Reciprocal regulation of gastrointestinal homeostasis by ... - Nature

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© 2002 Nature Publishing Group http://www.nature.com/naturemedicine

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 1

Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria, Australia Department of Medicine, University of Melbourne, Western Hospital, Footscray, Victoria, Australia 3 Department of Pathology, Peter MacCallum Institute, East Melbourne, Victoria, Australia 4 Laboratoire 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] 2

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

The intracellular signaling mechanisms that specify tissue-specific responses to the interleukin-6 (IL-6) family of cytokines are not well understood. Here, we evaluated the functions of the two major signaling pathways, the signal transducers and activators of transcription 1 and 3 (STAT1/3) and the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK, emanating from the common signal transducer, gp130, in the gastrointestinal tract. Gp130757F mice, with a ‘knock-in’ mutation abrogating SHP2-Ras-ERK signaling, developed gastric adenomas by three months of age. In contrast, mice harboring the reciprocal mutation ablating STAT1/3 signaling (gp130∆STAT), or deficient in IL-6-mediated gp130 signaling (IL-6–/– mice), showed impaired colonic mucosal wound healing. These gastrointestinal phenotypes are highly similar to the phenotypes exhibited by mice deficient in trefoil factor 1 (pS2/TFF1) and intestinal trefoil factor (ITF)/TFF3, respectively, and corresponded closely with the capacity of the two pathways to stimulate transcription of the genes encoding pS2/TFF1 and ITF/TFF3. We propose 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 and SHP2-Ras-ERK pathways is disrupted.

Cytokines are characterized both by their functional redundancy and tissue-specific activities1. Redundancy among members of the interleukin-6 (IL-6) cytokine family, which comprises IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor and cardiotrophin-1, is attributed to the common use of the transmembrane receptor β-subunit gp130. The tissue-specific activities of these cytokines are thought to arise from differential responsiveness of target cells to individual signaling cascades that emanate from discrete signaling modules in the cytosolic domain of the receptors2. In gp130, at least two such modules have been characterized. One encompasses the four membrane-distal phospho-tyrosine (pY) binding sites for the SH2-domain of the latent transcription factors, the signal transducer and activator of transcription 1 (STAT1) and STAT3, with STAT3 having the predominant role in vitro and in vivo3. The other pathway comprises the membrane-proximal pY757 residue in gp130 responsible for engagement of the cytoplasmic Srchomology tyrosine phosphatase (SHP2) and subsequent activation of the Ras-ERK pathway3,4. Because both modules are necessary and sufficient for initiation of the corresponding intracellular signaling cascades, phenylalanine substitution of NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002

the appropriate Y-residues in gp130 completely abolishes activation of the associated pathways5. After IL-6 and IL-11 bind to their respective ligand-binding α-chains, recruitment and homodimerization of gp130 receptor β-chains activates intracellular signaling. Meanwhile, all other IL-6 cytokine family members activate intracellular signaling through heterodimerization of gp130 with other structurally related β-chains3,4. In either case, β-chain dimerization results in the coordinated and simultaneous activation of the STAT1/3 and SHP2-Ras-ERK pathways which, at least in vitro, appear to transduce activities regulating cellular differentiation and apoptosis (STAT1/3) and mitogenesis (SHP2-Ras-ERK). Hence, it has been suggested that the two pathways may maintain cellular homeostasis in some dynamic cellular systems, such as the immune system, by balancing positive and negative signals6. The physiological function of the gastrointestinal mucosa is dependent on the persistent integrity of the intestinal epithelium, which is continually challenged by the harsh lumenal contents of the gut. This challenge is met by coordinated repair mechanisms that have the capacity to rapidly restore mucosal integrity by stimulating migration of epithelial cells over de1089


