The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

Effects of dietary iron deficiency or overload on bone: Dietary details matter.

PURPOSE: Bone is susceptible to fluctuations in iron homeostasis, as both iron deficiency and overload are linked to poor bone strength in humans. In mice, however, inconsistent results have been reported, likely due to different diet setups or genetic backgrounds. Here, we assessed the effect of different high and low iron diets on bone in six inbred mouse strains (C57BL/6J, A/J, BALB/cJ, AKR/J, C3H/HeJ, and DBA/2J). METHODS: Mice received a high (20,000 ppm) or low-iron diet (∼10 ppm) after weaning for 6-8 weeks. For C57BL/6J males, we used two dietary setups with similar amounts of iron, yet different nutritional compositions that were either richer ("TUD study") or poorer ("UCLA study") in minerals and vitamins. After sacrifice, liver, blood and bone parameters as well as bone turnover markers in the serum were analyzed. RESULTS: Almost all mice on the UCLA study high iron diet had a significant decrease of cortical and trabecular bone mass accompanied by high bone resorption. Iron deficiency did not change bone microarchitecture or turnover in C57BL/6J, A/J, and DBA/2J mice, but increased trabecular bone mass in BALB/cJ, C3H/HeJ and AKR/J mice. In contrast to the UCLA study, male C57BL/6J mice in the TUD study did not display any changes in trabecular bone mass or turnover on high or low iron diet. However, cortical bone parameters were also decreased in TUD mice on the high iron diet. CONCLUSION: Thus, these data show that cortical bone is more susceptible to iron overload than trabecular bone and highlight the importance of a nutrient-rich diet to potentially mitigate the negative effects of iron overload on bone.

Clonal hematopoiesis and its impact on the aging osteo-hematopoietic niche.

Clonal hematopoiesis (CH) defines a premalignant state predominantly found in older persons that increases the risk of developing hematologic malignancies and age-related inflammatory diseases. However, the risk for malignant transformation or non-malignant disorders is variable and difficult to predict, and defining the clinical relevance of specific candidate driver mutations in individual carriers has proved to be challenging. In addition to the cell-intrinsic mechanisms, mutant cells rely on and alter cell-extrinsic factors from the bone marrow (BM) niche, which complicates the prediction of a mutant cell's fate in a shifting pre-malignant microenvironment. Therefore, identifying the insidious and potentially broad impact of driver mutations on supportive niches and immune function in CH aims to understand the subtle differences that enable driver mutations to yield different clinical outcomes. Here, we review the changes in the aging BM niche and the emerging evidence supporting the concept that CH can progressively alter components of the local BM microenvironment. These alterations may have profound implications for the functionality of the osteo-hematopoietic niche and overall bone health, consequently fostering a conducive environment for the continued development and progression of CH. We also provide an overview of the latest technology developments to study the spatiotemporal dependencies in the CH BM niche, ideally in the context of longitudinal studies following CH over time. Finally, we discuss aspects of CH carrier management in clinical practice, based on work from our group and others.

Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Comparison of the effects of high dietary iron levels on bone microarchitecture responses in the mouse strains 129/Sv and C57BL/6J.

Iron is an essential nutrient for all living organisms. Both iron deficiency and excess can be harmful. Bone, a highly metabolic active organ, is particularly sensitive to fluctuations in iron levels. In this study, we investigated the effects of dietary iron overload on bone homeostasis with a specific focus on two frequently utilized mouse strains: 129/Sv and C57BL/6J. Our findings revealed that after 6 weeks on an iron-rich diet, 129/Sv mice exhibited a decrease in trabecular and cortical bone density in both vertebral and femoral bones, which was linked to reduced bone turnover. In contrast, there was no evidence of bone changes associated with iron overload in age-matched C57BL/6J mice. Interestingly, 129/Sv mice exposed to an iron-rich diet during their prenatal development were protected from iron-induced bone loss, suggesting the presence of potential adaptive mechanisms. Overall, our study underscores the critical role of genetic background in modulating the effects of iron overload on bone health. This should be considered when studying effects of iron on bone.

Thyroid hormone receptor Thra and Thrb knockout differentially affects osteoblast biology and thyroid hormone responsiveness in vitro.

