Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe.

Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate >2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation.

Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone.

Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis.