Genome-wide profiling reveals transcriptional repression of MYC as a core component of NR4A tumor suppression in acute myeloid leukemia.

Acute myeloid leukemias (AMLs) are a heterogeneous group of diseases that are sustained by relatively rare leukemia-initiating cells (LICs) that exhibit diverse genetic and phenotypic properties. AML heterogeneity presents a major challenge to development of targeted therapies, and effective treatment will require targeting of common molecular drivers of AML maintenance. The orphan nuclear receptors NR4A1 and NR4A3 are potent tumor suppressors of AML. They are silenced in all human AML LICs, irrespective of patient cytogenetics, and their deletion in mice leads to postnatal AML development. In the current report, we address the tumor-suppressive mechanisms and therapeutic potential of NR4As for AML intervention. We show that rescue of either NR4A1 or NR4A3 inhibits the leukemogenicity of AML cells in vivo and reprograms a subset of gene signatures that distinguish primary human LICs from normal hematopoietic stem cells (HSCs), irrespective of subtype. Central to NR4A reprogramming is the acute suppression of an LIC submodule that includes the transcriptional repression of MYC. Additionally, we show that upregulation of MYC is an acute preleukemic consequence of NR4A deletion and that MYC suppression functionally contributes to NR4A antileukemic effects. Collectively, these results identify NR4As as novel targets for AML therapeutic intervention and reveal molecular targets of NR4A tumor suppression, including the suppression of MYC.

Progesterone signaling in mammary gland development.

The mammary gland undergoes extensive epithelial expansion and differentiation during pregnancy, leading ultimately to the development of functional milk-producing alveolar lobules. This phase of mammary gland remodeling is controlled primarily by the cooperative interplay between hormonal signals initiated by both progesterone and prolactin. Abrogation of mammary epithelial expression of receptors for either one of the hormones results in failure of alveologenesis and an absence of pregnancy-induced tertiary ductal side branches in the case of progesterone receptor-null (PRKO) mammary glands. By combining gene array approaches to identify PR- and prolactin (PRL)-dependent downstream signaling pathways and by using genetic mouse models to address the consequences of abrogation and/or misexpression of potential downstream genes, recent studies have begun to illuminate key signaling pathways that mediate the morphogenic effects of these hormones during pregnancy-induced mammary gland remodeling. Analysis of deregulated expression of PR-dependent gene transcripts in PRKO mammary glands has revealed that convergence between progesterone and prolactin signaling occurs in part through progesterone-dependent induction of mammary epithelial PRL receptors to prime the mammary epithelium to respond to PRL. Additional genes activated by PRs encode epithelial paracrine growth factor signals that regulate ductal and alveolar epithelial proliferation and survival, lineage-restricted transcription factors that control luminal and alveolar cell fate establishment and maintenance, and gap junction proteins that play a critical role in alveolar morphogenesis by establishment of epithelial cell polarity. Finally, two distinct isoforms of PRs (PR-A and PR-B) are coexpressed in the mammary gland and display extensively overlapping but partially distinct gene regulatory properties in relaying the progesterone signal.

Multifunctional roles of lactoferrin: a critical overview.

Lactoferrin (LF) is a member of the transferrin family that is expressed and secreted by glandular epithelial cells and is found in the secondary granules of neutrophils. Originally viewed as an iron-binding protein in milk, with bacteriostatic properties, it is becoming increasingly evident that LF is a multifunctional protein to which several physiological roles have been attributed. These include regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis. While iron binding is likely central to some of the biological roles of LF, other activities, including specific interactions with mammalian receptors and microbial components, also contribute to the pleoitropic functional nature of this protein. In this article, recent advances in the understanding of these functions at the cellular and molecular level are discussed.

p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis.

Full-term pregnancy early in reproductive life is protective against breast cancer in women. Pregnancy also provides protection in animals against carcinogen-induced breast cancer, and this protection can be mimicked by using the hormones estrogen and progesterone. The molecular mechanisms that form the basis for this protective effect have not been elucidated. On the basis of our results, we propose a cell-fate hypothesis. At a critical period in adolescence the hormonal milieu of pregnancy affects the developmental fate of a subset of mammary epithelial cells and its progeny, which results in persistent differences in molecular pathways between the epithelial cells of hormone-treated and mature virgin mammary glands. These changes in turn dictate the proliferative response to carcinogen challenge and include a block in carcinogen-induced increase in mammary epithelial cell proliferation and an increased and sustained expression of nuclear p53 in the hormone-treated mammary gland. This hormone-induced nuclear p53 is transcriptionally active as evidenced by increased expression of mdm2 and p21 (CIP1/WAF1). Importantly, exposure to perphenazine, a compound that induces mammary gland differentiation but does not confer protection, does not induce p53 expression, indicating that p53 is not a differentiation marker. The proliferative block and induction of p53 are operative in both rats and mice, results that support the generality of the proposed hypothesis.

Antiinflammatory activities of lactoferrin.

Lactoferrin is a non-heme iron binding glycoprotein produced during lactation and by epithelial cells at mucosal surfaces. The protein is a prominent component of the first line of mammalian host defense and its expression is upregulated in response to inflammatory stimuli. In this paper, the antibacterial and immune modulatory properties of lactoferrin that contribute to host defense are reviewed. In addition, the results of recent preclinical and clinical studies demonstrating that lactoferrin acts as an inhibitor of dermal inflammatory cytokine production are summarized. The results indicate that lactoferrin may act as a potent anti-inflammatory protein at local sites of inflammation including the respiratory and gastrointestinal tracts.

Early exposure of the rat mammary gland to estrogen and progesterone blocks co-localization of estrogen receptor expression and proliferation.

An early single full-term pregnancy induces a long-lasting protective effect against mammary tumor development in humans and rodents. This protective effect can be mimicked in rats by short-term administration of estrogen and progesterone hormones prior to carcinogen administration. The hormones of pregnancy are able to induce a proliferative block upon carcinogen challenge that is not observed in the age-matched virgin. We wished to determine whether carcinogen is needed to induce a paracrine-to-autocrine shift of proliferation in steroid receptor positive cells or if such a cell population already exists in the age-matched virgin mammary gland. Here we show that estrogen receptor positive (ER+) proliferating cells are rare in the developing mammary gland of the virgin rat but represent the majority of the proliferating cells in the mature (96-day-old) mammary gland of the virgin rat. As the majority of the proliferating cells before carcinogen challenge were ER positive, the ER+ proliferating cells in the mature mammary gland may represent the target cells for carcinogen-induced transformation. Importantly, prior exposure of the mammary gland to pregnancy levels of estrogen/progesterone blocked this positive association. This ability to block the proliferation of the ER+ cells may be one factor by which pregnancy induces protection against breast cancer.

Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice.

Progesterone plays a central coordinate role in diverse reproductive events associated with establishment and maintenance of pregnancy. In humans and other vertebrates, the biological activities of progesterone are mediated by two proteins, A (PR-A) and B (PR-B) that arise from the same gene and function as progesterone activated transcription factors that exhibit different transcription regulatory activities in vitro. Mice lacking both PR isoforms (PRKO mice) exhibit pleiotropic reproductive abnormalities. To address the physiological role of the individual isoforms, we have selectively ablated PR-A expression in mice (PRAKO). We have demonstrated that PR-B mediates a subset of the reproductive functions of P. Ablation of PR-A does not affect responses of the mammary gland or thymus to P but results in severe abnormalities in ovarian and uterine function. Analysis of urine function of PRAKP mice reveals an unexpected P-dependent proliferative activity of PR-B in the epithelium and provides evidence that the tissue-specific reproductive effects of this isoform are due to specificity of target gene transactivation rather than differences in tissue-specific expression relative to PR-A. Taken together, our data indicate that PR-A and PR-B act in vivo as two functionally distinct transcription factors.

Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin-releasing hormone expression and actions in human inflammatory arthritis.

