Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene...

NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002 1089

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Cytokines are characterized both by their functional redun-dancy and tissue-specific activities1. Redundancy amongmembers of the interleukin-6 (IL-6) cytokine family, whichcomprises IL-6, IL-11, leukemia inhibitory factor (LIF), onco-statin M, ciliary neurotrophic factor and cardiotrophin-1, isattributed to the common use of the transmembrane receptorβ-subunit gp130. The tissue-specific activities of these cy-tokines are thought to arise from differential responsiveness oftarget cells to individual signaling cascades that emanate fromdiscrete signaling modules in the cytosolic domain of the re-ceptors2. In gp130, at least two such modules have been char-acterized. One encompasses the four membrane-distalphospho-tyrosine (pY) binding sites for the SH2-domain ofthe latent transcription factors, the signal transducer and acti-vator of transcription 1 (STAT1) and STAT3, with STAT3 hav-ing the predominant role in vitro and in vivo3. The otherpathway comprises the membrane-proximal pY757 residue ingp130 responsible for engagement of the cytoplasmic Src-homology tyrosine phosphatase (SHP2) and subsequent acti-vation of the Ras-ERK pathway3,4. Because both modules arenecessary and sufficient for initiation of the corresponding in-tracellular signaling cascades, phenylalanine substitution of

the appropriate Y-residues in gp130 completely abolishes acti-vation of the associated pathways5.

After IL-6 and IL-11 bind to their respective ligand-bindingα-chains, recruitment and homodimerization of gp130 recep-tor β-chains activates intracellular signaling. Meanwhile, allother IL-6 cytokine family members activate intracellular sig-naling through heterodimerization of gp130 with other struc-turally related β-chains3,4. In either case, β-chain dimerizationresults in the coordinated and simultaneous activation of theSTAT1/3 and SHP2-Ras-ERK pathways which, at least in vitro,appear to transduce activities regulating cellular differentiationand apoptosis (STAT1/3) and mitogenesis (SHP2-Ras-ERK).Hence, it has been suggested that the two pathways may main-tain cellular homeostasis in some dynamic cellular systems,such as the immune system, by balancing positive and nega-tive signals6.

The physiological function of the gastrointestinal mucosa isdependent on the persistent integrity of the intestinal epithe-lium, which is continually challenged by the harsh lumenalcontents of the gut. This challenge is met by coordinated repairmechanisms that have the capacity to rapidly restore mucosalintegrity by stimulating migration of epithelial cells over de-

Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in

gp130 mutant mice

NIALL C. TEBBUTT1, ANDREW S. GIRAUD2, MELISSA INGLESE1, BRENDAN JENKINS1, PAUL WARING3, FIONA J. CLAY1, SINA MALKI2, BARBARA M. ALDERMAN2, DIANNE GRAIL1,

FRÉDÉRIC HOLLANDE4, JOAN K. HEATH1 & MATTHIAS ERNST1

1Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia2Department of Medicine, University of Melbourne, Western Hospital, Footscray, Victoria, Australia

3Department of Pathology, Peter MacCallum Institute, East Melbourne, Victoria, Australia4Laboratoire de Signalisation Cellulaire Normale et Tumorale, Faculté de Pharmacie, Montpellier, France

N.C.T. and A.S.G. contributed equally to this study.Correspondence should be addressed to M.E.; email: [email protected]

Published online: 9 September 2002, doi:10.1038/nm763

The intracellular signaling mechanisms that specify tissue-specific responses to the inter-leukin-6 (IL-6) family of cytokines are not well understood. Here, we evaluated the functionsof the two major signaling pathways, the signal transducers and activators of transcription 1and 3 (STAT1/3) and the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK, emanatingfrom the common signal transducer, gp130, in the gastrointestinal tract. Gp130757F mice, witha ‘knock-in’ mutation abrogating SHP2-Ras-ERK signaling, developed gastric adenomas bythree months of age. In contrast, mice harboring the reciprocal mutation ablating STAT1/3signaling (gp130∆STAT), or deficient in IL-6-mediated gp130 signaling (IL-6–/– mice), showed im-paired colonic mucosal wound healing. These gastrointestinal phenotypes are highly similarto the phenotypes exhibited by mice deficient in trefoil factor 1 (pS2/TFF1) and intestinal tre-foil factor (ITF)/TFF3, respectively, and corresponded closely with the capacity of the twopathways to stimulate transcription of the genes encoding pS2/TFF1 and ITF/TFF3. We pro-pose a model whereby mucosal wound healing depends solely on activation of STAT1/3,whereas gastric hyperplasia ensues when the coordinated activation of the STAT1/3 andSHP2-Ras-ERK pathways is disrupted.

