Guo, Ryan Price and Hydar Ali
Hariharan Subramanian, Kshitij Gupta, Qiang
INTERNALIZATION DESENSITIZATION, AND RECEPTOR PHOSPHORYLATION,Mast Cells: RESISTANCE TO
Antimicrobial Peptide LL-37 in Human Protein-coupled Receptor for the
Mas-related Gene X2 (MrgX2) Is a Novel G
Signal Transduction:
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2011, 286:44739-44749.
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Mas-related Gene X2(MrgX2)Is a Novel G Protein-coupled Receptor for the Antimicrobial Peptide LL-37in Human Mast Cells
RESISTANCE TO RECEPTOR PHOSPHORYLATION,DESENSITIZATION,AND INTERNALIZATION *
Received for publication,June 29,2011,and in revid form,October 29,2011Published,JBC Papers in Press,November 8,2011,DOI 10.1074/jbc.M111.277152
Hariharan Subramanian,Kshitij Gupta,Qiang Guo,Ryan Price,and Hydar Ali 1donna summer
From the Department of Pathology,School of Dental Medicine,University of Pennsylvania,Philadelphia,Pennsylvania 19104
Human LL-37is a multifunctional antimicrobial peptide that promotes inflammation,angiogenesis,wound healing,and tumor metastasis.Most effects of LL-37are mediated via the activation of the cell surface G protein-coupled receptor FPR2on leukocytes and endothelial cells.Although LL-37induces chemotaxis,degranulation,and chemokine produc-tion in mast cells,the receptor involved and the mechanism of its regulation remain unknown.MrgX2is a member of Mas-related genes that is primarily expresd in human dorsal root ganglia and mast cells.We found that a human mast cell line LAD2and CD34؉cell-derived primary mast cells,which natively express MrgX2,responded to LL-37for sustained Ca 2؉mobilization and substantial degranulation.However,an immature human mast cell line,HMC-1,that lacks func-tional MrgX2did not respond to LL-37.shRNA-mediated knockdown of MrgX2in LAD2mast cell line and primary CD34؉cell-derived mast cells caud a substantial reduction in LL-37-induced degranulation.Furthermore,mast cell lines stably expressing MrgX2responded to LL-37for che-motaxis,degranulation,and CCL4production.Surprisingly,MrgX2was resistant to LL-37-induced phosphorylation,densitization,and internalization.In addition,shRNA-me-diated knockdown of the G protein-coupled receptor kinas (GRK2and GRK3)had no effect on LL-37-induced mast cell degranulation.This study identified MrgX2as a novel G pro-tein-coupled receptor for the antibacterial peptide LL-37and demonstrated that unlike most G protein-coupled receptors it is resistant to agonist-induced receptor phosphorylation,densitization,and internalization.
Antimicrobial peptides such as defensins and cathelicid-ins are creted by activated epithelial cells as well as by invading leukocytes and play an important role in host defen (1).Cathelicidins consist of a putative N-terminal signal peptide,a highly conrved cathelin-like domain,and a C-terminal antimicrobial domain corresponding to the mature antibacterial peptide.About 30cathelicidin mem-bers have been identified in mammals.However,only one cathelicidin,hCAP18(human cationic antibacterial protein of 18kDa),has been found in humans thus far,and its C-ter-minal mature antibacterial peptide (LL-37),comprising 37amino acid residues,has direct antibacterial effects against Gram-positive and Gram-negative bacteria (2).In addition,LL-37displays immunomodulatory properties via the recruitment of monocytes and T cells (3,4).LL-37stimulates angiogenesis to promote wound healing and tumor invasive-ness (5–8).Most effects of LL-37appear to be mediated via the activation of G protein-coupled formyl peptide receptor 2(FPR2;earlier known as FPRL1)on monocytes,T cells,and endothelial cells (3,8–11).LL-37also activates chemokine receptor CXCR2in human neutrophils (12),purinergic receptor P2X7in fibroblasts (13),and insulin growth factor receptors in epithelial cells (14).
