Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.
The olfactory sensory neurons (OSNs) of the olfactory epithelium (OE) exhibit a remarkable regenerative capability, which protects the population against environmental insult and enables adjustment to new odors. The lifespan of OSNs is still open to question, with estimates ranging from 1 month to at least 1 year. However, the estimates come with some caveats, including low labeling efficiency and a focus solely on newborn neurons. We revisited the issue via the use of OMP-tTA; TetO-Cre; Rosa26fl(stop)-Tdtomato (OMP-tTA;TdT) mice, which allowed us to selectively label ∼95% of the OMP(+) OSN population that reach maturity by a given time and, by switching to doxycycline chow, to "chase" this preexisting OSN population. Two loading protocols were used: conception to 2 months old and conception to 4.5 months old. Surviving OSNs were common up to 6 months chase time in both groups, but more neurons survived when loading for 4.5 months as compared with 2 months. A spatial difference was evident: higher percentages of OSNs survived in the dorsomedial OE as compared with ventrolateral and in posterior versus anterior OE regions. Finally, proliferation rates anticorrelated with the spatial differences in OSN survival; higher proliferation rates were observed ventrally. Together, these results demonstrate spatial and temporal differences in OSN survival, highlighting it as a dynamic system that can be studied for factors affecting neuronal survival.
Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >;3 standard deviations above or >;1.5 standard deviations below the mean of the other chemicals, while not adversely affecting cell viability. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. On retesting assays, we confirmed 22 of the initial positives (69%) and 1 of the initial negatives as regulators of Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti‐inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 4 kinase inhibitors, and 4 with other or unknown functions. We are now evaluating the mechanisms of action in human hepatocytes and in a zebrafish model. The best candidates will subsequently be tested in mouse models of iron overload syndromes. Funding: NIDDK R01 DK085250–01A1.
Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing internalization and degradation of the iron exporter ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, Hepcidin is a potential drug target. We previously conducted a chemical screen in human hepatocytes (HepG2 cells) to identify small molecules that upregulate Hepcidin transcript levels. One of the small molecules that we identified was ipriflavone, a synthetic isoflavone, which has been used to treat osteoporosis in postmenopausal women. To evaluate ipriflavone’s effect on iron homeostasis in a mammalian model, we placed groups of 5-week old C57BL/6 male mice on a soy-free, iron-sufficient diet, AIN-93G containing 220 mg/kg iron and 0, 250, 500 or 750 mg ipriflavone per kg of food (n=4 or 5 per group) for 50 days, then sacrificed the animals for analysis of changes in gene expression by quantitative realtime RT-PCR, liver and spleen iron content, blood indices, and intestinal ferroportin expression. While producing less than a two-fold increase in liver hepcidin transcript levels, ipriflavone supplementation was associated with a significant decrease in liver iron content (mean±SE): 39.22±2.06 µg iron/g tissue, p=0.0033, and 44.10±2.58 µg iron/g tissue, p=0.0124, at 500 and 750 mg ipriflavone per kg of food, respectively, vs 57.77±3.39 µg iron/g tissue in mice that did not receive ipriflavone. Ferroportin expression detected in intestinal epithelial cells by immunohistochemistry was notably decreased in mice receiving ipriflavone: 66.6% and 80% of the animals receiving 500 and 750 mg/kg groups, respectively, exhibited decreased ferroportin staining versus 40% of the group not receiving ipriflavone. There was no significant difference in hemoglobin, hematocrit, or spleen iron among the groups. In conclusion, we have shown that orally administered ipriflavone is effective in decreasing liver iron content and intestinal ferroportin expression in vivo. Future experiments will evaluate ipriflavone’s effects on iron homeostasis and hematopoiesis in genetic models of iron overload disorders. Disclosures No relevant conflicts of interest to declare.
611 Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 3 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate from the Harvard Institute of Chemistry and Cell Biology library for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by a nonlytic fluorescence assay. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti-inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 6 kinase inhibitors, and 12 with other or unknown function. Of the positive modulators, two were flavones, consistent with our prior discovery that the isoflavone genistein upregulates Hepcidin expression. Of the negative regulators, one was a kinase inhibitor and two were of unknown function. Experiments are underway to characterize the mechanism of action of these regulators. The best candidates will subsequently be tested in mouse models of iron overload syndromes with the intention of developing new therapies for diseases in which Hepcidin is inappropriately regulated. Disclosures: No relevant conflicts of interest to declare.
Iron overload causes the generation of reactive oxygen species, which can lead to lasting organ damage, particularly to the liver. In patients with hereditary hemochromatosis, transfusion-dependent anemias, and hemoglobinopathies, iron overload is a major cause of mortality. A deeper understanding of iron regulation and the biological pathways involved in maintaining homeostasis may reveal new therapeutic targets for patients with iron overload disorders. We designed this study to discover genes that are differentially expressed in nutritional and genetic models of iron overload. For the nutritional iron overload study, 5-week old male C57BL/6 mice were placed on a soy-free diet (AIN-93G) containing different amounts of iron per kilogram of food: iron-deficient (2.5 mg/kg, n=3), iron-sufficient (37.5 mg/kg, n=3), and iron-excess (750 mg/kg, n=3). In the second study, 5-week old male C57BL/6 mice that were either wild type or HJV knockout mice that exhibited severe early onset iron overload secondary to homozygous deficiency of the bone morphogenic protein coreceptor, hemojuvelin (HJV), were maintained on the iron-deficient (2.5 mg/kg iron) diet (n=2 per group). For both studies animals were sacrificed after 50 days and liver RNA was extracted and sequenced at 40-50 million reads per sample. The RNA integrity number (RIN) for each sample was >6 and assessments of read duplication, base call frequency, and read quality indicated excellent quality of the data. For the HJV knockout mice, we used a false discovery rate <0.05 and a mean-fold change >2, to reveal genes that were differentially expressed compared to wild type mice. For the dietary iron study, genes were grouped by self-organizing maps to identify transcripts whose level of expression trended with increased or decreased dietary iron intake. The resulting analysis identified 148 genes in nutritionally iron-overloaded mice and 688 genes in HJV knockout mice that exhibited significant changes in expression. Of these, 28 genes were differentially regulated in both nutritionally iron overloaded and HJV knockout mice, including expected genes, such as transferrin receptor, HAMP (hepcidin), and bone morphogenic protein 6, and unexpected genes such as cytochrome P450 17a1 (cyp17a1), an enzyme that catalyzes critical steps in steroid synthesis, and nicotinomide N-methyltransferase (nnmt), an enzyme that regulates drug metabolism and DNA methylation. We clustered the 688 differentially expressed genes from the HJV knockout mice into functional pathways using the Functional Analysis tool from DAVID Bioinformatics Resources 6.7 (NIAID). Clusters were considered significant if there were >2 genes in the pathway and the Benjamini-Hochberg P-value was <0.05. We found that the expression of genes involved with PPAR signaling (P=0.0086) was decreased, while expression of transcripts involved with Huntington’s disease (P=0.038) was increased in HJV knockout mice compared to wild-type mice. Our RNA sequencing analysis identified a variety of novel pathways that were differentially regulated in dietary and genetic models of iron overload. Further studies are underway to characterize the potential roles of these genes in iron homeostasis. Disclosures No relevant conflicts of interest to declare.
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