Iron-dependent regulation of hepcidin responds to circulating (holo-Tf) and hepatic iron. Although the signals that modulate hepcidin according to holo-Tf are still unknown, hepatic iron regulates hepcidin expression through transcriptional modulation of Bmp6. Bmp6 is controlled by iron only in the liver. Heterogeneous cell types with different functions in the liver include hepatocytes (HC) and non-parenchymal cells as Kupffer cells (KC), sinusoidal endothelial cells (LSEC), stellate cells. It has been reported that LSEC and KC, but not HC, upregulate Bmp6 independently of intracellular iron (Enns et al., Plos One 2013). Here we extend this observation analyzing Bmp6 regulation in isolated liver cells after acute and chronic changes of the iron status in wild type mice. In addition we studied pathogenic models of iron deregulation due to low (iron loaded Hjv KO mice) and high (iron deficient Tmprss6 KO mice) hepcidin.
Mice were maintained in accordance with the European Union guidelines. The study was approved by the IACUC of San Raffaele Scientific Institute, Milan, Italy. For chronic changes of the iron status, four weeks-old male mice were maintained an iron-replete (IB, 200 mg/kg iron), iron-deficient diet (ID, <3 mg /kg iron) or iron-loaded (IL, 8.3 g/kg iron) diet for 3 weeks. To induce acute iron changes, we used two approaches: iron dextran injection (1 g/kg body weight i.p.) or 2 weeks ID diet followed by 1, 3 and 6 days of IB, ID or IL diet. Hjv and Tmprss6 KO mice were studied in basal conditions at 7-8 weeks. Liver cells were isolated according to Liu et al. (Proteomics 2011) and cell purity validated by mRNA expression of specific genes. RNA was extracted using RNeasy Mini kit. Total RNA was retro-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystem). Transferrin saturation (TS) and liver iron content (LIC) was measured as previously described (Pagani et al. Blood 2011).
In wild type animals hepcidin in basal condition is expressed exclusively in HC, whereas Bmp6 is expressed in non-parenchymal cells, mainly LSEC. In all cell types, Bmp6 levels inversely correlate with Tfr1 expression, suggesting that cellular iron influences Bmp6. In mice chronically maintained an IB, ID and IL diet HC, LSEC and KC modulate Bmp6 according to both cellular iron and TS. To investigate which cell type first regulates Bmp6, we injected wild type mice with a single dose of iron dextran and separated cells 3 and 6 hrs post injection. At 6 hrs, when TS and LIC are increased and Hamp is activated in HC, only LSEC upregulate Bmp6. As for the pathogenic models, Bmp6 is upregulated in HC, LSEC and KC cells in Hjv KO mice and downregulated in Tmprss6 KO animals. However, at difference with HC, regulation of Bmp6 in LSEC and KC is independent on cellular iron (as shown by Tfr1 mRNA), and related to TS in both animal models. To distinguish between the contribution of cellular and circulating iron in the regulation of Bmp6, we fed mice previously treated with an ID diet, with an ID, IB and IL diet for 1-3-6 days. At day 1, TS increases but LIC does not change in IB and ID mice while total liver Bmp6 is modulated according to TS. These results will be analyzed in separated cells. In vitro, exogenous BMP6 transcriptionally activates BMP6 expression in hepatoma-derived cells, even at short time point, suggesting that a paracrine mechanism contributes to BMP6 regulation.
In our hands Bmp6 expression is modulated not only in LSEC and KC but also in HC in animals fed a variable iron content diet. LSEC are the first iron “sensor” that, through Bmp6, activates hepcidin expression in HC. Bmp6 in LSEC and KC responds mainly to TS changes. We also suggest that an autocrine/paracrine mechanism activates liver Bmp6 to amplify signaling for hepcidin increase.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.