Mast cells are known to play a critical role in innate immu-nity,and this function requires mast cell degranulation and subquent neutrophil recruitment (15,16).LL-37induces Ca 2ϩmobilization,chem
otaxis,and degranulation in rat peri-toneal mast cells (17,18).It also caus incread vascular per-meability in wild-type but not in mast cell-deficient rats (19).Thus,LL-37-induced mast cell activation could contribute to the innate immune function of mast cells.LL-37is also thought to be involved in chronic inflammatory dias.The level of LL-37in human skin increas dramatically from 1M in nor-mal individuals to ϳ304M in psoriatic patients (20,21).LL-37induces degranulation and pruritogenic cytokine generation in human mast cells (22,23).Although LL-37activates mast cells via pertussis toxin-nsitive G protein and phospholipa
*This work was supported,in whole or in part,by National Institutes of Health
Grant HL085774.
1
To whom correspondence should be addresd:Dept.of Pathology,Univer-sity of Pennsylvania School of Dental Medicine,240South 40th St.,Phila-delphia,PA 19104-6030.Tel.:215-573-1993;Fax:215-573-2050;E-mail:alih@upenn.edu.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.286,NO.52,pp.44739–44749,December 30,201
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1©2011by The American Society for Biochemistry and Molecular Biology,Inc.Printed in the U.S.A.
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C-mediated signaling pathway(18),the GPCRs2it utilizes have not been determined.
A large family of GPCRs called Mas-related genes(Mrgs,also known as nsory neuron-specific receptors)has recently been identified in rodents(24,25).The receptors are lectively expresd in small diameter nsory neurons of dorsal root gan-glia and are thought to be involved in the ns
ation and modu-lation of pain.Interestingly,a subgroup of the receptors (MrgX1–MrgX4)are expresd in human but not in murine neurons(24,26).Recent studies have shown that MrgX2is also expresd in human mast cells and activated by basics peptides (27,28).Given that LL-37also displays basic properties,we hypothesized that it activates human mast cells via MrgX2. Using shRNA-mediated knockdown of MrgX2in human mast cells,we provide the novel finding that LL-37caus sustained Ca2ϩmobilization and degranulation via MrgX2.Moreover, using transfected mast cell lines that do not natively express MrgX2(27),we demonstrate that LL-37promotes chemotaxis and induces chemokine,CCL4via MrgX2.
Agonist-occupied GPCRs are phosphorylated by a family of protein kinas,collectively known as G protein-coupled recep-tor kinas(GRKs)(29).Of the ven known GRKs,four(GRK2, GRK3,GRK5,and GRK6)are expresd ubiquitously.It is well established that GPCR phosphorylation by GRKs leads to the recruitment of-arrestin,which results in receptor densiti-zation and internalization(29).A goal of this study was to deter-mine the role of agonist-induced receptor phosphorylation on the regulation of MrgX2.Our studies clearly demonstrate that unlike most other known GPCRs,MrgX2is resistant to LL-37-induced receptor phosphorylation,densitization,and inter-nalization.It is noteworthy that LL-37-induced mast cell degranulation leads to the relea of try
pta,which degrades LL-37,rendering it inactive(23).Thus,in the abnce of MrgX2 receptor densitization,enhanced mast cell degranulation likely provides a novel feedback mechanism to regulate recep-tor function by limiting ligand availability for the receptor. EXPERIMENTAL PROCEDURES
Materials—Frozen human G-CSF-mobilized peripheral blood CD34ϩprogenitors were obtained from The Fred Hutchinson Cancer Center(Seattle,WA).All cell culture reagents and pertussis toxin were purchad from Invitrogen. Amaxa cell transfection kits and reagents were purchad from Lonza(Gaithersburg,MD).Anti-HA antibody(12CA5)and anti-HA(HA-7)-agaro beads were purchad from Roche Applied Science and Sigma,respectively.All recombinant human cytokines were purchad from PeproTech(Rocky Hill, NJ).Phorbol12-myristate13-acetate(PMA)was purchad from Calbiochem.Anti-human C3a receptor antibody was obtained from Santa Cruz Biotechnology(Santa Cruz,CA). Phycoerythrin(PE)-labeled donkey anti-mou IgG was pur-chad from eBioscience(San Diego).Bovine adrenal medulla docosapeptide(BAM-22P)and cortistatin-14(CST)were obtained from American Peptide(Vista,CA).Native comple-ment C3a was from Complement Technology(Tyler,TX). LL-37(Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser)was from Anaspec(Freemont,CA).
