Dipeptidyl peptidase-4 disturbs adipocyte differentiation via the negative regulation of the glucagon-like peptide-1/adiponectin-cathepsin K axis in mice under chronic stress conditions.

Exposure to chronic psychosocial stress is a risk factor for metabolic disorders. Because dipeptidyl peptidase-4 (DPP4) and cysteinyl cathepsin K (CTSK) play important roles in human pathobiology, we investigated the role(s) of DPP4 in stress-related adipocyte differentiation, with a focus on the glucagon-like peptide-1 (GLP-1)/adiponectin-CTSK axis in vivo and in vitro. Plasma and inguinal adipose tissue from non-stress wild-type (DPP4+/+), DPP4-knockout (DPP4-/-) and CTSK-knockout (CTSK-/-) mice, and stressed DPP4+/+, DPP4-/-, CTSK-/-, and DPP4+/+ mice underwent stress exposure plus GLP-1 receptor agonist exenatide loading for 2 weeks and then were analyzed for stress-related biological and/or morphological alterations. On day 14 under chronic stress, stress decreased the weights of adipose tissue and resulted in harmful changes in the plasma levels of DPP4, GLP-1, CTSK, adiponectin, and tumor necrosis factor-α proteins and the adipose tissue levels of CTSK, preadipocyte factor-1, fatty acid binding protein-4, CCAAT/enhancer binding protein-α, GLP-1 receptor, peroxisome proliferator-activated receptor-γ, perilipin2, secreted frizzled-related protein-4, Wnt5α, Wnt11 and ß-catenin proteins and/or mRNAs as well as macrophage infiltration in adipose tissue; these changes were rectified by DPP4 deletion. GLP-1 receptor activation and CTSK deletion mimic the adipose benefits of DPP4 deficiency. In vitro, CTSK silencing and overexpression respectively prevented and facilitated stress serum and oxidative stress-induced adipocyte differentiation accompanied with changes in the levels of pref-1, C/EBP-α, and PPAR-γ in 3T3-L1 cells. Thus, these findings indicated that increased DPP4 plays an essential role in stress-related adipocyte differentiation, possibly through a negative regulation of GLP-1/adiponectin-CTSK axis activation in mice under chronic stress conditions.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Craniofacial dysmorphism of osteogenesis imperfecta mouse and effect of cathepsin K knockout: Preliminary craniometry observations.

OBJECTIVES: In addition to bone fragility, patients with osteogenesis imperfecta (OI) type III have typical craniofacial abnormalities, such as a triangular face and maxillary micrognathism. However, in the osteogenesis imperfecta mouse (oim), a validated model of OI type III, few descriptions exist of craniofacial phenotype. Treatment of OI mostly consists of bisphosphonate administration. Cathepsin K inhibition has been tested as a promising therapeutic approach for osteoporosis and positive results were observed in long bones of cathepsin K knocked out oim (oim/CatK-/-). This craniometry study aimed to highlight the craniofacial characteristics of oim and Cathepsin K KO mouse. MATERIALS AND METHODS: We analyzed the craniofacial skeleton of 51 mice distributed in 4 genotype groups: Wt (control), oim, CatK-/-, oim/CatK-/-. The mice were euthanized at 13 weeks and their heads were analyzed using densitometric (pQCT), X-ray cephalometric, and histomorphometric methods. RESULTS: The craniofacial skeleton of the oim mouse is frailer than the Wt one, with a reduced thickness and mineral density of the cranial vault and mandibular ramus. Different cephalometric data attest a dysmorphism similar to the one observed in humans with OI type III. Those abnormalities were not improved in the oim/CatK-/- group. CONCLUSION: These results suggest that oim mouse could serve as a complete model of the human OI type III, including the craniofacial skeleton. They also suggest that invalidation of cathepsin K has no impact on the craniofacial abnormalities of the oim model.

Treatment with an inhibitor of matrix metalloproteinase 9 or cathepsin K lengthens embryonic lower jaw bone.

