GDF11 contributes to hepatic hepcidin (HAMP) inhibition through SMURF1-mediated BMP-SMAD signalling suppression.

Hepcidin (HAMP) synthesis is suppressed by erythropoiesis to increase iron availability for red blood cell production. This effect is thought to result from factors secreted by erythroid precursors. Growth differentiation factor 11 (GDF11) expression was recently shown to increase in erythroid cells of ß-thalassaemia, and decrease with improvement in anaemia. Whether GDF11 regulates hepatic HAMP production has never been experimentally studied. Here, we explore GDF11 function during erythropoiesis-triggered HAMP suppression. Our results confirm that exogenous erythropoietin significantly increases Gdf11 as well as Erfe (erythroferrone) expression, and Gdf11 is also increased, albeit at a lower degree than Erfe, in phlebotomized wild type and ß-thalassaemic mice. GDF11 is expressed predominantly in erythroid burst forming unit- and erythroid colony-forming unit- cells during erythropoiesis. Exogeneous GDF11 administration results in HAMP suppression in vivo and in vitro. Furthermore, exogenous GDF11 decreases BMP-SMAD signalling, enhances SMAD ubiquitin regulatory factor 1 (SMURF1) expression and induces ERK1/2 (MAPK3/1) signalling. ERK1/2 signalling activation is required for GDF11 or SMURF1-mediated suppression in BMP-SMAD signalling and HAMP expression. This research newly characterizes GDF11 in erythropoiesis-mediated HAMP suppression, in addition to ERFE.

GDF11 upregulation independently predicts shorter overall-survival of uveal melanoma.

Growth differentiation factor 11 (GDF11), is a member of the transforming growth factor-beta (TGF-ß) superfamily and bone morphogenetic protein (BMP) subfamily. In this study, we aimed to assess the expression profile of GDF11, its prognostic value in terms of OS, as well as the potential mechanisms leading to its dysregulation in uveal melanoma. A retrospective study was conducted using our primary data and genetic, clinicopathological and overall survival (OS) data from the Cancer Genome Atlas-Uveal Melanoma (TCGA-UVM). Results showed that GDF11 expression was significantly higher in tumor tissues compared with that in adjacent normal tissues. High GDF11 expression was associated with uveal melanoma in advanced stages (IV), epithelioid cell dominant subtype, as well as extrascleral extension. Univariate analysis showed that older age, epithelioid cell dominant, with extrascleral extension and increased GDF11 expression were associated with unfavorable OS. Multivariate analysis confirmed that GDF11 expression was an independent prognostic indicator of unfavorable OS (HR: 1.704, 95%CI: 1.143-2.540, p = 0.009), after adjustment of age, histological subtypes and extrascleral extension. Among the 80 cases of uveal melanoma, only 3 cases had low-level copy gain (+1) and 2 cases had heterozygous loss (-1). No somatic mutations, including SNPs and small INDELs were observed in GDF11 DNA. The methylation of these four CpG sites had weakly (cg22950598 and cg23689080), moderately (cg09890930), or strongly (cg05511733) negative correlation with GDF11 expression. In addition, the patients with high methylation of these four sites had significantly better OS compared to the group with low methylation. Based on these findings, we infer that methylation modulated GDF11 expression might be a valuable prognostic biomarker regarding OS in uveal melanoma.

Expression of Bone Morphogenetic Proteins in Multiple Sclerosis Lesions.

Bone morphogenetic proteins (BMPs) are secreted proteins that belong to the transforming growth factor-ß superfamily. In the adult brain, they modulate neurogenesis, favor astrogliogenesis, and inhibit oligodendrogenesis. Because BMPs may be involved in the failure of remyelination in multiple sclerosis (MS), we characterized the expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMP type II receptor (BMPRII); and phosphorylated SMAD (pSMAD) 1/5/8 in lesions of MS and other demyelinating diseases. A total of 42 MS lesions, 12 acute ischemic lesions, 8 progressive multifocal leukoencephalopathy lesions, and 10 central nervous system areas from four nonneuropathological patients were included. Lesions were histologically classified according to the inflammatory activity. The expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified by immunostaining, and colocalization studies were performed. In MS lesions, astrocytes, microglia/macrophages, and neurons expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8. Oligodendrocytes expressed BMP-2 and BMP-7 and pSMAD1/5/8. The percentage of cells that expressed BMPs, BMPRII, and pSMAD1/5/8 correlated with the inflammatory activity of MS lesions, and changes in the percentage of positive cells were more relevant in MS than in other white matter-damaging diseases. These data indicate that BMPs are increased in active MS lesions, suggesting a possible role in MS pathogenesis.

