Expression of the Synaptotagmin I Gene Is Enhanced by Binding of the Pituitary-Specific Transcription Factor, POU1F1

The POU1F1 transcription factor (also known as Pit-1/GHF1) is required for development of pituitary cells that secrete prolactin, GH, and TSH. Presumably, POU1F1 regulates the expression of multiple genes required for expansion and differentiation of these pituitary cell lineages. However, only a few genes regulated by POU1F1 have been identified. In the present studies we have identified synaptotagmin I (Syt1) as a target gene for POU1F1 in GH3 pituitary cells. Chromatin immunoprecipitation assays have provided evidence that POU1F1 binds close to the Syt1 exon that contains the initiation codon. Although this exon has previously been considered to be located far from the transcription initiation site, transcript mapping in GH3 cells indicates that Syt1 mRNA synthesis is initiated close to the mapped POU1F1-binding site. POU1F1 knockdown studies using a short hairpin RNA vector have provided evidence that POU1F1 plays a role in stimulating expression of the endogenous Syt1 gene. Transfection studies with a Syt1-luciferase reporter gene are consistent with the presence of an internal, POU1F1-regulated promoter in the Syt1 gene. In vitro binding studies have provided further evidence for a POU1F1-binding site within this region of the Syt1 gene. Overall the studies provide evidence that Syt1 is a target gene regulated by POU1F1 in GH3 pituitary cells. Because SYT1 has been extensively studied as an important transducer of Ca2+ signaling in regulated secretion, it seems likely that activation of Syt1 gene expression is part of a mechanism mediating POU1F-induced differentiation of pituitary cells

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2’s role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR–Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf–TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to effectively compete with holo-Tf. Tf binding to a TfR2 chimera containing the TfR1 helical domain indicates that the differences in the helical domain account for differences in the on rate of Tf, and nonconserved inter-receptor interactions are necessary for the stabilization of the complex. Conserved residues at one possible site of stabilization, the apical arm junction, are not important for TfR1–Tf binding but are critical for the TfR2–Tf interaction. Our results highlight the differences in Tf interactions with the two TfRs

The Tumor Suppressor, P53, Decreases the Metal Transporter, ZIP14

Loss of p53’s proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14’s mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells

Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv(-/-) mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2(-/-) mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.National Institutes of Health [R01DK102791, R01DK072166, R00DK104066]12 month embargo; Published online: 18 September 2017This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]