Bone Morphogenetic Protein Antagonist Research Articles

SMAD6 is frequently mutated in nonsyndromic radioulnar synostosis

PurposeRadioulnar synostosis (RUS) can be syndromic or nonsyndromic. The genetic basis for several RUS syndromes have been reported. However, the genetic cause of nonsyndromic RUS (nsRUS) remains unknown. MethodsWe performed Giemsa (GTG) banding, Sanger sequencing, and exome sequencing on patients (n = 140) and families(n = 11) who suffered from RUS. ResultsGTG banding identified 10% RUS sporadic cases affected by sex chromosome aneuploidy. Sanger sequencing on candidate genes revealed noggin(NOG) rarely mutated in nsRUS. Exome sequencing identified 16 loss-of-function (LOF) and 6 missense variants (minor allele frequency [MAF] < 0.0001) in 22/117 nsRUS sporadic patients. Genetic association analysis found a significant association between SMAD6-LOF variants and nsRUS risk (odds ratio[OR] = 430, 95% confidence interval [CI]: 238–780, P < 0.000001). SMAD6 mutated in nsRUS was further confirmed by direct Sanger sequencing of SMAD6-coding regions on other unrelated cohorts of nsRUS cases or families. In summary, we detected 27 SMAD6 rare variants in nsRUS, most of which were LOF variants, 4 were de novo, and 3 were transmitted in families with autosomal dominant inheritance. ConclusionAs an intracellular bone morphogenetic protein (BMP) antagonist gene, SMAD6 is frequently mutated in nsRUS.NOG, which encodes an extracellular BMP antagonist, is rarely mutated in nsRUS. This work is the first genetic study on nsRUS.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

IntroductionAdipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes.MethodsTo examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12–97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5–90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs.ResultsIn adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/β-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis.ConclusionsThe GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

Gremlin-1 potentiates the dedifferentiation of VSMC in early stages of atherosclerosis

Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to pro-atherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. ResultsComparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22-alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4–6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. ConclusionOur data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.

Chrysosplenetin promotes osteoblastogenesis of bone marrow stromal cells via Wnt/\u03b2-catenin pathway and enhances osteogenesis in estrogen deficiency-induced bone loss

BackgroundChrysosplenetin is an O-methylated flavonol compound isolated from the plant Chamomilla recutita and Laggera pterodonta. The aim of our research is to evaluate the function of Chrysosplenetin on osteogenesis of human-derived bone marrow stromal cells (hBMSCs) and inhibition of estrogen deficiency-induced osteoporosis via the Wnt/β-catenin signaling pathway.MethodhBMSCs are cultured and treated by Chrysosplenetin in the absence or presence of Wnt inhibitor dickkopf-related protein 1 (DKK1) or bone morphogenetic protein 2 (BMP2) antagonist Noggin. RT-qPCR is taken to identify the genetic expression of target genes of Wnt/β-catenin pathway and osteoblast-specific markers. The situation of β-catenin is measured by western blot and immunofluorescence staining. An ovariectomized (OVX) mouse model is set up to detect the bone loss suppression by injecting Chrysosplenetin. Micro-CT and histological assay are performed to evaluate the protection of bone matrix and osteoblast number. Serum markers related with osteogenesis are detected by ELISA.ResultsIn the present study, it is found that Chrysosplenetin time-dependently promoted proliferation and osteoblastogenesis of hBMSCs reaching its maximal effects at a concentration of 10 μM. The expressions of target genes of Wnt/β-catenin pathway and osteoblast-specific marker genes are enhanced by Chrysosplenetin treatment. Furthermore, the phosphorylation of β-catenin is decreased, and nuclear translocation of β-catenin is promoted by Chrysosplenetin. Osteogenesis effects mentioned above are founded to be blocked by DKK1 or BMP2 antagonist Noggin. In vivo study reveals that Chrysosplenetin prevents estrogen deficiency-induced bone loss in OVX mice detected by Micro-CT, histological analysis, and ELISA.ConclusionsOur study demonstrates that Chrysosplenetin improves osteoblastogenesis of hBMSCs and osteogenesis in estrogen deficiency-induced bone loss by regulating Wnt/β-catenin pathway.

