Constitutive Activation of Nrf2 in Mice Expands Enterogenesis in Small Intestine Through Negative Regulation of Math1.

BACKGROUND & AIMS: Notch signaling coordinates cell differentiation processes in the intestinal epithelium. The transcription factor Nrf2 orchestrates defense mechanisms by regulating cellular redox homeostasis, which, as shown previously in murine liver, can be amplified through signaling crosstalk with the Notch pathway. However, interplay between these 2 signaling pathways in the gut is unknown. METHODS: Mice modified genetically to amplify Nrf2 in the intestinal epithelium (Keap1f/f::VilCre) were generated as well as pharmacological activation of Nrf2 and subjected to phenotypic and cell lineage analyses. Cell lines were used for reporter gene assays together with Nrf2 overexpression to study transcriptional regulation of the Notch downstream effector. RESULTS: Constitutive activation of Nrf2 signaling caused increased intestinal length along with expanded cell number and thickness of enterocytes without any alterations of secretory lineage, outcomes abrogated by concomitant disruption of Nrf2. The Nrf2 and Notch pathways in epithelium showed inverse spatial profiles, where Nrf2 activity in crypts was lower than villi. In progenitor cells of Keap1f/f::VilCre mice, Notch downstream effector Math1, which regulates a differentiation balance of cell lineage through lateral inhibition, showed suppressed expression. In vitro results demonstrated Nrf2 negatively regulated Math1, where 6 antioxidant response elements located in the regulatory regions contributed to this repression. CONCLUSIONS: Activation of Nrf2 perturbed the dialog of the Notch cascade though negative regulation of Math1 in progenitor cells, leading to enhanced enterogenesis. The crosstalk between the Nrf2 and Notch pathways could be critical for fine-tuning intestinal homeostasis and point to new approaches for the pharmacological management of absorptive deficiencies.

Current Landscape of NRF2 Biomarkers in Clinical Trials.

The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) plays a critical role in the maintenance of cellular redox and metabolic homeostasis, as well as the regulation of inflammation and cellular detoxication pathways. The contribution of the NRF2 pathway to organismal homeostasis is seen in many studies using cell lines and animal models, raising intense attention towards targeting its clinical promise. Over the last three decades, an expanding number of clinical studies have examined NRF2 inducers targeting an ever-widening range of diseases. Full understanding of the pharmacokinetic and pharmacodynamic properties of drug candidates rely partly on the identification, validation, and use of biomarkers to optimize clinical applications. This review focuses on results from clinical trials with four agents known to target NRF2 signaling in preclinical studies (dimethyl fumarate, bardoxolone methyl, oltipraz, and sulforaphane), and evaluates the successes and limitations of biomarkers focused on expression of NRF2 target genes and others, inflammation and oxidative stress biomarkers, carcinogen metabolism and adduct biomarkers in unavoidably exposed populations, and targeted and untargeted metabolomics. While no biomarkers excel at defining pharmacodynamic actions in this setting, it is clear that these four lead clinical compounds do touch the NRF2 pathway in humans.

AMPK is required for uterine receptivity and normal responses to steroid hormones.

We previously demonstrated that 5'-AMP-activated protein kinase (AMPK) is essential for normal reproductive functions in female mice. Conditional ablation of Prkaa1 and Prkaa2, genes that encode the α1 and α2 catalytic domains of AMPK, resulted in early reproductive senescence, faulty artificial decidualization, uterine inflammation and fibrotic postparturient endometrial regeneration. We also noted a delay in the timing of embryo implantation in Prkaa1/2d/d female mice, suggesting a role for AMPK in establishing uterine receptivity. As outlined in new studies here, conditional uterine ablation of Prkaa1/2 led to an increase in ESR1 in the uteri of Prkaa1/2d/d mice, resulting in prolonged epithelial cell proliferation and retention of E2-induced gene expression (e.g. Msx1, Muc1, Ltf) through the implantation window. Within the stromal compartment, stromal cell proliferation was reduced by five-fold in Prkaa1/2d/d mice, and this was accompanied by a significant decrease in cell cycle regulatory genes and aberrant expression of decidualization marker genes such as Hand2, Bmp2, Fst and Inhbb. This phenotype is consistent with our prior study, demonstrating a failure of the Prkaa1/2d/d uterus to undergo decidualization. Despite these uterine defects, ovarian function seemed to be normal following ablation of Prkaa1/2 from peri-ovulatory follicles in which ovulation, luteinization and serum progesterone levels were not different on day 5 of pregnancy or pseudopregnancy between Prkaa1/2fl/fl and Prkaa1/2d/d mice. These cumulative findings demonstrate that AMPK activity plays a prominent role in mediating several steroid hormone-dependent events such as epithelial cell proliferation, uterine receptivity and decidualization as pregnancy is established.

A functional role for AMPK in female fertility and endometrial regeneration.

Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved heterotrimeric complex that acts as an intracellular energy sensor. Based on recent observations of AMPK expression in all structures of the female reproductive system, we hypothesized that AMPK is functionally required for maintaining fertility in the female. This hypothesis was tested by conditionally ablating the two catalytic alpha subunits of AMPK, Prkaa1 and Prkaa2, using Pgr-cre mice. After confirming the presence of PRKAA1, PRKAA2 and the active phospho-PRKAA1/2 in the gravid uterus by immunohistochemistry, control (Prkaa1/2 fl/fl ) and double conditional knockout mice (Prkaa1/2 d/d ) were placed into a six-month breeding trial. While the first litter size was comparable between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice (P = 0.8619), the size of all subsequent litters was dramatically reduced in Prkaa1/2 d/d female mice (P = 0.0015). All Prkaa1/2 d/d female mice experienced premature reproductive senescence or dystocia by the fourth parity. This phenotype manifested despite no difference in estrous cycle length, ovarian histology in young and old nulliparous or multiparous animals, mid-gestation serum progesterone levels or uterine expression of Esr1 or Pgr between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice suggesting that the hypothalamic-pituitary-ovary axis remained unaffected by PRKAA1/2 deficiency. However, an evaluation of uterine histology from multiparous animals identified extensive endometrial fibrosis and disorganized stromal-glandular architecture indicative of endometritis, a condition that causes subfertility or infertility in most mammals. Interestingly, Prkaa1/2 d/d female mice failed to undergo artificial decidualization. Collectively, these findings suggest that AMPK plays an essential role in endometrial regeneration following parturition and tissue remodeling that accompanies decidualization.

Conditional Ablation of Progesterone Receptor Membrane Component 1 Results in Subfertility in the Female and Development of Endometrial Cysts.

Progesterone (P4) is essential for female fertility. The objective of this study was to evaluate the functional requirement of the nonclassical P4 receptor (PGR), PGR membrane component 1, in regulating female fertility. To achieve this goal, the Pgrmc1 gene was floxed by insertion of loxP sites on each side of exon 2. Pgrmc1 floxed (Pgrmc1(fl/fl)) mice were crossed with Pgr(cre) or Amhr2(cre) mice to delete Pgrmc1 (Pgrmc1(d/d)) from the female reproductive tract. A 6-month breeding trial revealed that conditional ablation of Pgrmc1 with Pgr(cre/+) mice resulted in a 40% reduction (P = .0002) in the number of pups/litter. Neither the capacity to ovulate in response to gonadotropin treatment nor the expression of PGR and the estrogen receptor was altered in the uteri of Pgrmc1(d/d) mice compared with Pgrmc1(fl/fl) control mice. Although conditional ablation of Pgrmc1 from mesenchymal tissue using Amhr2(cre/+) mice did not reduce the number of pups/litter, the total number of litters born in the 6-month breeding trial was significantly decreased (P = .041). In addition to subfertility, conditional ablation of Pgrmc1 using either Amhr2(cre/+) or Pgr(cre/+) mice resulted in the development of endometrial cysts starting around 4 months of age. Interestingly, pregnancy attenuated the formation of these uterine cysts. These new findings demonstrate that PGR membrane component 1 plays an important role in female fertility and uterine tissue homeostasis.

Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors.

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors.