Bone Morphogenetic Protein Pathway Activity Research Articles

Kinome-wide analysis of the effect of statins in colorectal cancer.

Epidemiological studies and meta-analyses show an association between statin use and a reduced incidence of colorectal cancer (CRC). We have shown that statins act on CRC through bone morphogenetic protein (BMP) signalling, but the exact cellular targets and underlying mechanism of statin action remain elusive. In this study, we set out to assess the influence of statins on global cancer cell signalling by performing an array-based kinase assay using immobilised kinase substrates spanning the entire human kinome. CRC cells with or without Lovastatin treatment were used for kinome analysis. Findings on kinome arrays were further confirmed by immunoblotting with activity-specific antibodies. Experiments in different CRC cell lines using immunoblotting, siRNA-mediated knockdown and treatment with specific BMP inhibitor Noggin were performed. The relevance of in vitro findings was confirmed in xenografts and in CRC patients treated with Simvastatin. Kinome analysis can distinguish between non-specific, toxic effects caused by 10 µM of Lovastatin and specific effects on cell signalling caused by 2 µM Lovastatin. Statins induce upregulation of PTEN activity leading to downregulation of the PI3K/Akt/mTOR signalling. Treatment of cells with the specific BMP inhibitor Noggin as well as PTEN knockdown and transfection of cells with a constitutively active form of AKT abolishes the effect of Lovastatin on mTOR phosphorylation. Experiments in xenografts and in patients treated with Simvastatin confirm statin-mediated BMP pathway activation, activation of PTEN and downregulation of mTOR signalling. Statins induce BMP-specific activation of PTEN and inhibition of PI3K/Akt/mTOR signalling in CRC.

The Bone Morphogenetic Protein Signaling Inhibitor LDN-193189 Enhances Metastasis Development in Mice.

Breast cancer with bone metastasis is essentially incurable with current anticancer therapies. The bone morphogenetic protein (BMP) pathway is an attractive therapeutic candidate, as it is involved in the bone turnover and in cancer cell formation and their colonization of distant organs such as the bone. We previously reported that in breast cancer cells, the ZNF217 oncogene drives BMP pathway activation, increases the metastatic growth rate in the bone, and accelerates the development of severe osteolytic lesions in mice. In the present study, we aimed at investigating the impact of the LDN-193189 compound, a potent inhibitor of the BMP type I receptor, on metastasis development in vivo. ZNF217-revLuc cells were injected into the left ventricle of nude mice (n = 16) while control mice (n = 13) were inoculated with control pcDNA6-revLuc cells. Mice from each group were treated or not with LDN-193189 for 35 days. We found that systemic LDN-193189 treatment of mice significantly enhanced metastasis development, by increasing both the number and the size of metastases. In pcDNA6-revLuc-injected mice, LDN-193189 also affected the kinetics of metastasis emergence. Altogether, these data suggest that in vivo, LDN-193189 might affect the interaction between breast cancer cells and the bone environment, favoring the emergence and development of multiple metastases. Hence, our report highlights the importance of the choice of drugs and therapeutic strategies used in the management of bone metastases.

A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification

Neural progenitors produce neurons whose identities can vary as a function of the time that specification occurs. Here, we describe the heterochronic specification of two photoreceptor (PhR) subtypes in the zebrafish pineal gland. We find that accelerating PhR specification by impairing Notch signaling favors the early fate at the expense of the later fate. Using in vivo lineage tracing, we show that most pineal PhRs are born from a fate-restricted progenitor. Furthermore, sister cells derived from the division of PhR-restricted progenitors activate the bone morphogenetic protein (BMP) signaling pathway at different times after division, and this heterochrony requires Notch activity. Finally, we demonstrate that PhR identity is established as a function of when the BMP pathway is activated. We propose a novel model in which division of a progenitor with restricted potential generates sister cells with distinct identities via a temporal asymmetry in the activation of a signaling pathway.

NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification.

Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways. FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context‐dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ‐secretase‐independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co‐localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal‐subtype tumours, where it interacts with Notch to induce an epithelial–mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP–Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal‐subtype tumours promotes a synergistic BMP–Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal‐subtype CRC insensitive to γ‐secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Twisted Gastrulation BMP Signaling Modulator 1 Regulates Papillary Thyroid Cancer Cell Motility and Proliferation.