银杏什么意思Differentiation of Human Mast Cells from CD34ϩProgenitors and Culture of Human Mast Cell Lines—To obtain primary mast cells,human CD34ϩprogenitors were cultured in Stem-Pro-34medium supplemented with L-glutamine(2m M),peni-cillin(100IU/ml),streptomycin(100g/ml),rhSCF(100 ng/ml),rhIL-6(100ng/ml),and rhIL-3(30ng/ml)(1st week only).Hemidepletions were performed weekly with media con-taining rhSCF(100ng/ml)and rhIL-6(100ng/ml)(30).Cells were ud for experiments after7–10weeks in culture.HMC-1 cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with10%FCS,glutamine(2m M),penicillin(100 IU/ml),and streptomycin(100g/ml)(31).LAD2cells were maintained in complete StemPro-34medium supplemented with100ng/ml rhSCF(32).RBL-2H3and HEK-293T cells were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium(DMEM)supplemented with10%FBS,L-glu-tamine(2m M),penicillin(100IU/ml),and streptomycin(100g/ml)(33).
Lentivirus-mediated Knockdown of MrgX2,GRK2,and GKR3 in Human Mast Cells—MrgX2-targeted Mission shRNA lenti-viral plasmids were purchad from Sigma.The clone that gave the highest knockdown efficiency(TRCN0000009174)was ud.A scrambled control nontarget vector(SHC002),which does not bind to any known human mRNAs,was also pur-chad from Sigma.Lentivirus generation was performed according to the manufacturer’s manual.Cell transductio
n was conducted by mixing1.5ml of viral supernatant with3.5ml of LAD2(5ϫ106cells)or CD34ϩmast cells(3ϫ106cells).Eight hours post-infection,medium was changed to virus-free com-plete medium,and antibiotic(puromycin,2g/ml,Sigma) lection was initiated16h later.Knockdown of GRK2and GRK3in LAD2cells was performed as described previously (34).Cells were analyzed for MrgX2,GRK2,or GRK3knock-down and ud for subquent assays4days following initiation of puromycin lection.
Quantitative PCR—Total RNA from mast cells was extracted using TRIzol(Invitrogen),treated with DNa I,and rever-transcribed to cDNA using first strand cDNA synthesis kit(GE Healthcare).Gene expression was analyzed using real time PCR with TaqManfast universal PCR master mix on a TaqMan 7500fast real time PCR system(Applied Biosystems,Foster City,CA).TaqMan probes for hGAPDH,hMrgX2,and hGRK3 were ud for real time PCR to analyze the knockdown effi-ciency.The amplification conditions were as follows:initial denaturation at95°C for20s,followed by40cycles of amplifi-cation:95°C for3s and60°C for30s.Analysis was performed according to⌬⌬Ct method.The results are expresd as a ratio of MrgX2or GRK3to GAPDH(34,35).
Western Blotting—Protein extracts of control and GRK2 knockdown LAD2cells following lysis with RIPA buffer were parated by SDS-PAGE and transferred to Hybond ECL nitro-cellulo membrane
s(GE Healthcare).Blots were incubated
2The abbreviations ud are:GPCR,G protein-coupled receptor;CST,cortista-tin-14;Mrg,Mas-related gene;PTx,pertussis toxin;ANOVA,analysis of vari-ance;rh,recombinant human;PMA,phorbol12-myristate13-acetate;PE, phycoerythrin;GRK,G protein-coupled receptor kina.
