Differentiation of naive CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling of Th2 cytokine gene loci. Hyperacetylation of histone H3 on nucleosomes associated with the interleukin (IL)-4, IL-13 and IL-5 genes was observed in developing Th2 cells but not in Th1 cells. Histone hyperacetylation on IL-5 gene-associated nucleosomes was Th2-specific but occurred with delayed kinetics, and hyperacetylation on RAD50 gene-associated nucleosomes was T cell antigen receptor stimulation-dependent but not Th2-specific. The induction of the Th2-specific histone hyperacetylation was STAT6- and GATA3-dependent, and interestingly, it was accompanied by the expression of intergenic transcripts within the IL-13 and IL-4 gene loci. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream from the IL-13 gene, corresponding with the 5'-border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyltransferase complexes including CBP/p300, and RNA polymerase II. Also, the CGRE showed a significant enhancing effect on the Th2 cytokine gene promoters. Thus, the CGRE may play a crucial role for GATA3-mediated targeting and downstream spreading of core histone hyperacetylation within the IL-13 and IL-4 gene loci.

The loss of expression of the transcription factor GATA3 in breast tumors has been linked to aggressive tumor development and poor patient survival. In the present work, we address potential roles for GATA3 in breast tumor lung metastasis and progression. Using an aggressive breast cancer cell line, which metastasizes specifically to the lung, we show that GATA3 expression results in reduced tumor outgrowth in the mammary fat pad and lower lung metastatic burden in nude mice. Specifically, GATA3 expression inhibits breast cancer cell expansion inside the lung parenchyma. This phenotype correlates with the ability of GATA3 to negatively regulate the expression of several genes that promote breast cancer lung metastasis (ID1/-3, KRTHB1, LY6E and RARRES3). Conversely, the expression of genes encoding known inhibitors of lung metastasis (DLC1 (deleted in liver cancer 1) and PAEP (progestagen-associated endometrial protein)) is upregulated by GATA3. These data correlate with microarray data from human breast cancer patients, showing a strong correlation between high GATA3 expression and absence of metastases specifically to the lungs. We conclude that GATA3 inhibits primary breast tumor outgrowth and reduces lung metastatic burden by regulating key genes involved in metastatic breast tumor progression.

Acute lymphoblastic leukemia (ALL) is the major pediatric cancer diagnosed in economically developed countries with B-cell precursor (BCP)-ALL, accounting for approximately 70% of ALL. Recent genome-wide association studies (GWAS) have provided the first unambiguous evidence for common inherited susceptibility to BCP-ALL, identifying susceptibility loci at 7p12.2, 9p21.3, 10q21.2, and 14q11.2. To identify additional BCP-ALL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1658 cases and 4723 controls, with validation in 1449 cases and 1488 controls. Combined analysis identified novel loci mapping to 10p12.2 (rs10828317, odds ratio [OR] = 1.23; P = 2.30 × 10(-9)) and 10p14 marked by rs3824662 (OR = 1.31; P = 8.62 × 10(-12)). The single nucleotide polymorphism rs10828317 is responsible for the N215S polymorphism in exon 7 of PIP4K2A, and rs3824662 localizes to intron 3 of the transcription factor and putative tumor suppressor gene GATA3. The rs10828317 association was shown to be specifically associated with hyperdiploid ALL, whereas the rs3824662-associated risk was confined to nonhyperdiploid non-TEL-AML1 + ALL. The risk allele of rs3824662 was correlated with older age at diagnosis (P < .001) and significantly worse event-free survivorship (P < .0001). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to BCP-ALL and the influence of constitutional genotype on disease development.

Innate lymphocyte populations play a central role in conferring protective immunity at the mucosal frontier. In this study, we demonstrate that T cell factor 1 (TCF-1; encoded by Tcf7), a transcription factor also important for NK and T cell differentiation, is expressed by multiple innate lymphoid cell (ILC) subsets, including GATA3(+) nuocytes (ILC2) and NKp46(+) ILCs (ILC3), which confer protection against lung and intestinal inflammation. TCF-1 was intrinsically required for the differentiation of both ILC2 and NKp46(+) ILC3. Loss of TCF-1 expression impaired the capacity of these ILC subsets to produce IL-5, IL-13, and IL-22 and resulted in crippled responses to intestinal infection with Citrobacter rodentium. Furthermore, a reduction in T-bet expression required for Notch-2-dependent development of NKp46(+) ILC3 showed a dose-dependent reduction in TCF-1 expression. Collectively, our findings demonstrate an essential requirement for TCF-1 in ILC2 differentiation and reveal a link among Tcf7, Notch, and Tbx21 in NKp46(+) ILC3 development.

