BMP-2 Levels Research Articles

Clinical and biological significance of adamantinomatous craniopharyngioma with CTNNB1 mutation.

The Wnt/β-catenin signaling pathway is strongly implicated in the pathogenesis of adamantinomatous craniopharyngioma (adaCP). However, there is no evidence that the CTNNB1 mutation activates the target gene of Wnt/β-catenin signaling, and it is unknown whether it affects the tumorigenesis of adaCP. To assess the effect of the CTNNB1 mutation of adaCP, the authors analyzed the correlation between the mutation and clinical, radiological, pathological, and biological findings. Between 2003 and 2015, 42 patients (24 male and 18 female, median age 42 years) with either papillary craniopharyngioma (papCP) or adaCP underwent tumor resection at the authors' institution. BRAF V600E and CTNNB1 in papCP and adaCP samples were sequenced by next-generation sequencing and the Sanger method, and mRNA expression levels of Axin2 and BMP4 were evaluated by RT-PCR. Axin2, BMP4, β-catenin, and BRAF expression were evaluated by immunohistochemistry. Other data were collected from clinical reports. The BRAF V600E mutation was detected in all 10 cases of papCP (100%). CTNNB1 exon 3 mutations were detected in 21 of 31 (68%) cases of adaCP, excluding 1 case for which there were no available sequence data. The mRNA expression level of Axin2 was significantly higher in adaCPs with a CTNNB1 mutation than in those without (p < 0.05). The immunohistochemical findings of Axin2 and BMP4 did not correlate with CTNNB1 mutation positivity. When patients who received adjuvant radiation therapy were excluded, progression-free survival was shorter in the mutation-positive group than in the mutation-negative group (log-rank test, p = 0.031). Examination of clinical characteristics and immunohistochemical findings of adaCPs showed that there was no significant correlation between CTNNB1 mutation positivity and age, sex, tumor volume, gross-total resection, optic tract edema, calcification, or T1 signal intensity of cyst fluid on MRI, β-catenin, and MIB-1 index. These results raise the possibility that the CTNNB1 mutation in adaCP may be associated with disease recurrence, and genes related to the Wnt/β-catenin signaling pathway might represent a therapeutic target.

S1627 THE HEMOCHROMATOSIS PROTEINS HJV AND TFR2 ARE DISPENSABLE FOR ACUTE BMP2‐MEDIATED HEPCIDIN UPREGULATION IN MICE

Background:Hepcidin is the liver hormone that maintains body iron homeostasis. In hemochromatosis (HH), a hereditary disease due to mutations in HFE, TFR2, HJV and hepcidin itself, iron accumulates in several tissues and organs because levels of hepcidin are inappropriately low. Hepcidin is regulated by the liver BMP‐SMAD pathway, whose activation requires BMP type I (ALK2 and ALK3) and type II receptors (the redundant BMPR2 and ACVR2A), hemochromatosis proteins (HFE, TFR2 and the BMP‐coreceptor HJV), and BMP ligands (BMP2 and BMP6). BMP2 signals preferentially through ALK3 for basal hepcidin activation, whereas BMP6, increased by body iron, signals via ALK2. The current model considers that the HH proteins functionally interact with ALK3 and are part of ALK3‐BMP2 branch of the pathway, thus forming a multiprotein complex important for basal hepcidin modulation. Moreover the characterization of Bmp2 LSECKO , Bmp6 KO , HH proteins KO models and double knock out animals (Bmp6 KO ‐HH KO with worsen phenotypes), indicates that the two branches of BMP‐SMAD signaling do not compensate each other and require different ligands.Aims:Since HH proteins interact mainly with ALK3, we investigated the effect of BMP2 on hepcidin regulation in murine models of hereditary hemochromatosis (Hjv KO and Tfr2 KO).Methods:In vivo studies were performed on Hjv KO and Tfr2 KO mice, compared to wild type controls, treated with a single BMP2 injection (24 mg/mouse) and sacrificed 4 hrs later. We analyzed: iron parameters (TS%, LIC, SIC, serum hepcidin); BMP‐SMAD target genes (Hamp, Id1), ligands (Bmp6, Bmp2) and inflammation target genes (Saa1, Il6, Il1β, Tnfα, Ifnγ, Tgfβ) expression with qRT‐PCR; SMAD1/5/8 and STAT3 phosphorylation with standard western blot technique. We studied the effect of BMP2 treatment ex vivo on cultured primary hepatocytes isolated from wt, Hjv KO, and Tfr2 KO.Results:The treatment protocol did not change iron parameters of wild type, Hjv KO and Tfr2 KO mice. Hamp mRNA and serum hepcidin were both increased by BMP2 in HH mice and the BMP‐SMAD pathway was activated as in WT mice. Interestingly, although Bmp6 levels were increased in iron loaded HH mice, Bmp2 levels were not, and the expression of both ligands was not affected by BMP2 injection. Systemic administration of BMP2 activated an inflammatory response hallmarked by increased expression of the liver acute phase protein Saa1 in WT and HH mice through STAT3 phosphorylation. Serum IL‐6 as well as spleen Il‐6, Tnfα, IfnY and Tgfβ mRNA levels were not affected by BMP2. In contrast, Il1β was significantly upregulated in BMP2‐treated mice. To evaluate if an inflammatory signal contributed to hepcidin upregulation, we analyzed wt, Hjv KO and Tfr2 KO primary hepatocytes. Hamp and the BMP‐SMAD target gene Id1 were strongly increased in a BMP2 dose‐dependent manner both in WT and HH hepatocytes in the absence of Saa1 changes, excluding a direct effect of BMP2 on STAT3 in these cells.Summary/Conclusion:Overall our results demonstrate that: TFR2 and HJV are dispensable for BMP2‐dependent hepcidin activation in vivo in an acute setting The BMP2‐induced proinflammatory response is mediated by its action on non parenchymal cells, likely macrophages Bmp2

