Osteoblast-selective Erfe Deletion Confirms Cell-Autonomous Role of ERFE in the Bone homeostasis

We explore the incompletely understood crosstalk between erythropoiesis, bone homeostasis, and iron metabolism.  We anticipate that, because erythroferrone (ERFE) acts by sequestering bone morphogenetic proteins (BMPs) to inhibit the main signaling pathway in hepcidin regulation and BMPs are crucial for bone modeling and remodeling, ERFE plays a central role in this mutual regulation.  We discovered that osteoblasts express Erfe mRNA and functional ERFE is found in cultured osteoblast supernatants. Erfe-/- mouse osteoblasts displayed enhanced mineralization and osteoblastogenesis.  However, skeletal phenotyping revealed that Erfe-/- mice had low bone mineral density.  We thus generated conditional Erfe knockout (Erfe floxed) mice and analyzed progeny crossed with osteoblast-selective Cre (Col2.3-Cre). Our results demonstrate that Erfe expression is selectively lost in osteoblasts of Col2.3-Cre;Erfefl/fl mice but not erythroblasts and Col1a1 and Alp expression is increased in cultured osteoblasts from Col2.3-Cre;Erfefl/fl mice, consistent with enhanced osteoblast differentiation.  First, Col2.3-Cre;Erfefl/fl mice do not exhibit decreased bone mineral density. Next, parathyroid hormone treated Col2.3-Cre;Erfefl/fl mice demonstrate enhanced bone mineral density relative to Erfefl/fl mice, consistent with an advantage during bone formation when ERFE is lost selectively in osteoblasts.  As expected, no differences in spleen weight, hemoglobin, reticulocyte counts, and hepcidin expression are evident between Erfefl/fl and Col2.3-Cre;Erfefl/fl mice. Lastly, Col2.3-Cre;Erfefl/fl mice do not suppress hepcidin in response to phlebotomy.  Taken together, our results demonstrate the utility of the newly generated Erfefl/fl mice, supports our hypothesis that cell-autonomous osteoblast ERFE expression is relevant in bone homeostasis, and confirms a possible systemic contribution to canonical ERFE function.