Elevated Protein Kinase A Activity in Stomach Mesenchyme Disrupts Mesenchymal-epithelial Crosstalk and Induces Preneoplasia.

BACKGROUND & AIMS: Mesenchymal-epithelial crosstalk (MEC) in the stomach is executed by pathways such as bone morphogenetic protein (BMP) and extracellular signal-regulated kinase (ERK). Mis-regulation of MEC disrupts gastric homeostasis and causes tumorigenesis. Protein Kinase A (PKA) crosstalks with BMP and ERK signaling; however, PKA function(s) in stomach development and homeostasis remains undefined. METHODS: We generated a novel Six2-Cre+/-PKAcαRfl/wt (CA-PKA) mouse in which expression of constitutive-active PKAcαR was induced in gastric mesenchyme progenitors. Lineage tracing determined spatiotemporal activity of Six2-Cre in the stomach. For phenotyping CA-PKA mice histological, co-immunofluorescence, immunoblotting, mRNA sequencing, and bioinformatics analyses were performed. RESULTS: Lineage tracing showed that Six2-Cre activity in the stomach is restricted to the mesenchymal compartment. CA-PKA mice showed disruption of gastric homeostasis characterized by aberrant mucosal development and epithelial hyperproliferation; ultimately developing multiple features of gastric corpus preneoplasia including decreased parietal cells, mucous cell hyperplasia, spasmolytic peptide expressing metaplasia with intestinal characteristics, and dysplastic and invasive cystic glands. Furthermore, mutant corpus showed marked chronic inflammation characterized by infiltration of lymphocytes and myeloid-derived suppressor cells along with the upregulation of innate and adaptive immune system components. Striking upregulation of inflammatory mediators and STAT3 activation was observed. Mechanistically, we determined there is an activation of ERK1/2 and downregulation of BMP/SMAD signaling characterized by marked upregulation of BMP inhibitor gremlin 1. CONCLUSIONS: We report a novel role of PKA signaling in gastric MEC execution and show that PKA activation in the gastric mesenchyme drives preneoplasia by creating a proinflammatory and proproliferative microenvironment associated with the downregulation of BMP/SMAD signaling and activation of ERK1/2.

Lessons Learned from ToxMSDT: A Pilot Innovative Toxicology Research Education Pipeline Program Targeting Underrepresented Undergraduate Students to the Field of Toxicology.

Toxicology, as a profession, lacks diversity. Undergraduate students, and especially underrepresented students, are not commonly introduced to toxicology at US colleges and universities. The Toxicology Mentoring and Skills Development Training Program (ToxMSDT) seeks to acquaint underrepresented undergraduates enrolled in STEM fields with toxicology fundamentals and skills to aid their entry into graduate programs and, ultimately, careers in toxicology. ToxMSDT is a collaboration among three universities. It is a year-long holistic training and mentoring program comprised of web resources accessible 24/7 and extensive one-to-one mentor-mentee interactions throughout the year. Evaluation of the two-year pilot program shows that students expressed a significant increase in knowledge about toxicology careers, networking with people involved in the field of toxicology, feelings of being part of the toxicology community, and seeing themselves as someone who will study toxicology, compared with their feelings prior to their participation in the ToxMSDT program. Thirty students have completed the ToxMSDT program and all 10 (100%) of those who have graduated have joined graduate school in toxicology or toxicology-related STEM fields. Of the 20 (66.6%) program alumni still enrolled as undergraduates, five (25%) are in the process of applying to graduate programs and medical schools as of August 2019.

Transcriptomic profile analysis of brain inferior colliculus following acute hydrogen sulfide exposure.

Hydrogen sulfide (H2S) is a gaseous molecule found naturally in the environment, and as an industrial byproduct, and is known to cause acute death and induces long-term neurological disorders following acute high dose exposures. Currently, there is no drug approved for treatment of acute H2S-induced neurotoxicity and/or neurological sequelae. Lack of a deep understanding of pathogenesis of H2S-induced neurotoxicity has delayed the development of appropriate therapeutic drugs that target H2S-induced neuropathology. RNA sequencing analysis was performed to elucidate the cellular and molecular mechanisms of H2S-induced neurodegeneration, and to identify key molecular elements and pathways that contribute to H2S-induced neurotoxicity. C57BL/6J mice were exposed by whole body inhalation to 700 ppm of H2S for either one day, two consecutive days or 4 consecutive days. Magnetic resonance imaging (MRI) scan analyses showed H2S exposure induced lesions in the inferior colliculus (IC) and thalamus (TH). This mechanistic study focused on the IC. RNA Sequencing analysis revealed that mice exposed once, twice, or 4 times had 283, 193 and 296 differentially expressed genes (DEG), respectively (q-value < 0.05, fold-change> 1.5). Hydrogen sulfide exposure modulated multiple biological pathways including unfolded protein response, neurotransmitters, oxidative stress, hypoxia, calcium signaling, and inflammatory response in the IC. Hydrogen sulfide exposure activated PI3K/Akt and MAPK signaling pathways. Pro-inflammatory cytokines were shown to be potential initiators of the modulated signaling pathways following H2S exposure. Furthermore, microglia were shown to release IL-18 and astrocytes released both IL-1ß and IL-18 in response to H2S. This transcriptomic analysis data revealed complex signaling pathways involved in H2S-induced neurotoxicity and may provide important associated mechanistic insights.