OBJECTIVES: Skeletal malocclusions are common, and severe malocclusions are treated by invasive surgeries. Recently, jaw bone length has been shown to be developmentally controlled by osteoclasts. Our objective was to determine the effect of inhibiting osteoclast-secreted proteolytic enzymes on lower jaw bone length of avian embryos by pharmacologically inhibiting matrix metalloproteinase-9 (MMP9) or cathepsin K (CTSK). METHODS: Quail (Coturnix coturnix japonica) embryos were given a single dose of an inhibitor of MMP9 (iMMP9), an inhibitor CTSK (iCTSK), or vehicle at a developmental stage when bone deposition is beginning to occur. At a developmental stage when the viscerocranium is largely calcified, the heads were scanned via micro-computed tomography and reproducible landmarks were placed on 3D-reconstructed skulls; the landmark coordinates were used to quantify facial bone dimensions. RESULTS: Approximately half of the quail given either iMMP9 or iCTSK demonstrated an overt lower jaw phenotype, characterized by longer lower jaw bones and a greater lower to upper jaw ratio than control embryos. Additionally, iMMP9-treated embryos exhibited a significant change in midface length and iCTSK-treated embryos had significant change in nasal bone length. CONCLUSION: MMP9 and CTSK play a role in osteoclast-mediated determination of lower jaw bone length. Pharmacological inhibition of MMP9 or CTSK may be a promising therapeutic alternative to surgery for treating skeletal jaw malocclusions, but more preclinical research is needed prior to clinical translation.

A SNP in the ovine cathepsin K (CTSK) gene is associated with yearling growth performance in a crossbred sheep population.

Cathepsin K (CTSK) is a lysosomal protease existent in the skeletal muscles which is involved in biochemical processes related to obesity. Several studies have reported the effects of CTSK gene on body weight and fat deposition in human, mice and pigs. However, information about its structure and functions in sheep is very limited. Thus, this study was performed to evaluate the association between CTSK gene variants and yearling growth performance in Afshari × Booroola-Merino crossbred sheep. A fragment of 500 bp in exon 6 and partial of intron 5 of CTSK gene was amplified with polymerase chain reaction (PCR). All animals were genotyped by single-stranded conformation polymorphism (SSCP) and further confirmed by sequencing. Association analysis using a fixed linear model indicated that g.106510225G > A SNP was significantly related to average daily weight gain (ADWG) per year, fat-tail weight to carcass weight ratio (FW/CW), muscle thickness (MT) and muscle cross-sectional area (MCSA) of animals (p < 0.05). Due to the low polymorphic information content (PIC <0.25) for targeted locus in studied population, more association studies are needed to confirm the CTSK gene effects on growth traits in sheep.

Dopamine D1 receptor stimulates cathepsin K-dependent degradation and resorption of collagen I in lung fibroblasts.

Matrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution.This article has an associated First Person interview with the first author of the paper.

Ectodysplasin A1 Deficiency Leads to Osteopetrosis-like Changes in Bones of the Skull Associated with Diminished Osteoclastic Activity.

Pathogenic variants of the gene Eda cause X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by structural abnormalities or lack of ectodermal appendages. Signs of dysplasia are not restricted to derivatives of the ectodermal layer, but mesodermal abnormalities, such as craniofacial dysmorphism, are also frequently observed, suggesting close reciprocal interactions between the ectoderm and mesoderm; however, a causal link has remained unsubstantiated. We investigated the functional impact of defective ectodysplasin A1 (Eda1) signaling on postnatal bone homeostasis in Eda1-deficient Tabby mice. Interestingly, Eda1 was detected in wild-type mouse calvariae throughout postnatal lifetime. In calvariae, bone-lining Osterix (Osx)+ osteoblasts stained positive for Eda1, and osteoclasts were revealed as Eda receptor (Edar)-positive. Moreover, adult Eda1-deficient calvarial bone showed osteopetrosis-like changes with significantly diminished marrow space, which was maintained during adulthood. Concomitantly with osteopetrosis-like changes, Tabby calvarial bone and Tabby bone marrow-derived osteoclasts had far less osteoclastic activity-associated co-enzymes including cathepsin K, Mmp9, Trap, and Tcirg1 (V-type proton ATPase a3 subunit) compared with wild-type calvariae in vivo or osteoclasts in vitro, indicating that Eda1 deficiency may affect the activity of osteoclasts. Finally, we confirmed that nuclear Nfatc1-positive osteoclasts were strongly diminished during mature osteoclastic differentiation under M-CSF and RANKL in the Tabby model, while Fc-EDA treatment of Tabby-derived osteoclasts significantly increased nuclear translocation of Nfatc1. Furthermore, we identified enhanced Nfatc1 and NF-κB transcriptional activity following Fc-EDA treatment in vitro using luciferase assays. Overall, the results indicate that diminished expressions of osteoclastic activity-associated co-enzymes may lead to disturbed bone homeostasis in Tabby calvariae postnatally.

[Icariin inhibits thioacetamide-induced osteoclast differentiation through RANKL-p38/ERK-NFAT pathway].

