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]

eGFP transgene expression is comparable between IP and RP preparation methods.

<p>Liver tissue was obtained from 129/S mice injected intraperitoneally with PBS, IP-rAAV8-CMV-eGFP, or RP-rAAV8-CMV-eGFP. Indicated doses for rAAV8 vectors are 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), and 7.50 x 10¹¹ vg/mouse (High). <b>(A)</b> Liver tissue lysate was analyzed by anti-GFP immunoblotting. Anti-<i>β</i> actin immunoblotting is shown as a loading control. <b>(B)</b> Isolated liver RNA was analyzed by quantitative RT-PCR for eGFP transcript level. Expression is represented as relative to the 0.75 x 10¹¹ vg RP-rAAV8-CMV-eGFP data set (<i>β</i> actin-normalized). Data are represented as scatter plot with mean ± S.E.M. bars (n = 3 to 6 mice). Unpaired t-tests with Welch’s correction were performed to determine p-values.</p

Schematic comparison of the iodixanol purification (IP) and rapid purification (RP) methods.

<p>Depicted is a general overview of the steps required to produce, purify, and titer rAAV vectors using the RP and IP methods. F-T = Freeze-Thaw.</p

Ultrafiltered recombinant AAV8 vector can be safely administered <i>in vivo</i> and efficiently transduces liver

<div><p>Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered <i>in vivo</i>. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.</p></div

Injection with RP-rAAV8-CMV-eGFP does not induce expression of inflammatory markers in mouse liver or systemic cytokines.

<p>129/S mice were injected intraperitoneally with PBS, IP-rAAV8-CMV-eGFP, or RP-rAAV8-CMV-eGFP. <b>(A)</b> Isolated liver RNA was used for quantitative RT-PCR using primers for TNFα, IL-6, Activin B, or Hepcidin. Expression is represented as relative to the PBS data set (<i>β</i> actin-normalized). Data are represented as scatter plot with mean ± S.E.M. bars (n = 3 to 6 mice). One-way ANOVA with Dunnett’s correction did not reveal any significant differences in the means of treatment groups compared to the PBS control group. Indicated doses for rAAV8 vectors are 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), or 7.50 x 10¹¹ vg/mouse (High). <b>(B)</b> Serum cytokine concentrations before and after rAAV injection with 7.50 x 10¹¹ vg/mouse were measured by immunoassay. Concentrations units are represented as mean ± S.E.M. (n = 4 mice). Multiple comparison of means using two-way ANOVA with Bonferroni correction did not detect any statistically significant differences between PBS and the treatment groups at each time point. No detectable expression was observed for the cytokines GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IP-10, KC, MCP-1, MIG, MIP-1α, and TNF-α.</p

Injection of IP and RP rAAV8-CMV-eGFP vectors induce dose-dependent neutralizing antibody responses.

<p>ID₅₀ neutralizing antibody assays were carried out on sera of mice that were injected with a 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), or 7.50 x 10¹¹ vg/mouse (High) dose of IP or RP rAAV8-CMV-eGFP. Data are represented as scatter plot of neutralizing anti-AAV8 response titers (ID₅₀) with mean ± S.E.M. bars (n = 3 to 6 mice). Unpaired t-tests with Welch’s correction were performed to determine p-values. Pre-injection, all mice in the rAAV treatment groups had a negligible anti-AAV8 response (titer <1:50).</p

Fairness and Equality in Insurance Classification&ast;

Insurance is to a large extent based on risk selection and classification. Legislators however are inclined to impose restrictions to these differentiations by banning those that are considered to be “discriminatory”. Risk selection and risk classification are not disallowed by law, but each such decision requires a well-funded, that is, fair justification. The conditions for reaching a fair insurance-differentiation scheme could be clarified by bridging the apparent conflict between an “individualistic” human rights approach and an insurance “group” approach to equality. Therefore, a number of considerations concerning the notion of subsidy-aversion should be taken into account in the legal justification of unequal treatment. These considerations concern the notion of controllability of risks, the (im)possibility of establishing a causal relation between risk variables and the risk itself, scepticism of adverse selection in case where price-inelastic markets are concerned and the influence of tracing costs on the choice of risk variables. The Geneva Papers (2006) 31, 190–211. doi:10.1057/palgrave.gpp.2510078