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.
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