This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.

Micro/nano topography of selective laser melting titanium inhibits osteoclastogenesis via mediation of macrophage polarization.

Selective laser melting (SLM) titanium (Ti) implants have shown good prospects for personalized clinical application, but further research is necessary to develop stabilized long-term properties. Since surface modification has been proven bioactive for osseointegration, conventional Ti surface treatment technologies, including sandblasting/acid-etching (SLA) and sandblasting/alkali-heating (SAH), were applied to construct micro and micro/nano surfaces. The SAH group with netlike nano-structure topography exhibited appropriate surface roughness and high hydrophilicity, and as expected, the osseointegration capacities in vivo of the three groups were in order of SAH > SLA > SLM. Besides, both in vivo and in vitro studies revealed that the SLA- and SAH-treated SLM Ti implants significantly inhibited osteoclast activity of peri-implants. Considering the close associations between osteoclasts and macrophages, the effects of Ti surface topography on macrophage polarization were detected. The results showed that the SLA- and SAH-treated SLM Ti implants, especially the latter, had the capacity to promote macrophage polarization to the M2 phenotype. Moreover, the cell culture supernatants of M2 macrophages and RAW264.7 cells seeded on SLA- and SAH-treated SLM Ti surfaces had an adverse effect on osteoclastogenesis. Collectively, this study demonstrated that micro/nano topographies of SLM Ti implants were effective for osseointegration promotion, and their inhibition of osteoclastogenesis might be attributed to macrophage polarization. Our findings shed some light on clinical application of SLM Ti implants and also prove a specific association between macrophage polarization and osteoclastogenesis.

Myelofibrosis osteoclasts are clonal and functionally impaired.

Bone marrow (BM) sclerosis is commonly found in patients with late-stage myelofibrosis (MF). Because osteoclasts (OCs) and osteoblasts play a key role in bone remodeling, and MF monocytes, the OC precursors, are derived from the neoplastic clone, we wondered whether decreased OC numbers or impairment in their osteolytic function affects the development of osteosclerosis. Analysis of BM biopsies from 50 MF patients showed increased numbers of multinucleated tartrate-resistant acid phosphatase (TRAP)/cathepsin K+ OCs expressing phosphorylated Janus kinase 2 (JAK2). Randomly microdissected TRAP+ OCs from 16 MF patients harbored JAK2 or calreticulin (CALR) mutations, confirming MF OCs are clonal. To study OC function, CD14+ monocytes from MF patients and healthy individuals were cultured and differentiated into OCs. Unlike normal OCs, MF OCs appeared small and round, with few protrusions, and carried the mutations and chromosomal abnormalities of neoplastic clones. In addition, MF OCs lacked F-actin-rich ring-like structures and had fewer nuclei and reduced colocalization signals, compatible with decreased fusion events, and their mineral resorption capacity was significantly reduced, indicating impaired osteolytic function. Taken together, our data suggest that, although the numbers of MF OCs are increased, their impaired osteolytic activity distorts bone remodeling and contributes to the induction of osteosclerosis.

The genotypic and phenotypic spectrum of pycnodysostosis in Saudi Arabia: Novel variants and clinical findings.

Pycnodysostosis is characterized by short stature, osteosclerosis, acro-osteolysis, increased tendency of fractures, and distinctive dysmorphic features. It is a rare autosomal recessive disease caused by biallelic CTSK mutations. The clinical details of 18 patients from Saudi Arabia were reviewed. Short stature, osteopetrosis, acro-osteolysis, and distinctive facial dysmorphism were documented in all cases. Our results highlight the significant complications associated with this disease. The large anterior fontanelle is one of the cardinal signs of this disease; however, half of our patients had small fontanelles and a quarter had craniosynostosis, which caused optic nerve compression. Sleep apnea was of the major complications in three patients. Bone fracture can be a presenting symptom, and in our patients it mainly occurred after the age of 3 years. Bone marrow suppression was seen in a single patient of our cohort who was misdiagnosed initially with malignant osteopetrosis. In this study, we also describe two novel (c.5G > A [p.Trp2Ter], c.538G > A [p.Gly180Ser]) and two reported (c.244-29 A > G, c.830C > T [p.Ala277Val]) CTSK mutations. Our results indicate that the recurrent intronic variant, c.244-29 A > G is likely to be a founder mutation, as it was found in 78% (14/18 patients) of our cohort belonging to the same tribe.

