for EMT and tumor metastasis

Ark Pharm, Inc (Arlington Heights, IL). Doxycycline(44). Inflammatory injury triggers EMT to promote

was from Wako Pure Chemical Industries, Ltd (Osaka,tissue-remodeling and the sustained EMT causes

Japan). BFA was from Cayman Chemical (Ann Arbor,

pathologic fibrosis (45). It would be interesting

MI). BX795, TMI-1 and CHX were from Sigma-

whether Fam3C is involved in tissue repair or fibrosis

Aldrich.

during STING-dependent inflammation. LRPAP1,
also known as RAP, interacts with members of the

Cell lines

LDL receptor family such as LRP1 within the ER to

Raw264.7 cells were cultured in DMEM

assist the folding of such receptor proteins (46).

containing 10% FCS. The CRISPR/Cas9-mediatedBecause of its high affinity binding, recombinantgene editing was used to establish Raw264.7 cells

LRPAP1 protein is used for LRP1 ligand and induces

lacking STING. Px330 vector expressing Cas9 andproinflammatory cytokine expression (47). However,sgRNA for mouse STING (16) was transfected into

whether ER chaperone protein LRPAP1 is secreted in

Raw264.7 cells by electroporation using the NEPA21

physiological condition was unclear. This is the firstsystem (135V, 10ms; Nepagene, Chiba, Japan). Afterevidence that LRPAP1 is secreted in response to

24-h incubation at 37 ºC, the cells were subjected toinflammatory stimuli.

limiting dilution, and the mutated clones were

So far, most studies have focused on IRF3-

identified by Western blotting. For rescue experiment,

dependent transcriptional program in the STING

the C-terminal Flag-tagged mouse STING (STING-

pathway, thus no one could find release of SEMA4D,

Flag) was stably expressed in STING deficient cells

Fam3C and LRPAP1, whose gene expression levels

using the PiggyBac transposon system. To generate theare not altered during STING activation (Fig. 3A).

inducible gene expression cells, Tet-on expression

This study provides new insight into the STING-

vector carrying STING-Flag WT or V146L mutant

associated inflammatory response and diseases.

was introduced into the STING deficient cells. The
V146L

mutant

STING

with

primers

5’-

recombinant

Reagents

CACCGGCCAGTGTGGTGGAAGATGCCATACTCmAb(#547406)

CAACCT-3’

and

using

constructed

Experimental procedures
Anti-SEMA4D

PCR

was

(BstXI-Start)

and

of

5’-

CAGCGGAAGTCTCTGCACTCTGTGAAGAAAA

biotinylated anti-CXCL10 polyAb were purchased

7

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In addition to membrane proteins cleaved by

5’-(Thermo Fisher Scientific). The peptides were

AACTTCTTTTCTTCACAGAGTGCAGAGACTTC

separated on a 75 μm inner diameter × 120 mm C18

CGCTG-3’

5’-reversed-phase column (Nikkyo Technos, Tokyo,

GCTAACCACTGTGCTGGCTACTTATCGTCGTC

Japan) with a linear gradient from 4–28% acetonitrile

ATCCTTGTAATCGATGAGGTCAGTGCGGAGTG(ACN) for min 0–150 followed by increase to 80%-3’ (Flag-Stop-BstXI).

ACN during min 150–170. The mass spectrometer was

GAAGTT-3’(V146L),(V146L)andandoperated in a data-dependent acquisition mode with a
top 10 MS/MS method. Raw data were directly

Sample preparation for secretome analysis

analyzed against the SwissProt database restricted to6(2.5×10 cells/well) and cultured overnight. After

M. musculus using Proteome Discoverer version 2.2

washing with PBS twice, the cells were cultured with

(Thermo Fisher Scientific) for identification and label-

FCS-free DMEM medium containing DMSO or 100

free precursor ion quantification. Normalization was

μg/ml DMXAA for 4 h. The conditioned medium was

performed such that the total sum of abundance values

collected and centrifuged at 400 g for 10 min at 4 ºC.