Ligustrum Japonicum fructus Induces Anti-adipogenetic and Pro-osteoblastogenic Activities in Human Bone Marrow-derived Mensencymal Stem Cells (P06-016-19)

ObjectivesMasenchymal stem cells (MSCs) have pluripotent differentiation properties that confirmed to differentiate into myocyte, adipocyte, osteoblast, neuron and chondrocyte when specific culture conditions and stimuli are applied. In bone, both adipocytes and osteoblasts are derived from bone marrow MSCs (BMSCs) and production of these cells has been reported as reciprocal processes. The bone mass disequilibrium causes osteoporosis, as a result of elevated adipogenic differentiation accompanied by reducing bone formation. Therefore, in this study the effect of Ligustrum japonicum fructus (Waxleaf privet) on the adipogenesis and osteoblast differentiation was investigated in BMSCs. MethodsThe fruits of L. japonicum were extracted with dichloromethane and methanol, and the combined extracts were concentrated. Differentiation of BMSCs was performed by changing the medium into adipocyte and osteoblast differentiation supplied by Promocell GmbH. The cellular lipid was stained with Oil Red O and the alkaline phosphatase (ALP) activity was measured using a colormetric assay kit (Biovision, Inc.). The relative protein levels were measured by immunoblotting assay. ResultsPresense of L. japonicum fructus extract (LJE) inhibited the cellular lipid accumulation in a dose-dependent manner. Consistent with the effects on lipid accumulation, the adipocyte specific genes including PPARγ, C/EBPα and SREBP1c was down regulated by treatment with LJE. Moreover, treatment with LJE enhanced osteoblast differentiation observed as increased ALP activity and upregulated the proteALP, BMP-2 and osteocalcine protein levels. ConclusionsThe results indicated that LJE may prevent bone loss by inhibiting adipogenesis while activating the osteogenic differentiation in BMSCs. Therefore, LJE possess the potential to be utilized as a source of nutraceutical agents against osteoporosis. Funding SourcesThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP).

