Expression Of Bone Morphogenetic Protein Receptors Research Articles

Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 μM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

Aberrant expression of bone morphogenetic proteins in the disease progression and metastasis of breast cancer.

Bone morphogenetic proteins (BMPs) play crucial roles in the tumorigenesis and metastasis of cancers. Controversy remains about the exact implications of BMPs and their antagonists in breast cancer (BC), due to their diverse and complex biological functions and signalling. A comprehensive study of the whole family and their signalling in breast cancer is provoked. Aberrant expression of BMP, BMP receptors and antagonists in primary tumours in breast cancer were analysed by using TCGA-BRCA and E-MTAB-6703 cohorts. Related biomarkers including ER, HER, proliferation, invasion, angiogenesis, lymphangiogenesis and bone metastasis were involved to identify the relationship with BMPs in breast cancer. The present study showed BMP8B was significantly increased in breast tumours, while BMP6 and ACVRL1 were decreased in breast cancer tissues. The expressions of BMP2, BMP6, TGFBR1 and GREM1 were significantly correlated with BC patients' poor overall survival. Aberrant expression of BMPs, together with BMP receptors, were explored in different subtypes of breast cancer according to ER, PR and HER2 status. Furthermore, higher levels of BMP2, BMP6 and GDF5 were revealed in triple negative breast cancer (TNBC) whilst BMP4, GDF15, ACVR1B, ACVR2B and BMPR1B were relatively higher in Luminal type BC. ACVR1B and BMPR1B were positively correlated with ERα but were inversely correlated with ERβ. High expression of GDF15, BMP4 and ACVR1B were associated with poorer overall survival in HER2 positive BC. BMPs also play dual roles in tumour growth and metastasis of BC. A shift pattern of BMPs was showed in different subtypes of breast cancer suggesting a subtype specific involvement. It provokes more research to shed light on the exact role of these BMPs and receptors in the disease progression and distant metastasis through a regulation of proliferation, invasion and EMT.

Endothelin-1 alters BMP signaling to promote proliferation of pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

Enhanced BMP signalling causes growth plate cartilage dysrepair in rats

Growth plate cartilage injuries often result in bony repair at the injury site and premature mineralisation at the uninjured region causing bone growth defects, for which underlying mechanisms are unclear. With the prior microarray study showing upregulated bone morphogenetic protein (BMP) signalling during the injury site bony repair and with the known roles of BMP signalling in bone healing and growth plate endochondral ossification, this study used a rat tibial growth plate drill-hole injury model with or without systemic infusion of BMP antagonist noggin to investigate roles of BMP signalling in injury repair responses within the injury site and in the adjacent "uninjured" cartilage. At days 8, 14 and 35 post-injury, increased expression of BMP members and receptors and enhanced BMP signalling (increased levels of phosphorylated (p)-Smad1/5/8) were found during injury site bony repair. After noggin treatment, injury site bony repair at days 8 and 14 was reduced as shown by micro-CT and histological analyses and lower mRNA expression of osteogenesis-related genes Runx2 and osteocalcin (by RT-PCR). At the adjacent uninjured cartilage, the injury caused increases in the hypertrophic zone/proliferative zone height ratio and in mRNA expression of hypertrophy marker collagen-10, but a decrease in chondrogenesis marker Sox9 at days 14 and/or 35, which were accompanied by increased BMP signalling (increased levels of pSmad1/5/8 protein and BMP7, BMPR1a and target gene Dlx5 mRNA). Noggin treatment reduced the hypertrophic zone/proliferative zone height ratio and collagen-10 mRNA expression, but increased collagen-2 mRNA levels at the adjacent growth plate. This study has identified critical roles of BMP signalling in the injury site bony repair and in the hypertrophic degeneration of the adjacent growth plate in a growth plate drill-hole repair model. Moreover, suppressing BMP signalling can potentially attenuate the undesirable bony repair at injury site and suppress the premature hypertrophy but potentially rescue chondrogenesis at the adjacent growth plate.

BMP signaling is a therapeutic target in ovarian cancer

BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.

The Phosphodiesterase-5 Inhibitor Vardenafil Improves the Activation of BMP Signaling in Response to Hydrogen Peroxide.

