Osteoblastic Bone Formation Research Articles

Effect of Ishophloroglucin A Isolated from Ishige okamurae on In Vitro Osteoclastogenesis and Osteoblastogenesis.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis.

Pyrroloquinoline quinone alleviates natural aging-related osteoporosis via a novel MCM3-Keap1-Nrf2 axis-mediated stress response and Fbn1 upregulation.

Age-related osteoporosis is associated with increased oxidative stress and cellular senescence. Pyrroloquinoline quinone (PQQ) is a water-soluble vitamin-like compound that has strong antioxidant capacity; however, the effect and underlying mechanism of PQQ on aging-related osteoporosis remain unclear. The purpose of this study was to investigate whether dietary PQQ supplementation can prevent osteoporosis caused by natural aging, and the potential mechanism underlying PQQ antioxidant activity. Here, we found that when 6-month-old or 12-month-old wild-type mice were supplemented with PQQ for 12 months or 6 months, respectively, PQQ could prevent age-related osteoporosis in mice by inhibiting osteoclastic bone resorption and stimulating osteoblastic bone formation. Mechanistically, pharmmapper screening and molecular docking studies revealed that PQQ appears to bind to MCM3 and reduces its ubiquitination-mediated degradation; stabilized MCM3 then competes with Nrf2 for binding to Keap1, thus activating Nrf2-antioxidant response element (ARE) signaling. PQQ-induced Nrf2 activation inhibited bone resorption through increasing stress response capacity and transcriptionally upregulating fibrillin-1 (Fbn1), thus reducing Rankl production in osteoblast-lineage cells and decreasing osteoclast activation; as well, bone formation was stimulated by inhibiting osteoblastic DNA damage and osteocyte senescence. Furthermore, Nrf2 knockout significantly blunted the inhibitory effects of PQQ on oxidative stress, on increased osteoclast activity and on the development of aging-related osteoporosis. This study reveals the underlying mechanism of PQQ's strong antioxidant capacity and provides evidence for PQQ as a potential agent for clinical prevention and treatment of natural aging-induced osteoporosis.

Low-intensity pulsed ultrasound regulates osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling for orthodontic alveolar bone remodeling.

Background: The limited regenerative potential of periodontal tissue remains a challenge in orthodontic treatment, especially with respect to alveolar bone remodeling. The dynamic balance between the bone formation of osteoblasts and the bone resorption of osteoclasts controls bone homeostasis. The osteogenic effect of low-intensity pulsed ultrasound (LIPUS) is widely accepted, so LIPUS is expected to be a promising method for alveolar bone regeneration. Osteogenesis is regulated by the acoustic mechanical effect of LIPUS, while the cellular perception, transduction mode and response regulation mechanism of LIPUS stimuli are still unclear. This study aimed to explore the effects of LIPUS on osteogenesis by osteoblast-osteoclast crosstalk and the underlying regulation mechanism. Methods: The effects of LIPUS on orthodontic tooth movement (OTM) and alveolar bone remodeling were investigated via rat model by histomorphological analysis. Mouse bone marrow mesenchymal stem cells (BMSCs) and bone marrow monocytes (BMMs) were purified and used as BMSC-derived osteoblasts and BMM-derived osteoclasts, respectively. The osteoblast-osteoclast co-culture system was used to evaluate the effect of LIPUS on cell differentiation and intercellular crosstalk by Alkaline phosphatase (ALP), Alizarin Red S (ARS), tartrate-resistant acid phosphatase (TRAP) staining, real-time quantitative PCR, western blotting and immunofluorescence. Results: LIPUS was found to improve OTM and alveolar bone remodeling in vivo, promote differentiation and EphB4 expression in BMSC-derived osteoblasts in vitro, particularly when cells were directly co-cultured with BMM-derived osteoclasts. LIPUS enhanced EphrinB2/EphB4 interaction between osteoblasts and osteoclasts in alveolar bone, activated the EphB4 receptor on osteoblasts membrane, transduced LIPUS-related mechanical signals to the intracellular cytoskeleton, and gave rise to the nuclear translocation of YAP in Hippo signaling pathway, thus regulating cell migration and osteogenic differentiation. Conclusions: This study shows that LIPUS modulates bone homeostasis by osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling, which benefits the balance between OTM and alveolar bone remodeling.