PS1315 CHANGES IN THE SECRETOME COMPOSITION OF MULTIPOTENT MESENCHYMAL STROMAL CELLS PATIENTS WITH DIFFUSE LARGE B‐CELL LYMPHOMA WITHOUT BONE MARROW INVOLVEMENT

Background:Bone marrow (BM) is not affected in most patients with Diffuse Large B‐cell Lymphoma (DLBCL). However, it has been shown that multipotent mesenchymal stromal cells (MSCs) in DLBCL patients differ from those in healthy donors. MSCs are involved in the formation of a niche for hematopoietic stem cells and secrete many regulatory proteins. Total cellular MSC production from DLBCL patients before and one month after treatment was 1.5 times higher than in healthy donors. At the same time, the relative expression level of BMP4, MMP2, FGFR1, ICAM1 genes was reduced in MSCs from primary patients.Aims:The aim of the study was to investigate the changes in proteome profiles of secretomes from MSCs, isolated from the BM of DLBCL patients and from healthy donors.Methods:The study included 28 DLBCL patients before and one month after treatment and 31 MSC control samples from the BM of healthy donors. MSCs were isolated from the BM by the standard method and secretomes were generated for 24 h. We performed LC‐MS/MS analysis of secretomes of MSCs for 5 patients before and after chemotherapy and 5 donors using Q Exactive HF.Results:We identified 599 up‐regulated proteins and 122 down‐regulated proteins in MSC secretomes from primary patients compared to donors. Among the up‐regulated proteins the molecules of the major HLA 1 class were observed. This may be due to the increased formation of microvesicles. Moreover, functional analysis of up‐regulated proteins demonstrated that the MSC secretomes from primary patients were enriched with proteins involved in vesicle transport (133); regulating of exocytosis (78); immune response (71); proteasome (14). 18 proteins have known functions for tumors; 10 are indicators of rheumatoid arthritis; 10 are associated with signaling pathways of IL‐17 and TNF; 80 proteins are associated with both intracellular and external transport and 30 proteins are associated with transport through the Golgi apparatus, which explains significant difference between the secretomes of MSCs from healthy donors and patients before treatment. 83 elevated proteins are associated with adhesion and its regulation. Patients’ MSCs have elevated proteins associated with signal transmission PDGFR, IL‐12, FGFR.Down‐regulated proteins were involved in the regulation of the immune response, wound healing and inflammation.Of these, 7 proteins are components of PI3K‐Akt signaling pathway; 5 were Rap1 signaling pathway proteins and 5 proteins were associated with Ras signaling pathway. Comparison of proteins before treatment with proteins after treatment revealed a decrease of 366 proteins and an increase of 270.Summary/Conclusion:We show for the first time, that the secretomes from DLBCL patient's MSCs were dramatically altered in comparison with donor's ones. We can conclude that the existence of a lymphoid tumor in an organism, even without BM involvement, leads to very strong changes in the stromal cells.Supported by grant from the Russian Foundation for Basic Research, Project № 17–00‐00170

Incremental effects of diabetes mellitus and chronic kidney disease in medial arterial calcification: Synergistic pathways for peripheral artery disease progression.

Diabetes mellitus (DM) and chronic kidney disease (CKD) separately are known to facilitate the progression of medial arterial calcification (MAC) in patients with symptomatic peripheral artery disease (PAD), but their combined effect on MAC and associated mediators of calcification is not well studied. The association of MAC and calcification inducer bone morphogenetic protein (BMP-2) and inhibitor fetuin-A, with PAD, is well known. Our aim was to investigate the association of MAC with alterations in BMP-2 and fetuin-A protein expression in patients with PAD with DM and/or CKD. Peripheral artery plaques (50) collected during directional atherectomy from symptomatic patients with PAD were evaluated, grouped into no-DM/no-CKD (n = 14), DM alone (n = 10), CKD alone (n = 12), and DM+CKD (n = 14). MAC density was evaluated using hematoxylin and eosin, and alizarin red stain. Analysis of inflammation, neovascularization, BMP-2 and fetuin-A protein density was performed by immunohistochemistry. MAC density, inflammation grade and neovessel content were significantly higher in DM+CKD versus no-DM/no-CKD and CKD (p < 0.01). BMP-2 protein density was significantly higher in DM+CKD versus all other groups (p < 0.01), whereas fetuin-A protein density was significantly lower in DM+CKD versus all other groups (p < 0.001). The combined presence of DM+CKD may be associated with MAC severity in PAD plaques more so than DM or CKD alone, as illustrated in this study, where levels of calcification mediators BMP-2 and fetuin-A protein were related most robustly to DM+CKD. Further understanding of mechanisms involved in mediating calcification and their association with DM and CKD may be useful in improving management and developing therapeutic interventions.

Can follicular Emmprin and BMP 4 levels predict ICSI outcome?

