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As a result of an unhealthy diet and limited physical activity, obesity has become a widespread pandemic worldwide and is an important predictor for the development of cardiovascular disease. Obesity is often characterized by a pro-inflammatory environment in white adipose tissue (WAT), mainly due to increased macrophage infiltration. These immune cells boost their lipid concentrations by accumulating the content of dying adipocytes. As the lysosome is highly involved in lipid handling, the progressive lipid accumulation may result in lysosomal stress and a metabolic shift. Recent studies have identified glycoprotein non-metastatic melanoma protein B (GPNMB) as a novel marker of inflammatory diseases. GPNMB is a type I transmembrane protein on the cell surface of various cell types, such as macrophages, dendritic cells, osteoblasts, and microglia, from which it can be proteolytically cleaved into a soluble molecule. It is induced by lysosomal stress via microphthalmia-associated transcription factor and thus has been found to be upregulated in many lysosomal storage disorders. In addition, a clear connection between GPNMB and obesity was recently established. GPNMB was shown to have protective and anti-inflammatory effects in most cases, preventing the progression of obesity-related metabolic disorders. In contrast, soluble GPNMB likely has the opposite effect and promotes lipogenesis in WAT. This review aims to summarize and clarify the role of GPNMB in the progression of obesity and to highlight its potential use as a biomarker for lipid-associated disorders. [ABSTRACT FROM AUTHOR]

The study examines the dynamics of a regulatory protein called osteoactivin in the blood serum of patients with osteoporosis and osteoporotic fractures. Patients applying to the Traumatology Department of the Azerbaijan Scientific Research Institute of Traumatology and Orthopedics were included in the research material. Patients in this study had osteoporosis diagnosis in anamnesis and frequent osteoporotic fractures, as well as signs of osteoporosis and related complaints. The diagnosis was confirmed by densitometry and X-ray. 68 people from 38 to 83 years of age who meet the admission criteria were selected and divided into 4 groups: Group I – control group – 14 practically healthy people, Group II – 14 patients with osteoporosis, Group III – 15 patients with non-osteoporotic fractures, Group IV – 25 patients with osteoporotic fractures. Patients were measured for serum osteoactivin 3 times: on the first day, 10 days later, and 1 month later. Results. According to the results, in the first and subsequent 10 days in 13 out of 14 patients with osteoporosis, there was an increase in concentration of GPNMB, while remaining one patient witnessed a decline in this indicator. After 1 month, 12 out of 14 patients showed an increase relative to initial levels. In all patients with fractures not associated with osteoporosis, the content of GPNMB in the first and in 10 days increased by a statistically significant value. At the same time, comparison of GPNMB concentrations in blood serum in the first day and after 1 month yielded a similar result. Nevertheless, there was no statistically significant difference in the dynamics of osteoactivin in patients with osteoporotic fractures (p>0,001). Following the dynamics for 1 month, it is impossible to think about the effectiveness of treatment during the recovery period. This requires more long-term and extensive research to monitor the dynamics of GPNBM during the full recovery of osteoporotic fractures.

Atherosclerosis has become prevalent not only in Western industrialized countries but all over the globe. Gpnmb, a transmembrane protein expressed by macrophages, has been detected in aortic lesions. We created an ApoE/Gpnmb-double knockout mouse using Crispr-Cas9 to examine the effect of Gpnmb deficiency on the development of atherosclerotic plaques. Feeding female mice a high cholesterol diet for 8 and 12 weeks, we detected an increased plaque size in aortic root sections of Gpnmb-deficient compared to control mice. However, the plaque area in whole thoracic and abdominal aorta was not different. Despite its strong expression in macrophages in aortic plaques, Gpnmb exerts only a minor effect on the growth of the atherosclerotic plaques in female mice. Future studies should examine plaque stability and include both sexes to elucidate the sex-specific function of Gpnmb in atherosclerosis. • Gpnmb is strongly expressed by macrophages of atherosclerotic lesions. • Gpnmb decreased size and macrophage content of atherosclerotic lesions. • Gpnmb decreased macrophage content in adipose tissue under high-cholesterol diet. [ABSTRACT FROM AUTHOR]

The musculoskeletal system consisting of bones and muscles have been recognized as endocrine organs secreting hormones that are involved in regulating metabolic and inflammatory pathways. Obesity and type 2 diabetes (T2D) are associated with several musculoskeletal system complications. We hypothesized that an interaction exists between adipomyokines namely, irisin and METRNL, and various molecules involved in bone remodeling in individuals with obesity and T2D. A total of 228 individuals were enrolled in this study, including 124 non-diabetic (ND) and 104 T2D. A Multiplex assay was used to assess the level of various osteogenic molecules namely osteoactivin, Syndecan, osteoprotegerin (OPG) and osteonectin/SPARC. Our data shows elevated levels of Osteoactivin, Syndecan, OPG and SPARC in T2D as compared to ND individuals (p ≤ 0.05). Using Spearman's correlation, a positive correlation was observed between irisin and Osteoactivin as well as OPG (p < 0.05). Similarly, a positive association was observed between METRNL and Osteoactivin (p < 0.05). The strong positive association shown in this study between irisin, METRNL and various molecules with osteogenic properties emphasize a possible interaction between these organs. This report suggests that having a dysregulation in the level of the aforementioned molecules could potentially affect the development of bone and muscle related complications that are associated with obesity and T2D. [ABSTRACT FROM AUTHOR]

The musculoskeletal system consisting of bones and muscles have been recognized as endocrine organs secreting hormones that are involved in regulating metabolic and inflammatory pathways. Obesity and type 2 diabetes (T2D) are associated with several musculoskeletal system complications. We hypothesized that an interaction exists between adipomyokines namely, irisin and METRNL, and various molecules involved in bone remodeling in individuals with obesity and T2D. A total of 228 individuals were enrolled in this study, including 124 non-diabetic (ND) and 104 T2D. A Multiplex assay was used to assess the level of various osteogenic molecules namely osteoactivin, Syndecan, osteoprotegerin (OPG) and osteonectin/SPARC. Our data shows elevated levels of Osteoactivin, Syndecan, OPG and SPARC in T2D as compared to ND individuals (p ≤ 0.05). Using Spearman’s correlation, a positive correlation was observed between irisin and Osteoactivin as well as OPG (p < 0.05). Similarly, a positive association was observed between METRNL and Osteoactivin (p < 0.05). The strong positive association shown in this study between irisin, METRNL and various molecules with osteogenic properties emphasize a possible interaction between these organs. This report suggests that having a dysregulation in the level of the aforementioned molecules could potentially affect the development of bone and muscle related complications that are associated with obesity and T2D.

Background and aims: Aging-induced bone loss is a multifactorial, age-related, and progressive phenomenon among the general population and may further progress to osteoporosis and increase the risk of fractures. Cycloastragenol (CAG), currently the only compound reported that activates human telomerase, is thought to be able to alleviate or delay the symptoms of aging and chronic diseases. Previous research has suggested that CAG may have the potential to alleviate age-related bone loss. However, to date, no research has specifically focused on this aspect. In this study, we aimed to investigate whether CAG could prevent senile osteoporosis, and further reveal its underlying mechanism. Methods: CAG treatment was administrated into two bone loss rat models (D-galactose administration and aging) for 20 weeks and 33 weeks, respectively. Serum biomarkers analyses, bone biomechanical tests, micro-computed tomography assessment, and bone histomorphometry analyses were performed on the bone samples collected at the endpoint, to determine whether CAG could prevent or alleviate age-related bone loss. Proteomic analysis was performed to reveal the changes in protein profiles of the bones, and western blot was used to further verify the identity of the key proteins. The viability, osteoblastic differentiation, and mineralization of MC3T3-E1 cells were also evaluated after CAG treatment in vitro. Results: The results suggest that CAG treatment improves bone formation, reduces osteoclast number, alleviates the degradation of bone microstructure, and enhances bone biomechanical properties in both d-galactose- and aging-induced bone loss models. CAG treatment promotes viability, osteoblastic differentiation, and mineralization in MC3T3-E1 cells. Proteomic and western blot analyses revealed that CAG treatment increases osteoactivin (OA) expression to alleviate bone loss. Conclusion: The results revealed that CAG alleviates age-related bone loss and improves bone microstructure and biomechanical properties. This may due to CAG-induced increase in OA expression. In addition, the results support preclinical investigations of CAG as a potential therapeutic medicine for the treatment of senile osteoporosis.