Blocking CCN2 Reduces Established Bone Loss Induced by Prolonged Intense Loading by Increasing Osteoblast Activity in Rats.

We have an operant model of reaching and grasping in which detrimental bone remodeling is observed rather than beneficial adaptation when rats perform a high-repetition, high-force (HRHF) task long term. Here, adult female Sprague-Dawley rats performed an intense HRHF task for 18 weeks, which we have shown induces radial trabecular bone osteopenia. One cohort was euthanized at this point (to assay the bone changes post task; HRHF-Untreated). Two other cohorts were placed on 6 weeks of rest while being simultaneously treated with either an anti-CCN2 (FG-3019, 40 mg/kg body weight, ip; twice per week; HRHF-Rest/anti-CCN2), or a control IgG (HRHF-Rest/IgG), with the purpose of determining which might improve the trabecular bone decline. Results were compared with food-restricted control rats (FRC). MicroCT analysis of distal metaphysis of radii showed decreased trabecular bone volume fraction (BV/TV) and thickness in HRHF-Untreated rats compared with FRCs; responses improved with HRHF-Rest/anti-CCN2. Rest/IgG also improved trabecular thickness but not BV/TV. Histomorphometry showed that rest with either treatment improved osteoid volume and task-induced increases in osteoclasts. Only the HRHF-Rest/anti-CCN2 treatment improved osteoblast numbers, osteoid width, mineralization, and bone formation rate compared with HRHF-Untreated rats (as well as the latter three attributes compared with HRHF-Rest/IgG rats). Serum ELISA results were in support, showing increased osteocalcin and decreased CTX-1 in HRHF-Rest/anti-CCN2 rats compared with both HRHF-Untreated and HRHF-Rest/IgG rats. These results are highly encouraging for use of anti-CCN2 for therapeutic treatment of bone loss, such as that induced by chronic overuse. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Assembling the Puzzle Pieces. Insights for in Vitro Bone Remodeling.

As a highly dynamic organ, bone changes during throughout a person's life. This process is referred to as 'bone remodeling' and it involves two stages - a well-balanced osteoclastic bone resorption and an osteoblastic bone formation. Under normal physiological conditions bone remodeling is highly regulated that ensures tight coupling between bone formation and resorption, and its disruption results in a bone metabolic disorder, most commonly osteoporosis. Though osteoporosis is one of the most prevalent skeletal ailments that affect women and men aged over 40 of all races and ethnicities, currently there are few, if any safe and effective therapeutic interventions available. Developing state-of-the-art cellular systems for bone remodeling and osteoporosis can provide important insights into the cellular and molecular mechanisms involved in skeletal homeostasis and advise better therapies for patients. This review describes osteoblastogenesis and osteoclastogenesis as two vital processes for producing mature, active bone cells in the context of interactions between cells and the bone matrix. In addition, it considers current approaches in bone tissue engineering, pointing out cell sources, core factors and matrices used in scientific practice for modeling bone diseases and testing drugs. Finally, it focuses on the challenges that bone regenerative medicine is currently facing.

The regulation and functions of the matricellular CCN proteins induced by shear stress.

