Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

Digit specific denervation does not inhibit mouse digit tip regeneration.

It is long-established that innervation-dependent production of neurotrophic factors is required for blastema formation and epimorphic regeneration of appendages in fish and amphibians. The regenerating mouse digit tip and the human fingertip are mammalian models for epimorphic regeneration, and limb denervation in mice inhibits this response. A complicating issue of limb denervation studies in terrestrial vertebrates is that the experimental models also cause severe paralysis therefore impairing appendage use and diminishing mechanical loading of the denervated tissues. Thus, it is unclear whether the limb denervation impairs regeneration via loss of neurotrophic signaling or loss of mechanical load, or both. Herein, we developed a novel surgical procedure in which individual digits were specifically denervated without impairing ambulation and mechanical loading. We demonstrate that digit specific denervation does not inhibit but attenuates digit tip regeneration, in part due to a delay in wound healing. However, treating denervated digits with a wound dressing that enhances closure results in a partial rescue of the regeneration response. Contrary to the current understanding of mammalian epimorphic regeneration, these studies demonstrate that mouse digit tip regeneration is not peripheral nerve dependent, an observation that should inform continued mammalian regenerative medicine approaches.

Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.

Microcomputed tomography staging of bone histolysis in the regenerating mouse digit.

Humans and mice have the ability to regenerate the distal digit tip, the terminal phalanx (P3) in response to amputation. What distinguishes P3 regeneration from regenerative failure is formation of the blastema, a proliferative structure that undergoes morphogenesis to regenerate the amputated tissues. P3 regeneration is characterised by the phases of inflammation, tissue histolysis and expansive bone degradation with simultaneous blastema formation, wound closure and finally blastemal differentiation to restore the amputated structures. While each regenerating digit faithfully progresses through all phases of regeneration, phase progression has traditionally been delineated by time, that is, days postamputation (DPA), yet there is widespread variability in the timing of the individual phases. To diminish variability between digits during tissue histolysis and blastema formation, we have established an in-vivo method using microcomputed tomography (micro CT) scanning to identify five distinct stages of the early regeneration response based on anatomical changes of the digit stump. We report that categorising the initial phases of digit regeneration by stage rather than time greatly diminishes the variability between digits with respect to changes in bone volume and length. Also, stages correlate with the levels of cell proliferation, osteoclast recruitment and osteoprogenitor cell recruitment. Importantly, micro CT staging provides a means to estimate open versus closed digit wounds. We demonstrate two spatially distinct and stage specific bone repair/regeneration responses that occur during P3 regeneration. Collectively, these studies showcase the utility of micro CT imaging to infer the composition of radiolucent soft tissues during P3 blastema formation. Specifically, the staging system identifies the onset of cell proliferation, osteoclastogenesis, osteoprogenitor recruitment, the spatial initiation of de novo bone formation and epidermal closure.

Effects of progesterone and interferon tau on ovine endometrial phosphate, calcium, and vitamin D signaling†.

Given recent reports of expression of postnatal mineral transport regulators at the maternal-conceptus interface during the peri-implantation period, this study tested the hypothesis that progesterone (P4) and interferon tau (IFNT) regulate phosphate, calcium, and vitamin D signaling in the ovine endometrium. Mature Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on day 7 of the estrous cycle. Ewes received daily intramuscular injections of 50 mg of P4 in corn oil vehicle and 75 mg of progesterone receptor antagonist (RU486) in corn oil from days 8 to 15, and twice-daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/day) from days 11 to 15 resulting in four treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT. On day 16, ewes were hysterectomized. RU486 + P4 + CX treated ewes had lower concentrations of 25 (OH) D in plasma than P4 + CX treated ewes (P < 0.05). Endometria from ewes treated with IFNT had greater expression of FGF23 (P < 0.01), S100A9 (P < 0.05), and S100A12 (P = 0.05) mRNAs and lower expression of ADAM10 mRNA (P < 0.01) than of ewes treated with CX proteins. Expression of FGF23 mRNA was greater in endometria of ewes that received RU486 + P4 + IFNT than in ewes that received RU486 + P4 + CX (hormone × protein interaction, P < 0.05). The expression of S100G mRNA was greater in endometria of ewes that received P4 + IFNT compared to ewes that received RU486 + P4 + IFNT (P < 0.05; hormone × protein interaction, P < 0.01). These data implicate P4 and IFNT in the regulation of phosphate, calcium, and vitamin D signaling during the peri-implantation period of pregnancy and provide a platform for continued mechanistic investigations.

Effects of exogenous progesterone on the expression of mineral regulatory molecules by ovine endometrium and placentomes†.

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25 mg of progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Phosphate, Calcium, and Vitamin D: Key Regulators of Fetal and Placental Development in Mammals.