Functional reconstruction of critical-sized load-bearing bone defects using a Sclerostin-targeting miR-210-3p-based construct to enhance osteogenic activity.

A considerable amount of research has focused on improving regenerative therapy strategies for repairing defects in load-bearing bones. The enhancement of tissue regeneration with microRNAs (miRNAs) is being developed because miRNAs can simultaneously regulate multiple signaling pathways in an endogenous manner. In this study, we developed a miR-210-based bone repair strategy. We identified a miRNA (miR-210-3p) that can simultaneously up-regulate the expression of multiple key osteogenic genes in vitro. This process resulted in enhanced bone formation in a subcutaneous mouse model with a miR-210-3p/poly-l-lactic acid (PLLA)/bone marrow-derived stem cell (BMSC) construct. Furthermore, we constructed a model of critical-sized load-bearing bone defects and implanted a miR-210-3p/ß-tricalcium phosphate (ß-TCP)/bone mesenchymal stem cell (BMSC) construct into the defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. We also identified a new mechanism by which miR-210-3p regulates Sclerostin protein levels. This miRNA-based strategy may yield novel therapeutic methods for the treatment of regenerative defects in vital load-bearing bones by utilizing miRNA therapy for tissue engineering. STATEMENT OF SIGNIFICANCE: The destroyed maxillofacial bone reconstruction is still a real challenge for maxillofacial surgeon, due to that functional bone reconstruction involved load-bearing. Base on the above problem, this paper developed a novel miR-210-3p/ß-tricalcium phosphate (TCP)/bone marrow-derived stem cell (BMSC) construct (miR-210-3p/ß-TCP/BMSCs), which lead to functional reconstruction of critical-size mandible bone defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. In addition, we also found the mechanism of how the delivered microRNA activated the signaling pathways of endogenous stem cells, leading to the defect regeneration. This miRNA-based strategy can be used to regenerate defects in vital load-bearing bones, thus addressing a critical challenge in regenerative medicine by utilizing miRNA therapy for tissue engineering.

Meningioma and Bone Hyperostosis: Expression of Bone Stimulating Factors and Review of the Literature.

BACKGROUND: Several hypotheses have been proposed regarding the mechanisms underlying meningioma-related hyperostosis. In this study, we investigated the role of osteoprotegerin (OPG), insulin-like growth factor 1 (IGF-1), endothelin 1 (ET-1), and bone morphogenetic protein (BMP) 2 and 4. METHODS: A total of 149 patients (39 males and 110 females; mean age, 62 years) who underwent surgery were included. Depending on the relationship with the bone, meningiomas were classified as hyperostotic, osteolytic, infiltrative, or unrelated. Expression of OPG, and IGF-1, ET-1, BMP-2, and BMP-4 was evaluated by tissue microarray analysis of surgical samples. RESULTS: Our series comprised 132 cases of grade I, 14 cases of grade II, and 3 cases of grade III meningiomas, according to the World Health Organization classification. Based on preoperative computed tomography scan, the cases were classified as follows: hyperostotic, n = 11; osteolytic, n = 11; infiltrative, n = 15; unrelated to the bone, n = 108. Four cases were excluded from the statistical analysis. Using receiver operating characteristic curve analysis, we identified a 2% cutoff for the mean value of IGF-1 that discriminated between osteolytic and osteoblastic lesions; cases with a mean IGF-1 expression of <2% were classified as osteolytic (P = 0.0046), whereas those with a mean OPG expression of <10% were classified as osteolytic (P = 0.048). No other significant relationships were found. CONCLUSIONS: Expression of OPG and expression of IGF-1 were found to be associated with the development of hyperostosis. Preliminary findings suggest that hyperostosis can be caused by an overexpression of osteogenic molecules that influence osteoblast/osteoclast activity. Based on our results, further studies on hyperostotic bony tissue in meningiomas are needed to better understand how meningiomas influence bone overproduction.