Fibroblast-derived Gremlin1 localises to epithelial cells at the base of the intestinal crypt

Gremlin1 (GREM1) is a secreted glycoprotein member of the differential screening-selected gene in aberrant neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists, which binds to BMPs preventing their receptor engagement. Previous studies have identified that stage II colorectal cancer (CRC) patients with high levels of GREM1 gene expression in their tumour tissue have a poorer prognosis. Using a series of in silico and in situ methodologies, we demonstrate that GREM1 gene expression is significantly higher (p < 0.0001) in CRC consensus molecular subtype 4 (CMS4), compared to the other CMS subtypes and correlates (p < 0.0001) with levels of cancer-associated fibroblasts (CAFs) within the CRC tumour microenvironment (TME). Our optimised immunohistochemistry protocol identified endogenous GREM1 protein expression in both the muscularis mucosa and adjacent colonic crypt bases in mouse intestine, in contrast to RNA expression which was shown to localise specifically to the muscularis mucosa, as determined by in situ hybridisation. Importantly, we demonstrate that cells with high levels of GREM1 expression display low levels of phospho-Smad1/5, consistent with reduced BMP signalling. Taken together, these data highlight a novel paracrine signalling circuit, which involves uptake of mature GREM1 protein by colonic crypt cells following secretion from neighbouring fibroblasts in the TME.

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time

SUMMARYHow broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless—Su(H)—and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer’s spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)’s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos.

Vitamin D Receptor Signaling Regulates Craniofacial Cartilage Development in Zebrafish.

Vitamin D plays essential roles in supporting the skeletal system. The active form of vitamin D functions through the vitamin D receptor (VDR). A hereditary vitamin-D-resistant rickets with facial dysmorphism has been reported, but the involvement of VDR signaling during early stages of craniofacial development remains to be elucidated. The present study investigated whether VDR signaling is implicated in zebrafish craniofacial cartilage development using a morpholino-based knockdown approach. Two paralogous VDR genes, vdra and vdrb, have been found in zebrafish embryos. Loss-of-vdra has no discernible effect on cartilage elements, whereas loss-of-vdrb causes reduction and malformation of craniofacial cartilages. Disrupting both vdra and vdrb leads to more severe defects or complete loss of cartilage. Notably, knockdown of vdrb results in elevated expression of follistatin a (fsta), a bone morphogenetic protein (BMP) antagonist, in the adjacent pharyngeal endoderm. Taken together, these findings strongly indicate that VDR signaling is required for early craniofacial cartilage development in zebrafish.

PF466 POTENTIAL COMBINATIONAL THERAPY OF LEUKEMIC STEM CELLS USING BONE MORPHOGENETIC PROTEIN AND TYROSINE KINASE INHIBITORS