for each sample over all peptides is the same. The mass

The supernatants were concentrated to 100 μl using

spectrometry proteomics data have been deposited toAmicon Ultra 3K filter (Millipore, Burlington, MA)

the ProteomeXchange Consortium via the jPOSTand proteins were purified by methanol/chloroform

partner repository (https://repository.jpostdb.org) with

precipitation. The protein pellets were solubilized in

the dataset identifier PXD009169. The data were20 μl of 8 M urea, followed by reduction in 5 mM DTT

analyzed by a two-tailed Student’s t-test and theat 37 ºC for 30 min and alkylation in 27.5 mM IAA at

resulting p-values were adjusted using the Benjamini-room temperature for 30 min in the dark. After

Hochberg method for controlling the false discovery

reducing the urea concentration to 1 M with 50 mM

rate (FDR). A volcano plot was used for showing the

Tris-HCl (pH8.0), the proteins were digested with 50

fold changes and q-values (FDR corrected p-values) of

ng Trypsin/Lys-C Mix, Mass Spec Grade (Promega,each protein. To select significant proteins, we set

Madison, WI) at 37 ºC overnight. The peptides werefold-change threshold of 4 and q-value threshold ofdesalted using GL-Tip SDB (GL Sciences, Tokyo,

0.05.

Japan) according to the manufacturer’s protocol andthe eluates were evaporated in a Speed Vac

PRM analysis

Concentrator (Thermo Fisher Scientific, Waltham,

To quantify SEMA4D, LRPAP1, Fam3C,

MA). The residues were dissolved in 0.1% TFA.

SEMA4B, CCL9, IFN-, CXCL10, and TNF-, atleast three peptides per protein were measured by PRM,
an MS/MS-based targeted quantification method using

Data-dependent LC-MS/MS analysis
LC-MS/MS analysis of the resultant peptides

high-resolution mass spectrometry. Time alignment(200 ng each) was performed on an EASY-nLC 1200

and relative quantification of the transitions were

UHPLC connected to a Q Exactive Plus mass

performed with PinPoint version 1.4 (Thermo Fisher

spectrometer through a nanoelectrospray ion source

Scientific). The mass spectrometry proteomics data

8

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Raw264.7 cells were seeded in 6-well plateshave been deposited to the ProteomeXchange
Consortium via the jPOST partner repository with theReal-time PCR
Total RNA was isolated using RNAiso plus

dataset identifier PXD009170.

(TAKARA,Shiga,

Japan)

accordingtothemanufacturer’s protocol. The RNA was reverse-

SDS-PAGE and Western blotting

transcribed using the ReverTra Ace qPCR RT Master

FCS/DMEM when the cells were treated withMix (TOYOBO, Osaka, Japan), and real-time PCRDMXAA in the presence or absence of inhibitors, andwas performed with the Light-Cycler 96 (Rochethe conditioned media were concentrated by Amicon

Diagnostics, Basel, Switzerland) using the FastStart

Ultra 3K filter or Vivaspin 10K filter (Sartorius,

Essential DNA Green Master Mix (Roche Diagnostics).

Goettingen, Germany). At the same condition, the cells

The primers used for real-time PCR were as follows:

were lysed on ice for 10 min in lysis buffer (20 mM

IFN-, 5′-CCACCACAGCCCTCTCCATCAACTAT-

Tris-HCl [pH7.5], 150 mM NaCl, 1 mM EDTA, 1%

3′ and 5′-CAAGTGGAGAGCAGTTGAGGACATC-

NP-40, 10% glycerol, a protease inhibitor cocktail

3′;[Nacalai Tesque, Kyoto, Japan] and a phosphatase

CCATCAGCACCATGAACCCAAGT-3′

inhibitor cocktail [Nacalai Tesque]) and centrifuged at

CACTCCAGTTAAGGAGCCCTTTTAGACC-3′;

20,000 g for 15 min at 4 ºC. The concentrated media

SEMA4D, 5’-TTGGGCAGTGAACCCATCATC-3’

or the cell lysates were mixed with 5×SDS sample

and 5’-GGATCACGTCAGCAAAGACGA-3’; TNF-

buffer (200 mM Tris-HCl buffer [pH 6.8], 10% SDS,, 5’-CACAGAAAGCATGATCCGCGACGT-3’ and

25% glycerol, and 0.05% bromophenol blue) with 5%5’-CGGCAGAGAGGAGGTTGACTTTCT-3’;

-mercaptoethanol, and heated at 95°C for 5 min. The

actin, 5′-TGTGATGGTGGGAATGGGTCAG-3′ andsamples were separated by electrophoresis on a 6%5′-TTTGATGTCACGCACGATTTCC-3′.