ARTICLES nuded areas (‘restitution’), increasing mucus production and blood flow, and re-establishing epithelial proliferation and differentiation programs. These processes are regulated by multifunctional polypeptides, including transforming growth factor-β (TGF-β), the epidermal growth factor receptor ligands and trefoil (TFF) peptides7. Although their regulation and molecular mechanisms of action remain unclear, the cytoprotective TFF peptides promote healing in response to gastrointestinal injury in humans and animal models by inhibiting apoptosis of epithelial cells and enhancing the barrier function of mucus8. In mammals, three distinct TFF genes encode pS2/TFF1, spasmolytic polypeptide (SP)/TFF2 and intestinal trefoil factor (ITF)/TFF3, which are expressed by gastric-pit mucus cells, glandular mucus cells of the stomach and duodenum, and mucus-producing goblet cells in the small intestine and colon, respectively9. The IL-6 family of cytokines also have a pivotal role in maintaining the normal physiological function of the gastrointestinal tract. IL-6 enhances the highaffinity immune response of the gut mucosa10, and IL-11 protects the epithelial lining of the small intestine when exposed to cytoablative treatment11. To dissect the molecular pathways regulating gastrointestinal epithelial cell homeostasis by IL-6 family cytokines in vivo, we generated gp130 ‘knock-in’ mutant mice (gp130757F) incapable of activating the SHP2-Ras-ERK pathway in response to gp130 engagement. Notably, the intestinal phenotype of gp130757F mice (gastric hyperplasia) is radically different from that observed in gp130∆STAT mice (they display exaggerated acute intestinal colitis), in which the ability of gp130 to activate the STAT1/3 pathway is abolished12. These two pheno-

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types are phenocopied in mice deficient for either pS2/TFF1 (gastric hyperplasia)13 or ITF/TFF-3 (exaggerated colitis in response to sodium dextran sulfate (DSS)-mediated epithelial damage)14. Compound IL-6–/–IL-11Rα1–/– mice, which are effectively deficient in gut-specific gp130 signaling, exhibit enhanced colitis in response to DSS but maintain normal gastric pathology. We therefore conclude that the integrity of the colonic epithelium, in the face of environmental stress, is largely dependent on the magnitude of STAT1/3 signals. However, the maintenance of the integrity of the gastric epithelium seems to depend on more subtle sensor mechanisms, which gauge the output of both the STAT1/3 and SHP2-RasERK pathways in relation to each other. STAT and SHP2-Ras-ERK pathway in gp130 mutants We generated gp130757F mice carrying the Y757F and V760A mutations in order to destroy the pY757xxV760 SHP2-binding domain5, thereby abolishing the associated activation of the SHP2-Ras-ERK signaling cascade in response to IL-6 and IL-11 (Fig. 1a). Homozygous gp130757F mice were born close to the expected mendelian ratio (data not shown) and developed normally into superficially healthy adults. Biochemical analysis of gp130-mediated signaling in the livers of gp130757F mice, a classical target organ for IL-6-family cytokines15, confirmed the selective impairment of IL-6-dependent activation and phosphorylation of the SHP2-Ras-ERK, but not the STAT1/3 cascade (Fig. 1b). IL-6 induced the converse response in gp130∆STAT mice, which express a truncated version of gp130 that lacks all YxxQ STAT-binding sites and are incapable of transducing STAT1/3-mediated gene activation12. Furthermore,

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c 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 corresponding genomic structure with exons numbered13 and the structure of the mutation in gp130∆STAT mice12. The targeting vector contains Y757RHQ760 to FRHA substitutions and the endogenous translational stop codon at position 918 followed by a ribosomal re-entry site and the sequence encoding neomycin resistance (IRESneo). A diagnostic digest with HindIII (H) yields a fragment of 4.5-kb from the wild-type (wt) allele and a 7.5-kb fragment from the targeted allele when hybridized with a probe external to the targeting vector (*)12. S, SalI. TM, transmembrane domain. b, Biochemical analysis of the intracellular signaling pathway 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 of SHP2, the MAP kinases ERK1/2 and STAT3 by western-blot analysis. The membranes were reprobed for SHP2, ERK1/2 and STAT3, respectively, to assess equality of protein loading. c, Sustained activation of intracellular signaling pro1090

teins in the gastrointestinal tract of gp130 mutant mice. For the analysis of ERK activation in the colon, mice were injected with IL-6 (5 µg, i.v.), whereas analysis 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 or phosphorylated STAT3 and total ERK1/2 or STAT3, respectively. NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002

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we observed sustained phosphorylation and activation of SHP2 and ERK1/2 in tissues (including liver and colon) of IL-6treated gp130∆STAT mice when compared with wild-type mice (Fig. 1b and c). Conversely, STAT3 phosphorylation was enhanced in the liver and stomach of IL-6- or IL-11-treated gp130757F mice compared with wild-type mice. This indicates reciprocal negative regulation between the two signaling cascades emanating from gp130. gp130757F mice develop gastric adenomas Autopsy of gp130757F mice revealed age-dependent enlargement of the stomach, proximal small intestine and spleen with an onset of 6–8 weeks (Fig. 2a and b). Histological examination revealed hyperproliferative lesions within the antropyloric mucosa, often circumferential, and resulting in gastric outlet NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002