Urolithin A suppresses RANKL-induced osteoclastogenesis and postmenopausal osteoporosis by, suppresses inflammation and downstream NF-κB activated pyroptosis pathways.

Osteoporosis (OP) is characterized by decreased trabecular bone volume and microarchitectural deterioration in the medullary cavity. Urolithin A (UA) is a biologically active metabolite generated by the gut microbiota. UA is the measurable product considered the most relevant urolithin as the final metabolic product of polyphenolic compounds. Considering that catabolic effects mediated by the intestinal microbiota are highly involved in pathological bone disorders, exploring the biological influence and molecular mechanisms by which UA alleviates OP is crucial. Our study aimed to investigate the effect of UA administration on OP progression in the context of estrogen deficiency-induced bone loss. The in vivo results indicated that UA effectively reduced ovariectomy-induced systemic bone loss. In vitro, UA suppressed Receptor Activator for Nuclear Factor-κB Ligand (RANKL)-triggered osteoclastogenesis in a concentration-dependent manner. Signal transduction studies and sequencing analysis showed that UA significantly decreased the expression of inflammatory cytokines (e.g., IL-6 and TNF-α) in osteoclasts. Additionally, attenuation of inflammatory signaling cascades inhibited the NF-κB-activated NOD-like receptor signaling pathway, which eventually led to decreased cytoplasmic secretion of IL-1ß and IL-18 and reduced expression of pyroptosis markers (NLRP3, GSDMD, and caspase-1). Consistent with this finding, an NLRP3 inflammasome inhibitor (MCC950) was employed to treat OP, and modulation of pyroptosis was found to ameliorate osteoclastogenesis and bone loss in ovariectomized (OVX) mice, suggesting that UA suppressed osteoclast formation by regulating the inflammatory signal-dependent pyroptosis pathway. Conceivably, UA administration may be a safe and promising therapeutic strategy for osteoclast-related bone diseases such as OP.

Inhibitory effect of cathepsin K inhibitor (ODN-MK-0822) on invasion, migration and adhesion of human breast cancer cells in vitro.

Approximately 90% of patients with advanced breast cancer develop bone metastases; an event that results in severe decrease of quality of life and a drastic deterioration in prognosis. Therefore, to increase the survival of breast cancer patients, the development of new therapeutic strategies to impair metastatic process and skeletal complications is critical. Previous studies on the role of cathepsin K (CTSK) in metastatic spreading led to several strategies for inhibition of this molecule such as MIV-711 (Medivir), balicatib and odanacatib (ODN) which were on trial in the past. The present study intended to assess the anti-metastatic efficacy of ODN in breast cancer cells. Human breast cancer cell lines MDA-MB-231 were treated with different concentrations of ODN and performed invasion, adhesion and migration assays and, RT-PCR and western blot to evaluate the effect of ODN on the metastatic potential of breast cancer cells. ODN markedly decreased wound healing cell migration, invasion and adhesion at a dose dependent manner. ODN inhibits cell invasion by decreasing the matrix metalloproteinase (MMP-9) with the upregulation of TIMP-1 expression. ODN effectively inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal Kinase (JNK), and blocked the expression of ß-integrins and FAK proteins. ODN also significantly inhibited PI3K downstream targets Rac1, Cdc42, paxillin and Src which are critical for cell adhesion, migration and cytoskeletal reorganization. ODN exerts anti-metastatic action through inhibition of signaling pathway for MMP-9, PI3K and MAPK. This indicates potential therapeutic effects of ODN in the treatment of metastatic breast cancer.

The Amino Acid Transporter Mct10/Tat1 Is Important to Maintain the TSH Receptor at Its Canonical Basolateral Localization and Assures Regular Turnover of Thyroid Follicle Cells in Male Mice.

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gαq-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gαs-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk-/-/Mct8-/y/Mct10-/- mice, which implies prolonged Gαs-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk-/- and Mct8-/y mice, whereas its localization is restricted to vesicles in Mct10-/- thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk-/-/Mct10-/- mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10-/- mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8-/y, Mct8-/y/Mct10-/-, and Ctsk-/-/Mct8-/y/Mct10-/- mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Kalkitoxin Reduces Osteoclast Formation and Resorption and Protects against Inflammatory Bone Loss.

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.

Isoflavone-enriched soybean leaves attenuate ovariectomy-induced osteoporosis in rats by anti-inflammatory activity.

