YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.
Abstract
Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.
Full text
In e na ional Jou nal o
Molecula Sciences
A icle
YB-1 In e e es wi h TNFα–TNFR Binding and
Modula es P og anulin-Media ed Inhibi ion o
TNFαSignaling
Ch is ophe L. Hessman 1,†, Josephine Hildeb and 1,†, Ane i Shah 1, Sabine B and 1,
An onia Bock 1, Bjö n C. F ye 2,‡, U e Ra e sede 2, Robe Ge e s 3,
Monika C. B unne -Weinzie l 4, Be end Ise mann 5, Pe e R. Me ens 1,*
and Jona han A. Lindquis 1,*
1Clinic o Neph ology and Hype ension, Diabe es and Endoc inology, O o- on-Gue icke Uni e si y,
39120 Magdebu g, Ge many; [email p o ec ed] (C.L.H.); josephine.hildeb and @ eene .de (J.H.);
[email p o ec ed] (A.S.); [email p o ec ed] (S.B.); [email p o ec ed] (A.B.)
2
Depa men o Neph ology and Clinical Immunology, RWTH Aachen Uni e si y,
52074 Aachen, Ge many
;
bjoe n.ch is ian. ye@uniklinik- eibu g.de (B.C.F.); u a [email p o ec ed] (U.R.)
3Genome Analy ics Resea ch G oup, Helmhol z Cen e o In ec ion Resea ch,
38124 B aunschweig, Ge many; obe .ge [email p o ec ed]
4Depa men o Expe imen al Pedia ics, O o- on-Gue icke Uni e si y, 39120 Magdebu g, Ge many;
monika.b unne [email p o ec ed]
5Ins i u e o Labo a o y Medicine, Clinical Chemis y and Molecula Diagnos ics,
Uni e si y Hospi al Leipzig, 04103 Leipzig, Ge many; [email p o ec ed]
*Co espondence: Pe e [email p o ec ed] (P.R.M.); [email p o ec ed] (J.A.L.);
Tel.: +49-391-6713236 (P.R.M.); +49-391-6724703 (J.A.L.)
†These au ho s con ibu ed equally o his wo k.
‡P esen add ess: Depa men o Pneumology, Medical Cen e –Uni e si y Hospi al F eibu g,
79106 F eibu g, Ge many.
Recei ed: 4 Sep embe 2020; Accep ed: 22 Sep embe 2020; Published: 25 Sep embe 2020
Abs ac :
In lamma ion and an in lux o mac ophages a e common elemen s in many diseases.
Among p o-in lamma o y cy okines, umo nec osis ac o
α
(TNF
α
) plays a cen al ole by ampli ying
he cy okine ne wo k. P og anulin (PGRN) is a g ow h ac o ha binds o TNF ecep o s and in e e es
wi h TNF
α
-media ed signaling. Ex acellula PGRN is p ocessed in o g anulins by p o eases eleased
om immune cells. PGRN exe s an i-in lamma o y e ec s, whe eas g anulins a e p o-in lamma o y.
The ac o s coo dina ing hese ambi alen unc ions emain unclea . In ou s udy, we iden i y Y-box
binding p o ein-1 (YB-1) as a candida e o his immune-modula ing ac i i y. Using a yeas -2-hyb id
assay wi h YB-1 p o ein as bai , clones encoding o p og anulin we e selec ed using s ingen c i e ia
o s ong in e ac ion. We demons a e ha a physiological concen a ions, YB-1 in e e es wi h
he binding o TNF
α
o i s ecep o s in a dose-dependen manne using a low cy ome y-based
binding assay. We show ha YB-1 in combina ion wi h p og anulin in e e es wi h TNF
α
-media ed
signaling, suppo ing he unc ionali y wi h an NF-
κ
B luci e ase epo e assay. Toge he , we show
ha YB-1 displays immunomodula ing unc ions by a ec ing he binding o TNF
α
o i s ecep o s
and in luencing TNFα-media ed signaling ia i s in e ac ion wi h p og anulin.
Keywo ds:
Y-box binding p o ein-1 (YB-1); DNA binding p o ein-B (DbpB); p og anulin; umo
nec osis ac o alpha (TNFα); in lamma ion; immune modula ion; signaling; mac ophage
In . J. Mol. Sci. 2020,21, 7076; doi:10.3390/ijms21197076 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2020,21, 7076 2 o 17
1. In oduc ion
Y-box binding p o ein-1 (YB-1) is a mul i unc ional p o ein, which was ini ially iden i ied by
i s binding o Y-box mo i s (in e ed CCAAT box) wi hin he p omo e s o human MHC class II
genes [
1
]. YB-1 pe o ms a wide a ie y o cellula unc ions and is in ol ed in di e se biological
p ocesses, such as he egula ion o ansc ip ion/ ansla ion, modi ica ion o ch oma in, DNA epai ,
RNA packaging, and modula ing cellula s ess esponses [
2
]. YB-1 con ols cell cycle-dependen genes,
and inc eased nuclea exp ession has been ound in umo s [
3
–
5
]. In addi ion o i s in acellula
unc ions, YB-1 is sec e ed ia a non-classical pa hway ollowing cy okine s imula ion wi h ei he
ans o ming g ow h ac o
β
(TGF-
β
) o pla ele -de i ed g ow h ac o (PDGF) [
6
,
7
]. Ex acellula
YB-1 is ound in he se um and u ine o pa ien s su e ing om in lamma o y glome ula diso de s,
as well as in he se um and u ine o animals unde going expe imen al models o in lamma o y disease.
Sec e ed YB-1 was shown o be chemoa ac i e and pa icipa es in monocy e/mac ophage ec ui men ,
di e en ia ion, and unc ion a e lipopolysaccha ide (LPS) s imula ion
in i o
and in an animal model
o kidney in lamma ion
in i o
, suppo ing i s ole in immunomodula ion [
8
,
9
]. Recen ly, sec e ed
YB-1 was iden i ied as an impo an ac o in melanoma [
10
]. In a p e ious s udy, we iden i ied
and cha ac e ized an in e ac ion o ex acellula YB-1 wi h ecep o No ch-3, leading o in acellula
signaling [
7
]. No ch-3 was shown o be up egula ed in human kidney diseases and plays a ole
in modula ing in lamma ion and ib osis in ubuloin e s i ial kidney inju y [
11
]. Ex acellula YB-1
egula es No ch-3 ecep o exp ession and signaling [12].
