Bone Morphogenetic Protein Receptor 1A Research Articles

Correlation between intestinal stem cell niche changes and small bowel crypt failure in patients with treatment-naïve celiac disease.

We hypothesized that crypt failure in the small bowel results in villous flattening in patients with celiac disease (CeD). We investigated whether alterations in the stem cell niche (ISC) are responsible for this phenomenon. We included 92 duodenal (D2/3) biopsies from treatment-naive patients of CeD and 37 controls. All underwent screening for serum anti-tissue transglutaminase and endoscopic upper small bowel biopsy. Immunohistochemical markers were used to investigate ISC niche alterations, including LGR5 for crypt basal cells (CBC), Bmi1 for position 4+ cells, β-Defensin for Paneth cells, R-spondin1 as WNT activator, transcription factor-4 as WNT transcription factor, BMP receptor1A as WNT inhibitor, fibronectin-1 as periepithelial stromal cell marker, H2AX as apoptosis marker, and Ki67 as proliferation marker. We also analyzed IgA anti-tTG2 antibody deposits by using dual-color immunofluorescence staining. We found that in biopsies from patients with treatment-naive CeD with modified Marsh grade 3a-3c changes, the epithelial H2AX apoptotic index was upregulated than in controls. LGR5+ crypt basal cells were upregulated in all modified Marsh grades compared to controls. However, the Ki67 proliferation index, expressions of WNT-activator RSPO1, and position-4 cell marker Bmi1 did not significantly alter in patients' biopsies as compared to controls (P = 0.001). We also observed depletion of pericrypt stromal fibronectin-1 in patients with CeD compared to controls. In addition, we identified IgA anti-TG2 antibody deposits in pericrypt stroma. Our data suggests that ISC niche failure is a plausible hypothesis for villous flattening in patients with CeD, resulting from pericrypt IgA anti-TG2 antibody complex-mediated stromal depletion.

A26 BMP-SIGNALING IMPAIRED TELOCYTES CREATE A DISRUPTED NICHE GRADIENT FOSTERING COLITIS-ASSOCIATED CANCER

Abstract Background The colonic stem cell niche is established by a gradient of WNT, R-spondin, BMP factors and their antagonists along the colonic epithelial vertical axis. Telocytes (TCFoxL1+) are mesenchymal cell forming a 3D hub underneath the epithelium, identified as an important source of niche factors. Specifically, they express non-canonical (nc) WNT factors and are the richest source of BMPs. Disruption of the BMPs gradient has been shown to be related to the development of several gastrointestinal diseases like Inflammatory Bowel Diseases (IBD). Such chronic inflammation drives the onset of Colitis-Associated Cancer (CAC) in about 60% of IBD patients. We previously showed that following a chronic inflammatory stress, 50% of the KO mouse for the BMP receptor 1a in colon telocytes (Bmpr1a△FoxL1+) presented malignant epithelial transformations. These cancer-like regions showed an aberrant epithelial b-catenin localization and an enlargement of the double positive α-SMA+/Vimentin+ mesenchymal population. Purpose: Loss of BMP signaling in TCFoxL1+ affects the mesenchymal-epithelial crosstalk and makes the colonic epithelium vulnerable to injuries, promoting/perpetuating inflammation fostering CAC onset. Method Following a DSS-based chronic inflammatory challenge in mutant and control mice, TCFoxL1+ ultrastructure was analyzed using transmission electron microscopy. Expression levels of members of the WNT-BMP axis (BMPs, WNTs and associated antagonists) were evaluated by qPCR in tumor-like areas and adjacent tissue. YAP cellular localization was evaluated by immunofluorescence in colon after chronic DSS challenge in Bmpr1a△FoxL1+ mice and controls. To differentiate cancer-associated fibroblasts (CAFs) subtypes, myCAF (myofibroblastic) and iCAF (inflammatory), in tumor-like region and adjacent tissue, we used co-staining against gp38, ICAM, Tagln and αSMA. Result(s) Following a chronic DSS-challenge, electron microscopy analysis demonstrated that TCFoxL1+ in the control mice exhibited a shortening and erosion in their telopodes (Tp). TCFoxL1+ in Bmpr1a△FoxL1+ mice tumor-like regions presented an expanded endoplasmic reticulum with fragmented and dilated Tp. A significant increase in BMP 4, 5 and 7 and in Wnt5 (nc) was detected in Bmpr1a△FoxL1+ mice compared to controls. Confocal analysis revealed a strong nuclear accumulation of YAP in cancer-like regions in mutant mice compared to controls. Finally, tumour-like regions presented an heterogeneous distribution of iCAF and myCAF compared to controls. Conclusion(s) These results exposed that the disruption of TCFoxL1+ associated BMP signaling disturbs the WNT-BMP gradient essential for the optimal maintenance of the SC niche and thus impacting epithelial regeneration when under stress. Thus, defective TCFoxL1+ assume a key role in the poor regeneration process of the epithelium which in the end promotes the development and progression of CAC. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Regulation of Parietal Cell Homeostasis by Bone Morphogenetic Protein Signaling