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nuded areas (‘restitution’), increasing mucus production andblood flow, and re-establishing epithelial proliferation and dif-ferentiation programs. These processes are regulated by multi-functional polypeptides, including transforming growthfactor-β (TGF-β), the epidermal growth factor receptor ligandsand trefoil (TFF) peptides7. Although their regulation and mol-ecular mechanisms of action remain unclear, the cytoprotec-tive TFF peptides promote healing in response togastrointestinal injury in humans and animal models by in-hibiting apoptosis of epithelial cells and enhancing the barrierfunction of mucus8. In mammals, three distinct TFF genes en-code pS2/TFF1, spasmolytic polypeptide (SP)/TFF2 and intesti-nal trefoil factor (ITF)/TFF3, which are expressed by gastric-pitmucus cells, glandular mucus cells of the stomach and duode-num, and mucus-producing goblet cells in the small intestineand colon, respectively9. The IL-6 family of cytokines also havea pivotal role in maintaining the normal physiological func-tion of the gastrointestinal tract. IL-6 enhances the high-affinity immune response of the gut mucosa10, and IL-11 pro-tects the epithelial lining of the small intestine when exposedto cytoablative treatment11.

To dissect the molecular pathways regulating gastrointesti-nal epithelial cell homeostasis by IL-6 family cytokines in vivo,we generated gp130 ‘knock-in’ mutant mice (gp130757F) inca-pable of activating the SHP2-Ras-ERK pathway in response togp130 engagement. Notably, the intestinal phenotype ofgp130757F mice (gastric hyperplasia) is radically different fromthat observed in gp130∆STAT mice (they display exaggeratedacute intestinal colitis), in which the ability of gp130 to acti-vate the STAT1/3 pathway is abolished12. These two pheno-

types are phenocopied in mice deficient for either pS2/TFF1(gastric hyperplasia)13 or ITF/TFF-3 (exaggerated colitis in re-sponse to sodium dextran sulfate (DSS)-mediated epithelialdamage)14. Compound IL-6–/–IL-11Rα1–/– mice, which are effec-tively deficient in gut-specific gp130 signaling, exhibit en-hanced colitis in response to DSS but maintain normal gastricpathology. We therefore conclude that the integrity of thecolonic epithelium, in the face of environmental stress, islargely dependent on the magnitude of STAT1/3 signals.However, the maintenance of the integrity of the gastric ep-ithelium seems to depend on more subtle sensor mechanisms,which gauge the output of both the STAT1/3 and SHP2-Ras-ERK pathways in relation to each other.

STAT and SHP2-Ras-ERK pathway in gp130 mutantsWe generated gp130757F mice carrying the Y757F and V760A muta-tions in order to destroy the pY757xxV760 SHP2-binding do-main5, thereby abolishing the associated activation of theSHP2-Ras-ERK signaling cascade in response to IL-6 and IL-11(Fig. 1a). Homozygous gp130757F mice were born close to the ex-pected mendelian ratio (data not shown) and developed nor-mally into superficially healthy adults. Biochemical analysis ofgp130-mediated signaling in the livers of gp130757F mice, a clas-sical target organ for IL-6-family cytokines15, confirmed the se-lective impairment of IL-6-dependent activation andphosphorylation of the SHP2-Ras-ERK, but not the STAT1/3cascade (Fig. 1b). IL-6 induced the converse response ingp130∆STAT mice, which express a truncated version of gp130that lacks all YxxQ STAT-binding sites and are incapable oftransducing STAT1/3-mediated gene activation12. Furthermore,

Fig. 1 Altered intracellular signaling in mice with targeted gp130 mutations.a, Targeting strategy for the introduction of the gp130757F knock-in mutation.Cytoplasmic domain of mouse gp130 is depicted alongside the correspondinggenomic structure with exons numbered13 and the structure of the mutation ingp130∆STAT mice12. The targeting vector contains Y757RHQ760 to FRHA substitu-tions and the endogenous translational stop codon at position 918 followed bya ribosomal re-entry site and the sequence encoding neomycin resistance(IRESneo). A diagnostic digest with HindIII (H) yields a fragment of 4.5-kb fromthe wild-type (wt) allele and a 7.5-kb fragment from the targeted allele whenhybridized with a probe external to the targeting vector (*)12. S, SalI. TM, trans-membrane domain. b, Biochemical analysis of the intracellular signaling path-way in the liver of gp130 mutant mice. Mice were injected with IL-6 (5 µg, i.v.)and liver lysates were subsequently analyzed for phosphorylated species ofSHP2, the MAP kinases ERK1/2 and STAT3 by western-blot analysis. The mem-branes were reprobed for SHP2, ERK1/2 and STAT3, respectively, to assessequality of protein loading. c, Sustained activation of intracellular signaling pro-

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teins in the gastrointestinal tract of gp130 mutant mice. For the analysis of ERKactivation in the colon, mice were injected with IL-6 (5 µg, i.v.), whereas analy-sis of STAT3 activation in the antrum was done on IL-11-treated (5 µg, i.v.)mice. Lysates were analyzed by western blot for phosphorylated ERK1/2 orphosphorylated STAT3 and total ERK1/2 or STAT3, respectively.