YB-1 is comp ised o h ee domains wi h dis inc unc ions [
2
]. The N- e minal domain is
alanine/p oline- ich and associa es wi h ac in. The cold shock domain ea u es DNA/RNA binding
ac i i ies ha enable i s ansc ip ional and ansla ional ac i i ies. The C- e minal ail associa es wi h
o he YB-1 p o eins (homo-mul ime iza ion) and con ols he subcellula dis ibu ion o YB-1 wi hin
he cell [13–16].
Tumo nec osis ac o
α
(TNF
α
) p omo es in lamma ion and issue-des uc ion in a numbe o
in lamma o y diseases, including glome ulopa hies [
17
–
24
]. TNF
α
ac s h ough wo dis inc ecep o s.
TNF ecep o 1 (TNFR1) is widely exp essed. Signaling ia his ecep o p omo es p o-in lamma o y
esponses and induces cell dea h, bu i can also lead o cell su i al [
25
]. In he heal hy kidney,
TNFR1 is mainly exp essed on glome uli and pe i ubula endo helial cells. TNF ecep o 2 (TNFR2)
exp ession is mo e es ic ed. I is p ima ily exp essed on immune cells. In a ious kidney diseases,
TNFR2 exp ession is induced on enal cells [
26
–
28
]. Signaling ia TNFR2 is no comple ely unde s ood,
bu i appea s o ha e an immunomodula o y unc ion [29,30].
Based on ou cu en unde s anding o in lamma o y diseases, new ea men s a egies and d ugs
ha e been de eloped ha a ge key cy okines o in e e e wi h hei signaling cascades. Common
app oaches include cy okine blocking monoclonal an ibodies (Anakin a: IL-1
β
, In liximab: TNF
α
) and
soluble TNF ecep o s (E ane cep , One cep ) [
31
,
32
]. T ea men o heuma oid a h i is pa ien s wi h
TNF
α
-blocking d ugs imp o ed clinical symp oms. Pa ien s who also su e ed om ch onic kidney
disease demons a ed a bene icial e ec on kidney unc ion [
24
]. Al hough all an i-TNF
α
he apies
ha e well-demons a ed e icacy, he inc eased isk o lymphomas o eac i a ion o la en in ec ion
emains a d awback [
32
–
36
]. The e o e, d ug de elopmen has mo e ecen ly ocused on in e e ence
wi h cy okine ecep o s, he eby blocking he ac i a ion o cy okine-induced signaling pa hways.
2. Resul s
2.1. YB-1/PGRN In e ac ion
In acellula YB-1 has a numbe o known in e ac ing pa ne s (Figu e 1A) [
2
]. Howe e , less is
known abou ex acellula YB-1. In a p e ious e o o iden i y no el YB-1 in e ac ing p o eins,
we pe o med a yeas - wo hyb id (Y2H) sc een using a cDNA lib a y gene a ed om a human
mesangial cell line. Bo h ull-leng h YB-1 and a C- e minally unca ed
∆
YB-1, missing 30 amino acids
ha includes he dilysine mo i equi ed o sec e ion (K301/K304), we e used as bai (Figu e 1B) [
6
].
In . J. Mol. Sci. 2020,21, 7076 3 o 17
The unc ional ele ance o his app oach has been demons a ed o wo o he in e ac ing p o eins:
ecep o No ch-3 and splicing ac o SRp30c [
7
,
37
]. Among he p o eins iden i ied (see Figu e 1C),
p og anulin (PGRN) was o pa icula in e es , since i is hough o exe i s an i-in lamma o y ac i i ies
by modula ing he binding o TNF
α
o i s ecep o s [
38
,
39
]. The e o e, we hypo hesized ha YB-1 and
PGRN migh eam up o modula e TNFαac i i y.
In . J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 o 17
ecep o No ch-3 and splicing ac o SRp30c [7,37]. Among he p o eins iden i ied (see Figu e 1C),
p og anulin (PGRN) was o pa icula in e es , since i is hough o exe i s an i-in lamma o y
ac i i ies by modula ing he binding o TNFα o i s ecep o s [38,39]. The e o e, we hypo hesized ha
YB-1 and PGRN migh eam up o modula e TNFα ac i i y.
Figu e 1. Iden i ica ion o Y-box binding p o ein-1 in e ac ing p o eins. (A) Published in e ac ion
pa ne s o Y-box binding p o ein-1 (YB-1) and he co esponding p o ein sequences ha a e needed
o in e ac ion. Figu e modi ied om Elisee a e al. 2011 [2]. (B) A cDNA lib a y o human mesangial
cells was sc eened wi h a yeas -2-hyb id sc een o in e ac ion pa ne s o YB-1. A ull-leng h YB-1 o
a C- e minally unca ed cons uc ΔYB-1 we e used as bai . Iden i ied in e ac ions o YB-1 wi h
ecep o No ch-3 and SRp30c ha e been al eady published [7,37]. (C) Clones gene a ed in he yeas -
2-hyb id sc een ha we e posi i e o YB-1 and p og anulin (PGRN) in e ac ion we e picked and
sequenced, allowing us o iden i y he binding sequence o he p o ein. (D) RAW 264.7 mac ophages
we e p e-incuba ed wi h ecombinan YB-1 and/o ecombinan PGRN, s ained o binding o B -
TNFα (bio inyla ed umo nec osis ac o α) and analyzed by low cy ome y.
The inding ha YB-1 di ec ly binds o PGRN p omp ed us o in es iga e whe he YB-1/PGRN
show an added bene i o modula ing he TNFα–TNFR in e ac ion. The e o e, we pe o med a low
cy ome ic binding assay and es ed he e ec s o adding ei he ecombinan PGRN and/o
ecombinan human YB-1 ( hFlag-YB-1) on he TNFα–TNFR in e ac ion. The incuba ion o RAW
mac ophages wi h PGRN esul ed in a dec eased binding o bio inyla ed TNFα (B -TNFα) o TNFR,
con i ming ha PGRN compe es wi h TNFα o binding o i s ecep o s (Figu e 1D). Simila ly,
hFlag-YB-1 also compe es wi h TNFα o ecep o binding. Toge he , PGRN and YB-1 show an
enhanced inhibi o y e ec on TNFα binding. Hea dena u ing YB-1 ab oga ed he inhibi o y e ec ,
Figu e 1.
Iden i ica ion o Y-box binding p o ein-1 in e ac ing p o eins. (
A
) Published in e ac ion
pa ne s o Y-box binding p o ein-1 (YB-1) and he co esponding p o ein sequences ha a e needed o
in e ac ion. Figu e modi ied om Elisee a e al. 2011 [
2
]. (
B
) A cDNA lib a y o human mesangial
cells was sc eened wi h a yeas -2-hyb id sc een o in e ac ion pa ne s o YB-1. A ull-leng h YB-1 o a
C- e minally unca ed cons uc
∆
YB-1 we e used as bai . Iden i ied in e ac ions o YB-1 wi h ecep o
No ch-3 and SRp30c ha e been al eady published [
7
,
37
]. (
C
) Clones gene a ed in he yeas -2-hyb id
sc een ha we e posi i e o YB-1 and p og anulin (PGRN) in e ac ion we e picked and sequenced,
allowing us o iden i y he binding sequence o he p o ein. (
D
) RAW 264.7 mac ophages we e
p e-incuba ed wi h ecombinan YB-1 and/o ecombinan PGRN, s ained o binding o B -TNF
α
(bio inyla ed umo nec osis ac o α) and analyzed by low cy ome y.
The inding ha YB-1 di ec ly binds o PGRN p omp ed us o in es iga e whe he YB-1/PGRN
show an added bene i o modula ing he TNF
α
–TNFR in e ac ion. The e o e, we pe o med a low
cy ome ic binding assay and es ed he e ec s o adding ei he ecombinan PGRN and/o ecombinan
human YB-1 ( hFlag-YB-1) on he TNF
α
–TNFR in e ac ion. The incuba ion o RAW mac ophages
wi h PGRN esul ed in a dec eased binding o bio inyla ed TNF
α
(B -TNF
α
) o TNFR, con i ming
ha PGRN compe es wi h TNF
α
o binding o i s ecep o s (Figu e 1D). Simila ly, hFlag-YB-1 also
compe es wi h TNF
α
o ecep o binding. Toge he , PGRN and YB-1 show an enhanced inhibi o y
e ec on TNF
α
binding. Hea dena u ing YB-1 ab oga ed he inhibi o y e ec , hus demons a ing
In . J. Mol. Sci. 2020,21, 7076 4 o 17
he speci ici y o his in e ac ion. Su p isingly, hea -dena u ed PGRN showed enhanced inhibi ion,
sugges ing ha a change in con o ma ion migh imp o e i s abili y o inhibi TNF binding.
2.2. YB-1 Inhibi s TNFα-Binding o I s Recep o s
To con i m he obse a ion ha YB-1 alone compe es wi h TNF
α
o ecep o binding, we epea ed
he low cy ome ic TNF
α
binding assay. We es ed he e ec s o adding di e en doses o
ecombinan human YB-1 on he TNF
α
–TNFR in e ac ion. The incuba ion o RAW mac ophages
wi h bio inyla ed TNF
α
(B -TNF
α
) con i med ha TNF
α
shows a speci ic binding o i s ecep o s.
The addi ion o unlabeled TNF
α
could compe e away binding o he bio inyla ed ligand, while he
addi ion o an un ela ed bio inyla ed p o ein showed negligible binding (Figu e 2A,
uppe le panel
).
As demons a ed in Figu e 1D, hFlag-YB-1 also compe ed wi h TNF
α
o ecep o binding.
The inhibi o y e ec o YB-1 u ned ou o be dose-dependen , as shown in Figu e 2A (uppe
igh panel). The inhibi o y e ec was no cell-speci ic, since a simila dec ease in TNFαbinding was
seen using bo h human THP-1 cells and a mesangial cells (Figu e 2A, lowe panels).
In . J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 o 17
Figu e 2. YB-1 inhibi s TNFα binding o TNF ecep o s (TNFRs). (A) RAW 264.7 mac ophages we e
p e-incuba ed wi h inc easing doses o ecombinan Flag-YB-1 and s ained o B -TNFα-binding.
Binding o B -TNFα was analyzed wi h low cy ome y. (B) GST- agged YB-1 dele ion mu an s we e
used o iden i y he minimal sequence equi ed o in e ac ion. Wes e n Blo analysis o pu i ied GST
cons uc s: lane 1—GST- ull leng h YB-1, lane 2—GST-YB-1 Δ1, lane 3—GST-YB-1 Δ3, lane 4—GST-
YB-1 Δ4, lane 5—GST-YB-1 Δ5 and lane 6—GST- ag alone. (C) RAW 264.7 cells we e p e-incuba ed
wi h GST-YB-1 dele ion mu an s, s ained o he binding o B -TNFα, and analyzed wi h low
cy ome y. (D) RAW 264.7 mac ophages we e incuba ed wi h 15 µg ecombinan Flag-YB-1 and/o 50
ng/mL B -TNFα, washed, lysed and bound ligand de ec ed by Wes e n blo ing. GAPDH is included
as he loading con ol.