<h3>Background and Aims</h3> Loss of bone morphogenetic protein (BMP) signaling in the stomach, achieved by transgenic expression of the BMP inhibitor noggin (<i>H</i>⁺<i>/K</i>⁺<i>-Nog</i> mice), causes parietal cell (PC) loss, spasmolytic polypeptide-expressing metaplasia, a marker of preneoplasia, and activation of cell proliferation. We examined if specific inhibition of BMP signaling in PCs leads to aberrations in epithelial homeostasis. <h3>Methods</h3> Mice with floxed alleles of BMP receptor 1a (<i>Bmpr1a</i>^flox/flox mice) were crossed to <i>H</i>⁺<i>/K</i>⁺<i>-Cre</i> mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Cre;Bmpr1a</i>^flox/flox mice. Morphology of the mucosa was analyzed by hematoxylin and eosin staining. Distribution of H⁺/K⁺-ATPase-, IF-, and Ki-67-positive cells was analyzed by immunostaining. Expression of pit and neck cell mucins was determined by staining with the lectins Ulex Europaeus Agglutinin 1 and Griffonia (Bandeiraea) simplicifolia lectin II, respectively. Isolation of PCs from control and <i>Nog</i>-expressing mice was achieved by crossing <i>H</i>⁺<i>/K</i>⁺<i>-Nog</i> mice to <i>Rosa26-tdTomato</i> (Tom) mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice. <i>H</i>⁺<i>/K</i>⁺<i>-Cre</i> mice were then crossed to <i>H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Cre;H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice. Tom-labeled PCs were purified by flow cytometry. Changes in PC transcripts were measured by RNA-Seq. <h3>Results</h3> Six-month-old <i>H</i>⁺<i>/K</i>⁺<i>-Cre;Bmpr1a</i>^flox/flox mice exhibited increased epithelial cell proliferation, presence of transitional cells showing colocalization of IF with both Griffonia (Bandeiraea) simplicifolia lectin II-binding mucins and the H⁺/K⁺-ATPase, and expansion of Ulex Europaeus Agglutinin 1-positive cells. PC transcripts from Nog-expressing mice demonstrated induction of markers of Spasmolytic Polypeptide-Expressing Metaplasia. <h3>Conclusion</h3> PC-specific loss of BMP signaling alters the homeostasis of the gastric epithelium leading to the development of metaplasia.

Bta-miR-493 Inhibits Bovine Preadipocytes Differentiation by Targeting BMPR1A via the TGFβ/BMP and p38MAPK Signaling Pathways.

Fat deposition significantly impacts the meat yield and the meat quality of beef cattle, which is closely associated with the preadipocyte proliferation and differentiation. Bta-miR-493 is expressed differentially in the backfat of Qinchuan cattle of various ages, and it may be involved in the fat accumulation of beef cattle. However, the role and functional mechanism of bta-miR-493 in fat deposition are still unclear. Tissue-specific expression analysis showed that the level of bta-miR-493 was moderately abundant in the adipose tissues of beef cattle. Moreover, the expression of bta-miR-493 in perirenal fat, subcutaneous fat, and longissimus dorsi muscle of Japanese black cattle was significantly higher than that in Qinchuan cattle. EdU staining, cell counting assay, and Oil Red O staining indicated that bta-miR-493 promoted the proliferation of bovine preadipocytes but inhibited their differentiation. The dual luciferase assay and transcriptomic analysis confirmed that bone morphogenetic protein receptor 1A (BMPR1A) is a target gene of bta-miR-493. Moreover, rescue experiments showed that bta-miR-493 could promote bovine preadipocyte proliferation but inhibit their differentiation by targeting BMPR1A via the TGFβ/BMP and p38MAPK signaling pathways. Overall, our findings revealed a bta-miR-493-BMPR1A-TGFβ/BMP/p38MAPK regulatory axis that will serve as a theoretical foundation for the molecular breeding of beef cattle with high intramuscular fat deposition.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

