Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17ß is mediated by a PKC-ERK1/2-mTOR signaling pathway.

The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17ß (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3ß pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

Xenografted tissue models for the study of human endometrial biology.

The human endometrium undergoes extensive morphological, biochemical and molecular changes under the influence of female sex steroid hormones. Besides the fact that estrogen stimulates endometrial cell proliferation and progesterone inhibits this proliferation and induces differentiation, there is limited knowledge about precise molecular mechanisms underlying human endometrial biology. The importance of paracrine signaling in endometrial physiology explains why in vitro culture of endometrial cells has been challenging. Researchers, therefore, have developed alternative experimental in vivo models for the study of endometrial biology. The objective of this review is to summarize the recent developments and work on these in vivo endometrial research models. The in vivo recombinant tissue models in which wild-type endometrial cells are combined with endometrial cells from a gene-targeted mouse strain followed by xenografting to host mice have been critical in confirming the significance of paracrine signaling between the epithelium and stroma in the growth regulation of the endometrium. Additionally, these studies have uncovered differences between the mouse and human, emphasizing the need for the development of experimental models specifically of the human endometrium. Recently, xenotransplants of human endometrial fragments into the subcutaneous space of host mice and endometrial xenografts of dissociated and recombined epithelial and stromal cells beneath the kidney capsule of immunodeficient host mice have proven to be highly promising tools for in vivo research of endometrial functions. For the first time, the latter approach provides an immense opportunity for the application of genome engineering, such as targeted ablation of endometrial genes for example by using CRISPR/CAS9 system. This research will begin to elucidate the functional role of specific genes in this complex tissue. Another advantage of xenotransplantation and xenograft models of the human endometrium is their use to investigate endometrial effects of new compounds and drugs without needing to give them to women. Underpinning the molecular mechanisms underlying endometrial functions is critical to ultimately advance our understanding of endometrial pathophysiology and develop targeted therapies to prevent and cure endometrial pathologies as well as enhance endometrial function when it is desired for fertility.

The selective progesterone receptor modulator, telapristone acetate, is a mixed antagonist/agonist in the human and mouse endometrium and inhibits pregnancy in mice.

OBJECTIVE: To investigate the effect of the selective progesterone receptor modulator, telapristone acetate (CDB-4124), on endometrial biology and reproductive outcomes. Ovariectomized and hormone-treated CD1 female mice, CD1 female mice with xenotransplants of reconstructed human endometrial tissue, mated wildtype female mice, and cultured human endometrial stromal cells (hESCs) were treated with CDB-4124, followed by the assessment of endometrial cell deoxyribonucleic acid (DNA) proliferation, stromal decidual response, and embryo implantation. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENTS: Healthy volunteer women from the community were recruited for endometrial biopsies. ANIMALS: CD1 out-bred mice (Charles River Laboratories) and nude mice, NU/J (Jackson Laboratories, Bar Harbor, ME). INTERVENTION: Treatment of mice and hESCs with CDB-4124. MAIN OUTCOME MEASURE: The effect of CDB-4124 on endometrial cell morphology and DNA synthesis, decidual response, and mouse embryo implantation. RESULTS: CDB-4124 inhibited estradiol-induced epithelial DNA synthesis in the mouse uterus and xenotransplanted human endometrium. This antiproliferative effect was less than that of progesterone (P4) and was observed when CDB-4124 was administered alone or concomitantly with P4. In the uterine epithelium, CDB-4124 acted as a P4 agonist and partial antagonist. In contrast, CDB-4124 acted as a complete P4 antagonist in the uterine stroma, where it blocked P4's action to induce a decidual response in the pseudopregnant mouse uterus and wildtype mouse uterus after copulation. In mated female mice, CDB-4124 impaired embryo implantation. Similarly, CDB-4124 inhibited the morphological and biochemical transformations of hESCs to decidual cells in vitro. CONCLUSION: CDB-4124 exerts mixed P4 antagonistic/agonistic effects in the human and mouse endometrium, which result in failed embryo implantation because of the absence of stromal decidualization.

Colony-stimulating factor-1-dependent macrophage functions regulate the maternal decidua immune responses against Listeria monocytogenes infections during early gestation in mice.