OBJECTIVE: To examine the regulation and mode of action of peripheral corticotropin-releasing hormone (CRH) in human inflammatory arthritis. METHODS: CRH messenger RNA (mRNA) levels were measured in normal and inflamed synovial tissue and in primary synoviocytes prior to and following cytokine stimulation. Primary synoviocytes were transiently transfected with CRH promoter/reporter constructs, and promoter activity in response to cytokines was assessed. Immunohistochemical staining established CRH receptor expression, and Northern blot analysis confirmed that the nuclear transcription factors NUR77 and NURR1 contributed to synovial CRH receptor-mediated signaling. Primary synoviocytes were treated with pro- and antiinflammatory mediators, and the time course of NURR1 and NUR77 modulation was examined. Nuclear extracts were analyzed by electrophoretic mobility shift assay to determine NURR1 binding to the CRH promoter/enhancer. RESULTS: CRH mRNA was up-regulated in the synovial tissue in rheumatoid arthritis (RA), psoriatic arthritis (PsA), and sarcoid arthritis, but not in normal synovium. Inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, enhanced the transcriptional activity of the human CRH promoter and increased levels of CRH mRNA in primary synoviocytes. Synovial CRH functioned in a paracrine manner to induce NURR1 and NUR77. NURR1 was abundantly expressed in the inflammatory cells of both RA and PsA synovium. NURR1 and NUR77 were differentially regulated, and NURR1 was the major cytokine-regulated member of the NURR subfamily as well as the mediator of cytokine- and CRH-dependent inflammatory responses in synovium. Furthermore, glucocorticoids dramatically suppressed cytokine- and CRH-induced synovial NURR1 mRNA. CONCLUSION: These data demonstrate the involvement of the transcription factor NURR1 in the regulation of CRH expression and activity in human inflammatory arthritis.

Progesterone receptors in reproduction: functional impact of the A and B isoforms.

Progesterone (P) is a key regulator of female reproductive activity. The effects of P are mediated by two progesterone receptor (PR) proteins, termed A and B, that arise from a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes. Null mutation of the PR gene in mice (PRKO) leads to pleiotropic reproductive abnormalities. This paper will review the reproductive functions of PRs delineated using the PRKO mouse. Further, we will summarize the structure and functional properties of PRs and discuss how functional differences between the PR-A and PR-B isoforms are likely to impact on the overall physiological role of the receptor in reproductive systems.

The dual function steroid receptor coactivator/ubiquitin protein-ligase integrator E6-AP is overexpressed in mouse mammary tumorigenesis.

Steroid receptor coactivator and corepressor proteins are important mediators of steroid receptor function. Changes in the expression or activity of these limiting cofactors can contribute to the etiology of steroidal cancers. Using a mouse mammary model of multistage tumorigenesis we have examined whether the expression of select steroid receptor coactivators is altered. The 10 kb transcript of the novel dual function steroid receptor coactivator/ubiquitin protein-ligase integrator E6-AP is overexpressed 2.5-4.5 fold in the mammary tumors but not in the precursor steps of tumorigenesis; that is, immortal ductal and alveolar hyperplastic outgrowths. The over expression is striking because the 10 kb transcript is expressed to variable levels in other wild type tissues like the uterus, ovary, testis, kidney and brain but is undetectable in normal virgin mammary gland and the prostate gland. The E6-AP overexpression in the mammary tumors is substantiated by western blot analysis and immunohistochemical analysis. Absence of ER and PR in these tumors in the presence of high levels of E6-AP could contribute to steroid receptor-independent function and tumorigenesis. There is no obvious correlation between p53 (a well-characterized substrate of E6-AP) status (wt vs. mutant) and levels of E6-AP in the mouse mammary tumors.

Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform.

Progesterone regulates reproductive function through two intracellular receptors, progesterone receptor-A (PR-A) and progesterone receptor-B (PR-B), that arise from a single gene and function as transcriptional regulators of progesterone-responsive genes. Although in vitro studies show that PR isoforms can display different transcriptional regulatory activities, their physiological significance is unknown. By selective ablation of PR-A in mice, we show that the PR-B isoform modulates a subset of reproductive functions of progesterone by regulation of a subset of progesterone-responsive target genes. Thus, PR-A and PR-B are functionally distinct mediators of progesterone action in vivo and should provide suitable targets for generation of tissue-selective progestins.

Regulation of epidermal Langerhans cell migration by lactoferrin.

