To identify genes that modify the severity of human iron disorders we pre-selected 74 genes from gene expression profiles of cells and tissues with altered iron levels and assessed whether siRNA-mediated knock-down of these genes affects uptake of transferrin, a key cellular process to acquire iron. This screen identifies the monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, as a critical suppressor of transferrin receptor mRNA expression in human cells. We next analyzed CCL2-deficient mice and demonstrate profound alterations of parameters of systemic iron homeostasis. Specifically, CCL2 knock-out mice show decreased serum iron levels and transferrin saturation, strong iron-overload in the spleen and duodenum as well as mild iron accumulation in the liver. Iron imbalance in CCL2−/− mice is unlikely explained by an impairment of the major control system of systemic iron homeostasis, the hepcidin/ferroportin regulatory system: hepcidin mRNA expression is unaltered and splenic ferroportin protein expression is strongly increased in CCL2−/− mice, as would be expected as a consequence of splenic iron overload. We speculate that increased iron absorption from the plasma, possibly mediated by inappropriately high levels of TfR1 in the spleen, duodenum and liver, may be responsible for tissue iron overload.
It is of note that CCL2 levels are strongly decreased in Hfe-deficient mice and patients with Hfe-associated Hereditary Hemochromatosis (HH). We therefore asked whether CCL2 levels could modify disease severity of HH. Analysis of 51 HH patients, all homozygous for the C282Y HFE mutation, confirms significantly lower MCP-1 levels in the serum compared to a group of 23 sex- and age-matched normal controls. Importantly, CCL2 levels in HH patients show a significant negative correlation with liver iron overload at the time point of diagnosis. Furthermore, low CCL2 concentrations are significantly associated with the HLA-A3 genotype and the CD8+ T lymphocyte phenotype, both traits previously shown to correlate with iron overload in HH patients. These patient data and the data from CCL2-deficient mice suggest that appropriate CCL2 expression is required to prevent iron overload.
Taken together our data demonstrate the power of siRNA screens to identify novel regulators of iron metabolism in human cells that are critically involved in maintaining systemic iron homeostasis in the mouse and that play a role in modifying hepatic iron overload in the frequent iron overload disorder Hereditary Hemochromatosis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.