The fibroblast growth factor receptor 2 (FGFR2) locus has been consistently identified as a breast cancer risk locus in independent genome-wide association studies. However, the molecular mechanisms underlying FGFR2-mediated risk are still unknown. Using model systems we show that FGFR2-regulated genes are preferentially linked to breast cancer risk loci in expression quantitative trait loci analysis, supporting the concept that risk genes cluster in pathways. Using a network derived from 2,000 transcriptional profiles we identify SPDEF, ERα, FOXA1, GATA3 and PTTG1 as master regulators of fibroblast growth factor receptor 2 signalling, and show that ERα occupancy responds to fibroblast growth factor receptor 2 signalling. Our results indicate that ERα, FOXA1 and GATA3 contribute to the regulation of breast cancer susceptibility genes, which is consistent with the effects of anti-oestrogen treatment in breast cancer prevention, and suggest that fibroblast growth factor receptor 2 signalling has an important role in mediating breast cancer risk.

A receptive endometrial environment requires adequate immunological tolerance to protect the implanting embryo from maternal immune rejection. Studies in mice implicate CD4+CD25+ T-regulatory (Treg) cells as essential mediators of immune tolerance in pregnancy. The aim of this study was to evaluate the link between Treg cells and fertility in women. Expression of Foxp3, a master regulator of Treg cell differentiation, was quantified in endometrial tissue from women experiencing primary unexplained infertility and normal fertile women. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women meeting rigorously defined criteria for unexplained infertility after experiencing repeated failed cycles of IVF treatment (infertile, n = 10), or women classified as proven fertile (control, n = 12). Expression of Foxp3 mRNA was reduced approximately two-fold in the tissue of infertile women. In contrast, mRNAs encoding T cell transcription factors T-bet and GATA3, associated with differentiation of Th1 and Th2 CD4+ T cells respectively, were unchanged. Treg cell differentiation is controlled by TGFbeta, but the relative abundance in endometrial tissue of TGFbeta1, TGFbeta2, TGFbeta3 mRNAs was not changed in infertile women. Cytokines influencing Th1 and Th2 cell differentiation, including IFNgamma, IL-2, IL-4, IL-5, IL-10 and IL-12p40, as well as dendritic cell-regulating cytokines IL-1alpha, IL-1beta, IL-6, LIF, GM-CSF and TNFalpha were also expressed similarly regardless of fertility status. The finding of reduced endometrial Foxp3 implicates impaired differentiation of uterine T cells into the Treg phenotype as a key determinant of fertility in women. The factors underpinning this aberration in the immune response remain to be identified.

BACKGROUND

The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA).

METHODS

We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1).

RESULTS

After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo.

CONCLUSIONS

Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).

Various therapeutic advances have led to a paradigm shift in the clinical management of patients with IBD. The introduction of immunosuppressive (such as azathioprine) and biologic agents (such as TNF blockers) has markedly reduced the need to use corticosteroids for therapy. Furthermore, the α4β7 integrin blocker vedolizumab has been introduced for clinical IBD therapy. Moreover, various new inhibitors of cytokines (for example, IL-6-IL-6R and IL-12-IL-23 blockers or apremilast), modulators of cytokine signalling events (for example, JAK inhibitors or SMAD7 blocker), inhibitors of transcription factors (for example, GATA3 or RORγt) and new anti-adhesion and anti-T-cell-activation and migration strategies (for example, β7 integrin, sphingosine 1-phosphate receptors and MAdCAM1 inhibitors, regulatory T-cell therapy and stem cells) are currently being evaluated in controlled clinical trials. This Review aims to provide a comprehensive overview about current and future therapeutic approaches for IBD therapy. Furthermore, potential mechanisms of action of these therapeutic approaches and their implications for clinical therapy in IBD are discussed.