The association between extreme-prematurity births and intrauterine infection emphasizes the importance of understanding the host immune responses against uterine-invading microbes during early pregnancy to the prevention of preterm births. Listeria monocytogenes, a clinically relevant intracellular bacterium, has a predilection for replication at the maternofetal interface during pregnancy. Here, using mice carrying the recessive null osteopetrotic mutation in the colony-stimulating factor-1 (CSF-1) gene, we show that CSF-1-dependent macrophage functions are required for the maternal decidua immune responses against L. monocytogenes infections during early gestation in mice. In the absence of CSF-1, pregnant mice were more susceptible to uterine infection by L. monocytogenes; their inability to control the expansion of colonized bacteria in the pregnant uterus led to decidual cell death, tissue disintegration, and resorption of the developing embryo. However, CSF-1-deficient mice were able to produce significant levels of both Th1 cytokines and neutrophil chemoattractants and to recruit neutrophils to the decidual tissue in response to Listeria infection. Depletion of macrophages in hormonally induced pseudopregnant mice resulted in higher uterine bacterial levels after L. monocytogenes infection. These data suggest that the anti-Listeria responses in the maternal decidual tissue are dependent on CSF-1-regulated macrophages.

Lithium chloride treatment induces epithelial cell proliferation in xenografted human endometrium.

BACKGROUND: In mouse endometrium, glycogen synthase kinase-3beta (GSK3beta) is a key enzyme controlling nuclear localization of cyclin D1. We developed a functional model of xenografted human endometrium to test whether similar mechanisms are operative in the human by using Lithium chloride (LiCl), an inhibitor of GSK3beta. METHODS: Human endometrial samples were obtained from normal volunteers, then implanted under the kidney capsule of nude mice, and treated with estradiol-17beta (E2) or LiCl. Xenografts were assessed for protein expression of MKI-67, mini-chromosome maintenance protein-2, estrogen receptor (ER), progesterone receptor (PR) and cyclin D1. RESULTS: Both E2 and LiCl induced a robust proliferative response in the epithelium. Only lithium treatment produced clear nuclear localization of cyclin D1 consistent with the proliferative response observed. Regenerated endometrium had detectable ER and PR expression. CONCLUSION: Xenografted human endometrium provides a dynamic model of uterine biology. Administration of LiCl in the absence of E2 induced epithelial proliferation, supporting the hypothesis that human and murine endometrial proliferation may share key regulatory pathways. These data suggest a possible link between the increased menstrual disturbances in women with affective disorders taking lithium and the consequent potential for the development of endometrial proliferative disorder.

Macrophages regulate the angiogenic switch in a mouse model of breast cancer.

The development of a tumor vasculature or access to the host vasculature is a crucial step for the survival and metastasis of malignant tumors. Although therapeutic strategies attempting to inhibit this step during tumor development are being developed, the biological regulation of this process is still largely unknown. Using a transgenic mouse susceptible to mammary cancer, PyMT mice, we have characterized the development of the vasculature in mammary tumors during their progression to malignancy. We show that the onset of the angiogenic switch, identified as the formation of a high-density vessel network, is closely associated with the transition to malignancy. More importantly, both the angiogenic switch and the progression to malignancy are regulated by infiltrated macrophages in the primary mammary tumors. Inhibition of the macrophage infiltration into the tumor delayed the angiogenic switch and malignant transition whereas genetic restoration of the macrophage population specifically in these tumors rescued the vessel phenotype. Furthermore, premature induction of macrophage infiltration into premalignant lesions promoted an early onset of the angiogenic switch independent of tumor progression. Taken together, this study shows that tumor-associated macrophages play a key role in promoting tumor angiogenesis, an essential step in the tumor progression to malignancy.

GSK-3beta mediates in the progesterone inhibition of estrogen induced cyclin D2 nuclear localization and cell proliferation in cyclin D1-/- mouse uterine epithelium.

We report that glycogen synthase kinase (GSK)-3beta is phosphorylated at ser9 and inactivated in uterine epithelial cells from E(2)-treated cyclin D1 null mutant mice. Simultaneous administration of P(4) together with E(2) blocked this effect. Pharmacological inhibition of GSK-3beta activity in mice treated with P(4)E(2) reversed the nuclear exclusion of cyclin D2 in the uterine epithelial cells and this caused phosphorylation of Rb protein and progression of cells towards S-phase. Our results indicate that GSK-3beta is a major target of E(2) and P(4) in regulation of cyclin D2 localization in the mouse uterine epithelium.