The glycoprotein nonmetastatic melanoma protein B (GPNMB, also known as osteoactivin) is highly expressed in many cell types and regulates the homeostasis in various tissues. In different physiological contexts, it functions as a melanosome‐associated protein, membrane‐bound surface receptor, soluble ligand, or adhesion molecule. Therefore, GPNMB is involved in cell differentiation, migration, inflammation, metabolism, and neuroprotection. Because of its various involvement in different physiological conditions, GPNMB has been implicated in many diseases, including cancer, neurological disorders, and more recently immune‐mediated diseases. This review summarizes the regulation and function of GPNMB in normal physiology, and discusses the involvement of GPNMB in disease conditions with a particular focus on its potential role and therapeutic implications in autoimmunity. [ABSTRACT FROM AUTHOR]

Backgrounds Dexamethasone (Dex) is widely used in autoimmune diseases and inflammation treatment. A sever side effect of prolonged exposure to Dex is increased risk of osteoporosis (OP) or even femoral head necrosis, which would cause much suffer to patients. To reveal the mechanism behind this phenomenon, provide therapeutic guidance and potential target, we analyzed the inhibitory mechanism of Dex on osteogenesis of rat-BMSC. Methods Rat BMSC were obtained and characterized with FACS analysis. Osteogenesis and adipogenesis abilities were detected with Oil-O-Red staining, Alizarin Red staining and ALP activity analysis. These BMSC were then treated with Dex in combination with recombinant OA or not and detected for osteogenesis related gene expression with qRT-PCR. Protein interaction and expression were detected by Co-Immunoprecipitation and western blot. Results Osteoactivin (OA) could promote integrin β 1 expression and interact with this protein physically, leading to ERK activation and promoting osteogenesis related genes’ expression including Runx2, Col1a and OCN in BMSC. Dex, however, could block expression of several upstream genes of OA and decrease OA mRNA and protein level, and eventually suppress integrin β1-ERK activation and lead to decreased osteogenesis, which could finally develop into OP. Conclusion Recombinant OA treated BMSC exerted better osteogenesis potency even with Dex administration. This is because additional OA in medium counter-acts with Dex’s influence and rescued osteoblast differentiation via up-regulating integrin β1 and activate ERK/MAPK pathway which promotes osteogenesis. Hence, OA/integrin β1 could serve as potential therapeutic target for OP. [ABSTRACT FROM AUTHOR]

We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18 weeks on the radius and ulna, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2 h/day, 3 days/week. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal metaphyseal radius, decreased anabolic indices in this same bone region (e.g., decreased osteoblasts and bone formation rate), and increased catabolic indices (e.g., microcracks, increased osteocyte apoptosis, secreted sclerostin, RANKL, and osteoclast numbers), compared to controls. Distal metaphyseal trabeculae in the ulna of High-ForceHR rats showed a non-significant decrease in bone volume, some catabolic indices (e.g., decreased trabecular numbers) yet also some anabolic indices (e.g., increased osteoblasts and trabecular thickness). In contrast, the mid-diaphyseal region of High-ForceHR rats' radial and ulnar bones showed few to no microarchitecture differences and no changes in apoptosis, sclerostin or RANKL levels, compared to controls. In further contrast, Low-ForceHR rats showed increased trabecular bone volume in the radius in the distal metaphysis and increased cortical bone area its mid-diaphysis. These changes were accompanied by increased anabolic indices, no microcracks or osteocyte apoptosis, and decreased RANKL in each region, compared to controls. Ulnar bones of Low-ForceHR rats also showed increased anabolic indices, although fewer than in the adjacent radius. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-, region-, and bone-dependent, with high force loads at high repetition rates inducing region-specific increases in bone degradative changes that were most prominent in distal radial trabeculae, while low force task loads at high repetition rates induced adaptive bone responses. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSC) are critical in regulating immune responses by suppressing antigen presenting cells (APC) and T cells. We previously observed that incubation of peripheral blood monocytes with interleukin (IL)-10 during their differentiation to monocyte-derived dendritic cells (moDCs) results in the generation of an APC population with a CD14+HLA-DRlowphenotype (IL-10-APC) with reduced stimulatory capacity similar to human MDSC. Co-incubation experiments now revealed that the addition of IL-10-APC to moDC caused a reduction of DC-induced T-cell proliferation, of the expression of maturation markers, and of secreted cytokines and chemokines such as TNF-α, IL-6, MIP-1α and Rantes. Addition of IL-10-APC increased the immunosuppressive molecule osteoactivin and its corresponding receptor syndecan-4 on moDC. Moreover, CD14+HLA-DRlow MDSC isolated from healthy donors expressed high levels of osteoactivin, which was even further upregulated by the auxiliary addition of IL-10. Using transcriptome analysis, we identified a set of molecules and pathways mediating these effects. In addition, we found that IL-10-APC as well as human isolated MDSC expressed higher levels of programmed death (PD)-1, PD-ligand-1 (PD-L1), glucocorticoid-induced-tumor-necrosis-factor-receptor-related-protein (GITR) and GITR-ligand. Inhibition of osteoactivin, syndecan-4, PD-1 or PD-L1 on MDSC by using blocking antibodies restored the stimulatory capacity of DC in co-incubation experiments. Activation of MDSC with Dectin-1 ligand curdlan reduced the expression of osteoactivin and PD-L1. Our results demonstrate that osteoactivin/syndecan-4 and PD-/PD-L1 are key molecules that are profoundly involved in the inhibitory effects of MDSC on DC function and might be promising tools for clinical application.

Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring. [ABSTRACT FROM AUTHOR]

Background: Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA's expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats.Methods: Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines.Results: Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6.Conclusion: The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse. [ABSTRACT FROM AUTHOR]

Glycoprotein nonmelanoma protein B (GPNMB, alias osteoactivin), a type I transmembrane glycoprotein, is cleaved by extracellular proteases, resulting in release of an extracellular fragment (ECF). GPNMB is widely expressed by neurons within the CNS, including the hippocampus; however, its function in the brain remains unknown. Here, we investigated the role of GPNMB in memory and learning by using transgenic (Tg) mice over-expressing GPNMB (Tg mice on a BDF-1 background) and ECF-treated mice. In the hippocampus of both wild-type and Tg mice, GPNMB was highly expressed in neurons and astrocytes. Tg mice exhibited memory improvements in two types of learning tasks but were impaired in a passive-avoidance test. In Tg mice, the hippocampus displayed increased levels of the a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluA1. Intracerebroventricular administration of ECF (50 ng) to Institute of Cancer Research (ICR) mice also improved memory in a passive-avoidance test and increased hippocampal GluA1 levels 24 h after treatment. In Tg mice and ECF (0.25 ig/mL)- treated hippocampal slices, long-term potentiation was promoted. These findings suggest that GPNMB may be a novel target for research on higher order brain functions. [ABSTRACT FROM AUTHOR]

Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a tissue-restricted glycoprotein with substantial sequence homology to PMEL, but no known function, and was proposed to localize to non-fibrillar domains of distinct melanosome subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in melanocytes and He La cells, respectively, and is largely absent from fibrils. Using domain swapping, the unique PMEL localization is ascribed to its polycystic kidney disease ( PKD) domain, whereas the homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its sorting function. These results reveal the molecular basis for the distinct trafficking and morphogenetic properties of PMEL and GPNMB and support a deterministic function of the PMEL PKD domain in both protein sorting and amyloidogenesis. [ABSTRACT FROM AUTHOR]