2.3. YB-1/PGRN Inhibi s TNFα-Media ed Signaling
To in es iga e whe he YB-1/PGRN in luences TNFR signaling, bone ma ow-de i ed
mac ophages (BMDMs) we e s imula ed wi h mu ine TNFα, PGRN, and YB-1 alone o in
combina ion. Cell signaling was analyzed o he exp ession and ac i a ion o ex acellula signal-
egula ed kinase (ERK), p38, and nuclea ac o kappa-B (NF-κB) (Figu e 3A). S imula ion o wild-
ype BMDMs wi h TNFα induced a apid phospho yla ion o ERK, p38, and NF-κB p65. A simila
le el o ac i a ion was also obse ed o TNFα in combina ion wi h ei he YB-1 o PGRN. Howe e ,
s imula ion wi h all h ee ligands combined (mTNFα, YB-1, and PGRN) inhibi ed he ac i a ion o
ERK, p38, and NF-κB p65 in compa ison o he TNFα-induced signal (Figu e 3A).
To u he alida e ou esul s, we u ilized a comme cial NF-κB epo e assay. S imula ion o
he ans ec ed human emb yonic kidney cells wi h ecombinan TNFα showed a obus induc ion
Figu e 2.
YB-1 inhibi s TNF
α
binding o TNF ecep o s (TNFRs). (
A
) RAW 264.7 mac ophages we e
p e-incuba ed wi h inc easing doses o ecombinan Flag-YB-1 and s ained o B -TNF
α
-binding.
Binding o B -TNF
α
was analyzed wi h low cy ome y. (
B
) GST- agged YB-1 dele ion mu an s
we e used o iden i y he minimal sequence equi ed o in e ac ion. Wes e n Blo analysis o
pu i ied GST cons uc s: lane 1—GST- ull leng h YB-1, lane 2—GST-YB-1
∆
1, lane 3—GST-YB-1
∆
3,
lane 4—GST-YB-1
∆
4, lane 5—GST-YB-1
∆
5 and lane 6—GST- ag alone. (
C
) RAW 264.7 cells we e
p e-incuba ed wi h GST-YB-1 dele ion mu an s, s ained o he binding o B -TNF
α
, and analyzed wi h
low cy ome y. (
D
) RAW 264.7 mac ophages we e incuba ed wi h 15
µ
g ecombinan Flag-YB-1 and/o
50 ng/mL B -TNF
α
, washed, lysed and bound ligand de ec ed by Wes e n blo ing. GAPDH is included
as he loading con ol.
In . J. Mol. Sci. 2020,21, 7076 5 o 17
To de e mine he egion(s) wi hin YB-1 esponsible o binding o he TNFR, we made use o
p e iously published GST- agged YB-1 dele ion mu an s o iden i y he minimal sequence equi ed
o he YB-1–TNFR in e ac ion (Figu e 2B) [
40
]. Flow cy ome ic analysis (Figu e 2C) e ealed ha
dele ion o he N- e minal domain YB-1
∆
1 had no nega i e a ec on he abili y o YB-1 o inhibi TNF
α
binding. Simila ly, dele ing he N- e minal and cold shock domain (CSD), i.e., GST-YB-1
∆
3, shows a
simila abili y o inhibi TNF
α
binding, indica ing ha he C- e minal domain is su icien o achie ing
a comple e compe i ion o TNF
α
-binding o he TNFRs. The dele ion mu an GST-YB-1
∆
4 con aining
bo h he N- e minal and cold shock domains shows a educed abili y o inhibi TNF
α
compa ed o he
ull-leng h cons uc , whe eas GST-YB-1
∆
5, con aining only he cold shock domain, has los he abili y
o inhibi TNF
α
binding. Dena u ing YB-1 ab oga ed i s inhibi o y e ec , hus demons a ing he
speci ici y o his in e ac ion. Thus, i appea s ha la gely he C- e minal domain o YB-1 con ibu es
o TNFR binding; ne e heless, a mino e ec is also seen wi h he p o ein N- e minus. The cold shock
domain i sel does no appea o con ibu e o ecep o in e ac ions.
To isualize his e ec , we incuba ed RAW 264.7 mac ophages wi h bio inyla ed human TNF
α
(B -TNF
α
), which se ed as a posi i e con ol o he binding assay, and/o ecombinan human YB-1.
Figu e 2D demons a es ha mac ophages a e able o bind TNF
α
o YB-1, indica ing ha hese p o eins
bind o speci ic ecep o s on he cell su ace. When bo h p o eins a e added oge he , he amoun
o TNF
α
eco e ed is s ongly educed, con i ming ha YB-1 compe es wi h TNF
α
o binding o
i s ecep o .
2.3. YB-1/PGRN Inhibi s TNFα-Media ed Signaling
To in es iga e whe he YB-1/PGRN in luences TNFR signaling, bone ma ow-de i ed mac ophages
(BMDMs) we e s imula ed wi h mu ine TNF
α
, PGRN, and YB-1 alone o in combina ion. Cell signaling
was analyzed o he exp ession and ac i a ion o ex acellula signal- egula ed kinase (ERK), p38,
and nuclea ac o kappa-B (NF-
κ
B) (Figu e 3A). S imula ion o wild- ype BMDMs wi h TNF
α
induced
a apid phospho yla ion o ERK, p38, and NF-
κ
B p65. A simila le el o ac i a ion was also obse ed o
TNF
α
in combina ion wi h ei he YB-1 o PGRN. Howe e , s imula ion wi h all h ee ligands combined
(mTNF
α
, YB-1, and PGRN) inhibi ed he ac i a ion o ERK, p38, and NF-
κ
B p65 in compa ison o he
TNFα-induced signal (Figu e 3A).
To u he alida e ou esul s, we u ilized a comme cial NF-
κ
B epo e assay. S imula ion o
he ans ec ed human emb yonic kidney cells wi h ecombinan TNF
α
showed a obus induc ion o
luci e ase ac i i y (Figu e 3B). Simila ly, s imula ion wi h ei he YB-1 o PGRN alone also induced
NF-
κ
B luci e ase ac i i y, howe e , o a lesse ex en o app oxima ely 2- old (Figu e 3B). In ag eemen
wi h he abo e esul s, adding ei he PGRN o YB-1 inhibi ed he TNF-induced epo e ac i i y by 30%
and 50% espec i ely, while combined s imula ion wi h YB-1 and PGRN shows maximal inhibi ion
(≈65%).
