Proapoptotic Bak and Bax guard against fatal systemic and organ-specific autoimmune disease.

Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak(-/-)bax(-/-) mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.

Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia.

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

SOCS3 deletion in B cells alters cytokine responses and germinal center output.

B cell behavior is fine-tuned by internal regulatory mechanisms and external cues such as cytokines and chemokines. Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of STAT3-dependent cytokine responses in many cell types and has been reported to inhibit CXCL12-induced retention of immature B cells in the bone marrow. Using mice with SOCS3 exclusively deleted in the B cell lineage (Socs3(Δ/Δ)mb1cre(+)), we analyzed the role of SOCS3 in the response of these cells to CXCL12 and the STAT3-inducing cytokines IL-6 and IL-21. Our findings refute a B cell-intrinsic role for SOCS3 in B cell development, because SOCS3 deletion in the B lineage did not affect B cell populations in naive mice. SOCS3 was strongly induced in B cells stimulated with IL-21 and in plasma cells exposed to IL-6. Its deletion permitted excessive and prolonged STAT3 signaling following IL-6 stimulation of plasma cells and, in a T cell-dependent immunization model, reduced the number of germinal center B cells formed and altered the production of Ag-specific IgM and IgE. These data demonstrate a novel regulatory signal transduction circuit in plasma cells, providing, to our knowledge, the first evidence of how these long-lived, sessile cells respond to the external signals that mediate their longevity.

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm.

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.

A complementary role for the tetraspanins CD37 and Tssc6 in cellular immunity.

The cooperative nature of tetraspanin-tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37(-/-) mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize "double knockout" mice (CD37(-/-)Tssc6(-/-)) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37(-/-)Tssc6(-/-) mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37(-/-)Tssc6(-/-) mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37(-/-)Tssc6(-/-) mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37(-/-) or Tssc6(-/-) mice, demonstrating a complementary role for these two molecules in cellular immunity.

Neuregulin 1 sustains the gene regulatory network in both trabecular and nontrabecular myocardium.

RATIONALE: The cardiac gene regulatory network (GRN) is controlled by transcription factors and signaling inputs, but network logic in development and it unraveling in disease is poorly understood. In development, the membrane-tethered signaling ligand Neuregulin (Nrg)1, expressed in endocardium, is essential for ventricular morphogenesis. In adults, Nrg1 protects against heart failure and can induce cardiomyocytes to divide. OBJECTIVE: To understand the role of Nrg1 in heart development through analysis of null and hypomorphic Nrg1 mutant mice. METHODS AND RESULTS: Chamber domains were correctly specified in Nrg1 mutants, although chamber-restricted genes Hand1 and Cited1 failed to be activated. The chamber GRN subsequently decayed with individual genes exhibiting decay patterns unrelated to known patterning boundaries. Both trabecular and nontrabecular myocardium were affected. Network demise was spatiotemporally dynamic, the most sensitive region being the central part of the left ventricle, in which the GRN underwent complete collapse. Other regions were partially affected with graded sensitivity. In vitro, Nrg1 promoted phospho-Erk1/2-dependent transcription factor expression, cardiomyocyte maturation and cell cycle inhibition. We monitored cardiac pErk1/2 in embryos and found that expression was Nrg1-dependent and levels correlated with cardiac GRN sensitivity in mutants. CONCLUSIONS: The chamber GRN is fundamentally labile and dependent on signaling from extracardiac sources. Nrg1-ErbB1/4-Erk1/2 signaling critically sustains elements of the GRN in trabecular and nontrabecular myocardium, challenging our understanding of Nrg1 function. Transcriptional decay patterns induced by reduced Nrg1 suggest a novel mechanism for cardiac transcriptional regulation and dysfunction in disease, potentially linking biomechanical feedback to molecular pathways for growth and differentiation.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis.

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis.

BACKGROUND: Histone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination. RESULTS: In ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted. CONCLUSION: These data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

The interleukin-6 family cytokine interleukin-11 regulates homeostatic epithelial cell turnover and promotes gastric tumor development.

BACKGROUND & AIMS: Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. METHODS: We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. RESULTS: IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor alpha significantly reduced HKbeta-/- mouse fundic hyperplasia and ablated gp130(757F/F) mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-beta, RegIII-gamma, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130(757F/F) and HKbeta-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. CONCLUSIONS: Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.

IL11 antagonist inhibits uterine stromal differentiation, causing pregnancy failure in mice.