Normal calcium and bone homeostasis in the adult is virtually fully explained by the interactions of several key regulatory hormones, including parathyroid hormone, 1,25 dihydroxy vitamin D3, fibroblast growth factor-23, calcitonin, and sex steroids (estradiol and testosterone). In utero, bone and mineral metabolism is regulated differently from the adult. During development, it is the placenta and not the fetal kidneys, intestines, or skeleton that is the primary source of minerals for the fetus. The placenta is able to meet the almost inexhaustible needs of the fetus for minerals by actively driving the transport of calcium and phosphorus from the maternal circulation to the growing fetus. These fundamentally important minerals are maintained in the fetal circulation at higher concentrations than those in maternal blood. Maintenance of these inordinately higher fetal levels is necessary for the developing skeleton to accrue sufficient minerals by term. Importantly, in livestock species, prenatal mineralization of the skeleton is crucial for the high levels of offspring activity soon after birth. Calcium is required for mineralization, as well as a plethora of other physiological functions. Placental calcium and phosphate transport are regulated by several mechanisms that are discussed in this review. It is clear that phosphate and calcium metabolism is intimately interrelated and, therefore, placental transport of these minerals cannot be considered in isolation.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein−umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Novel mineral regulatory pathways in ovine pregnancy: II. Calcium-binding proteins, calcium transporters, and vitamin D signaling.

Mineralization of the fetal mammalian skeleton requires a hypercalcemic gradient across the placenta from mother to fetus. However, the mechanisms responsible for maintaining the placental transport of calcium remain poorly understood. This study aimed to identify calcium and vitamin D regulatory pathway components in ovine endometria and placentae across gestation. Suffolk ewes were bred with fertile rams upon detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Calcium abundance was influenced by gestational day in uterine flushings and allantoic fluid (P<0.05). The expression of S100G, S100A9, S100A12, ATP2B3, ATP2B4, TRPV5, TRPV6, CYP11A1, CYP2R1, CYP24, and VDR mRNAs known to be involved in calcium binding, calcium transport, and vitamin D metabolism were quantified by qPCR. Mediators of calcium and vitamin D signaling were expressed by Day 17 conceptus tissue, and endometria and placentae across gestation. Gestational day influenced the expression of S100G, S100A9, S100A12, TRPV6, VDR, and CYP24 mRNAs in endometria and placentae (P<0.05). Gestational day influenced endometrial expression of ATP2B3, and placental expression of TRPV5, ATP2B4, and CYP11A1 (P<0.05). VDR protein localized to the endoderm and trophectoderm (Day 17 conceptus) and was expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile suggests a potential role of calcium and vitamin D in the establishment of pregnancy and regulation of fetal and placental growth, providing a platform for further mechanistic investigation.

Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters.

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortusΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

A Link between the Gut and Bone: Bone Health Impacted by Changes in Gut Microbiota.

This commentary highlights the article by Hathaway-Schrader et al that studies the impact of antibiotic-disruption of the gut microbiota on long-term bone development.

PTH and PTHrP Actions on Bone.

Parathyroid hormone (PTH), PTH-related peptide (PTHrP), PTHR, and their cognate G protein-coupled receptor play defining roles in the regulation of extracellular calcium and phosphate metabolism and in controlling skeletal growth and repair. Acting through complex signaling mechanisms that in many instances proceed in a tissue-specific manner, precise control of these processes is achieved. A variety of direct and indirect disease processes, along with genetic anomalies, can cause these schemes to become dysfunctional. Here, we review the basic components of this regulatory network and present both the well-established elements and emerging findings and concepts with the overall objective to provide a framework for understanding the elementary aspects of how PTH and PTHrP behave and as a call to encourage further investigation that will yield more comprehensive understanding of the physiological and pathological steps at play, with a goal toward novel therapeutic interventions.

Bisphosphonate use in the horse: what is good and what is not?

BACKGROUND: Bisphosphonates (BPs) are a family of molecules characterized by two key properties: their ability to bind strongly to bone mineral and their inhibitory effects on mature osteoclasts and thus bone resorption. Chemically two groups of BPs are recognized, non-nitrogen-containing and nitrogen-containing BPs. Non-nitrogen-containing BPs incorporate into the energy pathways of the osteoclast, resulting in disrupted cellular energy metabolism leading to cytotoxic effects and osteoclast apoptosis. Nitrogen-containing BPs primarily inhibit cholesterol biosynthesis resulting in the disruption of intracellular signaling, and other cellular processes in the osteoclast. BODY: BPs also exert a wide range of physiologic activities beyond merely the inhibition of bone resorption. Indeed, the breadth of reported activities include inhibition of cancer cell metastases, proliferation and apoptosis in vitro. In addition, the inhibition of angiogenesis, matrix metalloproteinase activity, altered cytokine and growth factor expression, and reductions in pain have been reported. In humans, clinical BP use has transformed the treatment of both post-menopausal osteoporosis and metastatic breast and prostate cancer. However, BP use has also resulted in significant adverse events including acute-phase reactions, esophagitis, gastritis, and an association with very infrequent atypical femoral fractures (AFF) and osteonecrosis of the jaw (ONJ). CONCLUSION: Despite the well-characterized health benefits of BP use in humans, little is known regarding the effects of BPs in the horse. In the equine setting, only non-nitrogen-containing BPs are FDA-approved primarily for the treatment of navicular syndrome. The focus here is to discuss the current understanding of the strengths and weaknesses of BPs in equine veterinary medicine and highlight the future utility of these potentially highly beneficial drugs.