Reduced expression of growth differentiation factor 11 promoted the progression of chronic obstructive pulmonary disease by activating the AKT signaling pathway.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease, which is associated with significant mortality and costs. The molecular mechanisms underlying the roles of cigarette smoke (an accepted risk factor for COPD) and growth differentiation factor 11 (GDF11), which is reduced in patients with COPD, in the occurrence of COPD are unclear. The aim of the present study was to explore the function of GDF11 in the progression of COPD. Western blotting analysis was used to determine the expression levels of GDF11 in serum and primary lung mesenchymal cells from patients with COPD and the healthy people, and the effect of cigarette smoke extract (CSE) on the expression of AKT, p-AKT (Ser473), p-AKT (Thr308) and GDF11 was examined. The correlations between the expression level of GDF11 and the ratio of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), as well as GDF11 and p-AKT (Ser473 and Thr308) in vivo and in vitro were examined. GDF11 expression was decreased in COPD patients' serum and cells when compared with that from the healthy people, and it was positively correlated with the FEV1/FVC ratio. Exposure to CSE reduced the expression of GDF11 but increased the expression of p-AKT (Ser473 and Thr308). Together, the results suggested that CSE promoted the progression of COPD by downregulating the expression of GDF11, which then activated the AKT signaling pathway. This study suggests that GDF11 may be a novel target for the diagnosis and treatment of COPD.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

The Microdamage and Expression of Sclerostin in Peri-implant Bone under One-time Shock Force Generated by Impact.

Osseointegration is the key to implant stability and occlusal support. Biomechanical response and remodeling of peri-implant bone occurs under impact loading. Sclerostin participates in bone formation and resorption through Wnt and RANKL pathways. However the mechanism of microdamage and expression of sclerostin in peri-implant bone under impact load is still unclear. In present study, specific impact forces were applied to the implants with favorable osseointegration in rabbits. The microdamage of peri-implant bone and the expression of sclerostin, ß-catenin and RANKL during the process of bone damage and remodeling were investigated by micro-CT, histology, immunofluorescence and RT-qPCR analysis. Interface separation and trabecular fracture were found histologically, which were consistent with micro-CT analyses. Throughout remodeling, bone resorption was observed during the first 14 days after impact, and osseointegration and normal trabecular structure were found by 28 d. The expression of sclerostin and RANKL increased after impact and reached a maximum by 14 d, then decreased gradually to normal levels by 28 d. And ß-catenin expression was opposite. Results indicated that sclerostin may involve in the peri-implant bone damage caused by impact and remodeling through Wnt/ß-catenin and RANKL/RANK pathways. It will provide a new insight in the diagnosis and treatment for patients suffering impact.

Supraphysiological levels of GDF11 induce striated muscle atrophy.

Growth and differentiation factor (GDF) 11 is a member of the transforming growth factor ß superfamily recently identified as a potential therapeutic for age-related cardiac and skeletal muscle decrements, despite high homology to myostatin (Mstn), a potent negative regulator of muscle mass. Though several reports have refuted these data, the in vivo effects of GDF11 on skeletal muscle mass have not been addressed. Using in vitro myoblast culture assays, we first demonstrate that GDF11 and Mstn have similar activities/potencies on activating p-SMAD2/3 and induce comparable levels of differentiated myotube atrophy. We further demonstrate that adeno-associated virus-mediated systemic overexpression of GDF11 in C57BL/6 mice results in substantial atrophy of skeletal and cardiac muscle, inducing a cachexic phenotype not seen in mice expressing similar levels of Mstn. Greater cardiac expression of Tgfbr1 may explain this GDF11-specific cardiac phenotype. These data indicate that bioactive GDF11 at supraphysiological levels cause wasting of both skeletal and cardiac muscle. Rather than a therapeutic agent, GDF11 should be viewed as a potential deleterious biomarker in muscle wasting diseases.