Chronic estrogen affects TIDA neurons through IL-1ß and NO: effects of aging.

Women are chronically exposed to estrogens through oral contraceptives, hormone replacement therapy or environmental estrogens. We hypothesized that chronic exposure to low levels of estradiol-17ß (E2) can induce inflammatory and degenerative changes in the tuberoinfundibular dopaminergic (TIDA) system leading to reduced dopamine synthesis and hyperprolactinemia. Young (Y; 3­4 months) and middle-aged (MA; 10­12 months) Sprague-Dawley rats that were intact or ovariectomized (OVX) were either sham-implanted or implanted with a slow-release E2 pellet (20 ng E2/day for 90 days). To get mechanistic insight, adult 3- to 4-month-old WT, inducible nitric oxide synthase (iNOS) and IL-1 receptor (IL-1R) knockout (KO) mice were subjected to a similar treatment. Hypothalamic areas corresponding to the TIDA system were analyzed. E2 treatment increased IL-1ß protein and nitrate levels in the arcuate nucleus of intact animals (Y and MA). Nitration of tyrosine hydroxylase in the median eminence increased with E2 treatment in both intact and OVX animals. There was no additional effect of age. This was accompanied by a reduction in dopamine levels and an increase in prolactin in intact animals. E2 treatment increased nitrate and reduced dopamine levels in the hypothalamus and increased serum prolactin in WT mice. In contrast, the effect of E2 on nitrate levels was blocked in IL-1R KO mice and the effect on dopamine and prolactin were blocked in iNOS KO animals. Taken together, these results show that chronic exposure to low levels of E2 decreases TIDA activity through a cytokine-nitric oxide-mediated pathway leading to hyperprolactinemia and that aging could promote these degenerative changes.

Prenatal programming: adverse cardiac programming by gestational testosterone excess.

Adverse events during the prenatal and early postnatal period of life are associated with development of cardiovascular disease in adulthood. Prenatal exposure to excess testosterone (T) in sheep induces adverse reproductive and metabolic programming leading to polycystic ovarian syndrome, insulin resistance and hypertension in the female offspring. We hypothesized that prenatal T excess disrupts insulin signaling in the cardiac left ventricle leading to adverse cardiac programming. Left ventricular tissues were obtained from 2-year-old female sheep treated prenatally with T or oil (control) from days 30-90 of gestation. Molecular markers of insulin signaling and cardiac hypertrophy were analyzed. Prenatal T excess increased the gene expression of molecular markers involved in insulin signaling and those associated with cardiac hypertrophy and stress including insulin receptor substrate-1 (IRS-1), phosphatidyl inositol-3 kinase (PI3K), Mammalian target of rapamycin complex 1 (mTORC1), nuclear factor of activated T cells -c3 (NFATc3), and brain natriuretic peptide (BNP) compared to controls. Furthermore, prenatal T excess increased the phosphorylation of PI3K, AKT and mTOR. Myocardial disarray (multifocal) and increase in cardiomyocyte diameter was evident on histological investigation in T-treated females. These findings support adverse left ventricular remodeling by prenatal T excess.

3-(2-Bromoethyl)-indole inhibits the growth of cancer cells and NF-κB activation.

Indole-3-carbinol (I3C) and diindolylmethane (DIM), found in cruciferous vegetables, have chemopreventive and anticancer properties. In the present study, 14 substituted indoles were tested for activity against SW480 colon cancer cells. Among these, 3-(2-bromoethyl)-indole, named BEI-9, showed the greatest inhibition. The effects of BEI-9 on cancer cells were analyzed by MTS and CellTiter-Glo assays for effects on cell viability, by microscopy for phenotypic changes, by scratch wound assays for effects on migration, by flow cytometry for changes in the cell cycle, by immunoblotting for cyclin D and A to assess effects on cell cycle regulation, and by NF-κB reporter assays for effects on basal and drug-induced NF-κB activation. BEI-9 inhibited the growth of SW480 and HCT116 colon cancer cells at concentrations of 12.5 and 5 µM, respectively. BEI-9 also inhibited cell motility as determined with scratch wound assays, and reduced the levels of cyclin D1 and A. Furthermore, in reporter cells, BEI-9 (0.8 µM) inhibited basal and induced NF-κB activation and increased cell death when combined with the cytokine TNFα or the drug camptothecin (CPT), both of which activate NF-κB. Preliminary experiments to identify a safe dose range for immunodeficient mice showed that BEI-9, administered intraperitoneally, was tolerable at doses below 10 mg/kg. Thus, BEI-9 and other indole derivatives may be useful in chemoprevention or as chemosensitizers. Since NF-κB activation is implicated in carcinogenesis and in reducing sensitivity to anticancer drugs, BEI-9 should be investigated in combination with drugs such as CPT, which activate NF-κB.

Kaiso overexpression promotes intestinal inflammation and potentiates intestinal tumorigenesis in Apc(Min/+) mice.