To evaluate the relationship of clinical pregnancy rates with bone morphogenetic proteins 2-4-7 (BMP 2, 4, 7), growth differentiation factor 9 (GDF 9), and Emmprin levels in follicular fluid of intracytoplasmic sperm injection patients. Follicular fluid of 77 patients who underwent ICSI procedure was collected during the oocyte retrieval procedure. And follicular fluid levels of BMP 2, BMP 4, BMP 7, GDF 9, and Emmprin (Basigin) were measured and compared for clinical pregnancy rates. Follicular levels of BMP 4 was significantly higher whereas Emmprin levels were lower in patients who had achieved clinically diagnosed pregnancy compared with those who did not achieve clinical pregnancy after ICSI procedure (P= 0.007 and P= 0.035, respectively). BMP 2, BMP 7, and GDF 9 levels were comparable for both groups. Clinical pregnancy rates after ICSI may be associated with follicular fluid levels of Emmprin and BMP 4. Follicular levels of Emmprin and BMP 4 can be used as a marker (as markers for predicting ICSI outcomes) for a better ICSI outcome.

NOD2 regulates distinct pathways of endogenous thymic regeneration after injury

Abstract Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Our recent studies have identified two key pathways for thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 from innate lymphoid cells (ILCs), which can be utilized individually as therapeutic strategies of immune regeneration. The specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Here we detail an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in orchestrating these distinct pathways of thymic regeneration. After acute injury, Nod2−/− mice exhibited superior overall thymic regeneration, consistent with increased levels of BMP4, IL-22, and Il-23, a key regulator of IL-22 secreted by dendritic cells (DCs). Furthermore, expression of miR29, a downstream effector of NOD2, was significantly reduced in ECs and DCs after injury, and miR29 mimics suppressed Bmp4 and Il23 expression in ECs or DCs, respectively. Consistent with a non-canonical role for NOD2 in sensing RhoGTPases, we found several RhoGTPase family members were downregulated after TBI; and modulation with small molecule inhibitors targeting Rho GTPases led to robust production of BMP4 and IL-23, with enhanced thymic regeneration after acute injury. These findings provide evidence not only of a novel master regulator of endogenous thymic regeneration, but also offer a superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by infection or cytoreductive therapy.

Expression of BMP-4 and Smad1 in patients with Hirschsprung disease and its clinical significance.

Expression and clinical significance of bone morphogenetic protein (BMP)-4 and Smad1 in patients with Hirschsprung disease (HD) were investigated. A retrospective analysis of 96 HD patients (experimental group) admitted to Xuzhou Children's Hospital, Xuzhou Medical University from June 2015 to June 2017 was performed. According to the samples, the experimental group was divided into the stenosis group, the transition group and the expansion group. Forty-seven children with colostomy due to intestinal obstruction were selected as the control group. The expression levels of BMP-4 and Smad1 proteins were detected by immunohistochemical staining. The expression levels of BMP-4 and Smad mRNA were detected by real-time quantitative PCR (RT-qPCR), and were quantified and compared. Via immunohistochemistry, BMP-4 and Smad1 proteins were detected in the samples of different parts of HD patients and children with intestinal obstruction. The positive expression levels of BMP-4 and Smad1 proteins in the transition group were decreased compared with those in the expansion and control groups (P<0.05), and the positive expression levels of BMP-4 and Smad1 proteins in the stenosis group were decreased compared with those in the transition, expansion, and control groups (P<0.05). Also, the gene expression levels of BMP-4 and Smad1 in the transition and stenosis groups were successively decreased, and the differences were statistically significant (P<0.05). In conclusion, the expression of BMP-4 and Smad1 in the intestinal plexus of HD lesions was significantly reduced, indicating that BMP-4 and Smad1 are closely related to the occurrence of HD, and it is suspected that they have a certain influence on the intestinal development of congenital digestive tract malformations.

