Runx3 regulates folliculogenesis and steroidogenesis in granulosa cells of immature mice.

We previously demonstrated that female Runx3 knockout (Runx3-/-) mice were anovulatory and their uteri were atrophic and that Runx3 mRNA was expressed in granulosa cells. To clarify how Runx3 regulates folliculogenesis and ovulation, we examine the effects of Runx3 knockout on the gene expression of growth factors associated with folliculogenesis and enzymes associated with steroidogenesis. In Runx3-/- mouse ovaries, the numbers of primary and antral follicles were lower than those in wild-type (wt) mice at 3 weeks of age, indicating that the loss of Runx3 affects folliculogenesis. The expression of genes encoding activin and inhibin subunits (Inha, Inhba and Inhbb) was also decreased in ovaries from the Runx3-/- mice compared with that in wt mice. Moreover, the expression of the genes Cyp11a1 and Cyp19a1 encoding steroidogenic enzymes was also decreased. In cultured granulosa cells from 3-week-old mouse ovaries, Cyp19a1 mRNA levels were lower in Runx3-/- mice than those in wt mice. Follicle-stimulating hormone (FSH) treatment increased Cyp19a1 mRNA levels in both wt and Runx3-/- granulosa cells in culture but the mRNA level in Runx3-/- granulosa cells was lower than that in wt ones, indicating that granulosa cells could not fully function in the absence of Runx3. At 3 weeks of age, gonadotropin α subunit, FSHß subunit and luteinizing hormone (LH) ß subunit mRNA levels were decreased in Runx3-/- mice. These findings suggest that Runx3 plays a key role in female reproduction by regulating folliculogenesis and steroidogenesis in granulosa cells.

Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours.

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

Core-binding factor ß and Runx transcription factors promote adaptive natural killer cell responses.

Natural killer (NK) cells are innate lymphocytes that have features of adaptive immunity such as clonal expansion and generation of long-lived memory. Interleukin-12 (IL-12) signaling through its downstream transcription factor signal transducer and activator of transcription 4 (STAT4) is required for the generation of memory NK cells after expansion. We identify gene loci that are highly enriched for STAT4 binding using chromatin immunoprecipitation sequencing for STAT4 and the permissive histone mark H3K4me3 in activated NK cells. We found that promoter regions of Runx1 and Runx3 are targets of STAT4 and that STAT4 binding during NK cell activation induces epigenetic modifications of Runx gene loci resulting in increased expression. Furthermore, specific ablation of Runx1, Runx3, or their binding partner Cbfb in NK cells resulted in defective clonal expansion and memory formation during viral infection, with evidence for Runx1-mediated control of a cell cycle program. Thus, our study reveals a mechanism whereby STAT4-mediated epigenetic control of individual Runx transcription factors promotes the adaptive behavior of antiviral NK cells.

Runx3 specifies lineage commitment of innate lymphoid cells.

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells.

A study of RUNX3, E-cadherin and ß-catenin in CagA-positive Helicobacter pylori associated chronic gastritis in Saudi patients.

OBJECTIVE: H. pylori is the most important risk factor for gastric carcinoma. CagA-positive H. pylori is associated with an increased risk for gastric cancer compared with negative strains. RUNX3 is a tumor suppressor gene, which is related to the genesis of gastric cancer. ß-catenin is integrated with E-cadherin in the cell membrane, and aberrant expression of the complex was reported in gastric carcinoma. Aim of this paper is to determine of the relation between RUNX3, E-cadherin and ß-catenin in chronic gastritis associated with cagA-positive H. pylori infection. PATIENTS AND METHODS: Retrospective study was done on formalin fixed paraffin embedded gastric biopsies blocks of 90 patients diagnosed as H. pylori associated chronic gastritis. H. pylori was detected using modified Giemsa stain. Nested PCR was used for detection of cagA, reverse transcription-PCR for detection of RUNX3 and immunohistochemistry for detection of E-cadherin and ß-catenin. RESULTS: Fifty percent of cases were found to be cagA positive. CagA was significantly associated with the intensity of mononuclear inflammation, the intensity of neutrophilic inflammation, the degree of mucosal atrophy and loss of RUNX3 but not with the density of H. pylori, intestinal metaplasia, E-cadherin or ß-catenin. There was significant relation between loss of RUNX3 and increasing density of H. pylori, intensity of neutrophilic inflammation, mucosal atrophy and intestinal metaplasia. RUNX3 was found to be significantly correlated with E-cadherin but not with ß-catenin. E-cadherin showed decreased expression in 36.7% of biopsies while, ß-catenin was decreased in 33% of biopsies. CONCLUSIONS: Loss of RUNX3, E-cadherin and ß-catenin was considered early events in the cascade of gastric carcinoma development. Loss of RUNX3 but neither E-cadherin nor ß-catenin was related to cagA positive H. pylori strains.