Molecularly targeted therapies are rapidly growing with respect to their clinical development and impact on cancer treatment due to their highly selective anti-tumor action. However, many aggressive cancers such as triple-negative breast cancer (TNBC) currently lack well-defined therapeutic targets against which such agents can be developed. The identification of tumor-associated antigens and the generation of antibody drug-conjugates represent an emerging area of intense interest and growth in the field of cancer therapeutics. Glycoprotein non-metastatic b (GPNMB) has recently been identified as a gene that is over-expressed in numerous cancers, including TNBC, and often correlates with the metastatic phenotype. In breast cancer, GPNMB expression in the tumor epithelium is associated with a reduction in disease-free and overall survival. Based on these findings, glembatumumab vedotin (CDX-011), an antibody-drug conjugate that selectively targets GPNMB, is currently being investigated in clinical trials for patients with metastatic breast cancer and unresectable melanoma. This review discusses the physiological and potential pathological roles of GPNMB in normal and cancer tissues, respectively, and details the clinical advances and challenges in targeting GPNMB-expressing malignancies. [ABSTRACT FROM AUTHOR]

The article focuses on a study which examines the regulatory mechanisms of the transmembrane protein osteoactivin. It mentions several techniques including liquid chromatography, polyacrylamide gel electrophoresis and mass spectrometry which are used as the research methodologies. The result reveals that osteoactivin is released by proteolytic cleavage at both the cell surface and the intramembrane space.

BACKGROUND: The goal is to analyze the role of Osteactivin (OA) in human prostate adenocarcinoma (CaP) cell lines, DU145 and PC3. OA plays a role in proliferation, adhesion, differentiation and protein synthesis in normal and malignant cells. Further, the OA expression is specific of activated mature osteoblasts. METHODS: DU145 and PC3 were maintained under recommended conditions and treated, respectively, with 50 ng/mL for 8 days and with 100 ng/mL for 15 days of Nerve Growth Factor (NGF). The NGF-induced reduction of invasive capacity was assessed by the BioCoat Matrigel Invasion Chambers technique. The gene expression was evaluated by Q-RT-PCR, while the protein expression by indirect immunofluorescence and by western blot. RESULTS: NGF treatment of DU145 and PC3 induced a reduction of 95% and 78% of the invasive capacity. NGF treatment decreased OA expression at both mRNA and protein levels. The OA was secreted into the culture medium. CONCLUSIONS: DU145 and PC3 cells expressed OA, that is strongly reduced by NGF treatment. Since it is known that OA is involved in the acquisition of the cell invasive capability, it could thus be hypothesized that OA could be a factor that contributes to the acquisition of invasive properties of PCa and could be proposed as a biomarker of cell tendency to metastatization. [ABSTRACT FROM AUTHOR]

Bateman JP, Safadi FF, Susin C, Wikesjö UME. Exploratory study on the effect of osteoactivin on bone formation in the rat critical-size Calvarial defect model. J Periodont Res 2012; 47: 243-247. © 2011 John Wiley & Sons A/S Background and Objective: Osteoactivin is a novel glycoprotein shown to exhibit an important role in regulating osteoblast differentiation and function. The aim of the present study was to evaluate the potential of osteoactivin to support bone regeneration using an established defect model. Material and Methods: Critical-size, 8-mm-diameter through-and-through calvarial osteotomy defects were created in 60 adult male Sprague-Dawley rats. Test animals received 0.1 mL of osteoactivin in phosphate-buffered saline (50 μg/mL) soak-loaded onto an absorbable collagen sponge. Controls received 0.1 mL of phosphate-buffered saline soak-loaded onto the absorbable collagen sponge or no further intervention (sham-surgery). The animals were euthanized 2 and 4 wk after treatment and histometric analyses were performed. Results: The absorbable collagen sponge control (mean ± standard deviation: 40.9 ± 26.9%) showed borderline significant greater bone fill compared with sham-surgery (22.9 ± 15.8%; p = 0.10) and osteoactivin (20.2 ± 11.8%; p = 0.07) treatments at 2 wk. In contrast, osteoactivin (84.7 ± 15.8%) showed significantly greater bone fill than sham-surgery (28.4 ± 9.6%; p < 0.001) and absorbable collagen sponge (41.8 ± 22.1%; p < 0.001) at 4 wk. No animals receiving sham-surgery or absorbable collagen sponge exhibited complete bone fill at 4 wk while 70% of the animals receiving osteoactivin showed complete bone fill. Conclusion: Osteoactivin demonstrates a significant potential to support bone regeneration/formation. Studies using discriminating large animal models are necessary to explore clinical application for periodontal and craniofacial indications. [ABSTRACT FROM AUTHOR]

