Hepatocytes sense systemic iron availability and regulate systemic iron fluxes. Plasma iron levels are controlled by 2 major hepatic signaling pathways: the HH-associated proteins (Hfe, Hjv, and Tfr2) and the Bmp/Smad signaling pathway, which control the hepcidin response to systemic iron availability, and proinflammatory cytokines and the Jak/Stat pathway, which mount the inflammatory response of hepcidin (56
). Activation of either of these signaling pathways increases hepcidin levels, which leads to decreased duodenal iron absorption and iron release from macrophages. In contrast, TMPRSS6 (58
) and the inhibitory Smad protein Smad7 (59
) suppress hepcidin levels.
In this study, we showed for the first time to our knowledge that systemic iron homeostasis is also controlled by a miRNA. Hepatic miR-122 expression was critical to prevent iron deficiency in plasma and liver, impairment of hematopoiesis caused by low iron availability, and extramedullary erythropoiesis in the spleen. Thus, decreased miR-122 expression, which was observed in Hfe–/– mice and HH patients (Figure , A and D), may be a compensatory response of the liver to limit iron uptake and counteract liver iron overload. However, the biological significance of the decreased miR-122 levels in Hfe–/– mice needs to be established, because hepatic miR-122 reduction in Hfe–/– mice failed to increase mRNA expression of the known miR-122 target gene Aldoa or to affect molecular pathways that reduce cholesterol levels in miR-122–depleted mice (data not shown). Thus, it may simply be a marker rather than a functionally consequential response.
Our findings demonstrated that hepatic miR-122 inhibition by LNA-modified PM_anti–miR-122 was highly specific: miR-122, and no other miRNA, was exclusively inhibited in the liver upon PM_anti–miR-122 injection (Supplemental Figure 2), and 2 mismatches within the LNA-modified anti–miR (2MM_anti–miR-122) were sufficient to prevent hepatic miR-122 inhibition and alterations in systemic iron homeostasis (Figure and Table ). Interestingly, hepatic miR-122 inhibition in the liver of WT mice and in primary murine hepatocytes caused increased mRNA expression of Hamp
and 3 of its transcriptional activators (Hfe
, and Bmpr1a
; Figures and ). Notably, increased Hfe
, and Bmpr1a
mRNA expression was also reported in 3 previous studies on miR-122 antagonism in mice (31
). This suggests that the observed stimulation of Hfe
mRNA expression is specific for miR-122 depletion and independent of the antisense chemistry applied. Elevated expression of Hfe
, and Bmpr1a
would be expected to stimulate the Bmp/Smad signaling pathway in mice (44
). However, increased Smad1/5/8 phosphorylation in protein extracts from PM_anti–miR-122–injected mice was not detected (Supplemental Figure 6). Consistently, mRNA expression of Id1
, a target gene of the Bmp/Smad signaling pathway (48
), remained unchanged in the liver of PM_anti–miR-122–injected mice (Supplemental Figure 3). High hepcidin levels would be expected to cause iron retention (e.g., in splenic macrophages) and decreased iron absorption by stimulating the internalization and degradation of the iron exporter ferroportin (4
). However, ferroportin protein levels in the spleen were not decreased in PM_anti–miR-122–injected mice (Supplemental Figure 7). We speculate that the iron demand for extramedullary erythropoiesis that occurs in the spleen of PM_anti–miR-122–injected mice needs to be satisfied, and thus hepcidin-independent regulatory mechanisms, such as BACH1- or NRF2-mediated transcriptional control of ferroportin (61
), may override the hepcidin response of ferroportin.
Extramedullary hematopoiesis in PM_anti–miR-122–injected mice is characterized by increased splenic mRNA expression of Tfr1
, enzymes involved in heme biosynthesis (Hmbs
, and Alas2
), and Hba-a1
as well as the hematopoietic miR-451. Interestingly, overexpression of Hba-a1
mRNA in anemic mice correlates with rapid recovery from anemia (62
), which suggests that similar mechanisms may apply to PM_anti–miR-122–injected mice. Also of note is the elevated expression of miR-451, which was previously reported to be essential for erythropoiesis in mammals and zebrafish and shown to depend on Gata1
). Elevated miR-451 expression is responsible for decreased expression of Gata2
, a transcription factor required to maintain the erythroid precursor stage. Here we show that Gata1
and miR-451 expression increased and Gata2
mRNA levels tended to decrease in the spleen of PM_anti–miR-122–injected mice, which suggests that this regulatory network is maintained during murine extramedullary erythropoiesis in the spleen. It is intriguing to speculate that increased expression of miR-29b and miR-17 (Figure C) — which was also observed in miR-122–depleted mice and, to our knowledge, previously not linked to erythropoiesis — may also be relevant for this process. Taken together, our findings suggest that iron deficiency induced by miR-122 limitation triggers erythropoiesis in the spleen to avoid anemia.
We further uncovered a mechanistic link among miR-122 depletion, elevated hepcidin expression, and systemic iron deficiency. We showed that Hfe and Hjv, 2 genes mutated in the frequent iron overload disorder HH, contained functional miR-122 binding sites (Figure , Supplemental Figure 5, and Supplemental Table 3). Importantly, mutation of the predicted miR-122 binding sites prevented the suppressive effect of miR-122, which suggests that miR-122–dependent inhibition of Hfe and Hjv is specific.
The fatty acid and iron metabolic networks are interconnected; however, the identification of common regulatory elements has been elusive. Recently, several studies showed that miR-122 is required to maintain serum cholesterol levels in mice and nonhuman primates (30
). However, the miR-122 target gene responsible for this phenotype has not been discovered. In addition to its role in maintaining iron and cholesterol metabolism, miR-122 is critical for efficient HCV replication (33
). Inhibition of hepatic miR-122 in chimpanzees chronically infected with HCV led to long-lasting suppression of both HCV replication and viremia (64
). miR-122 expression is also reduced in hepatocellular carcinoma (28
) and is regulated by the circadian rhythm (60
), which suggests that miR-122 activity may be required to synchronize genes within overlapping metabolic pathways. In recognition of the circadian rhythmicity of miR-122 expression, samples from the mice analyzed in this study were always collected at the same time of the day and sacrificed in a random order. Furthermore, the link between miR-122 expression and important human diseases suggests that miR-122 could be a target for therapeutic intervention.
In summary, we propose the following model for the role of miR-122 in iron homeostasis: miR-122 regulates the expression of the HH-associated proteins Hfe, Hjv, and possibly others. Overexpression of Hfe
mRNA expression (66
). Elevated hepcidin levels will limit the iron export capacity from duodenal enterocytes and macrophages and cause plasma and liver iron deficiency. As a consequence, iron-deficient hematopoiesis develops, and extramedullary hematopoiesis is observed in the spleen. Future experiments are needed to clarify the signaling pathways that control the Hamp
response to elevated Hfe
expression in anti–miR-122–treated mice.