Shear stress is a frictional drag generated by the flow of fluid, such as blood or interstitial fluid, and plays a critical role in regulating cellular gene expression and functional phenotype. The matricellular CCN family proteins are dynamically regulated by shear stress of different flow patterns, and their expression significantly alters the microenvironment of cells. Secreted CCN proteins mainly bind to several cell surface integrin receptors to mediate their diverse functions in regulating cell survival, function, and behavior. Gene-knockout studies indicate major functions of CCN proteins in the cardiovascular and skeletal systems, the two primary systems in which CCN expressions are regulated by shear stress. In the cardiovascular system, the endothelium is directly exposed to vascular shear stress. Unidirectional laminar blood flow generates laminar shear stress, which promotes a mature endothelial phenotype and upregulates anti-inflammatory CCN3 expression. In contrast, disturbed flow generates oscillatory shear stress, which induces endothelial dysfunction through the induction of CCN1 and CCN2. Shear-induced CCN1 binds to integrin α6β1 and promotes superoxide production, NF-κB activation, and inflammatory gene expression in endothelial cells. Although the interaction between shear stress and CCN4-6 is not clear, CCN 4 exhibits a proinflammatory property and CCN5 inhibits vascular cell growth and migration. The crucial roles of CCN proteins in cardiovascular development, homeostasis, and disease are evident but not fully understood. In the skeletal system, mechanical loading on bone generates shear stress from interstitial fluid in the lacuna-canalicular system and promotes osteoblast differentiation and bone formation. CCN1 and CCN2 are induced and potentially mediate fluid shear stress mechanosensing in osteocytes. However, the exact roles of interstitial shear stress-induced CCN1 and CCN2 in bone are still not clear. In contrast to other CCN family proteins, CCN3 inhibits osteoblast differentiation, although its regulation by interstitial shear stress in osteocytes has not been reported. The induction of CCN proteins by shear stress in bone and their functions remain largely unknown and merit further investigation. This review discusses the expression and functions of CCN proteins regulated by shear stress in physiological conditions, diseases, and cell culture models. The roles between CCN family proteins can be compensatory or counteractive in tissue remodeling and homeostasis.

Sustainable Bioactive Composite of Glehnia littoralis Extracts for Osteoblast Differentiation and Bone Formation

Different bone-related diseases are mostly caused by the disruption of bone formation and bone resorption, including osteoporosis. Traditional medicinal literature has reported the possible anti-osteoporotic properties of Glehnia littoralis. However, the chemical compounds in extracts that are responsible for bone metabolism are poorly understood. The present study aimed to explore and compare the coumarin-based compounds present in G. littoralis extracts, the antioxidant activities, and the anti-osteoporotic properties of different extracts of G. littoralis (leaf and stem, fruit, whole plant, and root extracts) on bone metabolism. This study analyzed G. littoralis extract effects on the proliferation and osteoblastic differentiation of MC3T3-E1 osteoblasts. Among the different tested samples, stem extracts had the highest scopoletin (53.0 mg/g), and umbelliferone (1.60 mg/g). The significantly (p < 0.05) highest amounts of imperatorin (31.9 mg/g) and phellopterin (2.3 mg/g), were observed in fruit and whole plant extracts, respectively. Furthermore, the results confirmed alkaline phosphatase activity, collagen synthesis, mineralization, osteocalcin content, and osterix and RUNX2 expression. G. littoralis extracts at concentrations greater than 20 µg/mL had particularly adverse effects on MC3T3-E1 cell viability and proliferation. Notably, cell proliferation was significantly elevated at lower G. littoralis concentrations. Comparatively, 0.5 µg/mL stem had a higher osteocalcin content. Of the four extract types, stem showed a higher collagen synthesis effect at concentrations of 0.5–5 µg/mL. Except for fruit extracts, G. littoralis extract treatment significantly elevated osterix gene expression. All G. littoralis extracts increased RUNX2 gene expression. The results described here indicate that G. littoralis ethanolic extracts can effectively prevent osteoporosis.

The plasma levels of Dickkopf-1 elevated in patients with Juvenile Idiopathic Arthritis.

To explore the role of two major inhibitors of Wnt signal pathway, Dickkopf-1(DKK-1) and Sclerostin (SOST), in the pathogenesis of juvenile idiopathic arthritis (JIA). 88 patients with JIA, which including 49 patients with enthesitis-related arthritis (ERA), 21 oligoarthritis (oJIA) and 18 polyarthritis (pJIA), and 36 age-and sex-matched children as healthy controls (HC) were enrolled in this study. The plasma levels of DKK-1 and SOST, measured using commercially available ELISA kits, were analyzed the correlation between the levels of DKK-1/SOST and JIA, and were analyzed in 14 patients with JIA during before and after treatment. Plasma levels of DKK-1 were significantly higher in the patients with JIA than that in HC, the elevation of DKK-1 level was positively correlated with HLA-B27 positive JIA. DKK-1 levels dropped significantly in patient with JIA after treatment (P < 0.05). There was no significant change in SOST levels among different subtypes of JIA, patients with JIA during before and after treatment, and HC. It was suggested that the DKK-1 may have a certain correlation with the pathogenesis of JIA, and DKK-1 levels are more closely related to the HLA-B27 positive-ERA. The abnormally elevated levels of Dickkopf-1 (DKK-1) may be involved in the pathogenesis of juvenile idiopathic arthritis (JIA). DKK-1 levels were more closely related to the HLA-B27 positive-enthesitis-related arthritis (ERA). DKK-1 is an inhibitor of Wnt signaling pathway that promotes osteoblastic new bone formation; it is very rare for pediatric patients with HLA-B27 positive-ERA to manifest typical spondylitis, while sacroiliac arthritis is relatively common, which may be related to the high levels of DKK-1, which is consistent with the early stage of ankylosing spondylitis (AS).