MrgX2as Novel G Protein-coupled Receptor for LL-37
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with anti-human GRK2antibody(Santa Cruz Biotechnology) in blocking buffer(PBS,0.5%Tween20,5
%skim milk)followed by HRP-labeled goat anti-rabbit IgG(Thermo Scientific,1:5000 in blocking buffer)condary antibody.Bound antibody was detected using the SuperSignalWest Femto maximum nsi-tivity substrate kit(Thermo Scientific)according to the manu-facturer’s protocol.
Stable Transfection of RBL-2H3and HMC-1Cells—RBL-2H3cells stably expressing MrgX1and MrgX2were generated as described previously(27,36).For HMC-1cells,2ϫ106cells were transfected with plasmids encoding HA-tagged MrgX2 using the Amaxa nucleofector device and Amaxa kit V accord-ing to the manufacturer’s protocol.Following nucleofection, cells were cultured in the prence of G418(1mg/ml),and cells expressing equivalent receptors were sorted using an anti-HA-specific antibody12CA5/FITC-conjugated anti-mou IgG and ud for studies on Ca2ϩmobilization,chemotaxis,receptor internalization,and CCL4chemokine generation.
Calcium Mobilization—Ca2ϩmobilization was determined as described previously(37,38).Briefly,cells(human mast cells, 0.2ϫ106)and(RBL-2H3or HMC-1cells,1.0ϫ106)were loaded with1M indo-1AM for30min at room temperature. Cells were washed and resuspended in1.5ml of HEPES-buff-ered saline.Ca2ϩmobilization was measured in a Hitachi F-2500spectrophotometer with an excitation wavelength of 355nm and an emission wavelength of410nm(38). Degranulation—Human mast cells(5ϫ103)and RBL-2H3 cells(5ϫ104)were eded into
96-well plates in a total volume of50l of HEPES buffer containing0.1%BSA and expod to different concentrations of peptides.In some assays,cells were pretreated with pertussis toxin(100ng/ml;16h)or La3ϩ(lan-thanum chloride,1M;5min).For total-hexosaminida relea,unstimulated cells were lyd in50l of0.1%Triton X-100.Aliquots(20l)of supernatants or cell lysates were incubated with20l of1m M p-nitrophenyl-N-acetyl--D-glu-cosamine for1.5h at37°C.Reaction was stopped by adding250
l of a0.1M Na
2CO
3
sperian,0.1M NaHCO
3
buffer,and absorbance
was measured at405nm(33).
Chemotaxis Assay—LL-37or buffer(30l)was added to the lower wells of a96-well chemotaxis chamber(8-m pore size; NeuroProbe,Gaithersburg,MD).Mock-transfected HMC-1 cells or cells expressing MrgX2(0.5ϫ106in50l of buffer) were placed on the upper chamber.After3h of incubation at 37°C,the migrated cells were collected from the lower cham-bers.Triplicate wells were pooled,and the cells were resus-pended in30l of complete Iscove’s modified Dulbecco’s medium.The cells were counted with a hemocytometer,and the results are expresd as absolute number of cells that had migrated.
Receptor Internalization—Cells(0.25ϫ106)were expod to buffer or different agonists for indicated time intervals at37°C. Cells were washed twice with ice-cold FACS buffer(PBS con-taining2%FBS)and labeled with anti-HA antibody(12CA5)or isotype control and incubated on ice for30min.After washing twice with cold FACS buffer,cells were stained with PE-labeled anti-mou IgG antibody on ice for30min.Cells were washed twice and fixed in300l of2%formaldehyde solution.The
samples were acquired and analyzed using LSR II flow cytome-
ter(BD Biosciences).