The evaluation of embryotoxicity of Ligusticum chuanxiong on mice and embryonic stem cells

Ethnopharmacological relevanceChuanxiong (Chuanxiong Rhizoma, CR), the dried rhizome of Ligusticum chuanxiong Hort, has been used during pregnancy for more than 2000 years. However, the embryotoxicity of CR was not evaluated so far. The purpose of this study was to examine the safety and rational use of CR during pregnancy on mice and mouse embryonic stem cell (ES), and to explore the mechanism of embryotoxicity. Aim of the studyThis study was carried out to evaluate embryotoxicity of CR decoction in vivo and in vitro, and to explore the mechanism of embryotoxicity from the perspective of bone metabolism. Materials and methodsIn animal experiments, pregnant mice were randomly assigned into 5 groups, i.e. mice were orally treated with CR decoction at dosages of 0 (distilled water, as negative controls), 2, 8, 32 g/kg/d (low, medium and high-dose group), and vitamin A (as positive controls), respectively. Maternal and embryo-fetal parameters were registered after cesarean section. The fetal skeletal development was further assessed with the alizarin red S and Hematoxylin-Eosin staining (H&E staining) and fluorescent imaging. Meanwhile, the mouse embryonic stem cell test model (EST model) was established to objectively evaluate the toxicity of CR on the embryo development. The median inhibitory proliferation values (IC50) for both the mouse embryonic stem cell D3 (ES) and mouse embryonic fibroblast 3T3 (3T3) were detected with MTT assays. After removal of inhibiting factor (LIF), mouse embryonic stem cells spontaneously differentiated into cardiomyocytes, the expression of specific myosin heavy chain gene (β-MHC) contained in cardiomyocytes were detected by q-PCR quantitative analysis, and median inhibitory differentiation concentration (ID50) of ES was obtained. The development toxicity calculation formula was used to determine the embryotoxicity grade of CR decoction. finally, based on the successful induction of osteoblasts, the molecular mechanism of CR embryotoxicity was preliminarily studied based on BMP-Smads signal pathway. ResultsCompared with the negative control group, high, medium, and low doses of CR decoction had no significant effect on the maternal body weight and uterine weight (P > 0.05), as well as on the maternal liver, heart, and kidneys. The observation results showed that high dose of CR decoction significantly increase the number of absorbed fetuses (P < 0.05). The EST model was successfully established, the IC50 3T3, IC50 ES and ID50 ES of CR were 9.39 mg/mL, 18.78 mg/mL, and 10.20 mg/mL, respectively. CR was classified as weak embryonic development toxicity by the EST linear discriminant formula. Meanwhile, osteoblasts were successfully induced in vitro, the relative expression levels of BMP2, BMPR2, Smad1, and Smad5 were down-regulated in varying degrees after 3, 6, and 9 days of treatment with different concentration gradients of CR decoction. ConclusionsCombining in vivo and in vitro experiments, CR showed a potential embryotoxicity. The mechanism of embryotoxicity may be related to inhibiting the expression of key genes in the BMP-SMADs signaling pathway. In the clinical application, the normal dosage of CR is safe to a certain extent. However, when the dosage is too high (160 g/60 kg/d), there may be a risk of embryotoxicity.

Astragalin Promotes Osteoblastic Differentiation in MC3T3-E1 Cells and Bone Formation in vivo.

Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 μM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis.

YAP, ΔNp63, and β-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness.

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche’s distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and β-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/β-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

The concomitant loss of APC and HNF4α in adult hepatocytes does not contribute to hepatocarcinogenesis driven by β-catenin activation.

Loss of hepatocyte nuclear factor-4α (HNF4α), a critical factor driving liver development and differentiation, is frequently associated with hepatocellular carcinoma (HCC). Our recent data revealed that HNF4α level was decreased in mouse and human HCCs with activated β-catenin signalling. In addition, increasing HNF4α level by miR-34a inhibition slowed tumour progression of β-catenin-activated HCC in mice. We generated a Hnf4aflox/flox/ Apcflox/flox /TTR-CreERT2 (Hnf4a/Apc∆Hep ) mouse line and evaluated the impact of Hnf4a disruption on HCC development and liver homoeostasis. There was no significant impact of Hnf4a disruption on tumour onset and progression in Apc∆Hep model. However, we observed an unexpected phenotype in 28% of Hnf4a∆Hep mice maintained in a conventional animal facility, which presented disorganized portal triads, characterized by stenosis of the portal vein and increased number and size of hepatic arteries and bile ducts. These abnormal portal structures resemble the human idiopathic non-cirrhotic portal hypertension syndrome. We correlated the presence of portal remodelling with a higher expression of protein and mRNA levels of TGFβ and BMP7, a key regulator of the TGFβ-dependent endothelial-to-mesenchymal transition. These data demonstrate that HNF4α does not play a major role during β-catenin-driven HCC, thus revealing that the tumour suppressor role of HNF4α is far more complex and dependent probably on its temporal expression and tumour context. However, HNF4α loss in adult hepatocytes could induce abnormal portal structures resembling the human idiopathic non-cirrhotic portal hypertension syndrome, which may result from endothelial- and epithelial-to-mesenchymal transitions.

