Paolo Pignataro

The myokine irisin increases cortical bone mass

Significance Although exercise is a well known and potent stimulus for new bone formation, and weightlessness or muscle loss characteristically cause bone loss, it has remained unclear how muscle talks to bone, despite their close proximity. Here, we show that a molecule irisin derived from skeletal muscle in response to exercise has profound effects in enhancing mass and improving the geometry and strength specifically of cortical bone, the key function of which is to resist bending and torsion. Trabecular bone, which is a reservoir for bodily calcium, is remarkably spared. Irisin may therefore not only be the molecule responsible for muscle–bone connectivity, but could also become a therapy for sarcopenia and osteoporosis, which occur in tandem in the elderly. It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg−1. We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg−1 per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin–injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle–bone connectivity.

The death receptor DR5 is involved in TRAIL-mediated human osteoclast apoptosis

The number and activity of osteoclasts (OCs) are critical for maintaining normal bone turnover. The number is determined by the rates of cell differentiation and death. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis by interacting with its death receptors, (DR4, DR5). However, its activity can be modulated by two decoy receptors, (DcR1 and DcR2). In this paper we show that TRAIL treatment causes reduced OC viability as well as an increased apoptotic OC number. Loss of nuclei integrity and derangement of the actin microfilament were also induced by TRAIL in OCs. Moreover, we demonstrated the expression of all TRAIL receptors in both precursors and differentiated OCs, and the upregulation of DR5 during OC differentiation. Interestingly, DcR2 was upregulated in the early stage of osteoclastogenesis and downregulated at the end of the differentiation process. We showed that DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis, since the addition of anti-DR5 neutralizing antibodies restores the OC viability previously reduced by TRAIL. Furthermore, the intracellular pathway induced by TRAIL in OCs involves caspase-8 and Bid activation. In conclusion, our data highlight an important role for the TRAIL/TRAIL receptor system in the regulation of OC apoptosis.

Irisin prevents and restores bone loss and muscle atrophy in hind-limb suspended mice

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass. Here we report that treatment with recombinant Irisin prevented bone loss in hind-limb suspended mice when administered during suspension (preventive protocol) and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks (curative protocol). MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented a dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin protected against muscle mass decline in the hind-limb suspended mice, and maintained the fiber cross-sectional area. Notably, the decrease of myosin type II expression in unloaded mice was completely prevented by r-Irisin administration. Our data reveal for the first time that Irisin retrieves disuse‐induced bone loss and muscle atrophy. These findings may lead to development of an Irisin-based therapy for elderly immobile osteoporotic and physically disable patients, and might represent a countermeasure for astronauts subjected to microgravity-induced bone and muscle losses.

TRAIL Is Involved in Human Osteoclast Apoptosis

Abstract:  Control of osteoclast (OC) apoptosis has been recognized as a critical regulatory factor in bone remodeling. TRAIL, a member of the TNF superfamily, induces apoptosis in neoplastic and normal cells. However, few data are available on the effects of TRAIL on bone cells, thus in the present study we investigated TRAIL role on the apoptosis of human mature OCs. We show that TRAIL treatment causes reduced cell viability, loss of nuclei integrity, and derangement of the actin microfilament in OCs. We also demonstrated that the death receptor DR5, upregulated by TRAIL, could be the mediator of TRAIL‐induced OC apoptosis.