In . J. Mol. Sci. 2020,21, 7076 6 o 17
In . J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 o 17
o luci e ase ac i i y (Figu e 3B). Simila ly, s imula ion wi h ei he YB-1 o PGRN alone also induced
NF-κB luci e ase ac i i y, howe e , o a lesse ex en o app oxima ely 2- old (Figu e 3B). In
ag eemen wi h he abo e esul s, adding ei he PGRN o YB-1 inhibi ed he TNF-induced epo e
ac i i y by 30% and 50% espec i ely, while combined s imula ion wi h YB-1 and PGRN shows
maximal inhibi ion (≈65%).
Figu e 3. TNFα-media ed signaling is in luenced by YB-1 and PGRN. (A) Bone ma ow-de i ed
mac ophages (BMDMs) we e s imula ed wi h ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1
(28 nM) o 10 min. A e cell lysis, p o eins we e blo ed and analyzed o he exp ession o pERK,
pp38, NF-κB, pNF-κB, YB-1, and ac in as he loading con ol. Band in ensi ies we e quan i ied and
no malized o ac in. Values a e exp essed as ela i e band in ensi y (RBI), wi h he le el o ac i a ion
o TNFα-s imula ion se o 100%. (B) The pGL4.32 luci e ase epo e cons uc ha bo ing an NF-κB
esponsi e elemen was in oduced in o HEKLen iX cells by calcium phospha e p ecipi a ion.
Recombinan hTNFα was added a he indica ed concen a ions ollowing he ans e o cells in o
se um- ee s a ing medium. S imula ion wi h ecombinan p o eins was pe o med as indica ed o
5 h, and luci e ase ac i i y quan i ied acco ding he manu ac u e ’s ins uc ions. * p = 0.05, ** p = 0.01,
*** p = 0.001.
2.4. YB-1/PGRN Al e s he TNFα-Induced Gene Exp ession P o ile
To gain a comp ehensi e o e iew o he changes going on wi hin he cells, we ex ac ed RNA
and analyzed he gene exp ession p o iles. The compa ison o TNF-s imula ed mac ophages wi h
uns imula ed cells iden i ied mo e han 1124 di e en ially exp essed genes (cu o >1.5- old),
including 26 known TNF a ge s. Compa ison o he iple s imula ion wi h TNF alone iden i ied a
unique subse o 46 di e en ially exp essed genes (Figu e 4A). To alida e hese indings, we again
pu i ied RNA om s imula ed cells, ansc ibed i in o cDNA, and pe o med TaqMan exp ession
analysis o six candida e genes (Nos2, P gs2, Ccl2/MCP-1, Ccl3/MIP1α, Ccl5/RANTES, and Mmp9).
As seen in Figu e 4B, we con i med he esul s o wo candida es selec ed om he gene a ay, namely
Nos2 and P gs2, which bo h showed a u he up egula ion upon iple s imula ion in compa ison o
TNF alone. A simila end was seen o ma ix me allopep idase 9 (Mmp9), which is a known TNFR
Figu e 3.
TNF
α
-media ed signaling is in luenced by YB-1 and PGRN. (
A
) Bone ma ow-de i ed
mac ophages (BMDMs) we e s imula ed wi h ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1
(28 nM) o 10 min. A e cell lysis, p o eins we e blo ed and analyzed o he exp ession o pERK,
pp38, NF-
κ
B, pNF-
κ
B, YB-1, and ac in as he loading con ol. Band in ensi ies we e quan i ied
and no malized o ac in. Values a e exp essed as ela i e band in ensi y (RBI), wi h he le el o
ac i a ion o TNF
α
-s imula ion se o 100%. (
B
) The pGL4.32 luci e ase epo e cons uc ha bo ing
an NF-
κ
B esponsi e elemen was in oduced in o HEKLen iX cells by calcium phospha e p ecipi a ion.
Recombinan hTNF
α
was added a he indica ed concen a ions ollowing he ans e o cells in o
se um- ee s a ing medium. S imula ion wi h ecombinan p o eins was pe o med as indica ed o 5 h,
and luci e ase ac i i y quan i ied acco ding he manu ac u e ’s ins uc ions. * p=0.05,
** p=0.01,
*** p=0.001.
2.4. YB-1/PGRN Al e s he TNFα-Induced Gene Exp ession P o ile
To gain a comp ehensi e o e iew o he changes going on wi hin he cells, we ex ac ed RNA
and analyzed he gene exp ession p o iles. The compa ison o TNF-s imula ed mac ophages wi h
uns imula ed cells iden i ied mo e han 1124 di e en ially exp essed genes (cu o >1.5- old), including
26 known TNF a ge s. Compa ison o he iple s imula ion wi h TNF alone iden i ied a unique
subse o 46 di e en ially exp essed genes (Figu e 4A). To alida e hese indings, we again pu i ied
RNA om s imula ed cells, ansc ibed i in o cDNA, and pe o med TaqMan exp ession analysis o
six candida e genes (Nos2, P gs2, Ccl2/MCP-1, Ccl3/MIP1
α
, Ccl5/RANTES, and Mmp9). As seen in
Figu e 4B, we con i med he esul s o wo candida es selec ed om he gene a ay, namely Nos2 and
P gs2, which bo h showed a u he up egula ion upon iple s imula ion in compa ison o TNF alone.
A simila end was seen o ma ix me allopep idase 9 (Mmp9), which is a known TNFR as well
as YB-1 a ge gene [
41
,
42
]. In he mul iplex assay, we obse ed an induc ion o bo h Ccl3/MIP1
α
and Ccl5/RANTES. The e ec o YB-1/PGRN co-s imula ion was less p ominen . This is mos likely
because RNA is subjec o addi ional egula ion wi hin he cell a he le el o ansla ion. He e, YB-1 is
known o exe much o i s ac i i y [
43
]. YB-1 is able o bo h enhance and supp ess he ansla ion o
chemokine mRNAs, such as Ccl5/RANTES, depending upon he cellula ci cums ances [44–48].