The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons from embryonic day 12, when SG neurons first extend projections, up until postnatal day 15, after the onset of hearing. For comparison, we also analyzed the closely related vestibular ganglion (VG). Gene ontology analysis confirmed enriched expression of genes associated with gene regulation and neurite outgrowth at early stages, with the SG and VG often expressing different members of the same gene family. At later stages, the neurons transcribe more genes related to mature function, and exhibit a dramatic increase in immune gene expression. Comparisons of the two populations revealed enhanced expression of TGFβ pathway components in SG neurons and established new markers that consistently distinguish auditory and vestibular neurons. Unexpectedly, we found that Gata3, a transcription factor commonly associated with auditory development, is also expressed in VG neurons at early stages. We therefore defined new cohorts of transcription factors and axon guidance molecules that are uniquely expressed in SG neurons and may drive auditory-specific aspects of their differentiation and wiring. We show that one of these molecules, the receptor guanylyl cyclase Npr2, is required for bifurcation of the SG central axon. Hence, our dataset provides a useful resource for uncovering the molecular basis of specific auditory circuit assembly events.

The innate lymphoid cell (ILC) family has recently expanded with the discovery of type-2 innate lymphoid cells (ILC2). These cells arise from lymphoid progenitors in the bone marrow and, under the control of the transcriptional regulators RORα and Gata3, they mature to give rise to IL-5, IL-9 and IL-13 producing ILC2. These cells are critical components of the innate immune response to parasitic worm infections and have also been implicated in the pathogenesis of asthma and allergy. Recent advances in our understanding of the molecular regulation of ILC2 development and function now present the opportunity to develop new genetic models to assess ILC2 immune function and to investigate possible therapeutic interventions.

Dlx homeobox transcription factors regulate epidermal, neural and osteogenic cellular differentiation. Here, we demonstrate the central role of Dlx3 as a crucial transcriptional regulator of hair formation and regeneration. The selective ablation of Dlx3 in the epidermis results in complete alopecia owing to failure of the hair shaft and inner root sheath to form, which is caused by the abnormal differentiation of the cortex. Significantly, we elucidate the regulatory cascade that positions Dlx3 downstream of Wnt signaling and as an upstream regulator of other transcription factors that regulate hair follicle differentiation, such as Hoxc13 and Gata3. Colocalization of phospho-Smad1/5/8 and Dlx3 is consistent with a regulatory role for BMP signaling to Dlx3 during hair morphogenesis. Importantly, mutant catagen follicles undergo delayed regression and display persistent proliferation. Moreover, ablation of Dlx3 expression in the telogen bulge stem cells is associated with a loss of BMP signaling, precluding re-initiation of the hair follicle growth cycle. Taken together with hair follicle abnormalities in humans with Tricho-Dento-Osseous (TDO) syndrome, an autosomal dominant ectodermal dysplasia linked to mutations in the DLX3 gene, our results establish that Dlx3 is essential for hair morphogenesis, differentiation and cycling programs.

Lung Kruppel-like factor (LKLF) is a member of the Kruppel-like family of zinc finger transcription factors and is closely related to erythroid kruppel-like factor (EKLF), which is necessary for beta-globin gene expression. While EKLF is expressed exclusively in erythroid cells, LKLF is expressed temporally during early embryonic development and predominantly in the adult mouse lung. To understand the role this novel transcription factor plays in development as well as tissue differentiation and function, animals lacking LKLF were produced using gene targeting technology. Mice lacking LKLF die in utero between day 11.5 and 13.5 of embryonic life and exhibit retarded growth, craniofacial abnormalities, abdominal bleeding and signs of anaemia. Although the yolk sac erythropoiesis is normal in mutant embryos, in vitro fetal liver cultures of these embryos fail to give rise to erythroid cells. Expression of other erythroid specific genes such as EKLF, GATA1 and GATA3 is unaltered in these animals. These findings demonstrate the LKLF function is indispensable during normal embryonic development, and although both LKLF and EKLF recognize common DNA motifs, they do not substitute for each other.