BMP7-based peptide agonists of BMPR1A protect the left ventricle against pathological remodeling induced by pressure overload

Aortic stenosis (AS) exposes the left ventricle (LV) to pressure overload leading to detrimental LV remodeling and heart failure. In animal models of cardiac injury or hemodynamic stress, bone morphogenetic protein-7 (BMP7) protects LV against remodeling by counteracting TGF-β effects. BMP receptor 1A (BMPR1A) might mediate BMP7 antifibrotic effects. Herein we evaluated BMP7-based peptides, THR123 and THR184, agonists of BMPR1A, as cardioprotective drugs in a pressure overload model. We studied patients with AS, mice subjected to four-week transverse aortic constriction (TAC) and TAC release (de-TAC). The LV of AS patients and TAC mice featured Bmpr1a downregulation. Also, pSMAD1/5/(8)9 was reduced in TAC mice. Pre-emptive treatment of mice with THR123 and THR184, during the four-week TAC period, normalized pSMAD1/5/(8)9 levels in the LV, attenuated overexpression of remodeling-related genes (Col 1α1, β-MHC, BNP), palliated structural damage (hypertrophy and fibrosis) and alleviated LV dysfunction (systolic and diastolic). THR184 administration, starting fifteen days after TAC, halted the ongoing remodeling and partially reversed LV dysfunction. The reverse remodeling after pressure overload release was facilitated by THR184. Both peptides diminished the TGF-β1-induced hypertrophic gene program in cardiomyocytes, collagen transcriptional activation in fibroblasts, and differentiation of cardiac fibroblasts to myofibroblasts. Molecular docking suggests that both peptides bind with similar binding energies to the BMP7 binding domain at the BMPR1A. The present study results provide a preclinical proof-of-concept of potential therapeutic benefits of BMP7-based small peptides, which function as agonists of BMPR1A, against the pathological LV remodeling in the context of aortic stenosis.

Expression of GALNT8 and O-glycosylation of BMP receptor 1A suppress breast cancer cell proliferation by upregulating ERα levels

BackgroundMucin-type O-glycosylation is one of the most abundant types of O-glycosylation and plays important roles in various human carcinomas, including breast cancer. A large family of polypeptide N-acetyl-α-galactosaminyltransferases (GALNTs) initiate and define sites of mucin-type O-glycosylation. However, the specific mechanisms underlying GALNT8 expression and its roles in tumorigenesis remain poorly characterized. MethodsGALNT8 expression was assessed in 140 breast cancer patients. Immunofluorescence, immunoprecipitation, lectin blot and quantitative real-time PCR were used to investigate the expression of GALNT8 and its role in regulating estrogen receptor α (ERα) via bone morphogenetic protein (BMP) signaling. ResultsThe expression of GALNT8 was associated with breast cancer patient survival. GALNT8 downregulation was associated with a reduction in ERα levels, while GALNT8 overexpression elevated the transcription and protein levels of ERα and suppressed colony formation, suggesting an important role of GALNT8 in cancer cell proliferation. Conversely, GALNT8 knockdown led to the inhibition of BMP/SMAD/RUNX2 axis, which decreased ERα transcription. Further analysis suggested that BMP receptor 1A (BMPR1A) was O-GalNAcylated. Sites mutation of BMPR1A indicated that Thr137 and Ser37/Ser39/Ser44/Thr49 of BMPR1A were the main O-glycosylation sites. Although we cannot exclude the indirect effect of GALNT8, our results demonstrated that the expression of GALNT8 and O-glycosylation of BMPR1A play key roles in regulating the activity of BMP/SMAD/RUNX2 signaling and ERα expression. ConclusionThese findings suggest that GALNT8 expression and abnormal O-GalNAcylation of BMPR1A increase ERα expression and suppress breast cancer cell proliferation by modulating the BMP signaling pathway. General significanceOur results identify the involvement of GALNT8 in regulating ERα expression.