The pleiotropic roles of phosphodiesterase-5 inhibitors (PDE5is) in cardiovascular diseases have attracted attention. The effect of vardenafil (a PDE5i) is partly mediated through reduced oxidative stress, but it is unclear whether vardenafil protects against hydrogen peroxide (H2O2)-induced endothelial cell injury, and the molecular mechanisms that are involved remain unknown. We determined the protective role of vardenafil on H2O2-induced endothelial cell injury in cultured human umbilical vein endothelial cells (HUVECs). Vardenafil decreased the number of TUNEL-positive cells, increased the Bcl2/Bax ratio, and ameliorated the numbers of BrdU-positive cells in H2O2-treated HUVECs. The bone morphogenetic protein receptor (BMPR)/p-Smad/MSX2 pathway was enhanced in response to H2O2, and vardenafil treatment could normalize this pathway. To determine whether the BMP pathway is involved, we blocked the BMP pathway using dorsomorphin, which abolished the protective effects of vardenafil. We found that vardenafil improved the H2O2-induced downregulation of BMP-binding endothelial regulator protein (BMPER), which possibly intersects with the BMP pathway in the regulation of endothelial cell injury in response to oxidative stress. We demonstrated for the first time that exogenous H2O2 activates BMPR expression and promotes Smad1/5/8 phosphorylation. Additionally, vardenafil can attenuate H2O2-induced endothelial cell injury in HUVECs. Vardenafil decreases apoptosis through an improved Bcl-2/Bax ratio and increases cell proliferation. Vardenafil protects against endothelial cell injury through ameliorating the intracellular oxidative stress level and BMPER expression. The protective role of vardenafil on H2O2-induced endothelial cell injury is mediated through BMPR/p-Smad/MSX2 in HUVECs.

Deregulated bone morphogenetic proteins and their receptors are associated with disease progression of gastric cancer

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β superfamily (TGF-β). BMPs are involved in tumourigenesis and disease progression of certain malignancies. To date, the role played by BMPs in gastric cancer (GC) remains largely unknown. In the present study, we systematically analysed the expression and clinical significance of BMP and BMP receptors (BMPR) in TCGA gastric cancer database and GEO database and explored the possible mechanism of action. BMP5 is reduced in gastric cancer tissues, while ACVRL1, ACVR1, TGFBR1, and BMPR2 were significantly increased in the gastric tumours. BMP3, ACVR1, TGFBR1, BMPR1B (also known as ALK6), TGFBR2 and BMPR2 were significantly associated with poorer overall survival of GC patients. A negative correlation was seen between BMP/BMPR and proliferation markers which was supported by their correlation with the cell cycle promoters and inhibitors. More interestingly, further analyses showed that BMPs and their receptors are positively correlated with matrix metalloproteinases (MMPs), epithelial mesenchymal transition (EMT) markers and stemness in GC. Furthermore, positive correlations were also frequently seen between BMP receptors and markers/regulators of angiogenesis and lymphangiogenesis in the gastric tumours. Taken together, these findings suggest that BMPs play dual roles in GC. They may inhibit proliferation of GC cells. On the other hand, they can also promote disease progression through a promotion of invasion, EMT and stemness. The elevated expression of BMP receptors in GC were also highly associated with tumour associated angiogenesis and lymphangiogenesis which facilitate tumour growth, expansion and spread.

High BMPR2 expression leads to enhanced SMAD1/5/8 signalling and GDF6 responsiveness in human adipose-derived stem cells: implications for stem cell therapies for intervertebral disc degeneration

Stem cell–based regenerative strategies are promising for intervertebral disc degeneration. Stimulation of bone-marrow- and adipose-derived multipotent stem cells with recombinant human growth differentiation factor 6 (rhGDF6) promotes anabolic nucleus pulposus like phenotypes. In comparison to mesenchymal stem cells, adipose-derived multipotent stem cells exhibit greater NP-marker gene expression and proteoglycan-rich matrix production. To understand these response differences, we investigated bone morphogenetic protein receptor profiles in donor-matched human mesenchymal stem cells and adipose-derived multipotent stem cells, determined differences in rhGDF6 signalling and their importance in NP-like differentiation between cell populations. Bone morphogenetic protein receptor expression in mesenchymal stem cells and adipose-derived multipotent stem cells revealed elevated and less variable expression of BMPR2 in adipose-derived multipotent stem cells, which corresponded with increased downstream pathway activation (SMAD1/5/8, ERK1/2). Inhibitor studies demonstrated SMAD1/5/8 signalling was required for rhGDF6-induced nucleus-pulposus-like adipose-derived multipotent stem cell differentiation, while ERK1/2 contributed significantly to critical nucleus pulposus gene expression, aggrecan and type II collagen production. These data inform cell regenerative therapeutic choices for intervertebral disc degeneration regeneration and identify further potential optimisation targets.