Runt-related transcription factor 3 is involved in the altered phenotype and function in ThPok-deficient invariant natural killer T cells.

The interplay between the CD4-lineage transcription factor ThPok and the CD8-lineage transcription factor, runt-related transcription factor 3 (Runx3), in T-cell development has been extensively documented. However, little is known about the roles of these transcription factors in invariant natural killer T (iNKT) cell development. CD1d-restricted iNKT cells are committed to the CD4(+)CD8(-) and CD4(-)CD8(-) sublineages, which respond to antigen stimulation with rapid and potent release of T helper (Th) 1 and Th2 cytokines. However, previous reports have demonstrated a new population of CD8(+) NKT cells in ThPok-deficient mice. In the current study, we sought to determine whether Runx3 was involved in the re-expression of CD8 and function of iNKT cells in the absence of ThPok. We used mice lacking Runx3, ThPok or both and verified that Runx3 was partially responsible for the appearance of CD8(+) iNKT cells in ThPok knockout mice. Additionally, Runx3 participated in the immune response mediated by iNKT cells in a model of α-galactosylceramide-induced acute hepatitis. These results indicate that Runx3 is crucial for the phenotypic and functional changes observed in ThPok-deficient iNKT cells.

Transcriptional reprogramming of CD11b+Esam(hi) dendritic cell identity and function by loss of Runx3.

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam(hi) DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4(+)/CD11b(+) DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b(+)Esam(hi) DC. Mechanistically, loss of Runx3 alters Esam(hi) DC gene expression to a signature characteristic of WT Esam(low) DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3(-/-) Esam(hi) DC capacity to prime CD4(+) T cells, attesting to the significant role of Runx3 in specifying Esam(hi) DC identity and function.

Runx3 deficiency results in myeloproliferative disorder in aged mice.

The RUNX family genes encode transcription factors that are involved in development and human diseases. RUNX1 is one of the most frequently mutated genes in human hematological malignancies and is a critical factor for the generation and maintenance of hematopoietic stem cells. Another Runx family gene, Runx3, is known to be expressed in hematopoietic cells. However, its involvement in hematopoiesis remains unclear. Here we show the hematopoietic phenotypes in Runx3 conditional knockout (KO) mice (Runx3(fl/fl);Mx1-Cre(+)): whereas young Runx3 KO mice did not exhibit any significant hematopoietic defects, aged Runx3 KO mice developed a myeloproliferative disorder characterized by myeloid-dominant leukocytosis, splenomegaly, and an increase of hematopoietic stem/progenitor cells (HSPCs). Notably, Runx3-deficient cells showed hypersensitivity to granulocyte-colony stimulating factor, suggesting enhanced proliferative and mobilization capability of Runx3-deficient HSPCs when stimulated. These results suggest that, besides Runx1, Runx3 also plays a role in hematopoiesis.

Abnormal liver differentiation and excessive angiogenesis in mice lacking Runx3.

Runt-related transcription factor 3 (Runx3) is essential for normal mouse development, and Runx3 knock-out (KO) mice (FVB strain), which die within 24 h after birth, show various organ defects, such as lung hyperplasia. For proper early liver development, angiogenesis and liver cell differentiation mechanisms are necessary in mammals. Previous studies have reported that various signaling molecules, such as vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and cluster of differentiation 31 (CD31), are closely related to angiogenesis in the developing liver. Proper expression levels of molecules that induce liver cell differentiation, such as phosphorylated Smad2 (pSmad2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Wilms tumor-1 (WT-1) and CD90 (Thy-1), are necessary for fetal liver development. To confirm the pathogenesis of liver defects caused by the loss of function of Runx3, the localization of proliferating cells was examined in wild-type and Runx3 KO mouse livers at postnatal day 1 (PN1). Specimens were also stained for various liver differentiation markers to confirm the function of Runx3. Moreover, gene expression level was examined by real-time quantitative polymerase chain reaction (RT-qPCR). Our results indicate that VEGF, vWF, CD31, pSmad2, NF-kB, WT-1 and Thy-1 were markedly up-regulated by the loss of Runx3. Therefore, our results indicate that liver development is controlled by Runx3. Clarifying the mechanisms of angiogenesis and liver differentiation might aid in the design of efficient and safe antiangiogenic therapy and gene therapy for liver disorders.