Fig. 2 Histological analysis of gp130757F mice. a–c, External appearance of stomachs and spleens from 8-wk- (a and b) and 6-mo- (c) old homozygous gp130757F mice and wild-type littermates. Arrows indicate the antropyloric hyperplasia leading to nearly complete gastric obstruction in older mice (a and c). Scale bars, 1 cm. d–i, The antropyloric section of a 12wk-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 a normal (f) and hyperplastic (g) region of the stomach shows a marked decrease in neutral mucins (dark pink stain) and differentiated cell types in the hyperplastic lesions. By contrast, the wide-ranging staining for PCNA in these regions (i) when compared with unaffected regions (h) is indicative of a greatly expanded proliferative compartment. Du, duodenum; An, antrum. e is a magnification of box in d. j–o, Spontaneous formation 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, e and l indicate adenomatous lesions in the antro-pyloric region. Magnifications, ×20(d); ×200 (e,i); ×100 (f,g,h).

obstruction (Fig. 2c). The pseudopolypoid diffuse adenomatous lesions featured elongated pits and enlarged glandular structures resulting from additional branching and interglandular bridging (Fig. 2d and e). The lesions contained fewer mucusproducing cells as judged by Alcian blue/periodic acid-Schiff staining, and we observed an expanded mitotic compartment as revealed by staining for proliferative-cell nuclear antigen (PCNA) (Fig. 2f–i). Frequently, infiltrating lymphocytes were highly abundant in the thickened lamina propria and the intraepithelial compartments (Fig. 2e). We detected no gastric hyperplasia in age-matched gp130∆STAT, IL-6–/– (ref. 16), IL-11Rα1–/– (ref. 17) or compound IL6–/–IL-11Rα1–/– mice (Fig. 2k–o). As IL-6 and IL-11 are the IL-6cytokine family members with documented intestinal activity in vivo10,11, IL-6–/–IL-11Rα1–/– mice not only provide a model for 1091

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Fig. 3 Increased susceptibility to DSS-mediated intestinal injury in 12–16-wkold mice. a, Weight loss (expressed as percent of initial body weight) and associated death is greater after DSS treatment in gp130∆STAT mice (blue lines and symbols) 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. This response is phenocopied in IL-6–/– and IL-6–/–IL-11Rα1–/– compound mice, while the susceptibility of IL-11Rα1–/– mice is comparable with that of wild-type mice. In contrast, gp130757F mice are completely resistant to DSS-induced colonic injury. c–h, Representative images of the distal colon following 5 d treatment with 3.5% DSS of gp130∆STAT (d), IL-6–/– (f) and IL-6–/–IL-11Rα1–/– mice (h) showing extensive 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 absence of histological damage in colon of gp130757F mice (e). Magnification, ×20.

impaired gp130 signaling in the gut (Fig. 2p), but also serve as surrogates for gp130-null mice, which die in utero18. We therefore propose that the hyperproliferative lesions observed in the stomachs of gp130757F arise as a result of enhanced STAT3 activity in the absence of a counter-acting signal from the SHP2Ras-ERK pathway rather than from the complete absence of (IL-6- and IL-11-mediated) gp130 signaling. Impaired gastrointestinal wound-healing in gp130∆STAT mice Gp130∆STAT mice spontaneously developed intestinal ulceration at sites associated with repeated mechanical trauma (gastric pylorus and rectum)12, suggesting impaired epithelial wound healing. In order to assess the capacity of intestinal wound healing in these mice more directly, we administered DSS in drinking water, which induces reproducible acute colonic epithelial injury with ulceration in wild-type mice14,19. DSS exerts direct toxic effects on epithelial cells independently of the underlying gut immune system19,20, and its transient administration provides an experimental system to assess the critical first 1092

phase of wound healing. We observed more profound illness and reduced survival in gp130∆STAT mice treated with 5% DSS compared 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 moribund after removal of DSS from the drinking water (data not shown), suggesting impaired intestinal wound healing in gp130∆STAT mice. To identify the specific cytokine mediating the protective effect on the colonic mucosa, we assessed the susceptibility of gp130∆STAT, IL-6–/–, IL-11R1α–/– and IL-6–/–IL-11Rα1–/– mice to DSS-induced injury using a lower concentration (3.5%) of DSS to avoid death. IL-6–/–IL-11Rα1–/– and IL-6–/– mice developed frank bloody diarrhea and severe weight loss comparable with that observed in gp130∆STAT mice (Fig. 3b). The same treatment resulted in less severe illness in wild-type and IL-11Rα1–/– mice, whereas gp130757F mice were completely resistant to DSSassociated signs of illness (Fig. 3b). Compared with wild-type and IL-11Rα1–/– mice, the colonic mucosa of DSS-treated NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002