Abstract: The European InnoMed–PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with “omics” data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value. [Copyright &y& Elsevier]

Abstract: Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis. [Copyright &y& Elsevier]

Abstract: This study presents gene expression, protein expression, and in situ immunohistochemical evidence that osteoclasts express high levels of osteoactivin (OA), which had previously been reported to be an osteoblast-specific protein in bone. OA expression in osteoclasts was up-regulated upon receptor activator of NFκB ligand-induced differentiation. Suppression of functional activity of OA with neutralizing antibody reduced cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. OA was co-immunoprecipitated with integrin β3 and β1, indicating that OA co-localizes with integrin β3 and/or β1 in a hetero-polymeric complex in osteoclasts. These findings indicate that OA is a novel osteoclastic protein and plays a role in osteoclast differentiation and/or activity. [Copyright &y& Elsevier]

Abstract: Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts. [Copyright &y& Elsevier]

Abstract: In this study, we examined the expression of osteoactivin in the rat kidney after unilateral ureteral obstruction. Male Wistar rats were sacrificed at 6h, and on days 1, 2, 3 and 7 after the obstruction. The renal tubular lumens gradually dilated, and marked interstitial fibrosis was confirmed histologically on day 3 after the obstruction. The expressions of osteoactivin and collagen type III were examined by quantitative real-time RT-PCR. An 8-fold increase in osteoactivin mRNA expression as compared with that in the sham-operated group was observed at 6h after the obstruction, whereas no elevation of collagen type III mRNA expression was observed at this early stage. Furthermore, semi-quantitative RT-PCR was performed to identify upregulation of expression of matrix metalloproteinase-13 mRNA relative to that in the sham-operated control, and normalized to the expression level of β-actin. Intense osteoactivin expression localized in the dilated tubular epithelium and interstitial fibroblasts in the obstructive kidney was detected by immunohistochemistry and by in situ hybridization. These results suggested that the early-phase upregulation of osteoactivin expression in the tubular epithelium in response to renal injury might play a role in triggering renal interstitial fibrosis via activation of matrix metalloproteinase expression and collagen remodeling in rats. [Copyright &y& Elsevier]

Abstract: The role of osteoactivin (OA) in liver fibrogenesis remains unclear. After feeding wild-type (WT) and OA transgenic (OA-Tg) rats a choline-deficient, l-amino acid-defined (CDAA) diet for 12 weeks, we evaluated liver fibrosis. Hepatic fibrosis and expression of α-smooth muscle actin protein in OA-Tg rats were reduced in comparison to WT rats. Our examination of the expression of 31,100 genes by microarray analysis identified 177 and 256 genes that were upregulated and downregulated, respectively, by at least twofold in OA-Tg rat livers in comparison to WT rat livers. Of these genes, we confirmed a significant downregulation in the expression levels of tissue inhibitor of metalloproteinase-1 and -2, type I collagen, and platelet-derived growth factor receptor-α and -β in the livers of OA-Tg rats. These results indicate that transgenic OA expression attenuates the development of hepatic fibrosis in association with the suppression of specific genes involved in its pathogenesis. [Copyright &y& Elsevier]

Osteoactivin (OA) is a novel protein identified by mRNA differential display using bone from osteopetrotic versus normal rats. Bioinformatic analysis showed that OA cDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein. In this study, we localized OA protein an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute in primary osteoblast culture by immunofluorescent staining and Western blot analysis. Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrix formation from day 7 to 14, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at 3 weeks when osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deliver anti-OA antibody into the cells, we demonstrated that anti-OA antibody significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function. [ABSTRACT FROM AUTHOR]