The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo.

Cleft lip with or without cleft palate (CLP) usually results from a failure of the medial nasal prominences to fuse with the lateral and maxillary prominences. This failure inhibits facial morphogenesis regulated by several major morphogenetic signaling pathways. We hypothesized that CLP results from the failure of the Wnt signaling pathway. To examine whether Wnt signaling can influences upper jaw development, we applied beads soaked with Dickkopf-1 (Dkk-1), Alsterpaullone (AL) or Wnt3a to the right side of the maxillary prominence of the chick embryo. The embryo showed a defect of the maxilla on the treated side, and skeletal staining revealed hypoplasia of the premaxilla and palatine bone as a result of Dkk-1-soaked bead implantation. 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers in the treated maxillary prominence were significantly lower at both 24 and 48 hr after implantation. Down-regulation of the expression of Bmp4, Tbx22, Sox9, and Barx1 was confirmed in the maxillary prominence treated with Dkk-1, which indicated that the deformity of the maxillary bone was controlled by gene targets of the Wnt signaling pathway. Expression of N-cadherin was seen immunohistochemically in the maxillary prominences of embryos at 6 hr and increased at 24 hr after AL treatment. Wnt signaling enhanced by AL or Wnt3a up-regulated the expression levels of Msx1, Bmp4, Tbx22, Sox9, and Barx1. Our data suggest that the Wnt signaling pathway regulates maxillary morphogenesis and growth through Bmp4, Tbx22, Sox9, and Barx1. Wnt signaling might regulate N-cadherin expression via Msx1, resulting in cell aggregation for osteochondrogenesis.

Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity.

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

Zinc Improves Thymic Regeneration after Allogeneic HSCT By Stimulating BMP4 Production from Endothelial Cells

Delayed T cell recovery after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal immune reconstitution requires a functional thymus, however, at the present time strategies to improve thymic reconstitution are limited. Zinc (Zn) is the second most abundant trace metal in the body, binding to more than 300 proteins. Zn deficiency is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, the role of Zn in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we previously demonstrated that patients receiving oral Zn supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events. Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of Zn supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received Zn supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, in our model of Zn administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that Zn supplementation is activating the BMP4 pathway, thymic levels of BMP4 were increased at day +10 in zinc-treated mice (Fig. 2a, 2b), and, when stimulated in vitro for 24 hours with Zn sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 2c), suggesting a likely mechanism by which Zn supplementation promotes thymic reconstitution. EC also express on their surface the Zn receptor GPR39 (Fig 3a), which is able to translate extracellular Zn signals in intracellular changes, and upregulate GPR39 expression after damage (Fig 3b). In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

Bone morphogenetic protein (BMP) 7 expression is regulated by the E3 ligase UBE4A in diabetic nephropathy

Mesangial cells played a central role in the pathophysiology of diabetic nephropathy (DN). Our goal was to evaluate the molecular mechanism that regulates loss of BMP7 protein expression in DN. The mRNA and protein levels of BMP7 or UBE4A were detected using qRT-PCR and Western blot respectively. Mass spectrometry and co-immunoprecipitation were used to explore the E3 ligase which regulated BMP7 post-translationally. We initially confirmed that BMP7 protein, but not mRNA, is downregulated when cultured under high glucose mimicking DN conditions, which was rescued by MG-132 treatment. Proteomic analysis of NRK-52E cells ± MG-132 revealed a list of ubiquitin ligases associated with BMP7. Knockdown of the ubiquitin ligase UBE4A stabilized BMP7 expression in NRK-52E cells grown under high glucose conditions. Concurrent overexpression experiments confirmed that UBE4A is the ubiquitin ligase that degrades BMP7. Co-immunoprecipitation analysis confirmed that BMP7 and UBE4A interact. BMP7 expression in DN is regulated by post-translational mechanism.