Background:Chronic myeloid leukemia (CML) disease outcomes have improved since the introduction of tyrosine kinase inhibitors (TKIs), however a proportion of patients will have TKI resistance and TKIs are not curative in the majority of patients. Low‐levels of Bcr‐Abl transcripts persist and a major clinical aim is to understand mechanism underlying disease persistence and the role of leukemic stem cells (LSCs) in this. To date it has proved difficult to both understand how LSCs dominate and alter the niche, and to target LSCs with current therapies. Previous studies have highlighted the importance of the bone morphogenetic protein (BMP) pathway in CML LSCs with altered stem cell fate, persistence and the response to BMP antagonists affecting cell behavior with altered cell cycle, apoptosis and expansion (Laperrousaz, B. et al, Blood, 2013; Grockowiak, E. et al, Blood, 2017).Aims:To investigate the effect of BMP pathway inhibition in combination with TKIs in primary CML samples to improve insights and identify new potential therapeutic approaches.Methods:Primary CML CD34+cells were treated with Imatinib (IM) or Saracatinib (SC), a Src/Abl dual inhibitor, alone or in combination with BMP inhibitor Dorsomorphin (DOR). Additionally, cell cultures were supplemented with soluble BMP4 or co‐cultured with the human stromal cell line HS‐5. Apoptosis, cell cycle and proliferation were investigated by flow cytometry. RNA was harvested, cDNA generated and pre‐amplified prior to the performance of multiplex Real‐Time PCR (Fluidigm) on 89 lineage‐specific, cell cycle, proliferation, and survival genes. Furthermore, we investigated tyrosine kinase activity in cell lysates of primary CML patient samples post single or dual treatment of SC and DOR using a chip‐based microarray assay from PamGene containing 196 peptides. Kinase phosphorylation activities measured on peptides were further evaluated by relating them to kinases that are able to phosphorylate a particular site on the peptide sequence.Results:Combinatorial treatments targeting the BMP pathway together with TKIs display a synergistic mode of action. Increases in late apoptosis (p &lt; 0.0002), altered cell cycle (G2‐M, p = 0.0058), fewer cell divisions, and reduced numbers of CD34+cells were observed in dual treatments. BMP4 supplementation revealed similar differences whereas stroma co‐cultures displayed a protective effect. Furthermore, multiplex quantitative PCR has revealed significant deregulation of genes involved in cell cycle,apoptosis and differentiation, such as CDKN1a, CDKN2b, BCL6, GATA1 and SMAD5 showing differential expression in relation to response. Upstream kinase analysis using the PamGene platform, revealed kinases involved in cell growth, development, differentiation, apoptosis and cell‐cell adhesion. Amongst the top 20 potential differentially expressed kinases after SC treatment, were the expected members of the Src family kinases. DOR treatment revealed an up regulation of the largest known subfamily of receptor tyrosine kinases, the Ephrin receptors and Met, a single pass tyrosine kinase receptor that had already been identified as a potential therapy target in CML for IM resistant cells.Summary/Conclusion:Our results highlight the potential of a combined approach of targeting the BMP pathway with BMP antagonists or small inhibitors together with TKIs, which could open up new therapeutic possibilities. Future experiments will assess the potential of combinatorial treatment in a 3D bone marrow model, comprising magnetically levitated Mesenchymal Stem Cell spheroids embedded in medical‐grade collagen type I.

Noggin depletion in adipocytes promotes obesity in mice

ObjectiveObesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis. MethodsWe generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the AdiponectinCre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency. ResultsOur studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO2, VCO2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice. ConclusionsBMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity.

Nox1/Ref-1-mediated activation of CREB promotes Gremlin1-driven endothelial cell proliferation and migration

Pulmonary arterial hypertension (PAH) is a complex degenerative disorder marked by aberrant vascular remodeling associated with hyperproliferation and migration of endothelial cells (ECs). Previous reports implicated bone morphogenetic protein antagonist Gremlin 1 in this process; however, little is known of the molecular mechanisms involved. The current study was designed to test whether redox signaling initiated by NADPH oxidase 1 (Nox1) could promote transcription factor CREB activation by redox factor 1 (Ref-1), transactivation of Gremlin1 transcription, EC migration, and proliferation. Human pulmonary arterial EC (HPAECs) exposed in vitro to hypoxia to recapitulate PAH signaling displayed induced Nox1 expression, reactive oxygen species (ROS) production, PKA activity, CREB phosphorylation, and CREB:CRE motif binding. These responses were abrogated by selective Nox1 inhibitor NoxA1ds and/or siRNA Nox1. Nox1-activated CREB migrated to the nucleus and bound to Ref-1 leading to CREB:CRE binding and Gremlin1 transcription. CHiP assay and CREB gene-silencing illustrated that CREB is pivotal for hypoxia-induced Gremlin1, which, in turn, stimulates EC proliferation and migration. In vivo, participation of Nox1, CREB, and Gremlin1, as well as CREB:CRE binding was corroborated in a rat PAH model. Activation of a previously unidentified Nox1-PKA-CREB/Ref-1 signaling pathway in pulmonary endothelial cells leads to Gremlin1 transactivation, proliferation and migration. These findings reveal a new signaling pathway by which Nox1 via induction of CREB and Gremlin1 signaling contributes to vascular remodeling and provide preclinical indication of its significance in PAH.

