Search

Background:Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis.Methods:To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β–thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone.Results:We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss.Conclusions:Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.Funding:YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in β–thalassemic (Hbbth3/+) mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre + : Oxtr fl/fl mice recapitulate the low-bone mass phenotype of Oxtr +/− mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre + : Oxtr fl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre + : Oxtr fl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre + : Oxtr fl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt −/− and Oxtr −/− mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

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. Disclosures Fleming: Protagonist: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Consultancy; Ultragenyx: Consultancy. Rivella:Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.