CXCL10,

5′and

5′-

-

(for SEMA4D), 10% (for -Tubulin, phospho-TBK1
and STING) or 16% (for CXCL10 and TNF-)Immunostaining
The STING-null Raw264.7 cells expressing

polyacrylamide gel, transferred onto a PVDF
membrane (Millipore). After blocking, the membrane

Tet-on-STING-Flag

was incubated with primary antibodies in 5% skim

V146L were seeded on coverslips in a 12-well plate.

milk or in Signal Enhancer HIKARI (Nacalai Tesque)After treatment with doxycycline, the cells were fixed

overnight at 4°C, followed by incubation with HRP-

with 3.7% formaldehyde/PBS for 15 min at 37°C andconjugated secondary antibodies. Protein bands in thepermeabilized with 0.2% Triton X-100/PBS for 10 min

membrane was detected using ImageQuant LAS 4000

at room temperature. The coverslips were incubatedmini (GEHealthcare, Little Chalfont, United

with primary antibodies in 2% goat serum/PBS for 1 h

Kingdom) after incubation of the membrane withat 37°C. After washing with PBS three times, theClarity Western ECL Substrate (BIO-RAD, Hercules,

coverslips were incubated with Alexa Fluor-488 goatCA) or Immunostar LD (Wako Pure Chemicalanti mouse IgG and Alexa Fluor-555 goat anti rabbit

Industries, Ltd).

IgG for 1 h at room temperature in the dark. After

9

WT

or

Tet-on-STING-Flag

Downloaded from http://www.jbc.org/ at TOKUSHIMA UNIV (KIN/ACC) on April 8, 2018

The culture medium was replaced with 0.1%

washing with PBS three times and subsequent rinse

immunofluorescence images were obtained by awith distilled water, the coverslips were mounted on

confocalthe glass slides with ProLong Gold Antifade Mountant(Olympus, Tokyo, Japan).

with

DAPI

(ThermoFisherScientific).

laser

scanning

microscope

FV1200The

Downloaded from http://www.jbc.org/ at TOKUSHIMA UNIV (KIN/ACC) on April 8, 201810

Acknowledgments: We thank M. Kajimoto and M. Kawano for technical assistance. We also thank
M. Iwata for secretarial assistance.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents ofthis article.

MS/MS analysis. K.M. wrote the paper with H.K.

11

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Author contributions: K.M. designed and performed most experiments. H.K. performed LC-

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FOOTNOTESThis work was supported by the JSPS KAKENHI (Grant Numbers 15K19024 and 17K08661) andthe Takeda Science Foundation (to K.M.), and by the JSPS KAKENHI (Grand Number 17K08635)
and the Japan Foundation for Applied Enzymology (to H.K.).

The abbreviations used are: STING, stimulator of interferon genes; ER, endoplasmic reticulum;

metalloproteinase 17; cGAMP, cyclic GMP-AMP; cGAS, cGAMP synthase; IFN, interferon; ISG,
IFN-stimulated genes; CHX, cycloheximide; DMXAA, 5, 6-dimethylxanthenone-4-acetic acid; PSM,
peptide spectrum match; BFA, brefeldin A

18

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TBK1, TANK-binding kinase 1; SEMA4D, semaphorin-4D; ADAM17, a disintegrin andActivation of stimulator of interferon genes (STING) induces ADAM17-mediatedshedding of the immune semaphorin SEMA4D
Kou Motani and Hidetaka Kosako
J. Biol. Chem. published online April 4, 2018

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