Reduced levels of Coco in sera of multiple sclerosis patients: A potential role in neuro-regeneration failure

Demyelination, axonal loss and failure of tissue repair characterize MS lesions. Bone morphogenetic proteins (BMPs) signaling is associated with remyelination failure. Coco is one of the BMP antagonists. We found reduced Coco serum levels in relapsing-remitting MS (RR-MS) and primary progressive MS (PP-MS) patients compared to matched healthy controls (HC) and patients with rheumatoid arthritis. Exposure of P19 cells, in the presence of retinoic acid, BMP-2, or BMP-4 to Coco, at average sera level of MS patients failed to induce neuronal phenotype, in contrast to the average sera level of HC. Coco may be a player in the BMP dysregulation and the tissue repair failure in MS.

Febuxostat inhibits TGF‑β1‑induced epithelial‑mesenchymal transition via downregulation of USAG‑1 expression in Madin‑Darby canine kidney cells invitro.

Our previous study demonstrated that febuxostat, a xanthine oxidase inhibitor, can alleviate kidney dysfunction and ameliorate renal tubulointerstitial fibrosis in a rat unilateral ureteral obstruction (UUO) model; however, the underlying mechanisms remain unknown. Increasing evidence has revealed that epithelial-mesenchymal transition (EMT) is one of the key mechanisms mediating the progression of renal tubulointerstitial fibrosis in chronic kidney disease (CKD). Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific bone morphogenetic protein antagonist, is involved in the development of numerous types of CKDs. The present study aimed to investigate the role of febuxostat in the process of EMT in Madin-Darby canine kidney (MDCK) cells in vitro. Western blotting, reverse transcription-semiquantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression levels of bone morphogenetic protein 7, USAG-1, α-smooth muscle actin (α-SMA) and E-cadherin, respectively. The results demonstrated that the expression of USAG-1 and α-SMA increased, and that of E-cadherin decreased significantly in MDCK cells following treatment with transforming growth factor-β1 (TGF-β1). The application of small interfering RNA-USAG-1 potently inhibited TGF-β1-induced EMT. Subsequently, the effects of febuxostat on TGF-β1-induced EMT was investigated. The results demonstrated that febuxostat downregulated the expression of USAG-1, and reversed TGF-β1-induced EMT in MDCK cells. Furthermore, pretreatment with febuxostat significantly restored the decreased expression levels of phosphorylated Smad1/5/8 induced by TGF-β1 in MDCK cells. The results of the present study suggested that USAG-1 may be involved in the EMT process of MDCK cells induced by TGF-β1, and febuxostat inhibited EMT by activating the Smad1/5/8 signaling pathway via downregulating the expression of USAG-1 in MDCK cells.

Denosumab effects on serum levels of the bone morphogenetic proteins antagonist noggin in patients with transfusion-dependent thalassemia and osteoporosis

ABSTRACTIntroduction: Noggin is an antagonist of bone morphogenetic proteins (BMPs) and has a strong effect on osteogenesis. Osteoporosis is a common complication of transfusion dependent beta-thalassemia (TDT) and denosumab has been recently emerged as a promising therapeutic option. This was a post hoc investigation of serum noggin levels among TDT patients with osteoporosis who participated in a randomized, placebo-control, phase 2b study.Methods: Patients received either 60 mg denosumab (n = 32) or placebo (n = 31) every 6 months for 12 months. Noggin was measured, for the first time in thalassemia patients, at baseline and at 12 months, using a recently developed high sensitivity fluorescent immunoassay.Results: Both groups showed a significant increase in noggin serum levels (denosumab p < 0.001; placebo p < 0.0001). Interestingly, the increase was higher in the placebo group. Furthermore, we observed a strong correlation between noggin and wrist bone mineral density (r = −0.641, p = 0.002) only in the denosumab group.Conclusion: In conclusion, higher noggin levels reflected more BMP inhibition, since our assay detects free bioactive noggin, which in turn impaired bone formation in placebo group. Therefore, denosumab possibly regulates noggin and favours bone turnover in TDT patients with osteoporosis through a novel mechanism of action.Trial registration: ClinicalTrials.gov identifier: NCT02559648.

Gingival fibroblasts prevent BMP-mediated osteoblastic differentiation.

ObjectivesThe inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)‐induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation.MethodsGingival fibroblasts were co‐cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP‐inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT‐qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads.ResultsCo‐culture experiments showed GF‐secreted factors that inhibit BMP‐stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well‐described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity.ConclusionsFactors secreted by gingival fibroblasts inhibit BMP‐induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.

Upregulated BMP-Smad signaling activity in the glucuronyl C5-epimerase knock out MEF cells

Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce, in mice resulted in neonatal lethality with varied defects in organ development. To understand the underlying molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activitywhen cultured in osteogenic medium. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.

Knockdown GREM1 suppresses cell growth, angiogenesis, and epithelial-mesenchymal transition in colon cancer.

Gremlin 1 (GREM1), as a bone morphogenetic protein (BMP) antagonist and vascular endothelial growth factor receptor-2 (VEGFR2) novel agonist, has been confirmed as overexpressed in colorectal cancer (CRC) tissues but its role in carcinogenesis remains unclear. Here we reported that the GREM1 expression in mesenchymal-like colon cancer cells (SW620 and SW480) was significantly higher than that of epithelial-like colon cancer cells (Caco-2, HTC116, and HT29) and normal colon cell. Simultaneously, we analyzed two series of CRC transcriptomes from Gene Expression Omnibus (GEO) databases and found the great majority of primary CRC tissues expressed high level of GREM1 messenger RNA (mRNA) compared with adjacent normal tissues, and that the GREM1 mRNA expression is correlated with low histological grade development and stage 2 to 3 metastatic recurrence in CRC based on a data analysis of 104 different stage CRC tissue from the GEO databases. Functional studies showed that GREM1 silencing by short hairpin RNA (shRNA) significantly inhibited CRC cells proliferation, migration, the formation of vascular endothelial growth factor (VEGF)-induced capillary structure of human umbilical vein endothelial cells (HUVECs),and epithelial-mesenchymal transition in colon cancer cells by repressing phosphorylation levels of BMP downstream signal Smad1, vascular endothelial growth factor (VEGF) downstream signal matrix metallopeptidase 2 (MMP2), and metastasis-related factor C-X-C motif chemokine ligand 12 (CXCL12) expression. In addition, shGREM1 combined with VEGF inhibitor BAW2881 displayed more effective antiangiogenesis to inhibit the tube formation of HUVEC. Hence, these experiments demonstrated that GREM1 is involved in CRC development and procession and provide a new idea for CRC diagnosis, resistance therapy, and prognosis.

Astrocyte-Secreted Chordin-like 1 Drives Synapse Maturation and Limits Plasticity by Increasing Synaptic GluA2 AMPA Receptors

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes invivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an invivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.

Calcification Induced by Type I Interferon in Human Aortic Valve Interstitial Cells Is Larger in Males and Blunted by a Janus Kinase Inhibitor.

Objective- Calcific aortic valve disease is the most prevalent valvulopathy in Western countries. An unanticipated pathogenetic clue involving IFN (interferon) was disclosed by the finding of constitutive type I IFN activity associated with aortic valve calcification in children with the atypical Singleton-Merten syndrome. On this basis, the role of type I IFN on inflammation and calcification in human aortic valve interstitial cells (AVIC) was examined. Approach and Results- IFN-α was weakly proinflammatory but potentiated lipopolysaccharide-mediated activation of NF (nuclear factor)-κB and the ensuing induction of proinflammatory molecules in human AVIC. Stimulation with IFN-α and in combination with lipopolysaccharide promoted osteoblast-like differentiation characterized by increased osteoblastic gene expression, BMP (bone morphogenetic protein)-2 secretion, and ectopic phosphatase activity. Sex differences were observed. Likewise, IFN-α treatment of human AVICs in osteogenic medium resulted in increased formation of calcific nodules. Strikingly, IFN-α-mediated calcification was significantly higher in AVICs from males, and was blocked by tofacitinib, a JAK (Janus kinase) inhibitor, and by a BMP antagonist. A female-specific protective mechanism involving the activation of PI3K-Akt (protein kinase B) pathways and cell survival was disclosed. Females exhibited higher levels of BCL2 in valve cells and tissues and lower annexin V staining on cell stimulation. Conclusions- IFN-α acts as a proinflammatory and pro-osteogenic cytokine in AVICs, its effects being potentiated by lipopolysaccharide. Results also uncovered sex differences with lower responses in female AVICs and sex-specific mechanisms involving apoptosis. Data point to JAK/STAT (signal transducer and activator of transcription) system as a potential therapeutic target for calcific aortic valve disease.