Bone morphogenetic proteins (BMPs) are growth factors that have important functions in cell proliferation, migration and differentiation. To date, BMP pathway activation has been found in multiple human tumors and is associated with enhanced malignant tumor growth and metastasis. BMP activity is tightly regulated by a family of soluble extracellular secreted BMP modulators. Twisted gastrulation BMP signaling modulator 1 (TWSG1) is a direct BMP regulator that is required for the full signaling activity of BMPs. However, the functions and mechanisms of TWSG1 in papillary thyroid cancer (PTC) metastasis have not been reported. TWSG1 expression was detected in 44 PTC tissues with lymph node metastasis (LNM) and 56 PTC tissues without LNM using quantitative real-time polymerase chain reaction (qRT-PCR). Gain- and loss-of-function approaches were used to assess the biological function of TWSG1 in PTC cells. Matrigel assays demonstrated the effect of tumor cell-derived TWSG1 on endothelial cell function. Our results showed that TWSG1 expression was significantly enhanced in PTC with LNM compared to that in PTC without LNM. TWSG1 knockdown inhibited migration, invasion and proliferation of PTC cells. Additionally, TWSG1 suppression impaired the tumor cell-induced endothelial cell sprout formation. We found that TWSG1 signaling may be transduced by the BMP target transcription factor inhibitor of DNA binding 1 (Id1) and matrix metalloproteinases (MMPs) 2 and 9. In conclusion, TWSG1 was highly expressed in metastasized PTC; tumor growth, migration and invasion were dependent on TWSG1, and it may be a new diagnostic and therapeutic target for PTC.

Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function.

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER+/- mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.

Bone Morphogenetic Protein Signaling Is Required for Aortic Valve Calcification.

Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice. We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification. BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

TMOD-05MODELING ACVR1 AND HIST1H3B MUTATIONS IN DIFFUSE INTRINSIC PONTINE GLIOMAIN VIVOUSING THE SLEEPING BEAUTY SYSTEM

Diffuse intrinsic pontine glioma (DIPG) is a brainstem tumor that occurs in the pons in children; median age 6-7 years. The most frequent DIPG mutations affect the N-terminal tail of histone H3.3 and histone H3.1 and result in the change of a lysine to methionine at residue 27. In addition, six recurrent somatic activating mutations in ACVR1, which encodes for the bone morphogenetic protein (BMP) receptor kinase, have also been identified in DIPG tumors. We aim to use the sleeping beauty transposase system to generate spontaneous in vivo tumor models that will allow us to test the contributions of these genes to the pathogenesis of DIPG tumors. We have cloned ACVR1 G328V, and HIST1H3B K27M sequences into a plasmid that contains flanking inverted repeats (IR) which contain binding sequences for the sleeping beauty transposase, which will excise DNA between the IR sites and insert it into the host genome. Plasmids encoding a short hair pin for p53, oncogenic NRAS, the sleeping beauty transposase/luciferase were injected with group 1: ACVR1 G328V; group 2: HIST1H3B K27M; or group 3: ACVR1 G328V and HIST1H3B K27M into the subventricular zone of neonatal mice. Tumor development in mice is monitored by measuring luciferase activity in vivo; animals that have reached moribund stage are perfused and processed for histology. Our long-term goal is to understand the interplay between the ACVR1 activating mutation and the histone H3K27M mutation. The ACVR1 mutations result in constitutive BMP pathway activation and the H3K27M mutation has been shown to globally alter H3K27 methylation. Through this study we hope to elucidate how ACVR1 mutations and H3K27 mutations contribute to tumor progression in order to uncover novel therapeutic targets for DIPG which would improve prognosis for DIPG patients.

BMP‐pSmad1/5/8 Pathway Activation in Calcific Aortic Valve Disease

Calcific Aortic Valve Disease (CAVD) affects >2% of the elderly population for whom the standard of care is valve replacement surgery. Unfortunately, the pathogenic mechanisms of CAVD, which could serve as potential therapeutic targets, remain unknown. Klotho‐null mice exhibit accelerated aging and aortic valve (AoV) calcification similar to human CAVD. Notably, phosphorylation of Smads1/5/8, Bone Morphogenetic Protein (BMP) pathway effectors essential for bone calcification, precedes and later localizes with calcific nodules in Klotho‐null AoV. Our hypothesis is that BMP pathway activation promotes AoV calcification. Our goal is to determine the role of the BMP pathway in the pathogenesis of CAVD and to use a pharmacologic inhibitor of BMP signaling as a potential treatment for CAVD in vivo. Our current studies demonstrate that osteochondrogenic factors involved in bone and cartilage formation are significantly increased in Klotho‐null AoV. In addition, BMP pathway components, including BMP2/4, Smad6 and Noggin, are significantly up‐regulated in Klotho­‐null AoV. Moreover, in porcine AoV interstitial cells cultured in vitro, osteogenic media treatment induces osteochondrogenic gene expression and activation of the BMP‐pSmad1/5/8 pathway, as well as formation of calcific nodules. Interestingly, treatment with LDN‐193189, a BMP pathway inhibitor, decreases osteochondrogenic gene induction and prevents calcific nodule formation in the presence of osteogenic media. Together these data support an active role for BMP signaling during osteochondrogenic gene induction and the development of AoV calcification.AHA Pre‐doctoral Fellowship 13PRE1623006 ‐MVGNIH R01 HL114682 ‐KEY.