Thyroid hormones (TH) are important modulators of bone remodeling and thus, thyroid diseases, in particular hyperthyroidism, are able to compromise bone quality and fracture resistance. TH actions on bone are mediated by the thyroid hormone receptors (TR) TRα1 and TRß1, encoded by Thra and Thrb, respectively. Skeletal phenotypes of mice lacking Thra (Thra0/0 ) and Thrb (Thrb-/- ) are well-described and suggest that TRα1 is the predominant mediator of TH actions in bone. Considering that bone cells might be affected by systemic TH changes seen in these mutant mice, here we investigated the effects of TR knockout on osteoblasts exclusively at the cellular level. Primary osteoblasts obtained from Thra0/0 , Thrb-/- , and respective wildtype (WT) mice were analyzed regarding their differentiation potential, activity and TH responsiveness in vitro. Thra, but not Thrb knockout promoted differentiation and activity of early, mature and late osteoblasts as compared to respective WT cells. Interestingly, while mineralization capacity and expression of osteoblast marker genes and TH target gene Klf9 was increased by TH in WT and Thra-deficient osteoblasts, Thrb knockout mitigated the responsiveness of osteoblasts to short (48 h) and long term (10 d) TH treatment. Further, we found a low ratio of Rankl, a potent osteoclast stimulator, over osteoprotegerin, an osteoclast inhibitor, in Thrb-deficient osteoblasts and in line, supernatants obtained from Thrb-/- osteoblasts reduced numbers of primary osteoclasts in vitro. In accordance to the increased Rankl/Opg ratio in TH-treated WT osteoblasts only, supernatants from these cells, but not from TH-treated Thrb-/- osteoblasts increased the expression of Trap and Ctsk in osteoclasts, suggesting that osteoclasts are indirectly stimulated by TH via TRß1 in osteoblasts. In conclusion, our study shows that both Thra and Thrb differentially affect activity, differentiation and TH response of osteoblasts in vitro and emphasizes the importance of TRß1 to mediate TH actions in bone.

Transferrin receptor 2 deficiency promotes macrophage polarization and inflammatory arthritis.

OBJECTIVE: Rheumatoid arthritis is an inflammatory joint disease in which synovial iron deposition has been described. Transferrin receptor 2 (Tfr2) represents a critical regulator of systemic iron levels. Loss of Tfr2 function in humans and mice results in iron overload. As iron contributes to inflammatory processes, we investigated whether Tfr2-deletion affects the pathogenesis of inflammatory arthritis in an iron-dependent manner. METHODS: Using a global and conditional genetic disruption of Tfr2, we assessed the relevance of Tfr2 in K/BxN serum-transfer arthritis (STA) and macrophage polarization. RESULTS: Male Tfr2-/- mice subjected to STA developed pronounced joint swelling, and bone erosion as compared to Tfr2+/+ littermate-controls (P < 0.01). Furthermore, an increase of neutrophils and macrophages/monocytes was observed in the inflammatory infiltrate within the paws of Tfr2-/- mice. To elucidate whether Tfr2 in myeloid cells has a direct role in the pathogenesis of arthritis or whether the effects were mediated via the systemic iron overload, we induced STA in Tfr2fl/fl-LysMCre + mice, which showed normal iron-loading. Cre + female mice displayed increased disease development compared to Cre-controls. As macrophages regulate iron availability and innate immunity, we hypothesized that Tfr2-deficiency would polarize macrophages toward a pro-inflammatory state (M1) that contributes to arthritis progression. In response to IFN-γ stimulation, Tfr2-/- macrophages showed increased expression of M1-like cytokines, IFN-γ-target genes, nitric-oxide production, and prolonged STAT1 activation compared to Tfr2+/+ macrophages (P < 0.01), while pre-treatment with ruxolitinib abolished Tfr2-driven M1-like polarization. CONCLUSION: Taken together, these findings suggest a protective role of Tfr2 in macrophages on the progression of arthritis via suppression of M1-like polarization.

Osteoprogenitor SFRP1 prevents exhaustion of hematopoietic stem cells via PP2A-PR72/130-mediated regulation of p300.

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Dickkopf1 fuels inflammatory cytokine responses.

Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1-controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3-restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and non-malignant cells. Additional studies are required to mechanistically dissect cellular DKK1 trafficking and signaling pathways.

Compartment-specific mutational landscape of clonal hematopoiesis.

Clonal hematopoiesis (CH) is characterized by somatic mutations in blood cells of individuals without hematologic disease. While the mutational landscape of CH in peripheral blood (PB) has been well characterized, detailed analyses addressing its spatial and cellular distribution in the bone marrow (BM) compartment are sparse. We studied CH driver mutations in healthy individuals (n = 261) across different anatomical and cellular compartments. Variant allele frequencies were higher in BM than PB and positively correlated with the number of driver variants, yet remained stable during a median of 12 months of follow-up. In CH carriers undergoing simultaneous bilateral hip replacement, we detected ASXL1-mutant clones in one anatomical location but not the contralateral side, indicating intra-patient spatial heterogeneity. Analyses of lineage involvement in ASXL1-mutated CH showed enriched clonality in BM stem and myeloid progenitor cells, while lymphocytes were particularly involved in individuals carrying the c.1934dupG variant, indicating different ASXL1 mutations may have distinct lineage distribution patterns. Patients with overt myeloid malignancies showed higher mutation numbers and allele frequencies and a shifting mutation landscape, notably characterized by increasing prevalence of DNMT3A codon R882 variants. Collectively, our data provide novel insights into the genetics, evolution, and spatial and lineage-specific BM involvement of CH.

Iron effects versus metabolic alterations in hereditary hemochromatosis driven bone loss.

Hereditary hemochromatosis (HH) is a genetic disorder in which mutations affect systemic iron homeostasis. Most subtypes of HH result in low hepcidin levels and iron overload. Accumulation of iron in various tissues can lead to widespread organ damage and to various complications, including liver cirrhosis, arthritis, and diabetes. Osteoporosis is another frequent complication of HH, and the underlying mechanisms are poorly understood. Currently, it is unknown whether iron overload in HH directly damages bone or whether complications associated with HH, such as liver cirrhosis or hypogonadism, affect bone secondarily. This review summarizes current knowledge of bone metabolism in HH and highlights possible implications of metabolic dysfunction in HH-driven bone loss. We further discuss therapeutic considerations managing osteoporosis in HH.

Association between bone turnover markers and periodontitis: A population-based cross-sectional study.

AIM: To examine the associations between bone turnover markers and periodontitis in two cross-sectional population-based studies. MATERIALS AND METHODS: We used data from two independent adult samples (N = 4993), collected within the Study of Health in Pomerania project, to analyse cross-sectional associations of N-procollagen type 1 amino-terminal propeptide (P1NP), C-terminal cross-linking telopeptide, osteocalcin, bone-specific alkaline phosphatase (BAP), fibroblast growth factor 23, wingless-type mouse mammary tumour virus integration site family member 5a (WNT5A), and sclerostin values with periodontitis. Confounder-adjusted gamma and fractional response regression models were applied. RESULTS: Positive associations were found for P1NP with mean pocket probing depth (PPD; eß=1.008 ; 95% confidence interval [CI]: 1.001-1.015), mean clinical attachment loss (mean CAL; eß=1.027 ; 95% CI: 1.011-1.044), and proportion of sites with bleeding on probing (%BOP; eß=1.055 ; 95% CI: 1.005-1.109). Similar associations were seen for BAP with %BOP ( eß=1.121 ; 95% CI: 1.042-1.205), proportion of sites with PPD ≥4 mm (%PPD4) ( eß=1.080 ; 95% CI: 1.005-1.161), and sclerostin with %BOP ( eß=1.308 ; 95% CI: 1.005-1.704). WNT5A was inversely associated with mean PPD ( eß=0.956 ; 95% CI: 0.920-0.993) and %PPD4 ( eß=0.794 ; 95% CI: 0.642-0.982). CONCLUSIONS: This study revealed scattered associations of P1NP, BAP, WNT5A, and sclerostin with periodontitis, but the results are contradictory in the overall context. Associations reported in previous studies could not be confirmed.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes.

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

New Insights Into Osteoclast Biology.