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we observed sustained phosphorylation and activation of SHP2and ERK1/2 in tissues (including liver and colon) of IL-6-treated gp130∆STAT mice when compared with wild-type mice(Fig. 1b and c). Conversely, STAT3 phosphorylation was en-hanced in the liver and stomach of IL-6- or IL-11-treatedgp130757F mice compared with wild-type mice. This indicatesreciprocal negative regulation between the two signaling cas-cades emanating from gp130.

gp130757F mice develop gastric adenomasAutopsy of gp130757F mice revealed age-dependent enlargementof the stomach, proximal small intestine and spleen with anonset of 6–8 weeks (Fig. 2a and b). Histological examination re-vealed hyperproliferative lesions within the antropyloric mu-cosa, often circumferential, and resulting in gastric outlet

obstruction (Fig. 2c). The pseudopolypoid diffuse adenomatouslesions featured elongated pits and enlarged glandular struc-tures resulting from additional branching and interglandularbridging (Fig. 2d and e). The lesions contained fewer mucus-producing cells as judged by Alcian blue/periodic acid-Schiffstaining, and we observed an expanded mitotic compartmentas revealed by staining for proliferative-cell nuclear antigen(PCNA) (Fig. 2f–i). Frequently, infiltrating lymphocytes werehighly abundant in the thickened lamina propria and the in-traepithelial compartments (Fig. 2e).

We detected no gastric hyperplasia in age-matchedgp130∆STAT, IL-6–/– (ref. 16), IL-11Rα1–/– (ref. 17) or compound IL-6–/–IL-11Rα1–/– mice (Fig. 2k–o). As IL-6 and IL-11 are the IL-6-cytokine family members with documented intestinal activityin vivo10,11, IL-6–/–IL-11Rα1–/– mice not only provide a model for

Fig. 2 Histological analysis of gp130757F mice. a–c, Externalappearance of stomachs and spleens from 8-wk- (a and b) and6-mo- (c) old homozygous gp130757F mice and wild-type lit-termates. Arrows indicate the antropyloric hyperplasia lead-ing to nearly complete gastric obstruction in older mice (aand c). Scale bars, 1 cm. d–i, The antropyloric section of a 12-wk-old stomach shows severe hyperplasia and elongated pits(d) and an accumulation of infiltrating lymphocytes (e).Within the same 12-wk-old gp130757F mouse, PAS staining of anormal (f) and hyperplastic (g) region of the stomach shows amarked decrease in neutral mucins (dark pink stain) and dif-ferentiated cell types in the hyperplastic lesions. By contrast,the wide-ranging staining for PCNA in these regions (i) whencompared with unaffected regions (h) is indicative of a greatlyexpanded proliferative compartment. Du, duodenum; An,antrum. e is a magnification of box in d. j–o, Spontaneous for-mation of gastric adenomas is unique to gp130757F mice.Representative appearance of the stomach of age-matched,4-mo-old wild-type (j), gp130∆STAT (k), gp130757F (l), IL-6–/– (m),IL-11Rα1–/– (n) and IL-6–/–IL-11Rα1–/– mice (o). Arrows in c, eand l indicate adenomatous lesions in the antro-pyloric re-gion. Magnifications, ×20(d); ×200 (e,i); ×100 (f,g,h).

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impaired gp130 signaling in the gut (Fig. 2p), but also serve assurrogates for gp130-null mice, which die in utero18. We there-fore propose that the hyperproliferative lesions observed in thestomachs of gp130757F arise as a result of enhanced STAT3 activ-ity in the absence of a counter-acting signal from the SHP2-Ras-ERK pathway rather than from the complete absence of(IL-6- and IL-11-mediated) gp130 signaling.

Impaired gastrointestinal wound-healing in gp130∆STAT miceGp130∆STAT mice spontaneously developed intestinal ulcerationat sites associated with repeated mechanical trauma (gastric py-lorus and rectum)12, suggesting impaired epithelial woundhealing. In order to assess the capacity of intestinal woundhealing in these mice more directly, we administered DSS indrinking water, which induces reproducible acute colonic ep-ithelial injury with ulceration in wild-type mice14,19. DSS exertsdirect toxic effects on epithelial cells independently of the un-derlying gut immune system19,20, and its transient administra-tion provides an experimental system to assess the critical first

phase of wound healing. We observed more profound illnessand reduced survival in gp130∆STAT mice treated with 5% DSScompared with wild-type and gp130757F mutant mice (Fig. 3a).Moreover, and in marked contrast to wild-type and gp130757F

mutant mice, all remaining gp130∆STAT mice remained mori-bund after removal of DSS from the drinking water (data notshown), suggesting impaired intestinal wound healing ingp130∆STAT mice.