Tumor suppressor function of RUNX3 in breast cancer.

Emerging evidence indicates that RUNX3 is a tumor suppressor in breast cancer. RUNX3 is frequently inactivated in human breast cancer cell lines and cancer samples by hemizygous deletion of the Runx3 gene, hypermethylation of the Runx3 promoter, or cytoplasmic sequestration of RUNX3 protein. Inactivation of RUNX3 is associated with the initiation and progression of breast cancer. Female Runx3(+/-) mice spontaneously develop ductal carcinoma, and overexpression of RUNX3 inhibits the proliferation, tumorigenic potential, and invasiveness of breast cancer cells. This review is intended to summarize these findings and discuss the tumor suppressor function of RUNX3 in breast cancer.

Absence of Runx3 expression in normal gastrointestinal epithelium calls into question its tumour suppressor function.

The Runx3 transcription factor regulates cell fate decisions during embryonic development and in adults. It was previously reported that Runx3 is strongly expressed in embryonic and adult gastrointestinal tract (GIT) epithelium (Ep) and that its loss causes gastric cancer. More than 280 publications have based their research on these findings and concluded that Runx3 is indeed a tumour suppressor (TS). In stark contrast, using various measures, we found that Runx3 expression is undetectable in GIT Ep. Employing a variety of biochemical and genetic techniques, including analysis of Runx3-GFP and R26LacZ/Runx3(Cre) or R26tdTomato/Runx3(Cre) reporter strains, we readily detected Runx3 in GIT-embedded leukocytes, dorsal root ganglia, skeletal elements and hair follicles. However, none of these approaches revealed detectable Runx3 levels in GIT Ep. Moreover, our analysis of the original Runx3(LacZ/LacZ) mice used in the previously reported study failed to reproduce the GIT expression of Runx3. The lack of evidence for Runx3 expression in normal GIT Ep creates a serious challenge to the published data and undermines the notion that Runx3 is a TS involved in cancer pathogenesis.

Runx3 is required for full activation of regulatory T cells to prevent colitis-associated tumor formation.

Inflammation is increasingly recognized as an essential component of tumorigenesis, which is promoted and suppressed by various T cell subsets acting in different ways. It was shown previously in Runx3-deficient mice that differentiation of CD8 T and NK cells is perturbed. In this study, we show that Runx3 is also required for proper differentiation and function of regulatory T cells. In Runx3-deficient mice, T cells were unable to inhibit inflammation and to suppress tumor development. As expected, recombination activating gene 2-deficient mice bearing Runx3-deficient lymphocytes spontaneously developed colon tumors. However, tumor formation was completely blocked by transfer of either regulatory T cells or CD8 T cells derived from wild-type mice to mutant mice or by housing mutant mice in a specific pathogen-free condition. These results indicate that Runx3-deficient lymphocytes and microorganisms act together to induce inflammation and consequently induce the development of colon tumors.

Loss of runt-related transcription factor 3 expression associated with human hepatocellular carcinoma progression and prognosis.

BACKGROUND: This study aimed to evaluate the expression level of runt-related transcription factor 3 (RUNX3) in human primary hepatocellular carcinomas (HCCs) and its relationship with the clinic pathological features. METHODS: RUNX3 expression was analyzed by real-time fluorescent quantitative PCR, immunohistochemistry and Western blotting in HCC cells and tissues. RESULTS: RUNX3 RNA and protein expression was decreased in HCC tissues compared with the adjacent normal tissues (P< 0.001), mRNA frequently being down-regulated in HCC cell lines (66.67%, 4/6). Low expression of RUNX3 showed a significant correlation with cirrhosis (P = 0.028), histologic type (P = 0.000) and lymph node metastasis (P= 0.004). CONCLUSION: RUNX3 expression is deleted or decreased in HCCs and cell lines, in association with progression and prognosis.

Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer.