In . J. Mol. Sci. 2020,21, 7076 7 o 17
In . J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 o 17
as well as YB-1 a ge gene [41,42]. In he mul iplex assay, we obse ed an induc ion o bo h
Ccl3/MIP1α and Ccl5/RANTES. The e ec o YB-1/PGRN co-s imula ion was less p ominen . This is
mos likely because RNA is subjec o addi ional egula ion wi hin he cell a he le el o ansla ion.
He e, YB-1 is known o exe much o i s ac i i y [43]. YB-1 is able o bo h enhance and supp ess he
ansla ion o chemokine mRNAs, such as Ccl5/RANTES, depending upon he cellula ci cums ances
[44–48].
2.5. YB-1/PGRN-Media ed Inhibi ion Modula es TNF-Induced Cy okine/Chemokine Exp ession
Ha ing demons a ed ha YB-1/PGRN modula es TNFα-induced signaling, we nex asked how
his ansla es in o a unc ional ou come o he cell. Since TNFα is ega ded as he mas e egula o
o immune-media ed in lamma ion, we i s analyzed he cy okine/chemokine sec e ion o ou wild-
ype BMDMs using a mul iplex assay. As expec ed, 24 h a e TNFα s imula ion, we de ec ed
measu able le els o he p o-in lamma o y chemokines CXCL9/MIG, CCL3/MIP1a, CXCL1/KC,
CCL5/RANTES, and CCL2/MCP-1 in he cell supe na an s (Figu e 4C). Incuba ion wi h ei he YB-1
o PGRN alone showed no induc ion. Co-s imula ion o TNFα wi h ei he YB-1 o PGRN showed
sub le changes in chemokine sec e ion; howe e , he combined e ec o h ee s imuli showed clea
modula o y e ec s. The le els o MIP1α and KC we e mo e han 2- old enhanced, whe eas
CCL5/RANTES is abla ed and MCP-1 is supp essed.
Figu e 4. YB-1 and PGRN modula e TNF-induced cell esponses. (A) BMDMs we e s imula ed as
desc ibed abo e, lysed, and RNA ex ac ed. Gene a ay analysis o he RNA was used o iden i y
al e a ions in gene exp ession be ween s imula ion condi ions. Hea map isualiza ion illus a es
di e en ially exp essed genes be ween each o he s imula ion condi ions compa ed o he nega i e
con ol (i.e., uns imula ed cells). Addi ionally, he compa ison o TNFα alone wi h cells s imula ed
wi h TNFα, YB-1, and PGRN. Genes egula ed >1.5 old we e selec ed. Nos2 and P gs2 a e indica ed
(a ows). (B) BMDMs we e s imula ed wi h ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1
(28 nM) o 24 h. Cells we e lysed; RNA was pu i ied and ansc ibed in o cDNA. Taqman gene
exp ession analysis o NosS2, P gs2, MCP-1/CCL2, MIP1α/CCL3, RANTES/CCL5, and ma ix
me allopep idase 9 (MMP9) we e used o alida e he esul s. Gene exp ession le els a e illus a ed
as ela i e old induc ion, using he gene exp ession o he nega i e con ol as 1. (C) BMDMs we e
s imula ed wi h ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM) o 24 h. Cy okines
sec e ed in o he media we e measu ed wi h a cy ome ic bead a ay and analyzed by FACS. The
cy okines MIG/CXCL9, MIP1α/CCL3, MCP-1/CCL2, RANTES/CCL5, and KC/IL-8 we e ound o be
Figu e 4.
YB-1 and PGRN modula e TNF-induced cell esponses. (
A
) BMDMs we e s imula ed as
desc ibed abo e, lysed, and RNA ex ac ed. Gene a ay analysis o he RNA was used o iden i y
al e a ions in gene exp ession be ween s imula ion condi ions. Hea map isualiza ion illus a es
di e en ially exp essed genes be ween each o he s imula ion condi ions compa ed o he nega i e
con ol (i.e., uns imula ed cells). Addi ionally, he compa ison o TNF
α
alone wi h cells s imula ed wi h
TNF
α
, YB-1, and PGRN. Genes egula ed >1.5 old we e selec ed. Nos2 and P gs2 a e indica ed (a ows).
(
B
) BMDMs we e s imula ed wi h ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM)
o 24 h. Cells we e lysed; RNA was pu i ied and ansc ibed in o cDNA. Taqman gene exp ession
analysis o NosS2, P gs2, MCP-1/CCL2, MIP1
α
/CCL3, RANTES/CCL5, and ma ix me allopep idase
9 (MMP9) we e used o alida e he esul s. Gene exp ession le els a e illus a ed as ela i e old
induc ion, using he gene exp ession o he nega i e con ol as 1. (
C
) BMDMs we e s imula ed wi h
ecombinan mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM) o 24 h. Cy okines sec e ed in o he
media we e measu ed wi h a cy ome ic bead a ay and analyzed by FACS. The cy okines MIG/CXCL9,
MIP1
α
/CCL3, MCP-1/CCL2, RANTES/CCL5, and KC/IL-8 we e ound o be egula ed depending on
he s imulus condi ion. Values a e exp essed as ela i e old induc ion, using le els o TNF
α
-induced
cy okine sec e ion as 1.
2.5. YB-1/PGRN-Media ed Inhibi ion Modula es TNF-Induced Cy okine/Chemokine Exp ession
Ha ing demons a ed ha YB-1/PGRN modula es TNF
α
-induced signaling, we nex asked how
his ansla es in o a unc ional ou come o he cell. Since TNF
α
is ega ded as he mas e egula o o
immune-media ed in lamma ion, we i s analyzed he cy okine/chemokine sec e ion o ou wild- ype
BMDMs using a mul iplex assay. As expec ed, 24 h a e TNF
α
s imula ion, we de ec ed measu able
le els o he p o-in lamma o y chemokines CXCL9/MIG, CCL3/MIP1a, CXCL1/KC, CCL5/RANTES,
and CCL2/MCP-1 in he cell supe na an s (Figu e 4C). Incuba ion wi h ei he YB-1 o PGRN alone
showed no induc ion. Co-s imula ion o TNF
α
wi h ei he YB-1 o PGRN showed sub le changes in
chemokine sec e ion; howe e , he combined e ec o h ee s imuli showed clea modula o y e ec s.