• Cycloastragenol (CAG) prevents age-related bone loss in aging rat models. • CAG inhibits bone resorption and improves bone formation. • Anti-osteoporosis effects of CAG may be due to upregulation of osteoactivin expression Aging-induced bone loss is a multifactorial, age-related, and progressive phenomenon among the general population and may further progress to osteoporosis and increase the risk of fractures. Cycloastragenol (CAG), currently the only compound reported that activates human telomerase, is thought to be able to alleviate or delay the symptoms of aging and chronic diseases. Previous research has suggested that CAG may have the potential to alleviate age-related bone loss. However, to date, no research has specifically focused on this aspect. In this study, we aimed to investigate whether CAG could prevent senile osteoporosis, and further reveal its underlying mechanism. CAG treatment was administrated into two bone loss rat models (D-galactose administration and aging) for 20 weeks and 33 weeks, respectively. Serum biomarkers analyses, bone biomechanical tests, micro-computed tomography assessment, and bone histomorphometry analyses were performed on the bone samples collected at the endpoint, to determine whether CAG could prevent or alleviate age-related bone loss. Proteomic analysis was performed to reveal the changes in protein profiles of the bones, and western blot was used to further verify the identity of the key proteins. The viability, osteoblastic differentiation, and mineralization of MC3T3-E1 cells were also evaluated after CAG treatment in vitro. The results suggest that CAG treatment improves bone formation, reduces osteoclast number, alleviates the degradation of bone microstructure, and enhances bone biomechanical properties in both d -galactose- and aging-induced bone loss models. CAG treatment promotes viability, osteoblastic differentiation, and mineralization in MC3T3-E1 cells. Proteomic and western blot analyses revealed that CAG treatment increases osteoactivin (OA) expression to alleviate bone loss. The results revealed that CAG alleviates age-related bone loss and improves bone microstructure and biomechanical properties. This may due to CAG-induced increase in OA expression. In addition, the results support preclinical investigations of CAG as a potential therapeutic medicine for the treatment of senile osteoporosis. [ABSTRACT FROM AUTHOR]

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein first identified in low-metastatic human melanoma cell lines as a regulator of tumor growth. GPNMB is widely expressed in various tissues, where it is involved in cell differentiation, migration, inflammation/anti-inflammation, tissue regeneration, and neuroprotection. GPNMB is identified in microglia of adult rat brains, neurons and astrocytes of GPNMB transgenic (Tg) mouse brains, and motor neurons of amyotrophic lateral sclerosis (ALS) patients. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either TYROBP ( DAP12 ) or TREM2 . TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Pathologically, the brains of NHD patients exhibit leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes and the basal ganglia. At present, molecular mechanisms responsible for development of leukoencephaolpathy in NHD brains remain totally unknown. Recent evidence indicates that disease-associated microglia (DAM) that cluster around amyloid plaques express high levels of GPNMB in Alzheimer's disease (AD) brains. Because microglia act as a key regulator of leukoencephalopathy in NHD brains, it is proposed that GPNMB expressed on microglia might play a protective role in progression of leukoencephalopathy possibly via active phagocytosis of myelin debris. In the present study using immunohistochemistry, we have attempted to clarify the expression of GPNMB in NHD brains, compared with AD brains. We found that microglia accumulating in the white matter express an intense GPNMB immunoreactivity in both NHD and AD brains, suggesting that the accumulation of GPNMB-immunoreactive microglia is a general phenomenon in neurodegenerative brains.

Myeloid-derived suppressor cells (MDSC) are critical in regulating immune responses by suppressing antigen presenting cells (APC) and T cells. We previously observed that incubation of peripheral blood monocytes with interleukin (IL)-10 during their differentiation to monocyte-derived dendritic cells (moDCs) results in the generation of an APC population with a CD14+HLA-DRlowphenotype (IL-10-APC) with reduced stimulatory capacity similar to human MDSC. Co-incubation experiments now revealed that the addition of IL-10-APC to moDC caused a reduction of DC-induced T-cell proliferation, of the expression of maturation markers, and of secreted cytokines and chemokines such as TNF-α, IL-6, MIP-1α and Rantes. Addition of IL-10-APC increased the immunosuppressive molecule osteoactivin and its corresponding receptor syndecan-4 on moDC. Moreover, CD14+HLA-DRlowMDSC isolated from healthy donors expressed high levels of osteoactivin, which was even further upregulated by the auxiliary addition of IL-10. Using transcriptome analysis, we identified a set of molecules and pathways mediating these effects. In addition, we found that IL-10-APC as well as human isolated MDSC expressed higher levels of programmed death (PD)-1, PD-ligand-1 (PD-L1), glucocorticoid-induced-tumor-necrosis-factor-receptor-related-protein (GITR) and GITR-ligand. Inhibition of osteoactivin, syndecan-4, PD-1 or PD-L1 on MDSC by using blocking antibodies restored the stimulatory capacity of DC in co-incubation experiments. Activation of MDSC with Dectin-1 ligand curdlan reduced the expression of osteoactivin and PD-L1. Our results demonstrate that osteoactivin/syndecan-4 and PD-/PD-L1 are key molecules that are profoundly involved in the inhibitory effects of MDSC on DC function and might be promising tools for clinical application. [ABSTRACT FROM AUTHOR]