Chemokine CCL4Generation—Chemokine relea assay was
performed as described previously(38).Briefly,mock-or
MrgX2-transfected HMC-1cells(0.2ϫ106)were stimulated
学位英语考试真题with indicated concentrations of LL-37for6h.CCL4chemo-
kine levels in the supernatants were quantified by DuoSet
ELISA kit from R&D Systems(Minneapolis,MN)according to
the manufacturer’s protocol.
Receptor Expression and Phosphorylation—Transient trans-
fections were performed on80%confluent HEK-293T mono-
layers in60-mm dishes in4ml of OptiMEMmedium(Invit-
rogen)containing1g of plasmid DNA(encoding hemagglutinin(HA)-tagged human MrgX2and C3aR)and7l of Lipofectamine reagent(Invitrogen).To detect receptor
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expression,cells(0.5ϫ106)were incubated with anti-HA
(12CA5)antibody followed by PE-conjugated anti-mou IgG
as the condary antibody at4°C for30min and analyzed by
flow cytometry.
Receptor phosphorylation experiments were performed via
modification of procedures described previously(33,39).
Briefly,HEK-293T cells expressing HA-tagged human MrgX2
and C3aR were labeled with0.15mCi/ml[32P]orthophosphate
for90min and stimulated with indicated agonists at37°C for5
min.The reaction was stopped by adding3volumes of ice-cold
PBS,and cells were lyd in immunoprecipitation buffer(50
m M Tris,pH8.0,150m M NaCl,1.0%Nonidet P-40,0.5%deoxy-安排英文
cholate,0.1%SDS,5m M EDTA,and protea and phosphata
inhibitors).The pre-cleared cell lysate was incubated with15l of anti-HA-agaro beads for2h.Samples were washed three
times with lysis buffer and resolved by10%SDS-PAGE.Theshawna lenee
gels were dried,and32P-incorporated protein bands were
imaged with Kodak image film.
RESULTS
LL-37Induces Degranulation in Human Mast Cells via Per-
tussis Toxin-nsitive G Protein-dependent and-independent
Pathways—Previous studies demonstrated that LL-37caus
substantial degranulation in primary human lung mast cells in a
do-dependent manner within a concentration range of1–10M(23,40).As shown in Fig.1A,we also found that LL-37 stimulates degranulation in LAD2mast cells within the same
concentration range.In rat peritoneal mast cells,LL-37induces
a sustained Ca2ϩmobilization and degranulation,and both of
the respons are inhibited by pertussis toxin(PTx),indicat-
ing the involvement of a G
i
family of G proteins(18).However,
the signaling pathway by which LL-37induces degranulation in
human mast cells is unknown.We therefore tested the effects of
PTx on LL-37-induced Ca2ϩmobilization and degranulation in
LAD2mast cells.We found that LL-37caud sustained Ca2ϩ
mobilization in LAD2cells,but PTx had no effect on this
respon(Fig.1B).By contrast,PTx caud substantial inhibi-
tion of LL-37-induced degranulation(Fig.1C).La3ϩhas been
shown to inhibit both Ca2ϩinflux and mast degranulation(41,
42).As shown in Fig.1B,unlike PTx,La3ϩ(1M)almost com-pletely inhibited LL-37-induced Ca2ϩrespon,and this was associated with a substantial inhibition of LL-37-induced mast cell degranulation(Fig.1D).The findings suggest that in con-MrgX2as Novel G Protein-coupled Receptor for LL-37
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trast to the situation in rat peritoneal mast cells,LL-37caus degranulation in human mast cells via the synergistic interac-
tion of a G␣
i -independent Ca2ϩinflux and another G␣
i
-medi-
ated pathway(33).
