p65 signaling dynamics drive the developmental progression of hematopoietic stem and progenitor cells through cell cycle regulation.

Most gene functions have been discovered through phenotypic observations under loss of function experiments that lack temporal control. However, cell signaling relies on limited transcriptional effectors, having to be re-used temporally and spatially within the organism. Despite that, the dynamic nature of signaling pathways have been overlooked due to the difficulty on their assessment, resulting in important bottlenecks. Here, we have utilized the rapid and synchronized developmental transitions occurring within the zebrafish embryo, in conjunction with custom NF-kB reporter embryos driving destabilized fluorophores that report signaling dynamics in real time. We reveal that NF-kB signaling works as a clock that controls the developmental progression of hematopoietic stem and progenitor cells (HSPCs) by two p65 activity waves that inhibit cell cycle. Temporal disruption of each wave results in contrasting phenotypic outcomes: loss of HSPCs due to impaired specification versus proliferative expansion and failure to delaminate from their niche. We also show functional conservation during human hematopoietic development using iPSC models. Our work identifies p65 as a previously unrecognized contributor to cell cycle regulation, revealing why and when pro-inflammatory signaling is required during HSPC development. It highlights the importance of considering and leveraging cell signaling as a temporally dynamic entity.

Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases.

Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Estrogen receptor alpha signaling in dendritic cells modulates autoimmune disease phenotype in mice.

Estrogen is a disease-modifying factor in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) via estrogen receptor alpha (ERα). However, the mechanisms by which ERα signaling contributes to changes in disease pathogenesis have not been completely elucidated. Here, we demonstrate that ERα deletion in dendritic cells (DCs) of mice induces severe neurodegeneration in the central nervous system in a mouse EAE model and resistance to interferon beta (IFNß), a first-line MS treatment. Estrogen synthesized by extragonadal sources is crucial for controlling disease phenotypes. Mechanistically, activated ERα directly interacts with TRAF3, a TLR4 downstream signaling molecule, to degrade TRAF3 via ubiquitination, resulting in reduced IRF3 nuclear translocation and transcription of membrane lymphotoxin (mLT) and IFNß components. Diminished ERα signaling in DCs generates neurotoxic effector CD4+ T cells via mLT-lymphotoxin beta receptor (LTßR) signaling. Lymphotoxin beta receptor antagonist abolished EAE disease symptoms in the DC-specific ERα-deficient mice. These findings indicate that estrogen derived from extragonadal sources, such as lymph nodes, controls TRAF3-mediated cytokine production in DCs to modulate the EAE disease phenotype.

Single intranasal administration of 17ß-estradiol loaded gelatin nanoparticles confers neuroprotection in the post-ischemic brain.

Globally, ischemic stroke is a leading cause of death and adult disability. Previous efforts to repair damaged brain tissue following ischemic events have been hindered by the relative isolation of the central nervous system. We have developed a gelatin nanoparticle-mediated intranasal drug delivery system as an efficient, non-invasive method for delivering 17ß-estradiol (E2) specifically to the brain, enhancing neuroprotection, and limiting systemic side effects. Young adult male C57BL/6 J mice subjected to 30 min of middle cerebral artery occlusion (MCAO) were administered intranasal preparations of E2-GNPs, water soluble E2, or saline as control 1 h after reperfusion. Following intranasal administration of 500 ng E2-GNPs, brain E2 content rose by 5.24 fold (P<0.0001) after 30 min and remained elevated by 2.5 fold at 2 h (P<0.05). The 100 ng dose of E2-GNPs reduced mean infarct volume by 54.3% (P<0.05, n=4) in comparison to saline treated controls, demonstrating our intranasal delivery system's efficacy.

Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

Porcine intestinal lymphoid tissues synthesize estradiol.

Estradiol (17ß-estradiol) is synthesized primarily in the gonads of both sexes and regulates the development and function of reproductive organs. Recently, we reported that intestinal lymphocyte homeostasis is regulated by estradiol synthesized de novo in the endothelial cells of the high endothelial venules (HEVs) of mesenteric lymph nodes and Peyer's patches in mice. This observation prompted us to hypothesize that HEVs of intestinal lymphoid tissues are sites of estradiol synthesis across species. In this study, we examined whether estradiol is synthesized in the intestinal lymphoid tissues of adolescent piglets. Comparisons of estradiol levels in blood and tissue showed that estradiol concentrations in mesenteric lymph nodes and Peyer's patches were significantly higher than the level in serum. Reverse transcription polymerase chain reaction showed that porcine intestinal lymphoid tissues express mRNAs for steroidogenic enzymes (StAR, 17ß-Hsd,3ß-Hsd, Cyp17a1, and Cyp19a1), and immunohistochemical results in ilial tissue showed expression of aromatase (CYP19) in Peyer's patch-localized endothelial cells of HEVs. When mesenteric lymph node and Peyer's patch tissues were cultured in vitro, they produced estradiol. Taken together, the results indicate that mesenteric lymph nodes and Peyer's patches are sites of estradiol synthesis in adolescent piglets.

Prenatal Exposure to DEHP Induces Premature Reproductive Senescence in Male Mice.

Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used phthalate, and it is an endocrine-disrupting chemical. This study tested a hypothesis that prenatal exposure to DEHP lays the foundation for premature gonadal dysfunction and subsequent reproductive senescence in male mice. Pregnant female CD-1 mice were orally dosed with vehicle control (tocopherol-stripped corn oil) or with 20 µg/kg/day, 200 µg/kg/day, 500 mg/kg/day, or 750 mg/kg/day of DEHP from gestational day 11 to birth. Overall, the prenatal DEHP exposure did not cause any overt physical health problems in male offspring, as no significant differences in their body nor gonadal weight were seen up to the age of 23 months. However, an age- and dose-dependent gonadal dysfunction was observed. As early as 7 months of age, the 750 mg/kg/day group of mice exhibited significantly reduced fertility. At 19 months of age, 86% of the 750 mg/kg/day mice became infertile, whereas only 25% of the control mice were infertile. At this age, all of the DEHP-exposed mice had lower serum testosterone levels, higher serum estradiol levels, and higher LH levels compared with control mice. Histological evaluations showed that mice prenatally exposed to DEHP displayed a wide array of gonadal and epididymal abnormalities such as increased germ cell apoptosis, degenerative seminiferous tubules, oligozoospermia, asthenozoospermia, and teratozoospermia in comparison to age-matching control mice. In summary, this study shows that prenatal exposure to DEHP induces premature reproductive senescence in male mice.

Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

17ß-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17ß-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17ß-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17ß-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17ß-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17ß-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17ß-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17ß-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-ß had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17ß-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17ß-estradiol content, or steroidogenic capacity in these lymphoid organs.

Extra-gonadal sites of estrogen biosynthesis and function.

Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496].