ARTICLES gp130∆STAT, IL-6–/–IL-11Rα1–/– and IL-6–/– mice showed consistently more severe erosion of the epithelium, which was associated with the presence of inflammatory cells; however, we observed no tissue damage in the colons of gp130757F mice (Fig. 3c–h). These data suggest a protective role of gp130-mediated STAT1/3 signaling on intestinal epithelium, reminiscent of the function of STAT3 in epithelial migration during epidermal wound healing21. We conclude that IL-6 is likely to have an important role in the initial phase of intestinal wound healing. Furthermore, endogenous IL-11, unlike pharmacologically administered IL-11 (ref. 11), seemed to have negligible effects on the intestinal epithelium. Reciprocal induction of TFF genes in gp130 mutants The gastric pathology of gp130757F mice is essentially phenocopied in pS2/TFF1–/– mice13, which show severe hyperplasia of the mucosa of the gastric antrum and pylorus. In contrast, the impaired wound-healing observed in gp130∆STAT and IL-6–/– mice is highly reminiscent of exaggerated susceptibility to DSS-induced intestinal injury14 in mice carrying a disruption in the gene encoding intestinal ITF/TFF3. We therefore assessed TFF protein concentrations and found that colonic levels of ITF/TFF3 were reduced in adult gp130∆STAT and IL-6–/– mice but reproducibly elevated in gp130757F mice (Fig. 4a). In contrast, gp130757F mice displayed a 75% reduction of gastric pS2/TFF1 levels when compared with wild-type mice (Fig. 4b). The colocalization of immunoreactive gp130 and pS2/TFF1 suggests a cell-autonomous mechanism by which gp130signaling regulates pS2/TFF1 levels in the stomach (Fig. 4c). However, gp130-mediated promotion of intestinal wound healing in vivo seemed to correlate with the motogenic activity of TFF proteins on mucosal cells in vitro. For instance, IL-6 promoted migration of colon carcinoma–derived LIM1215 cells to a similar extent as recombinant TFF (Fig. 5a), and the stimulatory effect of IL-6 on cellular migration of LIM 1215 cells was refractory to treatment with neutralizing antibodies against TFF (data not shown). As shown above, there is a reciprocal correlation in the gastrointestinal tract between activation of the SHP2-Ras-ERK and STAT1/3 cascades and pS2/TFF1 and ITF/TFF3 levels in vivo. In order to investigate the requirement for the STAT pathway to promote migration of LIM1215 cells (presumably through the production of their major TFF peptide ITF/TFF3),

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Fig. 4 Tissue-specific regulation of trefoil peptide level in gp130 mutant mice. a and b, Peptide quantification by radioimmunoassay of intestinal ITF/TFF3 (a) and gastric pS2/TFF1 (b) after extraction from the colon and antrum of 10–12-wk-old mice. *, P < 0.05, significantly different from NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002