To identify the specific cytokine mediating the protectiveeffect on the colonic mucosa, we assessed the susceptibility ofgp130∆STAT, IL-6–/–, IL-11R1α–/– and IL-6–/–IL-11Rα1–/– mice toDSS-induced injury using a lower concentration (3.5%) of DSSto avoid death. IL-6–/–IL-11Rα1–/– and IL-6–/– mice developedfrank bloody diarrhea and severe weight loss comparable withthat observed in gp130∆STAT mice (Fig. 3b). The same treatmentresulted in less severe illness in wild-type and IL-11Rα1–/– mice,whereas gp130757F mice were completely resistant to DSS-associated signs of illness (Fig. 3b). Compared with wild-typeand IL-11Rα1–/– mice, the colonic mucosa of DSS-treated

Fig. 3 Increased susceptibility to DSS-mediated intestinal injury in 12–16-wk-old mice. a, Weight loss (expressed as percent of initial body weight) and asso-ciated death is greater after DSS treatment in gp130∆STAT mice (blue lines andsymbols) compared with wild-type (black) and gp130757F mice (red). b, Treatment with 3.5% DSS for 5 d results inincreased morbidity of gp130∆STAT

mice as assessed by weight loss, colonic blood content and crypt damage. Thisresponse is phenocopied in IL-6–/– and IL-6–/–IL-11Rα1–/– compound mice, whilethe susceptibility of IL-11Rα1–/– mice is comparable with that of wild-type mice.In contrast, gp130757F mice are completely resistant to DSS-induced colonic in-jury. c–h, Representative images of the distal colon following 5 d treatment with3.5% DSS of gp130∆STAT (d), IL-6–/– (f) and IL-6–/–IL-11Rα1–/– mice (h) showing ex-tensive mucosal injury, loss of surface epithelium and inflammation (arrows).Less severe damage is observed in sections of wild-type (c) and IL-11Rα1–/– mice(g) with areas of preserved microvillus architecture (arrowheads). Note absenceof histological damage in colon of gp130757F mice (e). Magnification, ×20.

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gp130∆STAT, IL-6–/–IL-11Rα1–/– and IL-6–/– mice showed consis-tently more severe erosion of the epithelium, which was asso-ciated with the presence of inflammatory cells; however, weobserved no tissue damage in the colons of gp130757F mice (Fig.3c–h). These data suggest a protective role of gp130-mediatedSTAT1/3 signaling on intestinal epithelium, reminiscent ofthe function of STAT3 in epithelial migration during epider-mal wound healing21. We conclude that IL-6 is likely to havean important role in the initial phase of intestinal woundhealing. Furthermore, endogenous IL-11, unlike pharmaco-logically administered IL-11 (ref. 11), seemed to have negligi-ble effects on the intestinal epithelium.

Reciprocal induction of TFF genes in gp130 mutantsThe gastric pathology of gp130757F mice is essentially pheno-copied in pS2/TFF1–/– mice13, which show severe hyperplasia ofthe mucosa of the gastric antrum and pylorus. In contrast, theimpaired wound-healing observed in gp130∆STAT and IL-6–/–

mice is highly reminiscent of exaggerated susceptibility toDSS-induced intestinal injury14 in mice carrying a disruptionin the gene encoding intestinal ITF/TFF3. We therefore as-sessed TFF protein concentrations and found that colonic lev-els of ITF/TFF3 were reduced in adult gp130∆STAT and IL-6–/–

mice but reproducibly elevated in gp130757F mice (Fig. 4a). Incontrast, gp130757F mice displayed a 75% reduction of gastricpS2/TFF1 levels when compared with wild-type mice (Fig. 4b).The colocalization of immunoreactive gp130 and pS2/TFF1suggests a cell-autonomous mechanism by which gp130-signaling regulates pS2/TFF1 levels in the stomach (Fig. 4c).However, gp130-mediated promotion of intestinal woundhealing in vivo seemed to correlate with the motogenic activ-ity of TFF proteins on mucosal cells in vitro. For instance, IL-6promoted migration of colon carcinoma–derived LIM1215cells to a similar extent as recombinant TFF (Fig. 5a), and thestimulatory effect of IL-6 on cellular migration of LIM 1215cells was refractory to treatment with neutralizing antibodiesagainst TFF (data not shown).

As shown above, there is a reciprocal correlation in the gas-trointestinal tract between activation of the SHP2-Ras-ERKand STAT1/3 cascades and pS2/TFF1 and ITF/TFF3 levels invivo. In order to investigate the requirement for the STATpathway to promote migration of LIM1215 cells (presumablythrough the production of their major TFF peptide ITF/TFF3),

we inserted cDNAs encoding the cytosolic domains of gp130fused to the ectodomain of the receptors for granulocytecolony–stimulating factor (GCSF) (Fig. 5b). Activation of thechimeric GCSFR/gp130 receptor (containing the wild-type cy-tosolic domain of gp130) promoted cellular migration to asimilar extent as ligand engagement of the endogenous wild-type gp130, whereas stimulation of GCSFR/∆STAT receptorsdid not transduce a motogenic response (Fig. 5a). Conversely,erythropoietin (Epo)-dependent activation of chimeric Epo-re-ceptor/gp130757F molecules (EpoR/757F) did not promote mi-gration of gastric epithelial immortalized gastric epithelium(IMGE5) cells22, which produce pS2/TFF1 as their predominantTFF peptide (data not shown). Activation of EpoR/gp130 re-ceptor, however, transduced a motogenic response.

Next, we used transient transfection assays of IMGE5 cells si-multaneously expressing full-length, wild-type EpoR/757F andGCSFR/∆STAT (Fig. 5b) to demonstrate a direct and reciprocaleffect of the two signaling cascades on the transcriptional acti-vation of the pS2/TFF1 and ITF/TFF3 genes. We performed theseassays in the presence of the soluble IL-6R α-chain (sIL-6R), assIL-6R-bound IL-6 results in gp130 activation in cells lackingthe transmembrane version of IL-6R, as observed in the gas-trointestinal tract23. The simultaneous activation of theSTAT1/3 and SHP2-Ras-ERK pathways, mediated through endogenous wild-type gp130 in response to IL-6, resulted in en-hanced luciferase (luc) activity from the pS2/TFF1-luc and theITF/TFF3-luc reporter constructs (Fig. 5c). Furthermore, activa-tion of GCSFR/∆STAT also resulted in induction of luciferase ac-tivity from the pS2/TFF1-luc reporter construct, whereasengagement of EpoR/757F activated luciferase activity from theITF/TFF3-luc reporter construct. In contrast, we observed no lig-and-dependent increase in luciferase activity following the reci-procal receptor activation; this suggests selective responsivenessof the pS2/TFF1 gene to the SHP2-Ras-ERK pathway and of theITF/TFF3 gene to the STAT1/3 cascade, respectively, in situa-tions where the reciprocal pathway cannot be engaged (Fig. 5c).Similiar results were obtained with the colon-carcinoma-de-rived SW1222 cells, and the corresponding promoterless lu-ciferase reporter constructs were unresponsive to gp130signaling (data not shown). Consistent with Ras-ERK-mediatedactivation of the pS2/TFF1 promoter24, GCSFR/∆STAT-mediatedpS2/TFF1-luc activation was impaired in the presence of the ERKinhibitor PD98059 and was not observed with a truncated

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Fig. 4 Tissue-specific regulation of trefoil peptide level in gp130 mutantmice. a and b, Peptide quantification by radioimmunoassay of intestinalITF/TFF3 (a) and gastric pS2/TFF1 (b) after extraction from the colon andantrum of 10–12-wk-old mice. *, P < 0.05, significantly different from

wild-type. c, Immunohistochemistry of the antrum of 5-wk-old gp130757F

mice. The positive stain for pS2/Tff1 (left) and gp130 (right) on adjacentsections colocalize towards the tip of the gastric pits (arrow).Magnification, ×100.

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pS2/TFF1-luc reporter plasmid lacking the AP1 binding sites atnucleotide position –281 (ref. 25) (Fig. 5d). Conversely,EpoR/757F-stimulated activation of the pTF2449 ITF/TFF3 re-porter plasmid was abolished in the presence of a dominantlyinterfering version of STAT3 (Fig. 5e). Gp130-mediated ITF/TFF3gene activation is likely to be mediated through STAT3, as a 5′-truncated ITF/TFF3 reporter plasmid (pTF2257), which lacks theα2-macroglobulin-type STAT3 binding site26 at nucleotide posi-tion –2287, remained unresponsive to gp130-dependent tran-scriptional activation. Similar results were obtained with thepRITF-luc reporter plasmids27 encompassing nucleotides –1671to +36 of the rat ITF/TFF3 promoter (data not shown).

DiscussionWe used mice with subtle knock-in mutations to dissect thephysiological functions of the two major intracellular signal-ing cascades emanating from gp130. We identified two classesof pathologies, namely those that are unique to the gp130 sig-naling-domain mutants and those that are duplicated in micewith null-mutations in genes encoding specific components ofthe IL-6 family cytokine network. For instance, gastric adeno-mas (gp130757F mice) and degenerative joint disease (gp130∆STAT

mice12) are not observed in IL-6–/–, IL-11Rα1–/– and IL-6–/–IL-11Rα1–/– mice or conditional gp130-null mice28. These patholo-gies have not been associated with mice simultaneouslytransgenic for IL-6 and sIL-6Rα, which display aberrant andubiquitous gp130 activation29,30. We therefore conclude thatthe gastric and joint pathologies are not generated in responseto the alterations in the signaling throughput of any one sig-

naling pathway per se, but arise as a result of disturbing the nor-mally simultaneous and coordinated activation of the STAT1/3and SHP2-Ras-ERK cascades (Fig. 6).

Enhanced STAT signaling appears to be the common mecha-nism triggering splenomegaly in IL-6–/–sIL-6Rα–/– transgen-ics29,30 and gp130757F mice6. Similarly, the absence ofSTAT1/3-mediated gp130 signaling resulted in enhanced sus-ceptibility to DSS-induced mucosal injury as well as impairedhigh-affinity immune and hepatic acute-phase responses asobserved in IL-6–/– (ref. 16) and gp130∆STAT mice12. Thus, pheno-types common to gp130-signaling domain mutants and mu-tants with disrupted gp130 signaling such as in IL-6–/– orIL-11Rα1–/– mice are likely to result from the altered signalingthroughput of the STAT1/3 cascade irrespective of that of theSHP2-Ras-ERK pathway.

