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Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 39 untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice.

[Objetive] The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis., [Materials and Methods] Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes., [Results] Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction., [Conclusions] Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 modulates iron homeostasis in cell lines and mice.