Roles of Bone Morphogenetic Protein Receptor 1A in Germinal Centers and Long-Lived Humoral Immunity.

In response to T-dependent Ag, germinal centers (GC) generate bone marrow-resident plasma cells (BMPC) and memory B cells (MBC). In this study, we demonstrate that the bone morphogenetic protein receptor 1A (BMPR1A) signaling pathway, which regulates differentiation and self-renewal in multiple stem cell populations, regulates GC dynamics and resultant establishment of BMPC and MBC. Expression studies using quantitative PCR and novel Bmpr1aIRES.EGFP reporter mice demonstrated that Bmpr1a expression is upregulated among GC B cells (GCBC) and subsets of MBC, bone marrow plasmablasts, and BMPC. In immunized mice carrying B cell-targeted Bmpr1a gene deletions, the GC response was initially diminished. Subsequently, the GCBC compartment recovered in size, concurrent with accumulation of GCBC that carried unmodified rather than deleted Bmpr1a alleles. Similarly, the resulting class-switched MBC and BMPC carried retained non-recombined alleles. Despite the strong selective pressure for "leaky" B cells that retained Bmpr1a, there was a permanent marked reduction in switched bone marrow Ab-forming cells (plasmablasts + plasma cells), BMPC, MBC, and Ag-specific serum IgM in mice carrying B cell-targeted Bmpr1a gene deletions. These findings demonstrate a novel role for BMPR1A in the modulation of the B cell response and in the establishment of long-term memory.

DNA Hybridization-Based Differential Peptide Display Identified Potential Osteogenic Peptides

A DNA hybridization-based differential peptide display (DPD) was developed for the screening of phage peptide library to find osteogenic peptides intended to bind to epigenetically induced osteogenic receptors on NIH/3T3 (3T3) cell surface. In the presence of DNA methylation inhibitor of 5-azacytidine (5AZC), an osteoblastic receptor of bone morphogenetic protein (BMP) receptor 1A (BMPR1A) was induced on the cell surface of NIH/3T3 fibroblasts. Cyclic heptamer-displaying phage library was screened against vehicle and 5AZC treated (Tx) 3T3 cells. Antisense oligo against library against library peptide coding DNA of control 3T3 cell bound phages were synthesized to subtract common binders from that of 5AZC-Tx 3T3 cell-bound phages that included 5AZC-induced receptor binders. The library peptide coding regions of conformational receptor binder-subtracted DPD were PCR-amplified and cloned into a plasmid vector specifically designed for short peptide expression. No unique binder was identified when 96 clones were randomly picked from the third round of panning against 5AZC-treated 3T3 cells, suggesting miscellaneous bindings to cell surface proteins. Unique binders showing homology to known function proteins were successfully identified when constitutive receptor binders were subtracted from 5AZC-induced protein binders. Some of identified peptides significantly increased alkaline phosphatase activity in 5AZC-Tx 3T3 cells. DPD can be a useful tool to screen functional peptide bindings to cell surface receptors.Graphic

Human osteoarthritis cartilage-derived stromal cells activate joint degeneration through TGF-beta lateral signaling.

Human osteoarthritis cartilage contains chondrocytes (OAC) and mesenchymal stromal cells (OA-MSC). Here, we found that TGF-β had different effects on OA-MSC and OAC, and revealed its lateral signaling mechanism in OA. RNAseq analysis indicated that OA-MSC expressed the same level of Bone Morphogenetic Protein (BMP) Receptor-1A as OAC but only 1/12 of Transforming Growth Factor beta (TGF-β) Receptor-1. While TGF-β specifically activated SMAD2 in OAC, it also activated BMP signaling-associated SMAD1 in OA-MSC. While TGF-β stimulated chondrogenesis in OAC, it induced hypertrophy, mineralization, and MMP-13 in OA-MSC. Inhibiting TGF-βR1 suppressed MMP-13 in OA-MSC but stimulated it in OAC. In contrast, by specifically targeting BMPR1A/ACVR1 in both cell types, LDN193189 inhibits cartilage degeneration through suppressing hypertrophy and MMP-13 in a mouse osteoarthritis model. Thus, LDN193189, a drug under development to inhibit constitutive BMP signaling during heterotopic ossification, may be re-purposed for OA treatment.