The le els o MIP1
α
and KC we e mo e han 2- old enhanced, whe eas CCL5/RANTES is abla ed and
MCP-1 is supp essed.
3. Discussion
Chemokinesec e ion is essen ial o he ec ui men o immune cells, including monocy es/mac ophages.
These cells a e no only impo an o he induc ion o in lamma ion, bu hey also con ibu e o wound
In . J. Mol. Sci. 2020,21, 7076 8 o 17
healing and he es o a ion o issue homeos asis. Indeed, we and o he s ha e ecen ly shown ha
YB-1 plays an essen ial ole in each o hese s ages o disease [9,46,49–53].
The cu en challenge lies in de ining bo h he cell in insic ac i i ies o YB-1 as well as i s ex insic
ac i i ies. YB-1 sec e ion is known o be induced by a numbe o p o-in lamma o y s imuli, such as
PDGF, TGF-
β
, as well as LPS, and ex acellula YB-1 possesses chemoa ac an ac i i y [
6
–
8
,
54
].
Indeed, i s abili y o bind o cell su ace ecep o s, such as ecep o No ch-3, ha e led us o p opose he
exis ence o an au o- egula o y loop [55].
He ein, we ha e iden i ied a new ac i i y o ex acellula YB-1, namely modula ing TNFR ac i i y.
TNF is conside ed a mas e egula o o in lamma ion, due o i s abili ies o induce he p oduc ion o a
numbe o in lamma o y media o s as well as p o eases, which con ibu e o issue damage as well as
cance ogenesis [56,57].
Simila o YB-1, TNF also possesses he abili y o au o- egula e i s own ac i i y. TNFR s imula ion
ac i a es NF-
κ
B, amongs o he ac o s, which induces he exp ession o TNF mRNA. Recen ly, we
iden i ied YB-1 as an essen ial componen o he TNFR signaling cascade leading o NF-
κ
B ac i a ion,
as TNF s imula ion o YB-1-de icien cells ailed o ac i a e NF-
κ
B, as was p e iously shown o bo h
IGF-1 and IL-1
β
signaling [
58
–
60
]. A ailu e o ac i a e YB-1 and hus NF-
κ
B, nega i ely impac s on
cell su i al in monocy es, mac ophages, and T cells [60–62].
Howe e , his is no he only le el whe e YB-1 may a ec TNF unc ionali y. The TNF mRNA
possesses he same egula o y sequences (class II AU- ich elemen s) ha a e ound in he mRNA o
GM-CSF, which is a known a ge o YB-1 [
63
–
66
]. Addi ionally, RNA binding p o eins,
such as TIA-1
,
TIAR, and HuR, which a e componen s o he s ess g anules, egula e TNF mRNA. Since YB-1
egula es s ess g anule o ma ion and is ound oge he wi h TIA-1, TIAR, and HuR wi hin hese
s uc u es, he e is a s ong possibili y ha YB-1 con ibu es o he egula ion o TNF mRNA [67–69].
In addi ion o enhancing he exp ession o TNF mRNA, NF-
κ
B also induces TRAF2 exp ession [
32
].
TRAF2 is a common signaling componen o bo h TNFR1 and TNFR2 ha plays an impo an ole in
egula ing canonical e sus non-canonical NF-
κ
B ac i a ion, as well as he balance be ween su i al
and dea h signaling [
70
]. Thus, an induc ion o TRAF2 would ampli y he canonical NF-
κ
B pa hway
(p50/p65). Since TRAF2 is essen ial o ac i a ing p65, YB-1 mus lay in be ween. The phospho yla ion
o TRAF2 by p o ein kinase C egula es i s abili y o ec ui and ac i a e IKK
α
, which in u n
phospho yla es I
κ
B
α
, a ge ing i o deg ada ion and he eby ac i a ing NF-
κ
B (p50/p65) [
70
,
71
].
In addi ion, i has ecen ly been epo ed ha TRAF2 media es he ec ui men o ubiqui in ligases o
he TNFR1 complex I he eby p omo ing NF-
κ
B ac i a ion [
72
]. Mo eo e , YB-1 has been shown o
di ec ly in e ac wi h p65 (RelA) and ac as a ansc ip ional co-ac i a o [
73
]. A numbe o kinases
(RSK, AKT, ERK, PKC, CKII) ha e been iden i ied ha phospho yla e YB-1 wi hin i s cold shock
domain, he eby inducing nuclea ansloca ion [
74
,
75
]. Thus, i is o eseeable ha he TNF ecep o
oligome iza ion induced by ligand binding esul s in he ec ui men and oligome iza ion o adap o s,
such as TRAF2, which in u n ec ui kinases and ubiqui in ligases ha ac i a e bo h NF-
κ
B and i s
ans-ac i a o YB-1.
In his s udy, we ocused on s imula ion o he TNFRs wi h soluble TNF, which ac i a es TNFR1 [
76
].
Recen ly, i was shown ha TNFR1 oligome iza ion is essen ial o induce signaling and ha his equi es
bo h dime iza ion o ecep o chains ia he p eligand assembly domain and ligand binding [
77
].
The e o e, i is concei able ha YB-1/PGRN in e e es wi h oligome o ma ion and he eby p e en s
signal ansduc ion. De e mining he s uc u e o hese in e ac ions is essen ial, as his may e eal
new a ge s o de elopmen as an i-TNF he apy, as cu en he apies al hough success ul ha e hei
limi a ions [78]. How YB-1/PGRN in luence memb ane-bound ligand ac i i y equi es u he s udy.