we inserted cDNAs encoding the cytosolic domains of gp130 fused to the ectodomain of the receptors for granulocyte colony–stimulating factor (GCSF) (Fig. 5b). Activation of the chimeric GCSFR/gp130 receptor (containing the wild-type cytosolic domain of gp130) promoted cellular migration to a similar extent as ligand engagement of the endogenous wildtype gp130, whereas stimulation of GCSFR/∆STAT receptors did not transduce a motogenic response (Fig. 5a). Conversely, erythropoietin (Epo)-dependent activation of chimeric Epo-receptor/gp130757F molecules (EpoR/757F) did not promote migration of gastric epithelial immortalized gastric epithelium (IMGE5) cells22, which produce pS2/TFF1 as their predominant TFF peptide (data not shown). Activation of EpoR/gp130 receptor, however, transduced a motogenic response. Next, we used transient transfection assays of IMGE5 cells simultaneously expressing full-length, wild-type EpoR/757F and GCSFR/∆STAT (Fig. 5b) to demonstrate a direct and reciprocal effect of the two signaling cascades on the transcriptional activation of the pS2/TFF1 and ITF/TFF3 genes. We performed these assays in the presence of the soluble IL-6R α-chain (sIL-6R), as sIL-6R-bound IL-6 results in gp130 activation in cells lacking the transmembrane version of IL-6R, as observed in the gastrointestinal tract23. The simultaneous activation of the STAT1/3 and SHP2-Ras-ERK pathways, mediated through endogenous wild-type gp130 in response to IL-6, resulted in enhanced luciferase (luc) activity from the pS2/TFF1-luc and the ITF/TFF3-luc reporter constructs (Fig. 5c). Furthermore, activation of GCSFR/∆STAT also resulted in induction of luciferase activity from the pS2/TFF1-luc reporter construct, whereas engagement of EpoR/757F activated luciferase activity from the ITF/TFF3-luc reporter construct. In contrast, we observed no ligand-dependent increase in luciferase activity following the reciprocal receptor activation; this suggests selective responsiveness of the pS2/TFF1 gene to the SHP2-Ras-ERK pathway and of the ITF/TFF3 gene to the STAT1/3 cascade, respectively, in situations where the reciprocal pathway cannot be engaged (Fig. 5c). Similiar results were obtained with the colon-carcinoma-derived SW1222 cells, and the corresponding promoterless luciferase reporter constructs were unresponsive to gp130 signaling (data not shown). Consistent with Ras-ERK-mediated activation of the pS2/TFF1 promoter24, GCSFR/∆STAT-mediated pS2/TFF1-luc activation was impaired in the presence of the ERK inhibitor PD98059 and was not observed with a truncated

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wild-type. c, Immunohistochemistry of the antrum of 5-wk-old gp130757F mice. The positive stain for pS2/Tff1 (left) and gp130 (right) on adjacent sections colocalize towards the tip of the gastric pits (arrow). Magnification, ×100. 1093

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Fig. 5 Reciprocal responsiveness of genes encoding pS2/TFF1 and ITF/TFF3 to the SHP2-Ras-ERK and STAT pathways activated by gp130-signaling domain mutants. a, IL-6 promotes migration of colon carcinoma–derived LIM1215 cells via the STAT1/3 pathway in vitro. IL-6 transduces motogenic activation to a similar extent as stimulation with recombinant TFF2 (left columns). GCSF-dependent activation of introduced GCSFR/wild-type chimeric receptors (G/gp130), but not of introduced GCSFR/gp130∆STAT chimeras (G/∆STAT), promotes migration with similar effectiveness as IL-6-mediated stimulation of the endogenous full-length gp130. b, Chimeric receptors encoding the extracellular domain of the Epo-receptor (EpoR) and GCSF-receptor (GCSFR) fused to the intracellular domains encoding mutant forms of gp130 were stably introduced in gastric IMGE5 cells in order to permit ligand-dependent activation of wild-type (in response to IL-6), gp130∆STAT (in response to GCSF) or gp130757F (in response to Epo) within the same cell population. c, Specificity of transcriptional activation of heterologous TFF-luc reporter constructs in gastric epithelial IMGE5 cells. Activation of the pS2/TFF1 promoter plasmid requires gp130-mediated activation of the SHP2-Ras-ERK pathway, as no reporter activity occurs after activation of the EpoR/gp130757F chimeric receptor. Conversely, ac-

pS2/TFF1-luc reporter plasmid lacking the AP1 binding sites at nucleotide position –281 (ref. 25) (Fig. 5d). Conversely, EpoR/757F-stimulated activation of the pTF2449 ITF/TFF3 reporter plasmid was abolished in the presence of a dominantly interfering version of STAT3 (Fig. 5e). Gp130-mediated ITF/TFF3 gene activation is likely to be mediated through STAT3, as a 5′truncated ITF/TFF3 reporter plasmid (pTF2257), which lacks the α2-macroglobulin-type STAT3 binding site26 at nucleotide position –2287, remained unresponsive to gp130-dependent transcriptional activation. Similar results were obtained with the pRITF-luc reporter plasmids27 encompassing nucleotides –1671 to +36 of the rat ITF/TFF3 promoter (data not shown). Discussion We used mice with subtle knock-in mutations to dissect the physiological functions of the two major intracellular signaling cascades emanating from gp130. We identified two classes of pathologies, namely those that are unique to the gp130 signaling-domain mutants and those that are duplicated in mice with null-mutations in genes encoding specific components of the IL-6 family cytokine network. For instance, gastric adenomas (gp130757F mice) and degenerative joint disease (gp130∆STAT mice12) are not observed in IL-6–/–, IL-11Rα1–/– and IL-6–/–IL11Rα1–/– mice or conditional gp130-null mice28. These pathologies have not been associated with mice simultaneously transgenic for IL-6 and sIL-6Rα, which display aberrant and ubiquitous gp130 activation29,30. We therefore conclude that the gastric and joint pathologies are not generated in response to the alterations in the signaling throughput of any one sig1094