Hormonal contraceptives are unsuitable for many women; thus, the development of new, nonhormonal contraceptives is of great interest. In women, uterine epithelial expression of interleukin 11 (IL11) and its receptor (IL11RA) suggests IL11 is critical for blastocyst attachment during implantation. Il11ra-deficient mice are infertile due to a defective decidualization response to the blastocyst, leading to total pregnancy loss. We examined the effect of administering a PEGylated IL11 antagonist, PEGIL11A (where PEG is polyethylene glycol), on pregnancy outcomes in mice and IL11 signaling in human endometrial epithelial cells (HES). PEGIL11A was detected in sera up to 72 h after intraperitoneal (IP) injection versus up to 2 h for the non-PEGylated antagonist. Following IP injection, PEGIL11A localized to uterine decidual cells and reduced immunoreactive cyclin D3 (IL11 decidual target). To inhibit IL11 action during early decidualization, PEGIL11A or control were administered IP on Days 3-6 (beginning just prior to maximal decidual Il11 expression). On Day 6, mesometrial decidualization was disturbed in PEGIL11A-treated animals with regions of hemorrhage visible in the mesometrial decidua. On Day 10, severe decidual destruction was visible: implantation sites contained significant hemorrhage, and the uterine luminal epithelium had reformed, suggesting a return to estrous cycling. These results demonstrate that PEGIL11A blocked IL11 action in the decidua during early decidualization, which totally abolished pregnancy and which is equivalent to the Il11ra(-/-) mouse. PEGIL11A significantly diminished STAT3 phosphorylation in HES cells in vitro (P < or = 0.05). This study provides valuable information for PEGIL11A that could lead to the development of this protein as a nonhormonal contraceptive.

Deletion of the SOCS box of suppressor of cytokine signaling 3 (SOCS3) in embryonic stem cells reveals SOCS box-dependent regulation of JAK but not STAT phosphorylation.

The mechanism by which Suppressor of Cytokine Signaling-3 (SOCS3) negatively regulates cytokine signaling has been widely investigated using over-expression studies in cell lines and is thought to involve interactions with both the gp130 receptor and JAK1. Here, we compare the endogenous JAK/STAT signaling pathway downstream of Leukemia Inhibitory Factor (LIF) signaling in wild type (WT) Embryonic Stem (ES) cells and in ES cells lacking either the entire Socs3 gene or bearing a truncated form of SOCS3 (SOCS3DeltaSB) lacking the C-terminal SOCS box motif (SOCS3(DeltaSB/DeltaSB)). In SOCS3(DeltaSB/DeltaSB) cells phosphorylated JAK1 accumulated at much higher levels than in WT cells or even cells lacking SOCS3 (SOCS3(-/-)). In contrast enhanced activation of STAT3 and SHP2 was seen in SOCS3(-/-) cells. Size exclusion chromatography of cell extracts showed that in unstimulated cells, JAK1 was exclusively associated with receptors but following cytokine stimulation hyperphosphorylated JAK1 (pJAK1) appeared to dissociate from the receptor complex in a manner independent of SOCS3. In WT and SOCS3(DeltaSB/DeltaSB) cells SOCS3 was associated with pJAK1. The data suggest that dissociation of activated JAK1 from the receptor results in separate targeting of JAK1 for proteasomal degradation through a mechanism dependent on the SOCS3 SOCS box thus preventing further activation of STAT3.

Dkk1 and Wnt3 interact to control head morphogenesis in the mouse.

Loss of Dkk1 results in ectopic WNT/beta-catenin signalling activity in the anterior germ layer tissues and impairs cell movement in the endoderm of the mouse gastrula. The juxtaposition of the expression domains of Dkk1 and Wnt3 is suggestive of an antagonist-agonist interaction. The downregulation of Dkk1 when Wnt3 activity is reduced reveals a feedback mechanism for regulating WNT signalling. Compound Dkk1;Wnt3 heterozygous mutant embryos display head truncation and trunk malformation, which are not found in either Dkk1(+/-) or Wnt3(+/-) embryos. Reducing the dose of Wnt3 gene in Dkk1(-/-) embryos partially rescues the truncated head phenotype. These findings highlight that head development is sensitive to the level of WNT3 signalling and that DKK1 is the key antagonist that modulates WNT3 activity during anterior morphogenesis.

The role of SOCS3 in modulating leukaemia inhibitory factor signalling during murine placental development.

Cytokines are an integral part of the adaptive and innate immune responses. The signalling pathways triggered by receptor engagement translate exposure to cytokine into a coordinated biological response. To contain these responses, the initiation, duration and magnitude of the signal is controlled at multiple levels. Suppressor of cytokine signalling (SOCS) proteins act in a negative feedback loop to inhibit signal transduction. Mice with a deletion of SOCS3 die at midgestion due to placental insufficiency. SOCS3-null placentae have increased numbers of mature trophoblast giant cells, disruption of the labyrinthine layer and a decrease in the spongiotrophoblast layer. Genetic crosses have revealed that the phenotype is due to dysregulation of signalling downstream of the leukaemia inhibitory factor (LIF) receptor alpha (LIFRalpha) and that the ligand responsible for this, LIF, is produced by embryonic tissues and acts in a paracrine fashion. These observations highlight the role of LIF as an extrinsic factor regulating trophoblast differentiation in vivo. The creation of mice with conditional deletion of SOCS3 in different tissues has also uncovered critical roles for SOCS3 in the regulation of IL-6, G-CSF and leptin signalling.