Recen ly, PGRN was epo ed o ac i a e No ch signaling pa hways in neu ons [
79
]. YB-1 shows
abundan exp ession in neu ons and is a ligand o ecep o No ch-3 [
7
,
80
]. Since TNF and TNFR
in e ac ions a e p oposed o play a ole in neu onal g ow h, he dis up ions o hese in e ac ions may
con ibu e o cogni i e impai men , as seen in pa ien s wi h p og anulin mu a ions [
81
,
82
]. Finally,
p og anulin was ecen ly iden i ied as a ligand o eph in ecep o A2; howe e , no in e ac ion wi h
In . J. Mol. Sci. 2020,21, 7076 9 o 17
TNFR1 was ound, sugges ing ha hese ecep o s unc ion independen ly, a leas wi h ega d o
ligand binding [83].
4. Ma e ials and Me hods
4.1. Recombinan P o eins
Recombinan human PGRN (AG-40A-0068 om Adipogen, San Diego, CA, USA) was used o
cell s imula ion expe imen s wi h TNF
α
, YB-1, and PGRN. Recombinan mu ine TNF
α
(mTNF
α
)
was pu chased om Pep o ech (315-01A, Hambu g, Ge many). Recombinan YB-1 was p oduced
in ou labo a o y by he ansien ans ec ion o HEK293T cells, which we e cul i a ed in DMEM
g ow h media (+10% e al cal se um (FCS), 1% penicillin/s ep omycin). Fo ans ec ion, 3
×
10
6
cells
we e seeded in 10 cm pe i dishes and g own o 70–80% con luence. Using he calcium phospha e
me hod, cells we e ans ec ed wi h Flag- agged human YB-1 (pcDNA3/Flag-YB-1), which was a
gi o K. Kohno [
84
]. Media was changed a e 5 h and cul i a ed o addi ional 48 h in 37
◦
C
and 5% CO
2
. Cells we e lysed o Flag-YB-1 pu i ica ion wi h RIPA lysis bu e (50 mM T is Base,
150 mM NaCl,
1 mM EDTA, 1% NP-40, 0.25% Sodium deoxychola e) supplemen ed wi h comple e
mini p o ease inhibi o cock ail (Roche) and Phos-S op (Roche, Mannheim, Ge many). Flag-YB-1 was
pu i ied wi h an i-DYKDDDDK G1 a ini y esin (L00432, Gensc ip , Pisca away, NJ, USA) ollowing
he ecommenda ion o he company. Flag-pep ide (100
µ
g/mL) was used o elu e Flag-YB-1 om
he a ini y esin. A e dialysis in polye hylene glycol solu ion (a e age molecula weigh 20 kDa),
pu i ied p o ein was shock ozen and s o ed a
−
80
◦
C un il use (see Supplemen a y Figu e S1).
Pu i ied p o ein was quan i ied using a Low y assay (BioRad, Feldki chen, Ge many). Ac i i y o
YB-1 was es ed using a sc a ch assay [6].
GST- agged YB-1 dele ion mu an s (a gi o K. Kohno) we e used o iden i y he minimal sequence
equi ed o he in e ac ion o YB-1 and TNFR-binding [
84
]. In con as o GST- ull leng h YB-1,
GST-YB-1
∆
1 is missing he N- e minal sequence o YB-1 (aa 1–50), he GST-YB-1
∆
3 cons uc is missing
he N- e minal sequence and he cold shock domain (aa 1–128), and he GST-YB-1
∆
4 is missing he
C- e minal sequence (aa 129–324). The GST-YB-1
∆
5 only con ains he cold shock domain (aa 51–128).
A GST- ag cons uc was used as con ol. GST- agged YB-1 dele ion mu an s we e exp essed in
BL21 E. coli bac e ia, bac e ia we e lysed, and he p o eins pu i ied wi h Glu a hione Sepha ose 4B
(GE Heal hca e, Chicago, IL, USA) and elu ed wi h inc easing glu a hione concen a ions, dialyzed,
and analyzed by Wes e n blo . Recombinan p o eins we e s o ed a −80 ◦C un il use.
4.2. Cell Cul u e
4.2.1. Bone Ma ow-De i ed Mac ophages (BMDMs)
BMDMs we e gene a ed om wild- ype mice by lushing emu and ibia wi h s e ile DPBS.
E y h ocy es we e lysed unde hypo onic condi ions and cells seeded wi h 2
×
10
6
cells/mL in
RPMI g ow h media supplemen ed wi h 10% FCS, 1% penicillin/s ep omycin, and 10 ng/mL mu ine
mac ophage colony-s imula ing ac o (M-CSF, 315-02, Pep o ech). Cells we e cul i a ed unde
humidi ied condi ions a 37
◦
C and 5% CO
2
. Cells we e ed e e y 2 days un il ull di e en ia ion
a e 7 days. Then, cells we e s a ed o e nigh and s imula ed wi h ei he 28 nM YB-1, 5 nM PGRN,
o 625 pM
TNF
α
alone o in combina ion o he ime pe iod indica ed. Following s imula ion, he cells
we e lysed in DISC lysis bu e (30 mM T is pH 7.4, 120 mM NaCl, 10% Glyce in, 1% T i on-X100)
con aining comple e mini p o ease inhibi o cock ail and Phos-S op. Then, lysa es we e sepa a ed by
SDS-PAGE and analyzed by Wes e n blo ing.
Mice we e housed acco ding o FELASA guidelines (Fede a ion o Eu opean Labo a o y Animal Science
Associa ion) in a 12 h/12 h ligh da k cycle a 22
◦
C in he Cen al Animal Facili y o he O o- on-Gue icke
Uni e si y Magdebu g unde speci ic pa hogen- ee (SPF) condi ions using indi idual en ila ed cages
(IVC, Techniplas , Buguggia e, I aly) wi h ood and wa e ad libi um. Expe imen s we e conduc ed
In . J. Mol. Sci. 2020,21, 7076 16 o 17
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Dela e, O.; E dokimo a, V.; e al. YB-1 egula es s ess g anule o ma ion and umo p og ession by
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