BACKGROUND: With an increasing aging population, postmenopausal osteoporosis has become a global public health problem. Previous evidence has shown that postmenopausal osteoporosis is a skeletal disease mainly caused by estrogen deficiency, generally accompanied by inflammation, and dietary isoflavones may ameliorate postmenopausal osteoporosis by anti-inflammatory activity. We have generated isoflavone-enriched soybean leaves (IESLs), but their anti-inflammatory activity and effect on attenuating osteoporosis are still obscure. Here, we determined the isoflavone profiles of IESLs and evaluated their anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 cells and anti-osteoporotic effects on ovariectomy-induced osteoporosis in rats. RESULTS: IESLs had a high content of total isoflavone. Hydrolysate of IESLs (HIESLs) was rich with the aglycones daidzein and genistein, and HIESLs can significantly inhibit lipopolysaccharide-induced inflammation by reducing messenger RNA expression of iNOS, COX-2, IL6, and IL1ß. Moreover, ovariectomized rats receiving aqueous extracts of IESLs (HIESLs) orally maintained more bone mass than control rats did, which was attributed to inhibition of osteoclastogenesis by downregulating the messenger RNA expression of the bone-specific genes RANKL/OPG, OC, and cathepsin K, and the inflammation-related genes IL6, NFκB, and COX-2. CONCLUSION: IESLs may attenuate postmenopausal osteoporosis by suppressing osteoclastogenesis with anti-inflammatory activity and be a potential source of functional food ingredients for the prevention of osteoporosis. © 2020 Society of Chemical Industry.

Function of Cathepsin K in the Central Nervous System of Male Mice is Independent of Its Role in the Thyroid Gland.

Cathepsin K deficiency in male mice (Ctsk-/-) results in decreased numbers of hippocampal astrocytes and altered neuronal patterning as well as learning and memory deficits. Additionally, cathepsin K carries essential roles in the thyroid gland where it contributes to the liberation of thyroid hormones (TH). Because TH are essential for brain development, in particular for the cerebellum, we investigated whether cathepsin K's function in the thyroid is directly linked to the brain phenotype of Ctsk-/- mice. Serum levels of thyroid stimulating hormone, brain concentrations of free TH, and deiodinase 2 (Dio2) activity in brain parenchyma as well as cerebellar development were comparable in Ctsk-/- and WT animals, suggesting regular thyroid states and TH metabolism. Despite unaltered transcript levels, protein expression of two TH transporters was enhanced in specific brain regions in Ctsk-/- mice, suggesting altered TH supply to these regions. Thyrotropin releasing hormone (Trh) mRNA levels were enhanced threefold in the hippocampus of Ctsk-/- mice. In the striatum of Ctsk-/- mice the mRNA for Dio2 and hairless were approximately 1.3-fold enhanced, while mRNA levels for monocarboxylate transporter 8 and Trh were reduced to 60% and 40%, respectively, pointing to altered striatal physiology. We conclude that the role of cathepsin K in the thyroid gland is not directly associated with its function in the central nervous system (CNS) of mice. Future studies will show whether the brain region-specific alterations in Trh mRNA may eventually result in altered neuroprotection that could explain the neurobehavioral defects of Ctsk-/- mice.

A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1.

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.

Synovial Fluid Interleukin-16 Contributes to Osteoclast Activation and Bone Loss through the JNK/NFATc1 Signaling Cascade in Patients with Periprosthetic Joint Infection.

Because of lipopolysaccharide (LPS)-mediated effects on osteoclast differentiation and bone loss, periprosthetic joint infection (PJI) caused by Gram-negative bacteria increases the risk of aseptic loosening after reimplantation. Synovial fluid interleukin-16 (IL-16) expression was higher in patients with PJI than in patients without joint infection. Thus, we explored the effects of IL-16 on bone. We investigated whether IL-16 modulates osteoclast or osteoblast differentiation in vitro. An LPS-induced bone loss mice model was used to explore the possible advantages of IL-16 inhibition for the prevention of bone loss. IL-16 directly activated p38 and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling and increased osteoclast activation markers, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and nuclear factor of activated T cells 1 (NFATc1). IL-16 directly caused monocytes to differentiate into TRAP-positive osteoclast-like cells through NFATc1 activation dependent on JNK/MAPK signaling. Moreover, IL-16 did not alter alkaline phosphatase activity or calcium deposition during osteoblastic differentiation. Finally, IL-16 inhibition prevented LPS-induced trabecular bone loss and osteoclast activation in vivo. IL-16 directly increased osteoclast activation through the JNK/NFATc1 pathway. IL-16 inhibition could represent a new strategy for treating infection-associated bone loss.