Erythroferrone Regulates Bone Remodeling in β-Thalassemia

Erythropoiesis normally occurs in the bone marrow within the pelvis and femur, and both erythropoiesis and bone metabolism are susceptible to changes in iron homeostasis. Thus, hematopoietic and osteoid systems require coordination of iron metabolism during stress or ineffective erythropoiesis. Recently, a more extensive understanding of the crosstalk between iron metabolism and erythropoiesis revealed that a bone marrow secreted protein, erythroferrone (ERFE), is a negative regulator of hepcidin [Kautz Nat Gen 2014]. Hepcidin in turn is the main negative regulator of iron absorption and recycling [Nemeth Science 2004] and its suppression enables an increase in iron availability during stress erythropoiesis. Diseases of ineffective erythropoiesis, such as β-thalassemia, with chronic erythroid expansion, are associated with thinning of cortical bone, leading to decreased bone mineral density [Haidar Bone 2011; Vogiatzi Bone 2006]. Mechanisms underlying coordination of erythropoiesis and bone metabolism are incompletely understood. However, because ERFE functions to suppress hepcidin by sequestering BMPs [Arezes Blood 2018], and because BMPs are crucially important for bone metabolism [Hogan Genes Dev 1996], we hypothesize that ERFE may be involved in coordinating iron metabolism, erythropoiesis, and bone homeostasis. Lastly, osteoblast expression of TfR2 was found to inhibit bone formation by activating BMP-p38MAPK signaling and expression of the Wnt inhibitor Sclerostin, protein product of the SOST gene [Rauner Nat Med 2019]. We thus propose to explore the role of ERFE in disordered bone metabolism in β-thalassemia. In vitro data demonstrates that osteoblasts from wild type (WT) mice express ERFE and this expression is enhanced by BMP2/6/7 (Figure 1a and 1b). Furthermore, osteoblasts from ERFE-/- mice exhibit enhanced bone mineralization (6.8-fold increased von Kossa staining, measured by image J) (Figure 1c), increased expression of osteoblast-specific markers (e.g. osterix (OSX))(Figure 1d), and higher SOST expression (Figure 1e) relative to WT osteoblasts. We anticipate that if TfR2 is central to bone metabolism, ERFE-/- osteoblasts may exhibit a decrease in TfR2; our results demonstrate only a trend toward decreased TfR2 in ERFE-/- osteoblasts (Figure 1f). In addition, we propose that ERFE is a negative regulator of osteoblast activity, predicting that ERFE loss in th3/+ mice would enhance bone mineral density. To this end, we analyzed bone mineral density and histomorphometry in WT, ERFE-/-, th3/+, and th3/+ERFE-/- mice. Surprisingly, although no differences are evident between WT, ERFE-/-, and th3/+ femora, th3/+ERFE-/- mice exhibit a decrease in bone mineral density and bone volume / total volume (BV/TV) (Figure 2a-2b) with a trend toward enhanced femoral mineral apposition rate (Figure 2c) relative to th3/+ mice. These results indicate enhanced osteoblast activity without increased bone formation. Because bone mineralization is a composite of the relative osteoblast and osteoclast activity, we hypothesize that osteoclast activity is further enhanced in th3/+ ERFE-/- mice. TRAP staining demonstrates a significantly increased number of osteoclasts in ERFE-/- relative to WT as well as th3/+ ERFE-/- relative to th3/+ femora (Figure 2d). Our studies demonstrate that ERFE, like other members of the TNFα superfamily [Lu J Bone Miner Res 2011], negatively regulates OSX which is critical for osteoblast function (Figure 3a). Thus, suppression of ERFE results in more OSX (Figure 1d), enhanced mineralization (Figure 1c), and higher SOST expression (Figure 1e) which results in the secretion of Sclerostin (Figure 3b). Sclerostin both feeds back to suppress Wnt signaling to decrease osteoblast function and increases RANKL production to stimulate osteoclast differentiation (Figure 3b). Taken together, ERFE functions as a negative regulator of both osteoblast and especially osteoclast activity such that its loss leads to more osteoclast activity and results in decreased bone mineral density in β-thalassemia. These findings provide novel insights into the complex interplay between regulation of iron metabolism and bone homeostasis in diseases of dysregulated erythropoiesis, when ERFE expression is increased, and support the rationale to further explore the role of ERFE and TfR2 in this crosstalk in β-thalassemia.

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