tivation of the ITF/TFF3 promoter (–2449 to –6) depends on gp130mediated activation of the STAT pathway, which does not occur following activation of the GCSFR/gp130∆STAT receptor. d, SHP2-Ras-ERKdependent activation of the pS2/TFF1 promoter is dependent on sequences between –496 (pGV496) and –243 (pGV243), which include an AP-1 site at position –281 (ref. 25), and is sensitive to the presence of the ERK inhibitor PD98059 (PD). e, STAT-dependent activation of the mouse ITF/TFF3 promoter (–2449 to –6; pTF2449) is inhibited in the presence of a dominant interfering version of STAT3, whereas a 5′-truncated promoter construct (–2257 to –6; pTF2257) lacking the α2-macroglobulintype STAT3 binding26 site is unresponsive to gp130 signaling. For a and c–e: , control; , IL-6 + sIL-6Rα; , TFF; , Epo; , GCSF.

naling pathway per se, but arise as a result of disturbing the normally simultaneous and coordinated activation of the STAT1/3 and SHP2-Ras-ERK cascades (Fig. 6). Enhanced STAT signaling appears to be the common mechanism triggering splenomegaly in IL-6–/–sIL-6Rα–/– transgenics29,30 and gp130757F mice6. Similarly, the absence of STAT1/3-mediated gp130 signaling resulted in enhanced susceptibility to DSS-induced mucosal injury as well as impaired high-affinity immune and hepatic acute-phase responses as observed in IL-6–/– (ref. 16) and gp130∆STAT mice12. Thus, phenotypes common to gp130-signaling domain mutants and mutants with disrupted gp130 signaling such as in IL-6–/– or IL-11Rα1–/– mice are likely to result from the altered signaling throughput of the STAT1/3 cascade irrespective of that of the SHP2-Ras-ERK pathway. Activation of epithelial-repair mechanisms by the proinflammatory cytokine IL-6 provides an early advantageous wound-healing response; however, paradoxically, IL-6 also perpetuates inflammation in chronic colitis models31 by providing STAT3-mediated anti-apoptotic signals to lamina propria T cells23. Thus, in acute situations where antigens breach the epithelial barrier, IL-6 enhances mucosal repair by epithelial restitution via increased TFF expression, while simultaneously facilitating inhibition of apoptosis in the lymphocyte compartment and thereby promoting immune clearance. The TFF gene-encoded mucus cell–secreted peptides are essential regulators for the restitution of the gastrointestinal mucosa and transduce similar biological activities when applied ectopically. Hence, we interpret the gastrointestinal abnormalNATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002