Activation of epithelial-repair mechanisms by the pro-inflammatory cytokine IL-6 provides an early advantageouswound-healing response; however, paradoxically, IL-6 also per-petuates inflammation in chronic colitis models31 by providingSTAT3-mediated anti-apoptotic signals to lamina propria T cells23. Thus, in acute situations where antigens breach theepithelial barrier, IL-6 enhances mucosal repair by epithelialrestitution via increased TFF expression, while simultaneouslyfacilitating inhibition of apoptosis in the lymphocyte compart-ment and thereby promoting immune clearance.

The TFF gene-encoded mucus cell–secreted peptides are es-sential regulators for the restitution of the gastrointestinal mu-cosa and transduce similar biological activities when appliedectopically. Hence, we interpret the gastrointestinal abnormal-

Fig. 5 Reciprocal responsiveness of genes encod-ing pS2/TFF1 and ITF/TFF3 to the SHP2-Ras-ERK andSTAT pathways activated by gp130-signaling do-main mutants. a, IL-6 promotes migration of coloncarcinoma–derived LIM1215 cells via the STAT1/3pathway in vitro. IL-6 transduces motogenic activa-tion to a similar extent as stimulation with recombi-nant TFF2 (left columns). GCSF-dependentactivation of introduced GCSFR/wild-type chimericreceptors (G/gp130), but not of introducedGCSFR/gp130∆STAT chimeras (G/∆STAT), promotesmigration with similar effectiveness as IL-6-mediated stimulation of theendogenous full-length gp130. b, Chimeric receptors encoding the ex-tracellular domain of the Epo-receptor (EpoR) and GCSF-receptor(GCSFR) fused to the intracellular domains encoding mutant forms ofgp130 were stably introduced in gastric IMGE5 cells in order to permitligand-dependent activation of wild-type (in response to IL-6),gp130∆STAT (in response to GCSF) or gp130757F (in response to Epo) withinthe same cell population. c, Specificity of transcriptional activation ofheterologous TFF-luc reporter constructs in gastric epithelial IMGE5 cells.Activation of the pS2/TFF1 promoter plasmid requires gp130-mediatedactivation of the SHP2-Ras-ERK pathway, as no reporter activity occursafter activation of the EpoR/gp130757F chimeric receptor. Conversely, ac-

tivation of the ITF/TFF3 promoter (–2449 to –6) depends on gp130-mediated activation of the STAT pathway, which does not occur follow-ing activation of the GCSFR/gp130∆STAT receptor. d, SHP2-Ras-ERK-dependent activation of the pS2/TFF1 promoter is dependent on se-quences between –496 (pGV496) and –243 (pGV243), which include anAP-1 site at position –281 (ref. 25), and is sensitive to the presence of theERK inhibitor PD98059 (PD). e, STAT-dependent activation of the mouseITF/TFF3 promoter (–2449 to –6; pTF2449) is inhibited in the presenceof a dominant interfering version of STAT3, whereas a 5′-truncated pro-moter construct (–2257 to –6; pTF2257) lacking the α2-macroglobulin-type STAT3 binding26 site is unresponsive to gp130 signaling. For a andc–e: �, control; , IL-6 + sIL-6Rα; �, TFF; �, Epo; �, GCSF.

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ities in the two gp130 mutant strains to result from inappropri-ate levels of TFF peptide at specific sites. In particular, colonicITF/TFF3 levels correlate with reduced or enhanced susceptibil-ity to DSS-mediated colonic injury in gp130∆STAT and gp130757F

mice, respectively. By contrast, the gastric mucosa seems to beparticularly sensitive to threshold concentrations of pS2/TFF1,which are too low in gp130757F mice to provide the signal nor-mally required to promote differentiation of mucus cells in thegastric pits32. In the complete absence of pS2/TFF1 expressionin the mouse, the gastric adenomas may further progress tocarcinomas13. The implied role for pS2/TFF1 as a gastric-specifictumor-suppressor gene is further supported by the observationthat approximately half of all human gastric cancers displaysomatic loss of heterozygosity at 21q22.3, encompassing thecluster of three human TFF genes33.