Oviductus Ranae protein hydrolyzate prevents menopausal osteoporosis by regulating TGFβ/BMP2 signaling.

It is known that menopausal osteoporosis (MOP) is the most typical form of osteoporosis, which is characterized by low bone mass and microstructure damage of the bone tissue, leading to increased bone fragility and risk of fracture. This study aimed to evaluate the protective effects of Oviductus Ranae protein hydrolyzate (ORPH) on the MOP in vivo. Osteoporosis model was induced by ovariectomy, treated with ORPH 150 or 75mgkg-1. Body weight and bone mineral density (BMD) of rats were measured at the beginning and the end of the experiment, and femoral maximum load was determined immediately after killing. The expression levels of alkaline phosphatase (ALP), Smad4, tartrate acid phosphatase (TRAP), BMP2, Runx2, CPB, ColI and osteocalcin were examined by RT-PCR or western-blotting. HE staining was used to observe the pathological changes in the femurs. Immunohistochemistry was used to detect the expression of ALP and BMP2. All data were analyzed by SPSS 13.0. The results revealed that ORPH had no effect on the weight of normal and osteoporotic rats. ORPH could significantly improve the femur BMD and increase the maximum load of the osteoporotic rats. ORPH could significantly upregulate the expression level of bone formation makers, ALP, osteocalcin, ColI, and Runx2, and downregulate the expression level of bone resorption marker, TRAP. In the ORPH group, the expression levels of BMP2, Smad4, and CPB of key proteins in the TGFβ/BMP2 signaling pathway were significantly upregulated. In addition, immunohistochemistry showed that ALP and BMP2 expression in femurs of the ORPH group was stranger. H&E staining showed that ORPH (150mgkg-1) significantly increased the thickness of trabeculae and decreased fracture risk. Collectively, ORPH plays a role in the prevention and treatment of osteoporosis, which may be a potential anti-osteoporosis drug.

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion.

The short half‐life and use of recombinant bone morphogenetic protein (BMP)2 in large doses poses major limitations in the clinic. Events regulating posttranslational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Toward identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin‐mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro‐BMP2 region and three in the mature region increased both BMP2 stability and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest that preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half‐life.

Infection of Rat Osteoblasts with Junin Virus Promotes the Expression of Bone Morphogenetic Protein 6, an Osteogenic Differentiation Inducer

Background: The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication. Objectives: We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication. Method: Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed. Results: Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 10⁵ PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 μM ATPγS reduced the stimulation of BMP-6 expression induced by the infection. Conclusions: These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process.

Cyclophilin B enhances the proliferation and differentiation of MC3T3-E1 cells via JAK2/STAT3 signaling pathway

The aim of this study was to investigate the effects of cyclophilin B on MC3T3-E1 cells and the underlying mechanism. CCK-8 assay was used to evaluate the cell activity, polymerase chain reaction to verify the overexpression of cyclophilin B, western blot to detect the protein levels, and clone formation assay to evaluate the proliferation ability. Alkaline phosphatase (ALP) activity was assessed by ALP assessment kit. The results showed that compared with the control group/negative control (NC) group, the cells in the cyclophilin B overexpression group possessed enhanced proliferation ability, higher ALP activity and enhanced mineralization. Compared with the control group, the levels of Oct4, Sox2, Runx2, collagen I, BMP2 and BMPR-II, as differentiation-related proteins, were increased by cyclophilin B. Compared to the NC group, the expression levels of p-JAK2 and p-STAT3 in the cyclophilin B overexpression group were higher, suggesting that cyclophilin B can activate the JAK2/STAT3 signaling pathway. The expression levels of p-JAK2 and p-STAT3 in the cyclophilin B overexpression group were higher than that in the cyclophilin B overexpression + JAK2 inhibitor group, further confirming the hypothesis that the JAK2/STAT3 signaling pathway was activated by cyclophilin B. Taken together, the results suggest that cyclophilin B promotes the proliferation and differentiation of MC3T3-E1 cells via the JAK2/STAT3 signaling pathway and may be a promising therapeutic target for bone injury.