ARTICLES functionally equivalent SHP2-binding impairment mutation (Y759F) in a mouse-human gp130 fusion protein; however, no gastric pathology has been reported in gp130759F mice6. Hence, it is highly unlikely that the completely penetrant gastric phenotype reported here in gp130757F mice results from a Th1-biased immune-cell development common to gp130759F (ref. 6) and gp130757F mice. Instead, the gastric hyperplasia in gp130757F mice may reflect a primary response of the epithelium to subtle differences in the gp130-signaling throughput between the two gp130-specific SHP2-Ras-ERK signaling impairment mutants. Collectively, our data show that tissue-specific interpretation of the net gp130-signaling throughput Fig. 6 Schematic of the consequences of impairing the reciprocal negative regulation bedetermines organ-specific responses to the IL-6 famtween the SHP2-Ras-ERK and STAT1/3 pathways. The coordinated and simultaneous signaling ily cytokines in vivo. The underlying mechanism of through the two pathways that occurs in wild-type mice is unaffected in situations of continucoordinated activation of the two gp130 signaling ous ligand activation (for instance, in IL-6/sIL-6Rα double transgenic (tg) mice) or in the abcascades is reinforced by our finding of reciprocal sence of ligand-dependent gp130 activation (for instance, in IL-6–/– or IL-11Rα1–/– mice). negative regulation between the STAT1/3 and SHP2However, altering the overall signaling throughput in these mutant mouse strains results in speRas-ERK pathways. The resulting negative feedback cific phenotypes16–18,27–29. A subset of these pathologies phenocopied in gp130-signaling domain mutants6,12, suggesting that they result from altering the signaling throughput through loops seem to converge on the gp130-receptor comthe STAT1/3 pathway. In contrast, phenotypes specific to gp130-signaling domain mutants are plex, as gp130∆STAT mice are characterized by imlikely to arise from the net effect of unbalancing the coordinated activation of the STAT1/3 and paired induction of the suppressor of cytokine SHP2-Ras-ERK signaling cascades. In particular, neither gastric adenomas nor joint disease mansignaling 1 (SOCS-1) protein12, which reduces ifest in situations of simultaneous lack or hyper-activation of both pathways (observed in IL-6–/–, kinase-dependent receptor Y-phosphorylation and IL-11Rα1–/– or IL-6+sIL-6Rα tg mice), but only occur in situations of activating one pathway to mediates ubiquitination of receptor associated Jak the exclusion of the other in gp130757F or gp130∆STAT mice. kinases36. Similarly, enhanced STAT3 activation in gp130757F mice is likely to result from impaired ities in the two gp130 mutant strains to result from inappropri- SOCS-3 binding to the mutated 757F residue37,38, as well as from ate levels of TFF peptide at specific sites. In particular, colonic the associated decrease in SHP2-specific Y-phosphatase activITF/TFF3 levels correlate with reduced or enhanced susceptibil- ity39. Hence, simultaneous and balanced activation of the two ity to DSS-mediated colonic injury in gp130∆STAT and gp130757F signaling cascades is not only essential for mediating tissue-spemice, respectively. By contrast, the gastric mucosa seems to be cific activities of IL-6 family cytokines, but has the potential for particularly sensitive to threshold concentrations of pS2/TFF1, fine tuning the balance between negative and positive signals which are too low in gp130757F mice to provide the signal nor- during biological responses to these cytokines. mally required to promote differentiation of mucus cells in the gastric pits32. In the complete absence of pS2/TFF1 expression Methods in the mouse, the gastric adenomas may further progress to Mice. The bicistronic p(757F) targeting vector contained exon 17 carcinomas13. The implied role for pS2/TFF1 as a gastric-specific (ref. 27) encoding amino acids 672–917 with the Y757F (TAC to TTC) and tumor-suppressor gene is further supported by the observation V760A (GTG to GCG) substitutions and an introduced AvrII restriction site as part of the endogenous stop codon. A cDNA encoding neomycin resisthat approximately half of all human gastric cancers display tance, preceded by an internal ribosomal entry site (IRES), was introduced somatic loss of heterozygosity at 21q22.3, encompassing the into the AvrII site as a XbaI-NheI fragment; the p(757F) vector also con33 cluster of three human TFF genes . tained 0.8 kb and 4.3 kb of sequence homologous to the final intron and In line with our proposed role for pS2/TFF1 in mediating the the 3′-flanking sequence, respectively12. Gene targeting in W9.5 ES cells gastric phenotype in gp130757F mice, the histological appear- (129/Sv) was done with the SalI-linearized p(757F) vector, correctly tarance and exclusive spatial restriction of the lesions to the dis- geted ES clones were identified from the hybridization pattern of HindIII 12 tal glandular part of the stomach differs from the gastric digested DNA, and germ-line chimeras were obtained as described . –/– –/– –/– Compound IL-6 IL-11Rα1 mice were generated from IL-6 (ref. 16) pathologies observed in mice with modified expression patand IL-11Rα1–/– (ref. 17) mice, and the construction of gp130∆STAT mice terns of growth factors. Transgenic mice over-expressing TGFhas been described4. All mouse strains were kept on a mixed 129/BL6 α, for instance, develop severe adenomatous hypertrophy of background. All animal experiments were approved by the Ludwig the gastric mucosa that is overtly analogous to the prominent Institute for Cancer Research/Department of Surgery, Royal Melbourne giant rugal folds characteristic of Ménétrier’s disease in hu- Hospital Animal Experimentation Ethics Committee. mans34. By contrast, the glandular hyperplasia in mice with a hemi-allelic loss of TGF-β1 is characterized by infiltration of Analysis of tissue lysates. Liver lysates (3 mg), prepared at timed interthe muscularis mucosa with glandular tissue similar to human vals after an intravenous injection of IL-6 (5 µg) or saline, were immunogastritis cystica profunda35. Notably, the phenotype in the lat- precipitated with SHP2 or STAT3 antisera (Santa Cruz Biotechnology, Santa Cruz, California). Samples were resolved by SDS–PAGE and the reter mutant mice is thought to arise secondarily to multifocal sulting blots were probed with anti-phosphotyrosine (UBI, Lake Placid, inflammation, which is somewhat reminiscent of the lympho- New York), followed by SHP2 or STAT3 antibodies to assess protein load757F cytic infiltration observed in gp130 mice. Splenomegaly, ing12. Phosphorylated STAT3 in the stomach was detected by western which is associated with enhanced B- and T-cell proliferation blotting with a phospho-STAT3 antibody (New England BioLabs, and/or cell survival, is also observed in mice which carry a Berverly, Massachusetts) on lysates (50 µg) prepared from antra of mice NATURE MEDICINE • VOLUME 8 • NUMBER 10 • OCTOBER 2002