Sclerostin stimulates angiogenesis in human endothelial cells

Sclerostin, negative regulator of bone formation, has been originally known as an osteocyte product. Recently, it has been also detected in hypertrophic chondrocytes, distinctive cells of avascular cartilage which is invaded by capillaries and then replaced by vascularized bone. Thus, we hypothesized that sclerostin, in addition to its role already known, may exert an angiogenic activity. We first proved that sclerostin increased the proliferation of human umbilical vein endothelial cells (HUVECs), and next, by using the chicken chorioallantoic membrane (CAM) in vivo assay, we demonstrated that it exerts an angiogenic activity similar to that of vascular endothelial growth factor (VEGF). This last finding was reinforced by several in vitro approaches. Indeed, we showed that sclerostin induced the formation of a network of anastomosing tubules, a significant increase in the percentage of tubule number, total tubule length and number of junctions, as well as the ability of sclerostin-stimulated HUVECs to organize capillary-like structures and closed-meshes similar to VEGF. The angiogenic response elicited by the protein may be due to the binding to its receptor, LRP6, which is highly expressed at mRNA and protein levels by sclerostin treated HUVECs and through the production of two well-known pro-angiogenic cytokines, VEGF and placental growth factor (PlGF). Finally, we demonstrated that sclerostin was also responsible for the recruitment of osteoclasts and their circulating monocyte progenitors. Overall, these findings showed for the first time the new angiogenic in vitro role of sclerostin which could be also considered as a novel molecule in angiogenesis-osteogenesis coupling.

Impairment of Bone Remodeling in LIGHT/TNFSF14-Deficient Mice: IMPAIRMENT OF BONE REMODELING IN LIGHT/TNFSF14-DEFICIENT MICE

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone‐forming osteoblasts and bone‐resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT‐deficient mice (Tnfsf14‐/‐) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti‐osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro‐osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14‐/‐mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T‐ and B‐cell‐deficient mice (Rag‐/Tnfsf14‐) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease.

Treatment with r-irisin prevents and recovers disuse-induced bone loss and muscle atrophy

Irisin is a hormone-like myokine secreted from skeletal muscle in response to exercise. We previously showed that treatment with recombinant Irisin (r-Irisin) in healthy mice improved cortical bone mass and geometry, supporting the idea that Irisin recapitulates some of the most important benefits of physical exercise on the skeleton and plays protective role on bone health (1). Here we show that treatment with r-Irisin prevented bone loss in hind-limb suspended mice when administered during suspension and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks. MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented the dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin inhibited muscle mass decline during unloading, keeping proper fiber cross-sectional area. Notably, the decrease in myosin type II expression (MyHC II) in vastus lateralis of unloaded mice treated with r-Irisin was completely prevented. Our data reveal that r-Irisin treatment protects from disuse induced bone loss and muscle atrophy in mice. If these results will translate to humans, they may support a promising clinical strategy for the prevention and treatment of both osteoporosis and sarcopenia, particularly applicable to those patients who cannot perform physical activity, as occurs during aging, immobility and microgravity during space flight missions.

Effects of recombinant Irisin on the musculoskeletal system of hind-limb suspended mice

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass, driving positive effects on cortical mineral density and geometry in vivo (1). Here we demonstrated that r-Irisin treatment prevents bone loss in hind-limb suspended mice when administered during suspension and recovers bone mass when mice were injected after a suspension period (4 weeks) during which they developed bone loss. Micro computed tomography of femurs showed that r-Irisin treatment positively affected both cortical and trabecular bone. As expected, unloaded mice treated with vehicle displayed a remarkable decrease of cortical and trabecular bone mineral density (BMD), whereas in Irisin-treated unloaded mice no loss of BMD was observed with respect to control mice kept under normal loading. Likewise, by treating mice after they already developed disuse-induced bone loss, r-Irisin was able to restore the damaged mineral component. Furthermore, trabecular bone volume fraction (BV/TV), which dramatically decreased in unloaded mice, was prevented by r-Irisin therapy. In particular, r-Irisin treatment preserved the number of trabeculae (Tb.N) and the fractal dimension, an index of optimal micro-architectural complexity of trabecular bone.We also showed that r-Irisin treatment protects muscle mass suffering from atrophy during unloading. Thus, unloaded mice treated with vehicle displayed a severe loss of muscle mass, as confirmed by ~ 60% decline of vastus lateralis weight and ~33% decrease of fiber cross-sectional area. Conversely, Irisin-treated unloaded mice showed no loss of muscle weight and similar fiber cross-sectional area to control mice. Our data reveal for the first time that r-Irisin treatment prevents and retrieves disuse-induced bone loss and muscle atrophy. These findings may lead to develop an Irisin-based therapy for the prevention and treatment of osteoporosis and sarcopenia in all patients who cannot perform physical activity, as occurs during aging and immobility, and it could also represent a countermeasure for astronauts exposed to microgravity during space flight missions.This work was supported in part by ERISTO grant (to M.G.), by MIUR grant ex60% (to M.G.) and by SIOMMMS grant (to G.C.).