2018 AHA Late-Breaking Basic Science Abstracts.

2018 AHA Late-Breaking Basic Science Abstracts.

Rapid initiation of guided bone regeneration driven by spatiotemporal delivery of IL-8 and BMP-2 from hierarchical MBG-based scaffold

Initiation of endogenous repair mechanisms, including key steps of stem cell recruitment and cartilage intermediate formation in endochondral ossification, is vital to regeneration of large bone defects. To biomimetically promote a rapid initiation and ensuing osteogenic stimulation, exogenous chemokine IL-8 and growth factor BMP-2 were orchestrated in a mesoporous bioactive glass (MBG)-based spatiotemporal delivery system, to achieve a rapid release of IL-8 followed by a long-term sustained release of BMP-2. The synergistic effect of IL-8 and BMP-2 on initiation stage of bone healing and underlying mechanism were thoroughly investigated in vitro and in vivo. Intriguingly, apart from its superiority in stem cell recruitment to BMP-2, IL-8 not only endowed a histological “prep-state” of endochondral ossification by up-regulating chondrogenic genes and inducing the formation of extensive cartilage tissues, facilitating rapid bone transformation by BMP-2, but also triggered a cellular “prep-state” with high expression of BMP receptors, enhancing the osteoinductivity of BMP-2. With the spatiotemporal delivery system, orchestrated signal stimuli of IL-8 and BMP-2 induced a rapid initiation including efficient stem cell recruitment and a “chondrogenic/osteogenic balance” at the first stage of endochondral ossification, and the scaffold facilitated sufficient osteoconductivity, together resulting in early extensive bone mineralization and an advanced regeneration throughout the repair of large bone defect. We believe this new idea could provide insights toward designing bone-repairing biomaterials with higher regenerative efficiency.

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7

Background: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells.Methods: The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5’-untranslated region (5’-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay.Results: All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5’-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

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

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

Bone mesenchymal stem cell-conditioned medium induces the upregulation of Smad6, which inhibits the BMP-4/Smad1/5/8 signaling pathway

Objectives: In this study, we aimed to investigate the effect of bone mesenchymal stem cells (BMSCs) conditioned medium on BMP4-induced activation of the Smad signaling pathway during the process of neural stem cells differentiation.Methods: We designed four experimental groups: (A) control group, (B) BMP-4 treatment group, (C) BMSC-CM group, and (D) BMSC-CM (BMSC-conditioned medium) combined with BMP-4 group. The expression of microtube-associated protein-2 (MAP-2) detected by immunocytochemical staining and western blot. The expression of glial fibrillary acidic protein (GFAP) and BMP receptors (BMPRII and BMPRIa) was determined by western blot, while SMAD1/5/8 and SMAD6 mRNA expression was detected by quantitative real-time polymerase chain reaction (Q-RT-PCR).Results: We found that the expression of microtube-associated protein-2 (MAP-2) increased in group B compared to group A. In group C, the expression of GFAP was increased and the expression of MAP-2 was decreased compared to group A. This phenomenon was weaker in group D. We also demonstrated that BMSC-CM could inhibit the activation of BMP signaling by downregulating the expression of BMPRII and BMPRIa proteins. Moreover, BMSC-CM could upregulate the expression of Smad6 mRNA and inhibit the activation of Smad1/5/8 mRNA.Conclusion: These observations suggest that BMSC-CM could neutralize the effect of BMP-4 on the differentiation of NSCs by upregulating the expression of Smad6 and decrease the expression of GFAP by inhibiting the BMP-4-SMAD1/5/8 signaling pathway, conversely increasing the generation of neurons.

Abstract 1188: Forced induction of differentiation in osteosarcoma tumor initiating cells

Abstract Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years, and similarly the five-year survival rate has remained a dismal ∼60%, largely due to metastatic spread to the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation. In previous work, our lab developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to the coding sequence for Green Fluorescent Protein (GFP). Clonally derived, stably transfected OS Oct4/GFP+ cells are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment. These conditions appear to reprogram the GFP+ cells, resulting in loss of reporter activity, reduced proliferation, and adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in normal physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth. To test our hypothesis, we investigated the expression of BMP receptors in two primary OS cell lines, using Mesenchymal Stem Cells and Human Fetal Osteoblasts as positive controls. Expression of BMP Receptors II, 1A, and 1B was confirmed using flow cytometry indicating that the OS cells could potentially respond to BMP stimulation. The functionality of the BMP signaling pathway was subsequently investigated after stimulation with BMP heterodimers, BMP2/7 and BMP4/7. Immunoblotting showed OS TICs are capable of activating canonical and non-canonical BMP signaling pathways after BMP stimulation, suggesting a functional and intact signaling network potentially able to induce differentiation. However, only cells treated with BMP4/7 showed changes in cell cycle distribution as visualized by DAPI. Furthermore, BMP4/7-stimulated TICs showed reduced tumor formation in our xenograft mouse model Our data suggests an inhibition of OS tumorigenicity due to a BMP4/7 induced differentiation based response. Currently, we are investigating if forced expression of BMPs using an OS specific recombinant Adeno-Associated Virus can inhibit tumor growth in vivo. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy. Citation Format: Margaret E. White, Emma Viktoria Marie Hyddmark, Ali Zarezadeh, Elham Nasri, Maria V. Guijarro, Padraic P. Levings, Glyn Palmer, Steven C. Ghivizzani, C. Parker Gibbs. Forced induction of differentiation in osteosarcoma tumor initiating cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1188.