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after an intravenous injection of IL-11 (5 µg). Active ERK kinase was identified on western blots of liver or colon lysates (50 µg) reacted with a phospho-ERK1/2-specific antibody (New England Biolabs), and the level of 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 other day. Colitis was assessed by assigning histological scores to 4 independent colonic sections per mouse20, and gross colonic blood content was scored using a scale from 0 to 3: 0, no blood; 1, blood in < 1/3 of the colon; 2, blood in < 2/3 of the colon; 3, blood in > 2/3 of the colon. Histology, immunohistochemistry and TFF determination. Paraffinembedded sections of formalin-fixed tissues were stained with hematoxylin. Mucin was visualized by periodic acid/Schiff staining. Proliferating cells were identified by immunohistochemistry with an antibody directed against the nuclear proliferation antigen PCNA (Sigma, St. Louis, Missouri). All sections were counterstained with eosin. Immunohistochemical analysis was performed with an antiserum directed against human pS2/Tff1 (ref. 40) or gp130 (Santa Cruz Biotechnology). The concentration of TFF in protein extracts from the antrum or colon was determined with a radioimmunoassay specific for pS2/TFF1 (ref. 41) and ITF/TFF3 (ref. 42). Cell lines. Gastric epithelial IMGE5 cells22, expressing a temperature sensitive mutant of large T antigen, were maintained at 33 °C in RPMI supplemented with 10% FCS, whereas colon carcinoma–derived LIM1215 and SW1222 cells were propagated at 37 °C in DME containing 10% FCS. For each of the receptor constructs, 3 independent cell lines were derived following stable expression of plasmids encoding, either individually or in the indicated combination, the chimeric receptors EpoR/wild-type (ref. 38), GCSF/wild-type (ref. 42), EpoR/gp130757F (ref. 38) or GCSF/gp130∆STAT (ref. 43) together with the resistance marker pPGKpuro. In vitro wound-healing assay. Monolayers of LIM1215 cells were maintained in serum-free medium containing 0.2 µg/ml mitomycin C. Gastrointestinal epithelial cells may not ubiquitously express the transmembrane IL-6Rα ligand-binding subunit, but IL-6 in the presence of sIL-6Rα initiates gp130-dependent signaling in cells irrespective of the presence of endogenous IL-6Rα. Because of this, we stimulated cells with IL-6 plus sIL6Rα (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 distance of cells relative to the edge of the wound was calculated from measurements 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 regions 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 to the transcriptional start site25) (–2449 to –6; –2257 to –6) promoters and inserted into pGL3basic (Promega, Madison, Wisconsin), together with 2 µg of the Srα-β-galactosidase plasmid using FuGENE6 (Roche Molecular Biochemicals, Castle Hill, Australia). Cultures were maintained in 1% FCS for 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 inhibitor PD98059 (ref. 42) before lysis with Reporter Lysis Buffer (Promega). Lysates were assayed for luciferase activity, which was normalized according to the β-galactosidase activity12.

Acknowledgments We thank T. Terada for the tff-luciferase promoter constructs pGV2192, pGV496 and pGV243; D. Podolsky for pRITF-luc; S. Nicholson for the expression construct encoding EpoR/gp130757F; L. Thim for recombinant human TFF2; Amgen (Thousand Oaks, California) for recombinant human GCSF and IL-6; R. Simpson for sIL-6R; L. Robb and A. Ramsay for IL-11Rα1–/– and IL-6–/– mice; V. Feakes, J. Moverley and M. Howlett for histology; and J.Stickland for photography. 1096

Competing interests statement The authors declare that they have no competing financial interests.

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