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3-/- mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3-/- bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3-/- epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/- mice ( approximately 18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/- mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

Requirement for Runx proteins in IgA class switching acting downstream of TGF-beta 1 and retinoic acid signaling.

IgA is a specific isotype required for mucosal immunity and is the most abundant Ab produced in vivo. Recently, several inductive signals for IgA class switch recombination have been identified; however, the molecular details of the action of these signals and the specific factors acting in B cells remain elusive. In this study, we show that combination of retinoic acid (RA) and TGF-beta1 with other factors induced a much higher frequency of IgA-switched cells than reported previously. In addition, IgA production is severely impaired in Runx2-Runx3 double-deficient mice. In Runx2-Runx3-deficient B cells, both RA- and TGF-beta1-dependent inductions of alpha germline transcription are completely blocked. These data suggest that Runx proteins play an essential role in IgA class switching acting downstream of RA and TGF-beta1 signaling.

Loss of Runx3 affects ovulation and estrogen-induced endometrial cell proliferation in female mice.

Runx3 is a transcription factor that belongs to the Runx family. We studied the function of Runx3 in the mouse ovary and uterus using the Runx3 knockout (Runx3(-/-)) mouse. Ovaries were collected from 8-week-old wild type (wt) and Runx3(-/-) mice. Histological studies showed that follicles were present at various developmental stages in the Runx3(-/-) and wt mouse ovaries. The numbers of primary, preantral and antral follicles in the Runx3(-/-) mice were significantly less than those in the wt mice while the number of primordial follicles in the Runx3(-/-) mice was not significantly different from that in the wt mice. Corpora lutea were not detected in the Runx3(-/-) mouse ovary. Gonadotropin treatment in immature female mice induced ovulation in Runx3(-/-) mice as well as in wt mice, indicating that ovaries of Runx3(-/-) mice respond to gonadotropin treatment as those in wt mouse ovaries. This suggests that failure of ovulation is due to dysfunction of regulatory mechanism of gonadotropin secretion. In addition, the uteri of Runx3(-/-) mice were atrophic, showed thin epithelial layers compared with those of the wt mice, and did not respond to estrogen in terms of DNA replication in endometrial epithelial cells. These results suggest that Runx3 takes part in the regulation of reproductive functions.

Runx3 regulates dendritic epidermal T cell development.

The Runx3 transcription factor regulates development of T cells during thymopoiesis and TrkC sensory neurons during dorsal root ganglia neurogenesis. It also mediates transforming growth factor-beta signaling in dendritic cells and is essential for development of skin Langerhans cells. Here, we report that Runx3 is involved in the development of skin dendritic epidermal T cells (DETCs); an important component of tissue immunoregulation. In developing DETCs, Runx3 regulates expression of the alphaEbeta7 integrin CD103, known to affect migration and epithelial retention of DETCs. It also regulates expression of IL-2 receptor beta (IL-2Rbeta) that mediates cell proliferation in response to IL-2 or IL-15. In the absence of Runx3, the reduction in CD103 and IL-2Rbeta expression on Runx3(-/-) DETC precursors resulted in impaired cell proliferation and maturation, leading to complete lack of skin DETCs in Runx3(-/-) mice. The data demonstrate the requirement of Runx3 for DETCs development and underscore the importance of CD103 and IL-2Rbeta in this process. Of note, while Runx3(-/-) mice lack both DETCs and Langerhans cells, the two most important components of skin immune surveillance, the mice did not develop skin lesions under pathogen-free (SPF) conditions.

Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.

Cell differentiation involves activation and silencing of lineage-specific genes. Here we show that the transcription factor Runx3 is induced in T helper type 1 (T(H)1) cells in a T-bet-dependent manner, and that both transcription factors T-bet and Runx3 are required for maximal production of interferon-gamma (IFN-gamma) and silencing of the gene encoding interleukin 4 (Il4) in T(H)1 cells. T-bet does not repress Il4 in Runx3-deficient T(H)2 cells, but coexpression of Runx3 and T-bet induces potent repression in those cells. Both T-bet and Runx3 bind to the Ifng promoter and the Il4 silencer, and deletion of the silencer decreases the sensitivity of Il4 to repression by either factor. Our data indicate that cytokine gene expression in T(H)1 cells may be controlled by a feed-forward regulatory circuit in which T-bet induces Runx3 and then 'partners' with Runx3 to direct lineage-specific gene activation and silencing.