Messages from the Mineral: How Bone Cells Communicate with Other Tissues.

Bone is a highly dynamic tissue, and the constant actions of bone-forming and bone-resorbing cells are responsible for attaining peak bone mass, maintaining bone mass in the adults, and the subsequent bone loss with aging and menopause, as well as skeletal complications of diseases and drug side-effects. It is now accepted that the generation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts is modulated by osteocytes, osteoblast-derived cells embedded in the bone matrix. The interaction among bone cells occurs through direct contact and via secreted molecules. In addition to the regulation of bone cell function, molecules released by these cells are also able to reach the circulation and have effects in other tissues and organs in healthy individuals. Moreover, bone cell products have also been associated with the establishment or progression of diseases, including cancer and muscle weakness. In this review, we will discuss the role of bone as an endocrine organ, and the effect of selected, osteoblast-, osteocyte-, and osteoclast-secreted molecules on other tissues.

Sympathetic Innervation Regulates Osteocyte-Mediated Cortical Bone Resorption during Lactation.

Bone undergoes constant remodeling by osteoclast bone resorption coupled with osteoblast bone formation at the bone surface. A third major cell type in the bone is osteocytes, which are embedded in the matrix, are well-connected to the lacunar network, and are believed to act as mechanical sensors. Here, it is reported that sympathetic innervation directly regulates lacunar osteocyte-mediated bone resorption inside cortical bone. It is found that sympathetic activity is elevated in different mouse models of bone loss, including lactation, ovariectomy, and glucocorticoid treatment. Further, during lactation elevated sympathetic outflow induces netrin-1 expression by osteocytes to further promote sympathetic nerve sprouting in the cortical bone endosteum in a feed-forward loop. Depletion of tyrosine hydroxylase-positive (TH+ ) sympathetic nerves ameliorated osteocyte-mediated perilacunar bone resorption in lactating mice. Moreover, norepinephrine activates β-adrenergic receptor 2 (Adrb2) signaling to promote secretion of extracellular vesicles (EVs) containing bone-degrading enzymes for perilacunar bone resorption and inhibit osteoblast differentiation. Importantly, osteocyte-specific deletion of Adrb2 or treatment with a β-blocker results in lower bone resorption in lactating mice. Together, these findings show that the sympathetic nervous system promotes osteocyte-driven bone loss during lactation, likely as an adaptive response to the increased energy and mineral demands of the nursing mother.

Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis

The bone marrow contains various populations of skeletal stem cells (SSCs) in the stromal compartment, which are important regulators of bone formation. It is well-described that leptin receptor (LepR)+ perivascular stromal cells provide a major source of bone-forming osteoblasts in adult and aged bone marrow. However, the identity of SSCs in young bone marrow and how they coordinate active bone formation remains unclear. Here we show that bone marrow endosteal SSCs are defined by fibroblast growth factor receptor 3 (Fgfr3) and osteoblast-chondrocyte transitional (OCT) identities with some characteristics of bone osteoblasts and chondrocytes. These Fgfr3-creER-marked endosteal stromal cells contribute to a stem cell fraction in young stages, which is later replaced by Lepr-cre-marked stromal cells in adult stages. Further, Fgfr3+ endosteal stromal cells give rise to aggressive osteosarcoma-like lesions upon loss of p53 tumor suppressor through unregulated self-renewal and aberrant osteogenic fates. Therefore, Fgfr3+ endosteal SSCs are abundant in young bone marrow and provide a robust source of osteoblasts, contributing to both normal and aberrant osteogenesis.