LL-37Activates Human Mast Cells via MrgX2—LL-37acti-vates neutrophils,monocytes,eosinophils,and T cells via FPR2 (3,9).Although mast cells express transcript for FPR2,the effects of LL-37do not appear to be mediated via this receptor (17,40).It is noteworthy t
hat mast cells are the only known cells outside the dorsal ganglia that express MrgX2(28).Further-more,this receptor is activated by basic peptides(27,28)Given that LL-37displays basic properties,we hypothesized that it could activate mast cells via MrgX2.Although LAD2cells endogenously express MrgX2,an immature human mast cell line,HMC-1cells do not express functional receptors(27).By contrast both cell lines express GPCR for C3a(C3aR)(43).We therefore ud the cell lines to determine whether there was a correlation between MrgX2expression and responsiveness to LL-37.As shown in Fig.2(A and D),C3a caud transient Ca2ϩmobilization in both LAD2and HMC-1cells.However,the known MrgX2neuropeptide ligand CST and LL-37caud sus-tained Ca2ϩrespons in LAD2cells(Fig.2,B and C)but not in HMC-1cells(Fig.2,E and F).The findings suggest that MrgX2could rve as a receptor for LL-37in LAD2mast cells. To confirm the role of MrgX2on LL-37-induced mast cell respons in human mast cells,we generated stable transfec-tants expressing this receptor in HMC-1cells.FACS analysis demonstrated cell surface expression of HA-tagged MrgX2in receptor-transfected but not in mock-transfected cells(Fig. 3A).As shown in Fig.3,B–D,LL-37induced sustained Ca2ϩmobilization,chemotaxis,and chemokine CCL4in cells expressing MrgX2but not in mock-transfected cells.Our next goal was to test the role of MrgX2on LL-37-induced mast cell degranulation.Becau HMC-1cells do not have the capacity to undergo degranulation,we ud rat basophilic leukemia cells,RBL-2H3,stably expressing human MrgX2(27).I
n this system,both CST and LL-37induced substantial mast cell degranulation(Fig.4A).In addition to MrgX2,human mast cells express MrgX1(27,36).To determine the specificity of LL-37for MrgX2,we also utilized RBL-2H3cells stably express-ing MrgX1(36).As shown in Fig.4B,cells expressing MrgX1 responded to its known ligand BAM-22P for degranulation,but they were resistant to LL-37.Moreover,cells expressing MrgX2 did not respond to BAM-22P.The findings support the notion that LL-37utilizes human MrgX2to activate mast cells. To further confirm the role of MrgX2on LL-37-induced mast cell degranulation,we ud lentiviral Mission shRNA to knock down the expression of MrgX2in LAD2cells.Cells were transduced with five different shRNA constructs targeting dif-ferent regions of MrgX2.For control,we ud a scrambled shRNA construct.After transduction and lection with puro-mycin,quantitative PCR was performed to determine the extent of MrgX2knockdown.We found that clone4 (TRCN0000009174)was the most effective in knocking down MrgX2expression.We therefore ud this clone for sub-quent studies.As shown in Fig.5A,we were able to knock down
FIGURE1.LL-37stimulates degranulation and Ca2؉mobilization in LAD2mast cells.A,LAD2mast cells were stimulated with different concentrations of LL-37(0.1–10M),and percent degranulation(-hexosaminida relea)was determined.B,cells were treated with or without PTx(100ng/ml,16h),loa
ded with Indo-1AM,and Ca2ϩmobilization in respon to LL-37(1M)was determined.Indo-1-loaded cells were also expod to La3ϩ(1M),and LL-37-induced Ca2ϩmobilization was determined.C and D,LAD2cells were pretreated with vehicle or PTx(100ng/ml)(C)or La3ϩ(1M;5min)(D)and stimulated with different concentrations of LL-37(1–10M),and percent degranulation was determined.Data are meanϮS.E.of three experiments.Statistical significance was determined by one-way ANOVA(A)or two-way ANOVA with Bonferroni’s post test(C and D).**indicates pϽ0.001.
MrgX2as Novel G Protein-coupled Receptor for LL-37
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