GATA2 is expressed in the pituitary during development and in adult gonadotropes and thyrotropes. It is proposed to be important for gonadotrope and thyrotrope cell fate choice and for TSH production. To test this idea, we produced a pituitary-specific knockout of Gata2, designed so that the DNA-binding zinc-finger region is deleted in the presence of a pituitary-specific recombinase transgene. These mice have reduced secretion of gonadotropins basally and in response to castration challenge, although the mice are fertile. GATA2 deficiency also compromises thyrotrope function. Mutants have fewer thyrotrope cells at birth, male Gata2-deficient mice exhibit growth delay from 3-9 wk of age, and adult mutants produce less TSH in response to severe hypothyroidism after radiothyroidectomy. Therefore, Gata2 appears to be dispensable for gonadotrope and thyrotrope cell fate and maintenance, but important for optimal gonadotrope and thyrotrope function. Gata2-deficient mice exhibit elevated levels of Gata3 transcripts in the pituitary gland, suggesting that GATA3 can compensate for GATA2.

The principal aim of the immune system is to establish a balance between defense against pathogens and avoidance of autoimmune disease. This balance is achieved partly through regulatory T cells (Tregs). CD4(+)CD25(+) Tregs are either naturally occurring or induced by antigens and are characterized by the expression of the X-linked forkhead/winged helix transcription factor, Foxp3. Here we report a previously unrecognized subset of CD4(+)CD25(+) Tregs derived from CD4(+)CD25(-) T cells induced by nitric oxide (NO). The induction of Tregs (NO-Tregs) is independent of cGMP but depends on p53, IL-2, and OX40. NO-Tregs produced IL-4 and IL-10, but not IL-2, IFNgamma, or TGFbeta. The cells were GITR(+), CD27(+), T-bet(low), GATA3(high), and Foxp3(-). NO-Tregs suppressed the proliferation of CD4(+)CD25(-) T cells in vitro and attenuated colitis- and collagen-induced arthritis in vivo in an IL-10-dependent manner. NO-Tregs also were induced in vivo in SCID mice adoptively transferred with CD4(+)CD25(-) T cells in the presence of LPS and IFNgamma, and the induction was completely inhibited by N(G)-monomethyl-L-arginine, a pan NO synthase inhibitor. Therefore, our findings uncovered a previously unrecognized function of NO via the NO-p53-IL-2-OX40-survivin signaling pathway for T cell differentiation and development.

GATA-3 is a master regulator of T helper type 2 (T(H)2) differentiation. However, the molecular basis of GATA-3-mediated T(H)2 lineage commitment is poorly understood. Here we identify the DNase I-hypersensitive site 2 (HS2) element located in the second intron of the interleukin 4 locus (Il4) as a critical enhancer strictly controlled by GATA-3 binding. Mice lacking HS2 showed substantial impairment in their asthmatic responses and their production of IL-4 but not of other T(H)2 cytokines. Overexpression of Gata3 in HS2-deficient T cells failed to restore Il4 expression. HS2 deletion impaired the trimethylation of histone H3 at Lys4 and acetylation of histone H3 at Lys9 and Lys14 in the Il4 locus. Our results indicate that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNgamma, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+ CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFbeta, TGFbetaRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFbeta and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a (dr)) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a "cochlear-gravistatic" endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

There is an emerging body of research demonstrating that the co-expression of key lineage-specifying transcription factors, commonly referred to as 'master regulators', affects the functional capabilities and flexibility of CD4(+) T cell subsets. Here, we discuss how the natural co-expression of these lineage-specifying transcription factors has challenged the concept that the expression of a single 'master regulator' strictly establishes an absolute CD4(+) T cell phenotype. Instead, it is becoming clear that the interplay between the lineage-specifying (or lineage-defining) transcription factors, including T-bet, GATA3, RORγt, BCL-6 and FOXP3, contributes to the fate and flexibility of CD4(+) T cell subtypes. This in turn has led to the realization that CD4(+) T cell phenotypes are more diverse than previously recognized.

We have developed an in vitro differentiation assay in which human naive CD4(+) cells are driven toward either the Th1 or Th2 phenotype. We have examined the interrelationships among the expression of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-gamma in individual cells using intracellular cytokine staining at various times during the differentiation process. We provide direct evidence that the Th2 cytokines IL-4, IL-5, and IL-13, unlike the other cytokines, are regulated by a coordinated mechanism. We also show that IL-10 is expressed by a different subset of cells that is prevalent at early stages of Th2 differentiation, but then diminishes. Additionally we demonstrate that while naive cells can express IL-2 upon activation, they cannot express GM-CSF. Commitment to GM-CSF expression occurs during differentiation in a Th1/Th2 subset-independent manner. Furthermore, we have examined the levels of GATA3, c-Maf, T-bet, and Ets-related molecule during human Th1/Th2 differentiation and suggest that differences in the levels of these critical transcription factors are responsible for commitment toward the Th1 or Th2 lineage.

Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 ->> Stop and Arg-367 ->> Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 ->> Arg and Asn-320 ->> Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast two-hybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.

BACKGROUND

Breast cancer is a complex and heterogeneous disease at the molecular level. Evolution is difficult to predict according to classical histoclinical prognostic factors. Different studies highlight the importance of large-scale molecular expression analyses to improve taxonomy of breast cancer and prognostic classification. Identification of new molecular markers that refine this taxonomy and improve patient management is a priority in the field of breast cancer research.Nectins are cell adhesion molecules involved in the regulation of epithelial physiology. We present here Nectin-4/PVRL4 as a new histological and serological tumor associated marker for breast carcinoma.

METHODS

Expression of Nectin-4 protein was measured on a panel of 78 primary cells and cell lines from different origins and 57 breast tumors by FACS analysis and immunohistochemistry (IHC), respectively. mRNA expression was measured by quantitative PCR. Serum Nectin-4 was detected by ELISA and compared with CEA and CA15.3 markers, on panels of 45 sera from healthy donors, 53 sera from patients with non-metastatic breast carcinoma (MBC) at diagnosis, and 182 sera from patients with MBC. Distribution of histological/serological molecular markers and histoclinical parameters were compared using the standard Chi-2 test.

RESULTS

Nectin-4 was not detected in normal breast epithelium. By contrast, Nectin-4 was expressed in 61% of ductal breast carcinoma vs 6% in lobular type. Expression of Nectin-4 strongly correlated with the basal-like markers EGFR, P53, and P-cadherin, and negatively correlated with the luminal-like markers ER, PR and GATA3. All but one ER/PR-negative tumors expressed Nectin-4. The detection of Nectin-4 in serum improves the follow-up of patients with MBC: the association CEA/CA15.3/Nectin-4 allowed to monitor 74% of these patients compared to 67% with the association CEA/CA15.3. Serum Nectin-4 is a marker of disease progression, and levels correlate with the number of metastases (P = 0.038). Serum Nectin-4 is also a marker of therapeutic efficiency and correlates, in 90% of cases, with clinical evolution.

CONCLUSIONS

Nectin-4 is a new tumor-associated antigen for breast carcinoma. Nectin-4 is a new bio-marker whose use could help refine breast cancer taxonomy and improve patients' follow-up. Nectin-4 emerges as a potential target for breast cancer immunotherapy.

The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8-E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5-10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5-13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling.

IL-17A is produced from Th17 cells, and is involved in many autoimmune and inflammatory diseases. IL-13R has not previously been reported to be functionally expressed on T cells; however, we found that purified BALB/c CD4(+) cells polarized to Th17 with TGF-beta, IL-6, and IL-23 have increased mRNA and protein expression of IL-13R alpha1 and mRNA expression of IL-4R alpha compared with Th0, Th1, or Th2 polarized cells. The addition of IL-13 at Th17 polarization negatively regulated IL-17A and IL-21 expression, and reduced the number of CD4(+) T cells producing IL-17A. Further, adding IL-13 at the time of Th17 cell restimulation attenuated IL-17A expression. CD4(+) Th17 polarized cells from IL-4 knockout (KO) mice also had IL-13-induced inhibition of IL-17A production, but this was not observed in IL-4R KO and STAT6 KO mice. Addition of IL-13 at polarization increased IL-13R expression in wild-type Th17 cells. Further, IL-13 administration during Th17 polarization down-regulated retinoic acid-related-gammaT, the transcription required for Th17 development; increased STAT6 phosphorylation, and up-regulated GATA3, the transcription factor activated during the development of Th2 cells. This IL-13-mediated effect was specific to Th17 cells as IL-13 neither decreased IFN-gamma expression by Th1 cells nor affected Th2 cell production of IL-4. Collectively, we have shown that Th17 cells express a functional IL-13R and that IL-13 negatively regulates IL-17A and IL-21 production by decreasing retinoic acid-related-gammaT expression and while increasing phosphorylation of STAT6 and GATA3 expression. Therefore, therapeutic intervention inhibiting IL-13 production could have adverse consequences by up-regulating Th17 inflammation in certain disease states.