In the spleens of Gaucher disease mice and patients, there is a striking elevation of expression of glycoprotein non-Metastatic Melanoma B (gpNMB). We conducted a study in a large cohort of patients with Gaucher disease to assess the utility of serum levels of soluble fragment of gpNMB as a biomarker of disease activity. There was >15-fold elevation of gpNMB in sera of untreated patients with Gaucher disease. gpNMB levels correlated with overall disease severity as well as the severity of individual organ compartments: liver, spleen, bone and hematological disease. Imiglucerase enzyme replacement therapy resulted in significant reduction of gpNMB. Serum levels of gpNMB were highly correlated with accumulation of bioactive lipid substrate of Gaucher disease, glucosylsphingosine as well as established biomarkers, chitotriosidase and chemokine, CCL18. Our results suggest utility of gpNMB as a biomarker of Gaucher disease to monitor individual patients and cohorts of patients for disease progression or response to therapy. Investigation of gpNMB in Gaucher disease pathophysiology is likely to illuminate our understanding disease mechanisms., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cardiac diseases are the leading cause of death. Available treatment approaches are not sufficient to reverse persistent cardiac damage after injury; thus, the search for new therapeutic targets is essential. Our microarray-based screening in rat hearts 24 h after myocardial infarction (MI) yielded glycoprotein nonmetastatic melanoma protein B (GPNMB), which is known to be involved in inflammation and fibrosis after tissue injury. However, its role in the heart was elusive. We found increased cardiac expression levels of GPNMB in rats and mice after MI. Analysis of DBA/2J mice, which lack functional GPNMB due to a spontaneous point mutation, showed that systemic GPNMB deficiency was associated with preserved cardiac function and less left ventricular dilation after MI compared with DBA/2J mice with reconstituted GPNMB expression. These improvements were associated with decreased expression of matrix metalloproteinase 9, the cardiac stress genes for natriuretic peptides (atrial natriuretic peptide and brain natriuretic peptide), and β-myosin heavy chain after MI. Moreover, GPNMB deficiency attenuated the dilated cardiomyopathy in muscle lim protein knockout mice but could not prevent cardiac hypertrophy induced by isoprenaline infusion. This is the first experimental study to show that GPNMB adversely influences myocardial remodeling.-Järve, A., Mühlstedt, S., Qadri, F., Nickl, B., Schulz, H., Hübner, N., Özcelik, C., Bader, M. Adverse left ventricular remodeling by glycoprotein nonmetastatic melanoma protein B in myocardial infarction., (© FASEB.)

Introduction: Osteoactivin (OA) was first discovered in an osteopetrotic rat model using mRNA differential display a decade ago and has been studied recently. OA in bone tissue can directly or indirectly regulate the differentiation of osteoblasts by influencing cell behaviours, such as proliferation and adhesion, as well as inducing serial signal cascades, which would be of great importance in the field of tissue engineering. The results of recent studies have further demonstrated that OA plays a critical role in the differentiation and function of cells, especially in bone formation and fracture healing. Areas covered: The discovery, structure, and function of OA as well as its therapeutic potential in tissue regeneration of bone defects, kidney injury, liver damage, and muscle atrophy. Expert opinion: OA has great potential in promoting the regeneration of damaged tissues, particularly bone tissue, which is supported by a large body of data. Future studies should focus on exploring the underlying mechanism of OA as well as pursuing the ideal form of OA-related regenerative medicine.