LTP of inhibition at PV interneuron output synapses requires developmental BMP signaling

Parvalbumin (PV)-expressing interneurons (PV-INs) mediate well-timed inhibition of cortical principal neurons, and plasticity of these interneurons is involved in map remodeling of primary sensory cortices during critical periods of development. To assess whether bone morphogenetic protein (BMP) signaling contributes to the developmental acquisition of the synapse- and plasticity properties of PV-INs, we investigated conditional/conventional double KO mice of BMP-receptor 1a (BMPR1a; targeted to PV-INs) and 1b (BMPR1a/1b (c)DKO mice). We report that spike-timing dependent LTP at the synapse between PV-INs and principal neurons of layer 4 in the auditory cortex was absent, concomitant with a decreased paired-pulse ratio (PPR). On the other hand, baseline synaptic transmission at this connection, and action potential (AP) firing rates of PV-INs were unchanged. To explore possible gene expression targets of BMP signaling, we measured the mRNA levels of the BDNF receptor TrkB and of P/Q-type Ca2+ channel α-subunits, but did not detect expression changes of the corresponding genes in PV-INs of BMPR1a/1b (c)DKO mice. Our study suggests that BMP-signaling in PV-INs during and shortly after the critical period is necessary for the expression of LTP at PV-IN output synapses, involving gene expression programs that need to be addressed in future work.

Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII- cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII- progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

BMPR1A is necessary for chondrogenesis and osteogenesis, whereas BMPR1B prevents hypertrophic differentiation.

BMP2 stimulates bone formation and signals preferably through BMP receptor (BMPR) 1A, whereas GDF5 is a cartilage inducer and signals preferably through BMPR1B. Consequently, BMPR1A and BMPR1B are believed to be involved in bone and cartilage formation, respectively. However, their function is not yet fully clarified. In this study, GDF5 mutants with a decreased affinity for BMPR1A were generated. These mutants, and wild-type GDF5 and BMP2, were tested for their ability to induce dimerization of BMPR1A or BMPR1B with BMPR2, and for their chondrogenic, hypertrophic and osteogenic properties in chondrocytes, in the multipotent mesenchymal precursor cell line C3H10T1/2 and the human osteosarcoma cell line Saos-2. Mutants with the lowest potency for inducing BMPR1A-BMPR2 dimerization exhibited minimal chondrogenic and osteogenic activities, indicating that BMPR1A is necessary for chondrogenic and osteogenic differentiation. BMP2, GDF5 and the GDF5 R399E mutant stimulated expression of chondrogenic and hypertrophy markers in C3H10T1/2 cells and chondrocytes. However, GDF5 R399E, which induces the dimerization of BMPR1B and BMPR2 more potently than GDF5 or BMP2, displayed reduced hypertrophic activity. Therefore, we postulate that stronger BMPR1B signaling, compared to BMPR1A signaling, prevents chondrocyte hypertrophy and acts as a cartilage stabilizer during joint morphogenesis.This article has an associated First Person interview with the first author of the paper.

Increased BMPR1A Expression Enhances the Adipogenic Differentiation of Mesenchymal Stem Cells in Patients with Ankylosing Spondylitis.

Objective To investigate the adipogenic differentiation capacity of mesenchymal stem cells (MSCs) from ankylosing spondylitis (AS) patients and explore the mechanism of abnormal MSC adipogenesis in AS. Methods MSCs from patients with AS (ASMSCs) and healthy donors (HDMSCs) were cultured in adipogenic differentiation medium for up to 21 days. Adipogenic differentiation was determined using oil red O (ORO) staining and quantification and was confirmed by assessing adipogenic marker expression (PPAR-γ, FABP4, and adiponectin). Gene expression of adipogenic markers was detected using qRT-PCR. Protein levels of adipogenic markers and signaling pathway-related molecules were assessed via Western blotting. Levels of bone morphogenetic proteins 4, 6, 7, and 9 were determined using enzyme-linked immunosorbent assays. Lentiviruses encoding short hairpin RNAs (shRNAs) were constructed to reverse abnormal bone morphogenetic protein receptor 1A (BMPR1A) expression and evaluate its role in abnormal ASMSC adipogenic differentiation. Bone marrow fat content was assessed using hematoxylin and eosin (HE) staining. BMPR1A expression in bone marrow MSCs was measured using immunofluorescence staining. Results ASMSCs exhibited a greater adipogenic differentiation capacity than HDMSCs. During adipogenesis, ASMSCs expressed BMPR1A at higher levels, which activated the BMP-pSmad1/5/8 signaling pathway and increased adipogenesis. BMPR1A silencing using an shRNA eliminated the difference in adipogenic differentiation between HDMSCs and ASMSCs. Moreover, HE and immunofluorescence staining showed higher bone marrow fat content and BMPR1A expression in patients with AS than in healthy donors. Conclusion Increased BMPR1A expression induces abnormal ASMSC adipogenic differentiation, potentially contributing to fat metaplasia and thus new bone formation in patients with AS.