The preleukemic state of mice reconstituted with Mixl1-transduced marrow cells.

Murine granulocytic cells, in becoming leukemic, need to acquire enhanced self-generation and a capacity for autocrine growth stimulation. Mice transplanted with bone marrow cells transduced with the Mixl1 homeobox gene develop a very high frequency of myeloid leukemia derived from the transduced cells. Preleukemic mice contained a high frequency of transduced clonogenic granulocytic cells. They exhibited an abnormally high capacity for self-replication and could generate immortalized granulocytic cell lines that remained absolutely dependent on either GM-CSF or IL-3 and were not leukemic. Organs from mice repopulated by marrow cells transduced either with Mixl1 or the control murine stem cell virus vector exhibited a capacity to produce IL-3 in vitro, activity being highest with the lungs, marrow, bladder, and thymus. Supporting evidence for the in vivo production of IL-3 was the frequent development of mast cells in the marrow. Overexpression of Mixl1 appears capable of inducing an abnormal self-renewal capacity in granulocytic precursors. Aberrant production of IL-3 was not present in the continuous Mixl cell lines and was therefore not in itself likely to be a leukemogenic change but it could support the enhanced survival and proliferation of the Mixl1 granulocytic populations until a final leukemogenic mutation occurs in them.

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: a nonhormonal contraceptive strategy.

Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.

Agm1/Pgm3-mediated sugar nucleotide synthesis is essential for hematopoiesis and development.

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

The SOCS box of suppressor of cytokine signaling-3 contributes to the control of G-CSF responsiveness in vivo.

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of granulocyte-colony stimulating factor (G-CSF) signaling in vivo. SOCS proteins regulate cytokine signaling by binding, via their SH2 domains, to activated cytokine receptors or their associated Janus kinases. In addition, they bind to the elongin B/C ubiquitin ligase complex via the SOCS box. To ascertain the contribution of the SOCS box of SOCS3 to in vivo regulation of G-CSF signaling, we generated mice expressing a truncated SOCS3 protein lacking the C-terminal SOCS box (SOCS3(Delta SB/Delta SB)). SOCS3(Delta SB/Delta SB) mice were viable, had normal steady-state hematopoiesis, and did not develop inflammatory disease. Despite the mild phenotype, STAT3 activation in response to G-CSF signaling was prolonged in SOCS3(Delta SB/Delta SB) bone marrow. SOCS3(Delta SB/Delta SB) bone marrow contained increased numbers of colony-forming cells responsive to G-CSF and IL-6. Treatment of the mice with pharmacologic doses of G-CSF, which mimics emergency granulopoiesis and therapeutic use of G-CSF, revealed that SOCS3(Delta SB/Delta SB) mice were hyperresponsive to G-CSF. Compared with wild-type mice, SOCS3(Delta SB/Delta SB) mice developed a more florid arthritis when tested using an acute disease model. Overall, the results establish a role for the SOCS box of SOCS3 in the in vivo regulation of G-CSF signaling and the response to inflammatory stimuli.

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation.

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.

Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis.

We have previously demonstrated that STAT3 hyperactivation via the interleukin 6 (IL-6) cytokine family receptor gp130 in gp130 (Y757F/Y757F) mice leads to numerous hematopoietic and lymphoid pathologies, including neutrophilia, thrombocytosis, splenomegaly, and lymphadenopathy. Because IL-6 and IL-11 both signal via a gp130 homodimer, we report here a genetic approach to dissect their individual roles in these pathologies. Neutrophilia and thrombocytosis were absent in gp130 (Y757F/Y757F) mice lacking either IL-6 (gp130 (Y757F/Y757F): IL-6 (-/-)) or the IL-11 receptor alpha subunit (gp130 (Y757F/Y757F): IL-11Ralpha1 (-/-)), and this was associated with a normalized bone marrow compartment. The elevated myelopoiesis and megakaryopoiesis in bone marrow of gp130 (Y757F/Y757F) mice was attributable to an increase by either IL-6 or IL-11 in the STAT3-driven impairment of transforming growth factor beta (TGF-beta) signaling, which is a suppressor of these lineages. In contrast, the absence of IL-6, but not IL-11 signaling, prevented the splenomegaly, abnormal lymphopoiesis, and STAT3 hyperactivation in lymphoid organs of gp130 (Y757F/Y757F) mice. Furthermore, hyperactivation of STAT3 in lymphoid organs was associated with increased expression of IL-6Ralpha, and IL-6Ralpha expression was reduced in gp130 (Y757F/Y757F): Stat3 (+/-) mice displaying normal levels of STAT3 activity. Collectively, these data genetically define distinct roles of IL-6 and IL-11 in driving pathologic hematopoietic and lymphoid responses mediated by STAT3 hyperactivation.

Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation.

The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer. To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (beta3-integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.