Bariatric Roux-En-Y Gastric Bypass Surgery: Adipocyte Proteins Involved in Increased Bone Remodeling in Humans.

PURPOSE: Bariatric surgery has been associated with bone remodeling changes. The action of adipokines on the expression of receptor activator of nuclear factor kappa ß ligand (RANKL) and osteoprotegerin (OPG) and on an increase in sclerostin could be related to these changes. MATERIALS AND METHODS: This study aimed to assess the repercussions of weight loss, fat mass (FM), and fat-free mass (FFM) loss and biochemical and hormonal changes on bone remodeling markers after Roux-en-Y gastric bypass (RYGB). Anthropometric data, parathyroid hormone (PTH), bone-specific alkaline phosphatase (BSAP), collagen type 1 C-telopeptide (CTX), 25-hydroxy vitamin D (25-OH-VitD), leptin, adiponectin, RANKL, OPG, and sclerostin of 30 menstruating women were measured preoperatively (Pre), and 3, 12, and 24 months (m) after RYGB. RESULTS: Leptin (34.4 (14.7; 51.9) vs. 22.5 (1.9; 52.7) ng/mL) and OPG (3.6 (1.1; 11.5) vs. 3.4 (1.5; 6) pmol/L) decreased, and adiponectin (7.4 (1.7; 18.4) vs. 13.8 (3.0; 34.6) µg/mL), CTX (0.2 (0.1; 2.2) vs. 0.6 (0.4; 6.0) ng/mL), RANKL (0.1 (0.0; 0.5) vs. 0.3 (0.0; 2.0) pmol/L), and sclerostin (21.7 (3.2; 75.1) vs. 34.8 (6.4; 80.5) pmol/L) increased after 3 m. BSAP increased after 12 m (10.1 (5.4; 18.9) vs. 13.9 (6.9; 30.2) µg/mL) (p < 0.005). CTX correlated positively with adiponectin at 24 m and inversely with leptin Pre; OPG at 3 m; weight, FM, FFM, and leptin at 24 m. RANKL correlated directly with weight at 3 m. Sclerostin correlated inversely with weight Pre and FM at 3 m. BSAP correlated negatively with 25-OH-VitD at 12 m, and positively with PTH at 24 m. CONCLUSIONS: RYGB induced weight loss, and biochemical, hormonal, and body composition changes are associated with higher bone remodeling.

Chondrocyte FGFR3 Regulates Bone Mass by Inhibiting Osteogenesis.

Chondrogenesis can regulate bone formation. Fibroblast growth factor receptor 3, highly expressed in chondrocytes, is a negative regulator of bone growth. To investigate whether chondrocyte FGFR3 regulates osteogenesis, thereby contributing to postnatal bone formation and bone remodeling, mice with conditional knock-out of Fgfr3 in chondrocytes (mutant (MUT)) were generated. MUT mice displayed overgrowth of bone with lengthened growth plates. Bone mass of MUT mice was significantly increased at both 1 month and 4 months of age. Histological analysis showed that osteoblast number and bone formation were remarkably enhanced after deletion of Fgfr3 in chondrocytes. Chondrocyte-osteoblast co-culture assay further revealed that Fgfr3 deficiency in chondrocytes promoted differentiation and mineralization of osteoblasts by up-regulating the expressions of Ihh, Bmp2, Bmp4, Bmp7, Wnt4, and Tgf-ß1, as well as down-regulating Nog expression. In addition, osteoclastogenesis was also impaired in MUT mice with decreased number of osteoclasts lining trabecular bone, which may be related to the reduced ratio of Rankl to Opg in Fgfr3-deficient chondrocytes. This study reveals that chondrocyte FGFR3 is involved in the regulation of bone formation and bone remodeling by a paracrine mechanism.

Sclerostin expression in bone tumours and tumour-like lesions.