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation.

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.

NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis.

Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4-/- mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4-/- mice also displayed decreased trabecular BV/TV and trabecular number compared with PF (P < 0.05). However, NOX4-/- mice were protected against EtOH-induced decreases in cortical thickness (P < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone (P < 0.05). In WT and NOX4-/- vertebral bone, EtOH suppressed expression of Wnt signaling components that promote osteoblast maturation. A role for NOX4 in EtOH inhibition of osteoblast differentiation was further demonstrated by protection against EtOH inhibition of osteoblastogenesis in ex vivo bone marrow cultures from NOX4-/-, but not p47phox-/- mice lacking active NADPH oxidase-2. However, bone marrow cultures from NOX4-/- mice formed fewer osteoblastic colonies compared with WT cultures (P < 0.05), suggesting a role for NOX4 in the maintenance of mesenchymal progenitor cell populations. These data suggest that NOX4 deletion is partially protective against EtOH effects on osteoblast differentiation, but may predispose bone to osteogenic impairments.

Hallmarks of Bone Metastasis.

Breast cancer bone metastasis develops as the result of a series of complex interactions between tumor cells, bone marrow cells, and resident bone cells. The net effect of these interactions are the disruption of normal bone homeostasis, often with significantly increased osteoclast and osteoblast activity, which has provided a rational target for controlling tumor progression, with little or no emphasis on tumor eradication. Indeed, the clinical course of metastatic breast cancer is relatively long, with patients likely to experience sequential skeletal-related events (SREs), often over lengthy periods of time, even up to decades. These SREs include bone pain, fractures, and spinal cord compression, all of which may profoundly impair a patient's quality-of-life. Our understanding of the contributions of the host bone and bone marrow cells to the control of tumor progression has grown over the years, yet the focus of virtually all available treatments remains on the control of resident bone cells, primarily osteoclasts. In this perspective, our focus is to move away from the current emphasis on the control of bone cells and focus our attention on the hallmarks of bone metastatic tumor cells and how these differ from primary tumor cells and normal host cells. In our opinion, there remains a largely unmet medical need to develop and utilize therapies that impede metastatic tumor cells while sparing normal host bone and bone marrow cells. This perspective examines the impact of metastatic tumor cells on the bone microenvironment and proposes potential new directions for uncovering the important mechanisms driving metastatic progression in bone based on the hallmarks of bone metastasis.

Activin A inhibits RANKL-mediated osteoclast formation, movement and function in murine bone marrow macrophage cultures.

The process of osteoclastic bone resorption is complex and regulated at multiple levels. The role of osteoclast (OCL) fusion and motility in bone resorption are unclear, with the movement of OCL on bone largely unexplored. RANKL (also known as TNFSF11) is a potent stimulator of murine osteoclastogenesis, and activin A (ActA) enhances that stimulation in whole bone marrow. ActA treatment does not induce osteoclastogenesis in stroma-free murine bone marrow macrophage cultures (BMM), but rather inhibits RANKL-induced osteoclastogenesis. We hypothesized that ActA and RANKL differentially regulate osteoclastogenesis by modulating OCL precursors and mature OCL migration. Time-lapse video microscopy measured ActA and RANKL effects on BMM and OCL motility and function. ActA completely inhibited RANKL-stimulated OCL motility, differentiation and bone resorption, through a mechanism mediated by ActA-dependent changes in SMAD2, AKT1 and inhibitor of nuclear factor κB (IκB) signaling. The potent and dominant inhibitory effect of ActA was associated with decreased OCL lifespan because ActA significantly increased activated caspase-3 in mature OCL and OCL precursors. Collectively, these data demonstrate a dual action for ActA on murine OCLs.

Myeloma cell-derived Runx2 promotes myeloma progression in bone.

The progression of multiple myeloma (MM) is governed by a network of molecular signals, the majority of which remain to be identified. Recent studies suggest that Runt-related transcription factor 2 (Runx2), a well-known bone-specific transcription factor, is also expressed in solid tumors, where expression promotes both bone metastasis and osteolysis. However, the function of Runx2 in MM remains unknown. The current study demonstrated that (1) Runx2 expression in primary human MM cells is significantly greater than in plasma cells from healthy donors and patients with monoclonal gammopathy of undetermined significance; (2) high levels of Runx2 expression in MM cells are associated with a high-risk population of MM patients; and (3) overexpression of Runx2 in MM cells enhanced tumor growth and disease progression in vivo. Additional studies demonstrated that MM cell-derived Runx2 promotes tumor progression through a mechanism involving the upregulation of Akt/ß-catenin/Survivin signaling and enhanced expression of multiple metastatic genes/proteins, as well as the induction of a bone-resident cell-like phenotype in MM cells. Thus, Runx2 expression supports the aggressive phenotype of MM and is correlated with poor prognosis. These data implicate Runx2 expression as a major regulator of MM progression in bone and myeloma bone disease.