Lactoferrin (LF) is a member of the transferrin family of iron-binding glycoproteins to which several anti-inflammatory functions have been ascribed. LF has been shown to down-regulate expression of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha), although the possibility has been raised that the activity of LF in this regard was indirect and secondary to its ability to bind to and inactivate the bacterial lipopolysaccharide (LPS) used to induce cytokine production. However, the identification of putative membrane receptors for LF raises the possibility that the interaction of LF with its receptor may be one important route through which this protein exerts anti-inflammatory activity. In the present investigations the biological properties of LF have been examined in a model of cutaneous immune function where the allergen-induced migration of epidermal Langerhans cells (LC) from the skin and their subsequent accumulation as dendritic cells (DC) in skin-draining lymph nodes are known to be dependent upon the de novo synthesis of TNF-alpha, but independent of exogenous LPS. Consistent with the protein having direct anti-inflammatory properties, it was found that the intradermal injection of recombinant murine LF (either iron-saturated or iron-depleted LF) inhibited significantly allergen (oxazolone) -induced LC migration and DC accumulation. That these inhibitory effects were secondary to the inhibition of local TNF-alpha synthesis was suggested by the findings that first, LF was unable to inhibit LC migration induced by intradermal injection of TNF-alpha itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1beta, which is also dependent upon TNF-alpha production, was impaired significantly by prior treatment with LF. Finally, immunohistochemical analyses demonstrated the presence of LF in skin, associated primarily with keratinocytes. Collectively these data support the possession by LF of direct immunomodulatory and/or anti-inflammatory activity, probably associated in this case with inhibition of cytokine production. Furthermore, the results suggest that as a constituent of normal skin, LF may play a role in homeostatic regulation of cutaneous immune function.

Reproductive functions of the progesterone receptor.

Progesterone plays a central coordinate role in regulating reproductive events associated with the establishment and maintenance of pregnancy including ovulation, uterine and mammary gland development and tumorigenesis, and neurobehavioral expression associated with sexual responsiveness. The effects of progesterone are mediated by two receptor proteins (PR), termed A and B, that arise from a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes. Null mutation of both proteins in mice leads to pleiotropic reproductive abnormalities. This review summarizes the structure and functional properties of the PR isoforms and how functional differences between these proteins are likely to impact on the overall physiologic role of the receptor in reproductive systems.

A reappraisal of progesterone action in the mammary gland.

The ovarian hormones estrogen and progesterone and their respective receptors are essential for maintenance of postnatal developmental plasticity of the mammary gland and play a key role in mammary tumorigenesis. Mouse models in which expression of the progesterone receptors was genetically ablated have recently become available. Studies of these models have demonstrated that progesterone is specifically required for pregnancy associated ductal proliferation and lobuloalveolar differentiation of the mammary epithelium, but not for immediate postpubertal ductal morphogenesis. Use of these mice in combination with mammary gland transplantation indicates that developmental regulation by progesterone appears to occur through a paracrine mechanism in which progesterone receptor (PR) positive cells represent a subset of non-proliferating epithelial cells that are capable of directing proliferation and/or differentiation of neighboring receptor negative cells. The hierarchical organization of these receptors in the epithelium and their segregation from proliferating cells is a conserved feature in rodent and human mammary tissue. The identification of paracrine mediators of the progesterone response is now an imminent goal as is the delineation of the individual contributions of the two PR isoforms using similar approaches.

Selective agenesis of mesencephalic dopaminergic neurons in Nurr1-deficient mice.

Nurr1, a member of the nuclear receptor superfamily of transcription factors, has been found to be essential for the development of ventral midbrain dopamine (DA)ergic neurons. To study the regional selectivity and phenotypic specificity of regulation by Nurr1 of the genesis of DAergic neurons, we examined DAergic, serotonin (5-HT)ergic, norepinephrine (NE)ergic, cholinergic, glutamate (GLU)ergic, and gamma-aminobutyric acid (GABA)ergic neurons in the brains of Nurr1-deficient mice by immunohistochemistry and biochemistry. We demonstrated that in homozygous Nurr1-deficient mice (Nurr1-/-), DAergic neurons were totally absent in substantia nigra and ventral tegmental area, but preserved in other regions including diencephalon and hypothalamus, olfactory bulb (OB). Levels of DA in Nurr1-/- mice were decreased by 98% in striatum (Str) and 65% in OB. NEergic neurons in locus ceruleus, 5-HTergic neurons in raphe nuclei, and cholinergic neurons in basal forebrain and other regions were not changed. A 30% reduction of NE was found in the Str of Nurr1-/- mice. The levels of GLU and GABA and the activity of choline acetyl transferase in the brains of Nurr1-/- mice were not significantly altered. Our results demonstrate a selective and specific deficit of DA and absence of DAergic neurons in the mesencephalic structures of Nurr1-deficient mice, which resembles the pattern similar to that seen in patients with Parkinson's disease (PD). This model may contribute to our understanding of the mechanisms influencing DAergic cell survival in PD.

Progesterone receptors in the thymus are required for thymic involution during pregnancy and for normal fertility.

Thymocyte development is reported to be inhibited by pregnancy, although the impact of this effect on fertility is unknown. We demonstrate, using progesterone receptor null mutant mice, that the inhibitory effects of pregnancy hormones on T cell development require the presence of functional progesterone receptor (PR). A combination of hysterectomy, thymic immunohistochemistry, and transplant studies reveals that local expression of PR in thymic stromal cells is specifically required for thymic involution to occur. These cells, under the influence of progesterone, block T cell development at the early pre-T cell (CD3(-)CD44(+) CD25(+)) stage of development via a paracrine mechanism. In addition, age-related thymic involution is shown to occur by a separate PR-independent mechanism. Finally, pregnancy studies with thymic transplants from progesterone receptor null mutant mice to wild-type female recipients demonstrate that thymic stromal PR is required for normal fertility. Together, these observations provide evidence for a PR-dependent paracrine mechanism that blocks very early T cell lymphopoiesis during pregnancy and is essential for normal fertility.

Reduced Nurr1 expression increases the vulnerability of mesencephalic dopamine neurons to MPTP-induced injury.

Mutation in the Nurr1 gene, a member of the nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in the midbrain of null mice. Homozygous Nurr1 knockout mice (Nurr1-/-) die 1 day after birth, but heterozygous mice (Nurr1 +/-) survive postnatally without obvious locomotor deficits. Although adult Nurr1 +/- mice show significantly reduced Nurr1 protein levels in the substantia nigra (SN), they display a normal range of tyrosine hydroxylase-positive neuron numbers in the SN and normal levels of dopamine in the striatum. The reduction in Nurr1 expression in Nurr1 +/- mice, however, confers increased vulnerability to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) compared with wild-type (Nurr1 +/+) mice. This study suggests that Nurr1 may play an important role in maintaining mature mesencephalic dopaminergic neuron function and that a defect in Nurr1 may increase susceptibility to SN injury.

Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus.

Populations of macrophages and neutrophils in the uterus are under the control of the female sex steroids estrogen and progesterone (P4). Their influx is induced by estrogen, while P4 can both stimulate and inhibit leukocyte influx depending on the timing of P4 with respect to estrogen. Regulation of leukocytes has been implicated in changes in uterine immune responses during the estrous cycle, pregnancy, and implantation. This work demonstrates that P4 given concurrently with estrogen to ovariectomized mice for 4 days antagonizes the ability of estrogen to recruit macrophages and neutrophils into the mouse uterus. Using progesterone receptor knockout (PRKO) mice, we show that this effect is dependent on progesterone receptors (PR). In the absence of PR, neutrophils recruited by estrogen were found to be degranulated, partially explaining the edema that is observed with long-term treatment of PRKO mice with estrogen and P4. Populations of B lymphocyte cells were shown to be unchanged by estrogen and P4 treatment in both wild-type and PRKO mice. The neutrophil chemotactic chemokine MIP-2 was examined for down-regulation by P4 but was found to be unaffected by hormonal treatment. Together, these observations demonstrate that PR has a strong anti-inflammatory role in the mouse uterus when estrogen and P4 are present together.