Microarray analysis of uterine epithelial gene expression during the implantation window in the mouse.

In mice, the uterus becomes transiently receptive to the hatched blastocyst on the day of implantation to allow its attachment to the luminal epithelium and subsequent invasion into the uterus. This uterine preparation for implantation is regulated by estradiol-17beta and progesterone, acting through their transcription factor receptors. Using ovariectomized mice treated with physiological regimens of these hormones, combined with methods to isolate RNA specifically from the uterine epithelium followed by transcriptome analysis on cDNA microarrays, 222 genes whose transcript abundance was specifically increased by estradiol-17beta and progesterone treatment were identified. Gene ontology analysis revealed an emphasis on genes involved with immune responses, extracellular matrix metabolism, and cell-to-cell communication. In situ hybridization to uterine sections isolated through the first 6 d of pregnancy identified novel sets of genes such as Bach, Myd88, Cd14, Isg20, and Lrp2 whose expression was restricted to the uterine epithelium during the implantation window. Particularly notable was the expression of the mRNA for members of the signaling pathway from the Toll-like receptors to its downstream targets such as Irg-1. The identification of these genes showing a cell type hormonally regulated pattern of expression in the uterus suggests novel functions for them during implantation.

Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase-->AKT-->GSK-3beta-->cyclin D1-->pRB pathway to block uterine epithelial cell proliferation.

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.

Colony-stimulating factor 1 regulation of neuroendocrine pathways that control gonadal function in mice.

Colony stimulating factor 1 (CSF-1) is the primary regulator of cells of the mononuclear phagocytic lineage. Consequently mice lacking CSF-1 (Csf1(op)/Csf1(op)) have depleted populations of macrophages in many tissues. In addition, both sexes have reduced fertility with females having extended estrus cycles and poor ovulation rates, whereas males have low circulating LH and T. In this study, we show that puberty was significantly delayed in Csf1(op)/Csf1(op) females compared with control littermates. Restoration of circulating CSF-1 over the first 2 wk of life accelerated puberty, and this treatment until puberty completely corrected the extended estrous cycles. In a standard LH surge induction protocol, Csf1(op)/Csf1(op) females showed diminutive negative and no positive feedback response to E2. These data, together with that from male Csf1(op)/Csf1(op) mice that showed normal release of LH with a GnRH agonist, indicate that the hypothalamus is the site of the primary defect causing fertility problems in CSF-1-deficient mice. In the hypothalamus, microglia are the only CSF-1 receptor-bearing cells, and the recruitment of a full complement these cells is slightly delayed in Csf1(op)/Csf1(op) mice. These data suggest a role for CSF-1 and its target cells, microglia, in establishing the feedback sensitivity to circulating steroid hormones in the hypothalamus of mice.

Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival.

Colony-stimulating factor 1 (CSF1) and interleukin-34 (IL-34) are functional ligands of the CSF1 receptor (CSF1R) and thus are key regulators of the monocyte/macrophage lineage. We discovered that systemic administration of human recombinant CSF1 ameliorates memory deficits in a transgenic mouse model of Alzheimer's disease. CSF1 and IL-34 strongly reduced excitotoxin-induced neuronal cell loss and gliosis in wild-type mice when administered systemically before or up to 6 h after injury. These effects were accompanied by maintenance of cAMP responsive element-binding protein (CREB) signaling in neurons rather than in microglia. Using lineage-tracing experiments, we discovered that a small number of neurons in the hippocampus and cortex express CSF1R under physiological conditions and that kainic acid-induced excitotoxic injury results in a profound increase in neuronal receptor expression. Selective deletion of CSF1R in forebrain neurons in mice exacerbated excitotoxin-induced death and neurodegeneration. We conclude that CSF1 and IL-34 provide powerful neuroprotective and survival signals in brain injury and neurodegeneration involving CSF1R expression on neurons.

Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1(op)) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.

The EGF/CSF-1 paracrine invasion loop can be triggered by heregulin beta1 and CXCL12.