Constitutive Wnt/ß-catenin signaling is a key contributor to colorectal cancer (CRC). Although inactivation of the tumor suppressor adenomatous polyposis coli (APC) is recognized as an early event in CRC development, it is the accumulation of multiple subsequent oncogenic insults facilitates malignant transformation. One potential contributor to colorectal carcinogenesis is the POZ-ZF transcription factor Kaiso, whose depletion extends lifespan and delays polyp onset in the widely used Apc(Min/+) mouse model of intestinal cancer. These findings suggested that Kaiso potentiates intestinal tumorigenesis, but this was paradoxical as Kaiso was previously implicated as a negative regulator of Wnt/ß-catenin signaling. To resolve Kaiso's role in intestinal tumorigenesis and canonical Wnt signaling, we generated a transgenic mouse model (Kaiso(Tg/+)) expressing an intestinal-specific myc-tagged Kaiso transgene. We then mated Kaiso(Tg/+) and Apc(Min/+) mice to generate Kaiso(Tg/+):Apc(Min/+) mice for further characterization. Kaiso(Tg/+):Apc(Min/+) mice exhibited reduced lifespan and increased polyp multiplicity compared to Apc(Min/+) mice. Consistent with this murine phenotype, we found increased Kaiso expression in human CRC tissue, supporting a role for Kaiso in human CRC. Interestingly, Wnt target gene expression was increased in Kaiso(Tg/+):Apc(Min/+) mice, suggesting that Kaiso's function as a negative regulator of canonical Wnt signaling, as seen in Xenopus, is not maintained in this context. Notably, Kaiso(Tg/+):Apc(Min/+) mice exhibited increased inflammation and activation of NFκB signaling compared to their Apc(Min/+) counterparts. This phenotype was consistent with our previous report that Kaiso(Tg/+) mice exhibit chronic intestinal inflammation. Together our findings highlight a role for Kaiso in promoting Wnt signaling, inflammation and tumorigenesis in the mammalian intestine.

Chronic exposures to low levels of estradiol and their effects on the ovaries and reproductive hormones: Comparison with aging.

Aging in female rats is characterized by a state called "constant estrous" in which rats are unable to ovulate, have polycystic ovaries and moderately elevated estrogen levels. We hypothesized that chronic exposure of young animals to low levels of E2 can produce reproductive changes similar to that seen in aging animals. Adult female rats were sham-implanted (control) or implanted with slow-release E2 (20 ng/day) pellets for 30, 60, or 90 days. Old constant estrous (OCE) rats were used for comparison. Estrous cyclicity was monitored periodically. At the end of treatment, animals were sacrificed, trunk blood was collected for hormone measurements and ovaries for immunohistochemistry. Young animals became acyclic with increasing duration of E2 exposure while OCE rats were in a state of acyclicity. Ovaries became increasingly more cystic with E2 exposure, and were comparable to OCE rats; however, there was a marked reduction in interstitial tissue with exogenous E2 treatment. Exogenous E2 also decreased Mullerian inhibiting substance expression, increased infiltration of macrophages without much impact on apoptosis in the ovaries. Serum testosterone levels decreased in E2-treated young animals, while it increased significantly in OCE rats. There was a marked reduction in LH but not FSH levels with E2 exposure in both young and old animals. These results indicate that even very low doses of E2 are capable of inducing aging-like changes in young animals.

Chronic estradiol exposure induces oxidative stress in the hypothalamus to decrease hypothalamic dopamine and cause hyperprolactinemia.

Estrogens are known to cause hyperprolactinemia, most probably by acting on the tuberoinfundibular dopaminergic (TIDA) system of the hypothalamus. Dopamine (DA) produced by TIDA neurons directly inhibits prolactin secretion and, therefore, to stimulate prolactin secretion, estrogens inhibit TIDA neurons to decrease DA production. However, the mechanism by which estrogen produces this effect is not clear. In the present study, we used a paradigm involving chronic exposure to low levels of estradiol-17ß (E(2)) to mimic prolonged exposures to environmental and endogenous estrogens. We hypothesized that chronic exposure to low levels of E(2) induces oxidative stress in the arcuate nucleus (AN) of the hypothalamus that contains TIDA neurons and causes nitration of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA. This results in a significant decrease in DA and consequently, hyperprolactinemia. To investigate this, adult, intact female cycling rats were implanted with slow-release E(2) pellets (20 ng/day) for 30, 60, or 90 days and were compared with old (16-18 mo old) constant estrous (OCE) rats. Chronic E(2) exposure significantly increased the expression of glial fibrillary acidic protein and the concentrations of interleukin-1ß (IL-1ß) and nitrate in the AN that contains perikarya of TIDA neurons and increased nitration of TH in the median eminence (ME) that contains the terminals. These levels were comparable to those seen in OCE rats. We observed a significant decrease in DA concentrations in the ME and hyperprolactinemia in an exposure-dependent manner similar to that seen in OCE rats. It was concluded that chronic exposure to low levels of E(2) evokes oxidative stress in the AN to inhibit TIDA neuronal function, most probably leading to hyperprolactinemia.