LIGHT/TNFSF14 affects basal bone remodeling

LIGHT (TNFSF14), expressed by different cells of the immune system, binds two trans-membrane receptors: HVEM and LTβR. It is over-expressed in erosive rheumatoid arthritis and lytic myeloma-bone disease and controversial data have been published on its role in osteoclast (OC) formation in vitro. Here, we investigated the role of LIGHT on in vitro murine osteoclastogenesis model and bone phenotype in LIGHT-/- mice. Firstly, we showed that murine macrophages stimulated with LIGHT alone did not differentiate into OCs. Interestingly, the presence of LIGHT and sub-optimal RANKL concentration displayed synergic effects on OC formation through the early and sustained activation of Akt, NFκB and JNK pathways. Secondly, by microCT we found that the femurs of LIGHT-KO mice exhibited a 30% (p<0.01) decrease in trabecular BV/TV due to a significant reduction in trabecular thickness and number as well as the increase in trabecular spaces respect to wild-type (WT) mice. Furthermore, a five fold increase of OC number/bone surface was found in femora from KO mice compared to WT (p<0.008). To investigate the possible molecular mechanism/s responsible for this bone phenotype in LIGHT-/- mice we studied OPG levels in whole bone marrow (BM) extracts from the femurs of these mice and demonstrated a significant reduction in OPG mRNA transcript respect to WT. Further investigations showed that BM CD8+ T cells and B cell subpopulations from KO mice expressed lower levels of OPG compared to those from WT mice. Consistently, LIGHT treatment in a dose dependent manner increase OPG expression in BM CD8+ T cells and B-cells. In conclusion, our results identified LIGHT as a new important regulator of bone remodeling and highlighted a new modulator of OPG expression.

Irisin injected mice display increased tibial cortical mineral density and polar moment of inertia

It has been recently reported that, after physical activity, the skeletal muscle releases Irisin, the newly identified myokine able of driving transition of white adipocytes into brown [1]. This result supported the role of skeletal muscle as endocrine organ, suggesting that it could target other tissues besides adipose tissue. In our previous work, we demonstrated that conditioned media collected from primary myoblasts of exercised mice were able to increase OB differentiation and this effect was Irisinmediated [2]. Here we show that Irisin has positive effect on cortical mineral density and geometry in vivo. Young male mice were injected with r-Irisin and cortical bone adaptation was analyzed by micro-CT at tibial midshaft. Our results show that cortical tissue mineral density is significantly increased in Irisin-injected mice compared to vehicle-injected littermates (+7.15%; p<0.01). Furthermore, this higher density of calcium hydroxyapatite at cortical site is accompanied by increase in periosteal circumference (+7.5%; p<0.03) and polar moment of Inertia (pMOI +19,21%; p<0,01). A greater pMOI indicates stronger resistance of a long bone to torsion and, together with higher bone mineral density, suggests higher protection against fracture. The effect of Irisin is fully comparable to the effect of physical activity that is widely accepted method for increasing bone mineral density and bone size in healthy populations. In view of further proving the involvement of Irisin in bone metabolism, we validate its direct effect on osteoblasts in vitro. Phosphorylation of the MAP kinase ERK and the expression of Atf4 were significantly increased after Irisin treatment, as well as ALP and pro-Collagen I mRNA expression. Our data highlight a novel link in muscle-fat-bone axis demonstrating that Irisin targets bone tissue directly, driving positive effects on cortical mineral density and geometry in vivo. These findings would expand the research of exercise-mimetic drugs that might be widely used to treat osteoporotic patients who are suffering from immobilization and cannot perform physical activity.