An important step in the process of metastasis from the primary tumor is invasive spread into the surrounding stroma. Using an in vivo invasion assay, we have previously shown that imposed gradients of epidermal growth factor (EGF) or colony-stimulating factor-1 (CSF-1) can induce invasion through an EGF/CSF-1 paracrine loop between cancer cells and macrophages. We now report that invasion induced by other ligands also relies on this EGF/CSF-1 paracrine invasive loop. Using an in vivo invasion assay, we show that MTLn3 breast cancer cells overexpressing ErbB3 exhibit enhanced invasion compared with control MTLn3 cells in response to the ErbB3 ligand HRG-beta1. The invasive response of both MTLn3-ErbB3 and transgenic MMTV-Neu tumors to HRG-beta1 is inhibited by blocking EGF receptor, CSF-1 receptor, or macrophage function, indicating that invasiveness to HRG-beta1 is dependent on the EGF/CSF-1 paracrine loop. Furthermore, we show that CXCL12 also triggers in vivo invasion of transgenic MMTV-PyMT tumors in an EGF/CSF-1-dependent manner. Although the invasion induced by HRG-beta1 or CXCL12 is dependent on the EGF/CSF-1 paracrine loop, invasion induced by EGF is not dependent on HRG-beta1 or CXCL12 signaling, showing an asymmetrical relationship between different ligand/receptor systems in driving invasion. Our results identify a stromal/tumor interaction that acts as an engine underlying invasion induced by multiple ligands.

Estradiol-17beta regulates mouse uterine epithelial cell proliferation through insulin-like growth factor 1 signaling.

Estradiol-17beta (E(2)) causes cell proliferation in the uterine epithelium of mice and humans by signaling through its transcription factor receptor alpha (ERalpha). In this work we show that this signaling is mediated by the insulin-like growth factor 1 receptor (IGF1R) expressed in the epithelium, whose activation leads to the stimulation of the phosphoinositide 3-kinase/protein kinase B pathway leading to cyclin D1 nuclear accumulation and engagement with the canonical cell cycle machinery. This cyclin D1 nuclear accumulation results from the inhibition of glycogen synthase kinase 3beta (GSK3beta) activity caused by an inhibitory phosphorylation by protein kinase B. Once the IGF1 pathway is activated, inhibition of ER signaling demonstrates that it is independent of ER. Inhibition of GSK3beta in the absence of E(2) is sufficient to induce uterine epithelial cell proliferation, and GSK3beta is epistatic to IGF1 signaling, indicating a linear pathway from E(2) to cyclin D1. Exposure to E(2) is the major risk factor for endometrial cancer, suggesting that downstream activation of this IGF1-mediated pathway by mutation could be causal in the progression to ER-independent tumors.

Conditional deletion of the colony stimulating factor-1 receptor (c-fms proto-oncogene) in mice.

Colony stimulating factor-1 (CSF-1) is the primary regulator of the mononuclear phagocytic lineage acting through its transmembrane tyrosine kinase receptor, CSF-1R, that is the product of the c-fms proto-oncogene. Null mutations in either the ligand or the receptor genes result in a severe osteopetrosis as well as a number of other phenotypes, including reproductive defects and perturbations in organ development. The CSF-1R is also expressed in oocytes, myoblast progenitors, decidual, and trophoblastic cells. To distinguish cell type specific phenotypes, we have created a conditional allele of the Csf1r by placing LoxP sites around Exon 5 of the Csf1r gene in mice. Excision of this floxed sequence results in a null allele that in the homozygous state gives a phenotype indistinguishable of the complete Csf1r null mutant mouse. This conditional allele will prove extremely valuable to study the spatial and temporal roles of CSF-1R.

Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.

Animal models are powerful tools to analyze the mechanism of the induction of human breast cancer. Here we report a detailed analysis of mammary tumor progression in one mouse model of breast cancer caused by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium, and its comparison to human breast tumors. In PyMT mice, four distinctly identifiable stages of tumor progression from premalignant to malignant stages occur in a single primary tumor focus and this malignant transition is followed by a high frequency of distant metastasis. These stages are comparable to human breast diseases classified as benign or in situ proliferative lesions to invasive carcinomas. In addition to the morphological similarities with human breast cancer, the expression of biomarkers in PyMT-induced tumors is also consistent with those associated with poor outcome in humans. These include a loss of estrogen and progesterone receptors as well as integrin-beta1 expression and the persistent expression of ErbB2/Neu and cyclinD1 in PyMT-induced tumors as they progress to the malignant stage. An increased leukocytic infiltration was also closely associated with the malignant transition. This study demonstrates that the PyMT mouse model is an excellent one to understand the biology of tumor progression in humans.