Osteoclasts are multinucleated cells that are characterized by their unique ability to resorb large quantities of bone. Therefore, they are frequently the target of therapeutic interventions to ameliorate bone loss. In an adult organism, osteoclasts derive from hematopoietic stem cells and differentiate into osteoclasts within a multistep process under the influence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Historically, the osteoclast life cycle has been defined as linear, whereby lineage-committed mononuclear precursors fuse to generate multinucleated highly specialized and localized bone phagocytic cells, which then undergo apoptosis within weeks. Recent advances through lineage tracing, single cell RNA sequencing, parabiosis, and intravital imaging approaches have challenged this dogma, revealing they have greater longevity and the capacity to circulate and undergo cell recycling. Indeed, these new insights highlight that under homeostatic conditions very few incidences of osteoclast apoptosis occur. More importantly, as we revisit the formation and fate of the osteoclast, novel methods to target osteoclast biology in bone pathology and regeneration are emerging. This review briefly summarizes the historical life cycle of osteoclasts and highlights recent discoveries made through advanced methodologies, which have led to a paradigm shift in osteoclast biology. These findings are discussed in light of both existing and emerging bone targeted therapeutics, bone pathologies, and communication between osteoclasts and cells resident in bone or at distant sites. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Prednisone prevents particle induced bone loss in the calvaria mouse model.

INTRODUCTION: Glucocorticoids are essential in the treatment of many chronic inflammatory and malignant diseases but are known to have detrimental effects on bone. This study aimed to investigate the effects of prednisone on osteoclast functioning in vivo in the calvaria particle-induced bone loss mouse model. METHODS: 12-week-old male C57BL6/J mice received subcutaneously implanted prednisone (2.5 mg/d, 60 day release (n = 14)) or placebo pellets (n = 10). Osteolysis of the calvaria bone was induced two weeks later by application of ultra-high-molecular-weight polyethylene- (UHMWPE) particles to the dome (vs sham operation). The extent of osteolysis was determined histologically and by micro-computer tomography. RESULTS: Prednisone significantly inhibited particle-induced osteolysis in the skull. No significant difference in osteoclast numbers was seen in mice with prednisone vs placebo treatment. Prednisone treatment alone without particle application did not reduce bone mineral density or deterioration in bone microarchitecture parameters. CONCLUSIONS: The calvaria particle-induced bone loss mouse model can be adapted to investigate osteoclast activity in vivo and the effect of prednisone on osteoclasts. In this preventive experimental design, the application of short-term low-dose prednisone has osteoprotective effects without measurable systemic side effects on bone parameters.

Shaping the bone through iron and iron-related proteins.

Well-controlled iron levels are indispensable for health. Iron deficiency is the most common cause of anemia, whereas iron overload, either hereditary or secondary due to disorders of ineffective erythropoiesis, causes widespread organ failure. Bone is particularly sensitive to fluctuations in systemic iron levels as both iron deficiency and overload are associated with low bone mineral density and fragility. Recent studies have shown that not only iron itself, but also iron-regulatory proteins that are mutated in hereditary hemochromatosis can control bone mass. This review will summarize the current knowledge on the effects of iron on bone homeostasis and bone cell activities, and on the role of proteins that regulate iron homeostasis, i.e. hemochromatosis proteins and proteins of the bone morphogenetic protein pathway, on bone remodeling. As disorders of iron homeostasis are closely linked to bone fragility, deeper insights into common regulatory mechanisms may provide new opportunities to concurrently treat disorders affecting iron homeostasis and bone.

New insights into the role of glycosaminoglycans in the endosteal bone microenvironment.

The bone microenvironment is a complex tissue in which heterogeneous cell populations of hematopoietic and mesenchymal origin interact with environmental cues to maintain tissue integrity. Both cellular and matrix components are subject to physiologic challenges and can dynamically respond by modifying cell/matrix interactions. When either component is impaired, the physiologic balance is lost. Here, we review the current state of knowledge of how glycosaminoglycans - organic components of the bone extracellular matrix - influence the bone micromilieu. We point out how they interact with mediators of distinct signaling pathways such as the RANKL/OPG axis, BMP and WNT signaling, and affect the activity of bone remodeling cells within the endosteal niche summarizing their potential for therapeutic intervention.