AIMS: To assess the immunophenotypic and mRNA expression of sclerostin in human skeletal tissues and in a wide range of benign and malignant bone tumours and tumour-like lesions. METHODS AND RESULTS: Sclerostin expression was evaluated by immunohistochemistry and quantitative polymerase chain reaction (PCR). In lamellar and woven bone, there was strong sclerostin expression by osteocytes. Osteoblasts and other cell types in bone were negative. Hypertrophic chondrocytes in the growth plate and mineralized cartilage cells in zone 4 of hyaline articular cartilage strongly expressed sclerostin, but most chondrocytes in hyaline cartilage were negative. In primary bone-forming tumours, including osteosarcomas, there was patchy expression of sclerostin in mineralized osteoid and bone. Sclerostin staining was seen in woven bone in fibrous dysplasia, in osteofibrous dysplasia, and in reactive bone formed in fracture callus, in myositis ossificans, and in the wall of solitary bone cysts and aneurysmal bone cysts. Sclerostin was expressed by hypertrophic chondrocytes in osteochondroma and chondroblasts in chondroblastoma, but not by tumour cells in other bone tumours, including myeloma and metastatic carcinoma. mRNA expression of sclerostin was identified by quantitative PCR in osteosarcoma specimens and cell lines. CONCLUSIONS: Sclerostin is an osteocyte marker that is strongly expressed in human woven and lamellar bone and mineralizing chondrocytes. This makes it a useful marker with which to identify benign and malignant osteogenic tumours and mineralizing cartilage tumours, such as chondroblastomas and other lesions in which there is bone formation.

HC-HA/PTX3 Purified From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.

Blockade of bone morphogenetic protein signaling potentiates the pro-inflammatory phenotype induced by interleukin-17 and tumor necrosis factor-α combination in rheumatoid synoviocytes.

INTRODUCTION: Bone morphogenetic proteins (BMPs) are multifunctional secreted growth factors regulating a broad spectrum of functions in numerous systems. An increased expression and production of specific BMPs have been described in the rheumatoid arthritis (RA) synovium. The aim of this study was to analyze the involvement of the BMP signaling pathway in RA synoviocytes in response to interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α). METHODS: The expression of components of the BMP signaling pathway (BMP receptors, BMP ligands, BMP signal transducers, and BMP antagonists) was analyzed by quantitative polymerase chain reaction before and after treatment of RA synoviocytes with TNF-α or IL-17 or both. Regulation was studied in the presence of the specific BMP inhibitor DMH1 (dorsomorphin homologue 1) or an exogenous BMP ligand, BMP6. Expression and production of pro-inflammatory cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor), chemokines (IL-8, CCL2, CCL5, and CXCL10), and matrix metalloproteinases (MMP-1, -2, -3, -9, and -13) were analyzed. RESULTS: RA synoviocytes express BMP receptors (mainly BMPRIA, ACTRIA, and BMPRII), signal transducers of the Smad family (Smad1 and 5 and co-Smad4), and different BMP antagonists. The modulation of the expression of the BMP target genes-Id (inhibitor of DNA-binding/differentiation) proteins and Runx (Runt-related transcription factor) transcription factors-after the addition of exogenous BMP shows that the BMP signaling pathway is active. RA synoviocytes also express BMP ligands (BMP2, BMP6, and BMP7) which are highly upregulated after activation with TNF-α and IL-17. Autocrine BMP signaling pathway can be blocked by treatment with the inhibitor DMH1, leading to an increase in the upregulated expression of pro-inflammatory cytokines, chemokines, and MMPs induced by the activation of RA synoviocytes with TNF-α and IL-17. Conversely, the additional stimulation of the BMP pathway with the exogenous addition of the BMP6 ligand decreases the expression of those pro-inflammatory and pro-destructive factors. CONCLUSION: The results indicate that the canonical BMP pathway is functionally active in human RA synoviocytes and that the inhibition of autocrine BMP signaling exacerbates the pro-inflammatory phenotype induced in RA synoviocytes by the stimulation with IL-17 and TNF-α.

Inhibition of GSK3ß Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation.