In line with our proposed role for pS2/TFF1 in mediating thegastric phenotype in gp130757F mice, the histological appear-ance and exclusive spatial restriction of the lesions to the dis-tal glandular part of the stomach differs from the gastricpathologies observed in mice with modified expression pat-terns of growth factors. Transgenic mice over-expressing TGF-α, for instance, develop severe adenomatous hypertrophy ofthe gastric mucosa that is overtly analogous to the prominentgiant rugal folds characteristic of Ménétrier’s disease in hu-mans34. By contrast, the glandular hyperplasia in mice with ahemi-allelic loss of TGF-β1 is characterized by infiltration ofthe muscularis mucosa with glandular tissue similar to humangastritis cystica profunda35. Notably, the phenotype in the lat-ter mutant mice is thought to arise secondarily to multifocalinflammation, which is somewhat reminiscent of the lympho-cytic infiltration observed in gp130757F mice. Splenomegaly,which is associated with enhanced B- and T-cell proliferationand/or cell survival, is also observed in mice which carry a

functionally equivalent SHP2-binding impairmentmutation (Y759F) in a mouse-human gp130 fusionprotein; however, no gastric pathology has beenreported in gp130759F mice6. Hence, it is highly un-likely that the completely penetrant gastric pheno-type reported here in gp130757F mice results from aTh1-biased immune-cell development common togp130759F (ref. 6) and gp130757F mice. Instead, thegastric hyperplasia in gp130757F mice may reflect aprimary response of the epithelium to subtle dif-ferences in the gp130-signaling throughput be-tween the two gp130-specific SHP2-Ras-ERKsignaling impairment mutants.

Collectively, our data show that tissue-specific in-terpretation of the net gp130-signaling throughputdetermines organ-specific responses to the IL-6 fam-ily cytokines in vivo. The underlying mechanism ofcoordinated activation of the two gp130 signalingcascades is reinforced by our finding of reciprocalnegative regulation between the STAT1/3 and SHP2-Ras-ERK pathways. The resulting negative feedbackloops seem to converge on the gp130-receptor com-plex, as gp130∆STAT mice are characterized by im-paired induction of the suppressor of cytokinesignaling 1 (SOCS-1) protein12, which reduces kinase-dependent receptor Y-phosphorylation andmediates ubiquitination of receptor associated Jakkinases36. Similarly, enhanced STAT3 activation ingp130757F mice is likely to result from impaired

SOCS-3 binding to the mutated 757F residue37,38, as well as fromthe associated decrease in SHP2-specific Y-phosphatase activ-ity39. Hence, simultaneous and balanced activation of the twosignaling cascades is not only essential for mediating tissue-spe-cific activities of IL-6 family cytokines, but has the potential forfine tuning the balance between negative and positive signalsduring biological responses to these cytokines.

MethodsMice. The bicistronic p(757F) targeting vector contained exon 17 (ref. 27) encoding amino acids 672–917 with the Y757F (TAC to TTC) andV760A (GTG to GCG) substitutions and an introduced AvrII restriction siteas part of the endogenous stop codon. A cDNA encoding neomycin resis-tance, preceded by an internal ribosomal entry site (IRES), was introducedinto the AvrII site as a XbaI-NheI fragment; the p(757F) vector also con-tained 0.8 kb and 4.3 kb of sequence homologous to the final intron andthe 3′-flanking sequence, respectively12. Gene targeting in W9.5 ES cells(129/Sv) was done with the SalI-linearized p(757F) vector, correctly tar-geted ES clones were identified from the hybridization pattern of HindIIIdigested DNA, and germ-line chimeras were obtained as described12.Compound IL-6–/–IL-11Rα1–/– mice were generated from IL-6–/– (ref. 16)and IL-11Rα1–/– (ref. 17) mice, and the construction of gp130∆STAT micehas been described4. All mouse strains were kept on a mixed 129/BL6background. All animal experiments were approved by the LudwigInstitute for Cancer Research/Department of Surgery, Royal MelbourneHospital Animal Experimentation Ethics Committee.

Analysis of tissue lysates. Liver lysates (3 mg), prepared at timed inter-vals after an intravenous injection of IL-6 (5 µg) or saline, were immuno-precipitated with SHP2 or STAT3 antisera (Santa Cruz Biotechnology,Santa Cruz, California). Samples were resolved by SDS–PAGE and the re-sulting blots were probed with anti-phosphotyrosine (UBI, Lake Placid,New York), followed by SHP2 or STAT3 antibodies to assess protein load-ing12. Phosphorylated STAT3 in the stomach was detected by westernblotting with a phospho-STAT3 antibody (New England BioLabs,Berverly, Massachusetts) on lysates (50 µg) prepared from antra of mice

Fig. 6 Schematic of the consequences of impairing the reciprocal negative regulation be-tween the SHP2-Ras-ERK and STAT1/3 pathways. The coordinated and simultaneous signalingthrough the two pathways that occurs in wild-type mice is unaffected in situations of continu-ous ligand activation (for instance, in IL-6/sIL-6Rα double transgenic (tg) mice) or in the ab-sence of ligand-dependent gp130 activation (for instance, in IL-6–/– or IL-11Rα1–/– mice).However, altering the overall signaling throughput in these mutant mouse strains results in spe-cific phenotypes16–18,27–29. A subset of these pathologies phenocopied in gp130-signaling do-main mutants6,12, suggesting that they result from altering the signaling throughput throughthe STAT1/3 pathway. In contrast, phenotypes specific to gp130-signaling domain mutants arelikely to arise from the net effect of unbalancing the coordinated activation of the STAT1/3 andSHP2-Ras-ERK signaling cascades. In particular, neither gastric adenomas nor joint disease man-ifest in situations of simultaneous lack or hyper-activation of both pathways (observed in IL-6–/–,IL-11Rα1–/– or IL-6+sIL-6Rα tg mice), but only occur in situations of activating one pathway tothe exclusion of the other in gp130757F or gp130∆STAT mice.