Luteinizing Hormone effect on the GDF-9 and BMPR-1a Expression of Bovine Granulosa Cells culture

Growth Differentiation Factor-9 (GDF-9) and Bone Morphogenetic Protein Receptor 1a (BMPR-1a) was a member of the transforming growth factor-β (TGF-β) superfamily known to regulated ovarian functions in mammals. In addition, Luteinizing Hormone (LH) also has an important role in ovarian function. The objectives of this research are to observe effect of LH on the GDF-9 and BMPR-1a expression in granulosa cells (GCs) culture. Bovine ovaries were obtained from a slaughterhouse and carried to the laboratory within 30 min in a container kept at 37 °C in saline containing gentamicin and amphotericin. The ovaries were washed with pre-warmed PBS supplemented with gentamicin and amphotericin. Granulosa cells were collected from follicles by aspiration using a syringe with needle (20G). The cells were centrifuged for 1 min at 800g at room temperature and washed twice in DMEM/F12 medium and filtered through a stainless steel filter (100 um) to get the GCs then seeded in 6 well culture plates. The cells were cultured at 37 °C in a 5% CO2 atmosphere for 24 h and then the wells were washed with PBS to remove unattached cells. The culture medium was replaced with serum-free medium supplemented with LH at 100 ng/mL and cultured for 24 h. Total RNA was extracted from GCs using TRIsure. The RNA quality and quantity were estimated by using spectrophotometer at 260/280 nm. Aliquots (1 ug) of total RNA from each pool of GCs were independently reverse-transcribed to cDNA. The mRNA expression of GDF-9 and BMPR-1a was estimated by the quantitative real-time PCR method. The results showed that LH added to the culture medium can increase the expression of GDF9 and BMPR-1a of granulosa cells.

Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury

Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease.

Abstract 2119: Bone Morphogenetic Protein Receptor 1a in myeloid cells regulates mouse prostate cancer growth

Abstract The Bone Morphogenetic Protein (BMP) pathway is a member of the TGFβ signaling family and has mirrored the complex roles in diverse contexts during cancer development. BMP signaling is rarely mutated and can be frequently overexpressed in many human cancers. The dichotomous role of BMPs as both tumor promoters and suppressors appears to be largely context based in both the cancer cell and the surrounding microenvironment. Myeloid cells including macrophages and neutrophils have been shown to be tumor promoting when stimulated from BMP signaling. We discovered that conditional deletion of BMPR1a in myeloid cells (LysM-Cre) would affect tumor progression and found smaller tumors in a syngeneic FVBn prostate cancer model. Specific changes occurred in myeloid cells both in tumor-bearing mice and non-tumor naïve mice throughout multiple tissues. We profiled myeloid subsets both in the bone marrow, spleen and primary tumor. We found that BMPR1a loss in myeloid cells altered the differentiation and lineage capability of distinct populations by histologic, flow cytometry and high-dimensional mass cytometry analysis. These results indicate a distinct genetic requirement for BMP signaling in myeloid cells during tumor progression. Citation Format: Desiree M. Straign, Sergey V. Novitskiy, Philip Owens. Bone Morphogenetic Protein Receptor 1a in myeloid cells regulates mouse prostate cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2119.

Transcriptome sequencing analysis reveals the effect of combinative treatment with low‑intensity pulsed ultrasound and magnesium ions on hFOB1.19 human osteoblast cells.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low-intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including ‘wound healing’, ‘transforming growth factor beta receptor signaling pathway’, ‘transcription, DNA-templated’, ‘receptor complex’, ‘nucleus’, ‘SMAD protein complex’, ‘DNA binding’, ‘metal ion binding’ and ‘GTPase activator activity’. Notably, the transforming growth factor (TGF)-β, mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription-quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c-Jun N-terminal kinase, doublecortin, paxillin and Jun proto-oncogene AP-1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+-transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage-gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF-β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.

Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli.

Background & AimsGastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP) signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation.MethodsThe H+/K+-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog) in the stomach (H+/K+-Nog mice). Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre) mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice). Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom). Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction.ResultsLgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, and with anti-IF, -CD44, -CD44v9, and -bromodeoxyuridine antibodies.ConclusionsInflammation and inhibition of BMP signaling activate Lgr5+ve cells, which give rise to metaplastic, dysplastic, proliferating lineages that express markers of mucus neck and zymogenic cell differentiation.

A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield.

Sir: We have read with great interest the article entitled “A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield.”1 In the article, Tevlin et al. developed a new method that allows a higher extraction rate of adipose-derived stem cells with similar differentiation capacities compared with the conventional method.2 We would like to thank the authors for the study because it is increasingly important to develop new techniques that can facilitate the extraction of adipose-derived stem cells for use in cell therapy. Nevertheless, some points raised questions and need some discussion. First, the percentage of adipose-derived stem cells present in the stromal vascular fraction obtained with the “conventional” method seems very low (1.95 percent). Indeed, in our experience and in the literature, the average proportion of adipose-derived stem cells present in the stromal vascular fraction is approximately 30 to 40 percent.3 Moreover, stromal vascular fraction is a heterogeneous mix of cells, and the percentage of immune cells modified the adipogenic differentiation capacities of stromal vascular fraction. Therefore, it would have been interesting to compare the percentage of erythrocytes, endothelial cells, and immune cells to determine whether this new method modified the extraction rate of other cells that could be responsible for the modification of the adipogenic differentiation capacities of the adipose-derived stem cells. Moreover, the authors said that they studied the function of the adipose-derived stem cells isolated with their new methods; however, it seems that they characterized the functions of the adipose-derived stem cells but also the function of all the cells that are present in the stromal vascular fraction. The authors demonstrated that the adipose-derived stem cells extracted with the new method expressed more bone morphogenetic protein (BMP) receptor 1a and 1b. It would be interesting to determine also the expression of BMP receptor 2, receptors of BMP4 and BMP7, inducers of osteogenic differentiation of adipose-derived stem cells. Moreover, it would be interesting to determine whether this new technique isolates a different population of adipose-derived stem cells based on this BMP receptor expression. A minor remark is that the study was realized with a human sample; therefore, it should be important to indicate the number of samples analyzed for this study knowing the interpatient variability. The authors claimed that their new method of stromal vascular fraction extraction is different with regard to two aspects compared with the conventional method. Foremost, this method uses a stronger and quicker shaking during digestion, and the modification of the digestion media. Indeed, the media are enriched with different products and thus it is really difficult to determine which product is responsible for the higher extraction rate. Moreover, the higher percentage of extracted adipose-derived stem cells could be simply linked to the concentration of collagenase, which is three times more important in the new digestion media compared with the conventional one. The development of rapid, simple, and enzyme-free procedures to isolate stromal vascular fraction is the new goal of cell therapy. Therefore, the use of digestion media containing more of the chemical products, even if they seem to not affect cell viability, could be a real issue for clinical use and from the regulatory framework point of view.4,5 DISCLOSURE None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this communication. The authors received no funding support for this work. Jérôme Laloze, M.D.Department of Plastic and Reconstructive SurgeryRangueil University HospitalToulouse, FranceDepartment of Maxillofacial and Reconstructive Surgeryand StomatologyCHU DupuytrenLimoges, France Nicolas Bertheuil, M.D.Department of Plastic, Reconstructive and Aesthetic SurgeryHospital SudUniversity of Rennes 1Rennes, France Julie Usseglio-Grosso, M.D.Department of Maxillofacial and Reconstructive Surgeryand StomatologyCHU DupuytrenLimoges, France Audrey Varin, Ph.D.STROMAlabUMR5273 CNRS/UPS/EFS - INSERM U1031Rangueil University HospitalToulouse, France Benoit Chaput, M.D.Department of Plastic and Reconstructive SurgeryRangueil University HospitalToulouse, France