Influence of Cholesterol on the Regulation of Osteoblast Function.

Bone is a dynamic tissue composed of cells, an extracellular matrix, and mineralized portion. Osteoblasts are responsible for proper bone formation and remodeling, and function. These processes are endergonic and require cellular energy in the form of adenosine triphosphate (ATP), which is derived from various sources such as glucose, fatty acids, and amino acids. However, other lipids such as cholesterol have also been found to play a critical role in bone homeostasis and can also contribute to the overall bioenergetic capacity of osteoblasts. In addition, several epidemiological studies have found a link between elevated cholesterol, cardiovascular disease, an enhanced risk of osteoporosis, and increased bone metastasis in cancer patients. This review focuses on how cholesterol, its derivatives, and cholesterol-lowering medications (statins) regulate osteoblast function and bone formation. It also highlights the molecular mechanisms underlying the cholesterol-osteoblast crosstalk.

Serum osteocalcin – A biochemical marker for pubertal growth assessment

Objectives: Growth evaluation methods have made great strides in the shift from radiologic to non-radiologic biomarkers. Osteocalcin (OC), a bone protein, has been reportedly used as a biomarker for osteoblastic bone formation. The present study aimed at evaluation of serum OC in Class II skeletal patterns for accurate assessment of pubertal growth spurt to facilitate functional jaw orthopedics during the growth period. Material and Methods: Eighty subjects, comprising 38 males and 42 females with skeletal Class II malocclusion in the age range of 11–18 years, were recruited for the study. Human serum OC was quantitatively assessed with enzyme-linked immunosorbent assay. The cervical vertebral stages were assessed from lateral cephalograms. Statistical analysis for gender-wise comparison of mean serum levels of OC at each cervical stage (CS) and in the intervals of the CSs was carried out using Kruskal–Wallis test and for intergroup comparisons, Mann–Whitney U-test with Bonferroni’s correction was done. Results: Gender-wise comparison of mean serum OC levels revealed that it was highest in CS2 in both males (72.24 ng/mL) and females (74.71 ng/mL) with another discernible peak in CS5 in males (66.82 ng/mL) and in CS6 in females (63.78 ng/mL), exhibiting thereby a circadian rhythm in bone modeling during the entire adolescent growth spurt. Conclusion: Despite a pre-pubertal and a late pubertal spike in both the genders, the mean OC serum levels actually exhibited a circadian rhythmicity across all the CSs, exhorting thereby the importance of bone remodeling during the complete circumpubertal growth period.

Persicae Semen Promotes Bone Union in Rat Fractures by Stimulating Osteoblastogenesis through BMP-2 and Wnt Signaling

Fractures cause extreme pain to patients and impair movement, thereby significantly reducing their quality of life. However, in fracture patients, movement of the fracture site is restricted through application of a cast, and they are reliant on conservative treatment through calcium intake. Persicae semen (PS) is the dried mature seeds of Prunus persica (L.) Batsch, and in this study the effects of PS on osteoblast differentiation and bone union promotion were investigated. The osteoblast-differentiation-promoting effect of PS was investigated through alizarin red S and Von Kossa staining, and the regulatory role of PS on BMP-2 (Bmp2) and Wnt (Wnt10b) signaling, representing a key mechanism, was demonstrated at the protein and mRNA levels. In addition, the bone-union-promoting effect of PS was investigated in rats with fractured femurs. The results of the cell experiments showed that PS promotes mineralization and upregulates RUNX2 through BMP-2 and Wnt signaling. PS induced the expression of various osteoblast genes, including Alpl, Bglap, and Ibsp. The results of animal experiments show that the PS group had improved bone union and upregulated expression of osteogenic genes. Overall, the results of this study suggest that PS can promote fracture recovery by upregulating osteoblast differentiation and bone formation, and thus can be considered a new therapeutic alternative for fracture patients.