Differential gene expression of BMP2 and BMP receptors in chick retina & choroid induced by imposed optical defocus.

Recent studies have demonstrated the defocus sign-dependent, bidirectional gene expression regulation of bone morphogenetic proteins, BMP2, 4 and 7 in chick RPE. In this study, we examined the effects of imposed positive (+10 D) and negative (-10 D) lenses on the gene expression of these BMPs and BMP receptors (BMPR1A, BMPR1B, BMPR2) in chick retina and choroid after monocular lens treatment for 2 or 48 h, as indicators of the roles of retinal and choroidal BMPs and receptors in postnatal eye growth regulation. In retina, although all genes were expressed, neither +10 nor -10 D lenses, worn for either 2 or 48 h, significantly altered gene expression. In contrast, treatment-related differential gene expression was detected in the choroid for both BMPs and their receptors, although interestingly, with the +10 D lens, BMP2 was up-regulated by 156.7 ± 19.7% after 2 h, while BMPR1A was down-regulated to 82.3 ± 12.5% only after 48 h. With the -10 D lens, only the gene expression of BMPR1B was significantly altered, being up-regulated by 162.3 ± 21.2% after 48 h. Untreated birds showed no difference in expression between their two eyes, for any of the genes examined. The finding that retinal gene expression for BMP2, 4, 7 and their receptors are not affected by short-term optical defocus contrasts with previous observations of sign-dependent expression changes for the same genes in the RPE. The latter changes were also larger and more consistent in direction than the choroidal gene expression changes reported here. The interrelationship between these various changes and their biological significance for eye growth regulation are yet to be elucidated.

Abstract B20: Activin receptor-like kinase 1 complexes with endoglin in TGFß-mediated signaling in angiogenesis

Abstract Background: Activin Receptor-Like Kinase 1 (ALK1), a type I transforming growth factor (TGFß) family receptor, and endoglin, a type III receptor, are expressed in endothelial cells (EC) and involved in angiogenesis. Control of angiogenesis is critical in diseases such as cancer. Four ligands, TGFß, activin, bone morphogenetic protein 9 (BMP-9), and BMP-10, have been shown to bind ALK-1. Thus angiogenesis may be mediated by different combinations of ligands and receptors. Purpose: We investigated the role of TGFß and ALK-1 in tube formation. We also screened EC and also cancer cell lines for expression of various TGFß family receptors to begin to address which ligands and receptors are involved in angiogenesis and endothelial-tumor interactions,. Results: We also show that fibroblast growth factor 2 (FGF-2) + TGFß increase ALK-1 but not ALK 5 type I receptor expression in endothelial cells after tube formation. We show data that the type I receptor Activin-Like Kinase 1 and the co-receptor endoglin can signal in endothelial cells via the TGFß ligand. Antisense RNA that targets ALK 1 (and also ALK5) also disrupts tube formation. We further show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFß. This suggests it may function to regulate control of ALK 1 activation. Further, ALK-1 and endoglin may also be involved in cell-cell interactions between endothelial and other cells. Thus we screened endothelial and cancer cells for TGFß family receptor expression by RT-PCR. Endothelial cells show similar expression of ALK-1, ALK-5, and endoglin and lower expression of BMP receptor II and TGFßRII. Cancer cells show much lower expression of type II receptors. Citation Format: James Hawker, Patricia Varacallo, Iain Duffy. Activin receptor-like kinase 1 complexes with endoglin in TGFß-mediated signaling in angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B20.

Involvement of PRIP (Phospholipase C‐Related But Catalytically Inactive Protein) in BMP‐Induced Smad Signaling in Osteoblast Differentiation

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma

The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease.

Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth

The bone morphogenetic protein (BMP) pathway belonging to the Transforming Growth Factor beta (TGFβ) family of secreted cytokines/growth factors is an important regulator of cancer. BMP ligands have been shown to play both tumor suppressive and promoting roles in human cancers. We have found that BMP ligands are amplified in human ovarian cancers and that BMP receptor expression correlates with poor progression-free-survival (PFS). Furthermore, active BMP signaling has been observed in human ovarian cancer tissue. We also determined that ovarian cancer cell lines have active BMP signaling in a cell autonomous fashion. Inhibition of BMP signaling with a small molecule receptor kinase antagonist is effective at reducing ovarian tumor sphere growth. Furthermore, BMP inhibition can enhance sensitivity to Cisplatin treatment and regulates gene expression involved in platinum resistance in ovarian cancer. Overall, these studies suggest targeting the BMP pathway as a novel source to enhance chemo-sensitivity in ovarian cancer.