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after an intravenous injection of IL-11 (5 µg). Active ERK kinase was iden-tified on western blots of liver or colon lysates (50 µg) reacted with aphospho-ERK1/2-specific antibody (New England Biolabs), and the levelof total ERK1/2 protein was assessed by blotting with ERK1/2 antibodies(Santa Cruz Biotechnology).

DSS model. Mice received DSS (40,000 kD; ICN Biochemicals, Irvine,California) in their drinking water for 5 d and were weighed every otherday. Colitis was assessed by assigning histological scores to 4 indepen-dent colonic sections per mouse20, and gross colonic blood content wasscored using a scale from 0 to 3: 0, no blood; 1, blood in < 1/3 of thecolon; 2, blood in < 2/3 of the colon; 3, blood in > 2/3 of the colon.

Histology, immunohistochemistry and TFF determination. Paraffin-embedded sections of formalin-fixed tissues were stained with hema-toxylin. Mucin was visualized by periodic acid/Schiff staining.Proliferating cells were identified by immunohistochemistry with an anti-body directed against the nuclear proliferation antigen PCNA (Sigma, St.Louis, Missouri). All sections were counterstained with eosin.Immunohistochemical analysis was performed with an antiserum directedagainst human pS2/Tff1 (ref. 40) or gp130 (Santa Cruz Biotechnology).The concentration of TFF in protein extracts from the antrum or colonwas determined with a radioimmunoassay specific for pS2/TFF1 (ref. 41)and ITF/TFF3 (ref. 42).

Cell lines. Gastric epithelial IMGE5 cells22, expressing a temperature sen-sitive mutant of large T antigen, were maintained at 33 °C in RPMI sup-plemented with 10% FCS, whereas colon carcinoma–derived LIM1215and SW1222 cells were propagated at 37 °C in DME containing 10% FCS.For each of the receptor constructs, 3 independent cell lines were derivedfollowing stable expression of plasmids encoding, either individually or inthe indicated combination, the chimeric receptors EpoR/wild-type (ref. 38), GCSF/wild-type (ref. 42), EpoR/gp130757F (ref. 38) orGCSF/gp130∆STAT (ref. 43) together with the resistance marker pPGKpuro.

In vitro wound-healing assay. Monolayers of LIM1215 cells were main-tained in serum-free medium containing 0.2 µg/ml mitomycin C.Gastrointestinal epithelial cells may not ubiquitously express the transmem-brane IL-6Rα ligand-binding subunit, but IL-6 in the presence of sIL-6Rα ini-tiates gp130-dependent signaling in cells irrespective of the presence ofendogenous IL-6Rα. Because of this, we stimulated cells with IL-6 plus sIL-6Rα (500 ng/ml each12), GCSF (100 ng/ml), TFF2 (0.5 mg/ml) or vehicle.Forty h later, photomicrographs were taken and the average migration dis-tance of cells relative to the edge of the wound was calculated from mea-surements of 100 cells in each of 4 randomly selected fields.

Reporter assay. IMGE-5 cells in 24-well plates were transfected with 0.3 µg of luciferase (luc) reporter construct, encompassing various re-gions of the mouse pS2/TFF1 (–2192 to +28; –496 to +28; –243 to +28;ref 18) or ITF/TFF3 (encompassing nucleotides –2449 to +28 relative tothe transcriptional start site25) (–2449 to –6; –2257 to –6) promoters andinserted into pGL3basic (Promega, Madison, Wisconsin), together with 2 µg of the Srα-β-galactosidase plasmid using FuGENE6 (Roche MolecularBiochemicals, Castle Hill, Australia). Cultures were maintained in 1% FCSfor 48 h in the presence of IL-6 plus sIL-6Rα (500 ng/ml), Epo (25 U/ml,Janssen-Cilag, Switzerland), GCSF (100 ng/ml) or 10 µM of the ERK in-hibitor PD98059 (ref. 42) before lysis with Reporter Lysis Buffer(Promega). Lysates were assayed for luciferase activity, which was nor-malized according to the β-galactosidase activity12.

AcknowledgmentsWe thank T. Terada for the tff-luciferase promoter constructs pGV2192,pGV496 and pGV243; D. Podolsky for pRITF-luc; S. Nicholson for the expres-sion construct encoding EpoR/gp130757F; L. Thim for recombinant humanTFF2; Amgen (Thousand Oaks, California) for recombinant human GCSFand IL-6; R. Simpson for sIL-6R; L. Robb and A. Ramsay for IL-11Rα1–/– andIL-6–/– mice; V. Feakes, J. Moverley and M. Howlett for histology; andJ.Stickland for photography.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 11 JULY; ACCEPTED 21 AUGUST 2002.

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Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene...

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