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3ß inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3ß inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

Acute bone response to whole body vibration in healthy pre-pubertal boys.

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton's response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such "stimulation-testing" in clinical practice.

Lazaroid U-74389G inhibits the osteoblastic differentiation of IL-1ß-indcued aortic valve interstitial cells through glucocorticoid receptor and inhibition of NF-κB pathway.

BACKGROUND: Aortic valve calcification is characterized as the active process of aortic valve interstitial cells (AVICs), and considered as an inflammatory disease. As an antioxidant, the anti-inflammatory activity of Lazaroid has been exhibited in various models. We hypothesized that Lazaroid U-74389G would inhibit the osteoblastic differentiation of AVICs induced by IL-1ß. METHODS: Normal tricuspid aortic valve leaflets were collected from patients with acute aortic dissection (Type A) undergoing the Bentall procedure. AVICs were isolated and stimulated with IL-1ß in presence or absence of U-74389G in culture. Cell lysates were analyzed for osteogenic markers and nuclear factor-κB using real-time PCR and Immunoblotting. Culture media was analyzed for IL-6 and IL-8 with enzyme-linked immunosorbent assay. Alizarin Red Staining was adopted to demonstrate the calcium deposition. RESULTS: The expression of alkaline phosphatase and bone morphogenetic protein, accompanied by the production of IL-6 and IL-8, was up-regulated in response to IL-1ß and was inhibited by the addition of U-74389G. The NF-κB pathway was activated by IL-1ß and involved in the suppression of U-74389G on osteoblastic differentiation in AVICs. The negative effects of U-74389G on ostengenic gene expression and mineralization of AVICs were blocked by glucocorticoid receptor antagonist mifepristone and the NF-κB inhibitor Bay 11-7082. CONCLUSIONS: U-74389G inhibits the pro-osteogenic response to IL-1ß stimulation in AVICs. The osteoblastic differentiation and mineralization of AVICs were inhabited by U-74389G though the modulation of NF-κB activation, and this pathway could be potential therapeutic targets for medical treatment of calcified aortic valve disease.

Paradoxical response to mechanical unloading in bone loss, microarchitecture, and bone turnover markers.

BACKGROUND: Sclerostin, encoded by the SOST gene, has been implicated in the response to mechanical loading in bone. Some studies demonstrated that unloading leads to up-regulated SOST expression, which may induce bone loss. PURPOSE: Most reported studies regarding the changes caused by mechanical unloading were only based on a single site. Considering that the longitudinal bone growth leads to cells of different age with different sensitivity to unloading, we hypothesized that bone turnover in response to unloading is site specific. METHODS: We established a disuse rat model by sciatic neurectomy in tibia. In various regions at two time-points, we evaluated the bone mass and microarchitecture in surgically-operated rats and control rats by micro-Computed Tomography (micro-CT) and histology, sclerostin/SOST by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (qPCR), tartrate resistant acid phosphatase 5b (TRAP 5b) by ELISA and TRAP staining, and other bone markers by ELISA. RESULTS: Micro-CT and histological analysis confirmed bone volume in the disuse rats was significantly decreased compared with those in the time-matched control rats, and microarchitecture also changed 2 and 8 weeks after surgery. Compared with the control groups, SOST mRNA expression in the diaphysis was down-regulated at both week 2 and 8. On the contrary, the percentage of sclerostin-positive osteocytes showed an up-regulated response in the 5 - 6 mm region away from the growth plate, while in the 2.5 - 3.5 mm region, the percentage was no significant difference. Nevertheless, in 0.5 - 1.5 mm region, the percentage of sclerostin-positive osteocytes decreased after 8 weeks, consistent with serum SOST level. Besides, the results of TRAP also suggested that the expression in response to unloading may be opposite in different sites or system. CONCLUSION: Our data indicated that unloading-induced changes in bone turnover are probably site specific. This implies a more complex response pattern to unloading and unpredictable therapeutics which target SOST or TRAP 5b.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis.

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett's intestinal differentiation; however, in mice, basal progenitor cell-specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.