INTEGRIN A2B1 DEFICIENCY REVEALS FASTER REGENERATION THROUGH BMP PATHWAY ACTIVATION IN A MURINE FRACTURE HEALING MODEL

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms.Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2-/-). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR.Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones.Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1’s E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/β receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Abstract 82: Role of MSC-derived CXCL1 in bone-metastatic prostate cancer

Abstract In 2022, the American Cancer Society estimates that over 34,000 men will die of PCa, largely due to metastatic PCa. Up to 90% of advanced prostate cancers metastasize to bone, and there are currently no curative therapies for bone metastatic prostate cancer (BM-PCa). BM-PCa cells interact with bone-forming osteoblasts and bone-resorbing osteoclasts to induce aberrant bone remodeling, which leads to skeletal related adverse events such as increased risk of fractures, bone pain, and poor quality of life. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into osteoblasts. Cook lab data show BM-PCa cells interact with MSCs and promote MSC differentiation into osteoblasts. Microarray analysis of MSC gene expression changes in response to BM-PCa show significant upregulation of CXCL8. To determine the importance of MSC-derived CXCL8 to BM-PCa, CXCL1 (the murine homolog of CXCL8) was genetically deleted in murine MSCs. While CXCL1 knock out (KO) had no significant impact on MSC proliferation, loss of CXCL1 decreased MSC migration, which suggests upregulation of MSC CXCL1 by BM-PCa cells functions to increase MSC migration into the tumor microenvironment. Additionally, CXCL1 KO MSCs have altered osteoblastic differentiation, whereas adipogenic differentiation was unchanged. RNA sequencing of MSCs stably expressing CXCL1-targeted shRNAs compared to scrambled controls show extensive gene expression changes, including altered expression of multiple osteogenic genes. In vivo data show intratibial co-injection of CXCL1 KO MSCs with murine RM1 BM-PCa cells in an immunocompetent mouse model increases tumor burden compared with co-injection of wildtype MSCs and BM-PCa cells, indicating a role for MSC-derived CXCL1 in suppressing BM-PCa tumor growth in bone. This project shows for the first time the importance of CXCL1 in MSC migration, osteogenesis, gene expression, suppression of BM-PCa progression in bone and tumor-induced bone remodeling. Citation Format: Catherine S. Johnson, Diane Costanzo-Garvey, Massar Alsamraae, Jeremy S. Frieling, Leah M. Cook. Role of MSC-derived CXCL1 in bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 82.

Abstract 1189: MERTK is a target in the microenviroenment of osteolytic bone metastasis

Abstract Metastatic bone disease often induces osteolytic lesions caused by an imbalance of osteoblastic bone formation and osteoclastic bone resorption. We could previously show that TAM family receptor tyrosine kinase MERTK exerts an oncogenic function in multiple myeloma, NSCLC and breast cancer bone metastasis, providing a rationale for using MERTK inhibitors in bone metastasis therapy. Furthermore, we reported a thus far unrecognized role of MERTK as a novel negative regulator of osteoblast function and showed that MERTK represents an osteoanabolic target in the tumor microenvironment of bone metastasis by targeting osteoblasts. It is described that MERTK exerts a prominent role in phagocytosis of macrophages. Nevertheless, the role of MERTK in osteoclasts is unknown. To assess a potential role of MERTK in physiological bone remodeling by osteoclasts in vivo, we performed microcomputed tomography (μCT) of tibia of LysM-cre+;Mertkflox/flox mice and observed increased bone volume. Histomorphometry revealed reduced osteoclast number in LysM-cre+;Mertkflox/flox mice, indicating that increased bone volume induced by loss of MERTK in the myeloid lineage is mediated by decreased osteoclast formation in vivo. Interestingly, analysis of TRAP+ mononuclear cells in the metaphyseal bone marrow showed decreased numbers and increased distance to closest bone surface, suggesting dysfunctional osteoclast precursor cell differentiation and migration in LysM-cre+;Mertkflox/flox mice. In vitro assays demonstrated that loss of Mertk results in decreased osteoclast precursor cell migration towards its ligand PROS1 and decreased osteoclast differentiation through a RHOA dependent mechanism. To elucidate if MERTK represents a target in the tumor microenvironment of osteolytic bone metastasis we utilized syngeneic EO771 breast cancer bone metastasis model and injected luciferase transduced EO771 cells into Mertkflox/flox and LysM-cre+;Mertkflox/flox C57BL/6J mice. Metastatic spread was monitored by bioluminescence imaging. Analysis of bone metastasis by μCT showed higher bone volume in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, osteolysis zones in the cortical bone were markedly decreased. Histomorphometry of TRAP/Hematoxylin staining indicated decreased osteoclast numbers in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, intratumoral TRAP+ mononuclear cells in breast cancer bone metastases were decreased, indicating that osteolytic bone metastasis recruit osteoclast precursors from its microenvironment in the bone marrow via MERTK to induce osteolysis. In summary, tumor-induced activation of MERTK suppresses and transforms osteoblasts, which counteracts bone formation and recruits osteoclast precursors to form osteoclasts and mediate osteolysis. Hence, MERTK is a novel target in the tumor microenvironment to inhibit osteolytic bone metastasis. Citation Format: Janik Engelmann, Jennifer Zarrer, Kristoffer Riecken, Jonas Waizenegger, Maria Elena Vargas-Delgado, Lara Meier, Carsten Bokemeyer, Klaus Pantel, Emily Alberto, Sourav Ghosh, Carla Rothlin, Eric Hesse, Hanna Taipaleenmäki, Isabel Ben-Batalla, Sonja Loges. MERTK is a target in the microenviroenment of osteolytic bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1189.