BMP-7/Smad expression in dedifferentiated Schwann cells during axonal regeneration and upregulation of endogenous BMP-7 following administration of PTH (1-34)

To determine the expression and distribution of bone morphogenetic protein (BMP)-7 and related molecules during peripheral nerve regeneration and to assess whether administration of parathyroid hormone (PTH) drug (1-34) potentiates the intrinsic upregulation of BMP-7/Smad signaling. The rat sciatic nerves were crushed with an aneurysm clip resulting in axonal degeneration. In the normal nerve, and at 1, 2, 4, and 8 weeks after injury, BMP-7, BMP receptors, p-Smad 1/5/8, and Noggin, the endogenous BMP antagonist, were evaluated. Additionally, the distribution of BMP-7 was assessed by fluorescent double immunostaining. In vitro studies were also performed to examine the effect of BMP-7 and PTH (1-34) administration on rat Schwann cells (SCs). Aneurysm clip made reliable animal model of the nerve injury with recovery at 8 weeks after the injury. BMP-7/Smad protein and mRNA were significantly upregulated on axon-SCs units at 1 week after injury, and this upregulated expression was maintained for 4 weeks. Besides, significant upregulation of Noggin's expression was observed on axon-SCs units at 2 weeks after injury. Moreover, fluorescent double immunostaining showed co-localization between expression of BMP-7 and p75NTR during axonal regeneration. In the in vitro study, administration of BMP-7 induced significant proliferation of SCs. Application of PTH (1-34) upregulated BMP-7 on SCs. BMPs were reported to be involved in protection and recovery after injury as well as in neurogenesis. Our current study showed that BMP/Smad signaling molecules were upregulated on dedifferentiated SCs after peripheral nerve injury and that administration of BMP-7 increased SC viability in vitro. These results suggested that axonal regeneration could be induced via upregulation of endogenous BMP-7 on SCs by PTH (1-34) administration.

Bisphosphonates reduce biomaterial turnover in healing of critical-size rat femoral defects.

Treatment of osteoporotic patients with bisphosphonates (BPs) preserves bone mass and microarchitecture. The high prescription rate of the drugs brings about increases in the numbers of fractures and bone defects requiring surgical interventions in these patients. Currently, critical-size defects are filled with biomaterials and healing is supported with bone morphogenetic proteins (BMP). It is hypothesized that BPs interfere with biomaterial turnover during BMP-supported repair of defects filled with β-tricalcium phosphate (βTCP) ceramics. To test this hypothesis, retired breeder rats were ovariectomized ( OVX). After 8 weeks, treatment with alendronate (ALN) commenced. Five weeks later, 6 mm diaphyseal femoral defects were applied and stabilized with locking plates. βTCP cylinders loaded with 1 μg and 10 μg BMP2, 10 μg L51P, an inhibitor of BMP antagonists and 1 μg BMP2/10 μg L51P were fitted into the defects. Femora were collected 16 weeks post-implantation. In groups receiving calcium phosphate implants loaded with 10 μg BMP2 and 1 μg BMP2/10 μg L51P, the volume of bone was increased and βTCP was decreased compared to groups receiving implants with 1 μg BMP2 and 10 μg L51P. Treatment of animals with ALN caused a decrease in βTCP turnover. The results corroborate the synergistic effects of BMP2 and L51P on bone augmentation. Administration of ALN caused a reduction in implant turnover, demonstrating the dependence of βTCP removal on osteoclast activity, rather than on chemical solubility. Based on these data, it is suggested that in patients treated with BPs, healing of biomaterial-filled bone defects may be impaired because of the failure to remove the implant and its replacement by authentic bone.