Abstract 553: SKP2 knockout induces macrophage infiltration in p53/Rb1 null transgenic mouse models of osteosarcoma and drives gene expression correlated with improved survival in patients

Abstract Background: Osteosarcoma (OS) is an aggressive cancer of the bone with a poor prognosis. OS often involve mutations of tumor suppressors RB1 and TP53. One putative proto-oncogene in OS is SKP2, which codes for a substrate-recognition factor of the SCF E3 ubiquitin ligase. We have demonstrated that germline knockout of SKP2 in a pre-clinical transgenic mouse model of OS doubly deficient in Rb1 and Trp53 improved survival, delayed tumorigenesis, reduced malignant cell proliferation, slowed tumor growth, diminished stemness, and induced apoptosis. However, the role that SKP2 plays in OS progression remains unclear. Method: We performed bulk RNA-sequencing on pre-clinical transgenic mouse models of OS. To model OS in mice, we generated a conditional double-knockout line of Trp53 and Rb1 mutations induced in cells of the bone-forming osteoblast lineage via Cre-Lox recombination (“DKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox). We compared this with a triple-knockout model, which additionally features a germline Skp2 knockout (“TKO”: Osx1-Cre;Rb1lox/lox;p53lox/lox;SKP2-/-). We compared tumors from the TKO and DKO models via bulk RNA-sequencing (n=3 per group) to study differential expression and immune infiltration. We used immunohistochemistry (IHC) and quantification to validate differences in immune markers between genotypes. Using the transcriptomic results from the mouse models, we analyzed a human OS patient cohort of 83 participants (NCI TARGET OS) with RNA-seq and clinical data via survival analysis to infer the impact of modulating SKP2 in OS patients. Result: We found a large difference in gene expression between the TKO and DKO models. Surprisingly, we observed a dramatic increase in the expression of genes related to immune microenvironment infiltration in TKO tumors compared to DKO. Macrophage transcriptomic signatures were the strongest signal detected (P&amp;lt;0.001), but significant increases in B cells, endothelial cells and T cell signatures were also observed. IHC also showed macrophage markers were higher in TKO (P = 0.08). In the NCI TARGET OS cohort, high expression of genes up-regulated in TKO was significantly correlated with 5-year overall survival (p=0.02). This effect was driven by macrophage transcriptomic signatures. This finding was supported by an immune microenvironment deconvolution analysis, which showed that macrophages and T cells were associated with improved overall survival in the patient cohort (p=0.01). We are currently performing single-cell RNA-sequencing analysis to study immune infiltration and cellular heterogeneity in more detail. Conclusion: Taken together, our new findings indicate that SKP2 modulation in OS may induce a vigorous anti-tumor immune activation especially in the form of macrophage and lymphocyte infiltration into the tumor microenvironment. Citation Format: Alexander Ferrena, Jichuan Wang, Ranxin Zhang, Rui Yang, David Geller, Bang Hoang, Deyou Zheng. SKP2 knockout induces macrophage infiltration in p53/Rb1 null transgenic mouse models of osteosarcoma and drives gene expression correlated with improved survival in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 553.