Antagonism and synergy between extracellular BMP modulators Tsg and BMPER to balance blood vessel formation

Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously acts proangiogenically on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators, especially Tsg, on endothelial cell behaviour and compare them to BMPER. In Matrigel assays, BMP modulators chordin and noggin had no stimulatory effect; however, gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As the activation dynamics of Tsg were similar to those of BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse Matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro, dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.

Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1.

The hereditary mixed polyposis syndrome (HMPS) was first described about 50 years ago in a large Ashkenazi Jewish family from St Mark’s Hospital, London. The family showed apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma in a high proportion of individuals. In the last 15 years, we have mapped the HMPS gene to chromosome 15q13.3 and identified an ancestral haplotype common to all the known HMPS families. Here, we have used genetic mapping, copy number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a large duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This mutation has no effect on SCG5 expression, but is associated with greatly increased, allele-specific GREM1 expression. Whilst GREM1 is expressed in intestinal sub-epithelial myofibroblasts in controls, HMPS patients predominantly express GREM1 in the epithelium of the large bowel. The HMPS duplication contains predicted transcriptional enhancer elements; we have shown that some of these interact with the GREM1 promoter and are capable of driving gene expression in vitro. Increased GREM1 expression is predicted to lead to reduced bone morphogenetic protein pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. The pathogenic mechanism in HMPS is extremely unusual in Mendelian cancer syndromes and highlights ectopic gene expression as a mechanism of tumorigenesis.

646 Statins Inhibit the PI3K/AKT/mTOR Pathway in Colorectal Cancer Through Upregulation of PTEN in a BMP Dependent Manner

Background: Colorectal cancer (CRC) is a major public health problem worldwide and incidence rates are increasing, especially in areas historically at low risk. Removal of cancer precursor lesions at colonoscopy is the foundation of CRC prevention, but offers far from perfect protection from CRC. CRC prevention using chemical or natural compounds (chemoprevention) represents a promising alternative or adjuvant preventative strategy. Epidemiological studies and meta-analyses have shown an association between the use of statins and a reduced incidence of CRC. Statins are generally well tolerated and with proven beneficial effects on cardiovascular outcomes improving any overall benefit/risk ratio, they are an attractive candidate for use as a chemopreventive agent in CRC. Their exact cellular targets and underlying mechanism of action have, however, remained elusive. We have shown that statins act on CRC through activation of Bone Morphogenetic Protein (BMP) signaling. However, statins have been shown by others to have a wide range of effects on many other signaling pathways. Aim & Methods: In this study, we set out to assess the influence of statins on global cell signaling in a hypothesis-free manner. We performed an array-based kinase assay using immobilized kinase substrates spanning the entire human kinome. We used HCT116 colon cancer cells with or without Statin treatment. We confirmed our findings from the array using immunoblotting with activity-specific antibodies. Subsequent experiments in different CRC cell lines using immunoblotting with activation-specific antibodies, RNAi and kinase inhibitors focused on the most consistent and pronounced changes. Experiments in xenografts and in patients treated with Statins were performed to confirm our In Vitro data In Vivo. Results: The clinically relevant concentration of 2μM Lovastatin proved to be optimal for kinome analysis. Treatment with statin led to widespread changes in cell signaling compatible with anti-tumor activity. The strongest effect was seen on the PI3K/Akt pathway. Statins induced upregulation of PTEN activity leading to downregulation of the PI3K/Akt pathway and downstream mTOR-mediated signaling. In Vitro experiments showed that the effect on PTEN is dependent on BMP signaling. Further experiments in xenografts and in patients treated with Statins confirmed the changes in BMP pathway and PTEN activity. Conclusion: Statin treatment rapidly leads to significant changes in signal transduction favoring anti-tumor activity. Statins induce downregulation of Akt/mTOR signaling through upregulation of PTEN activity in a BMP dependent manner; both In Vitro as In Vivo. These findings should ultimately facilitate more targeted use of statins in CRC either alone or in combination with other therapies.

Relay of retrograde synaptogenic signals through axonal transport of BMP receptors

Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. We show that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. We further demonstrate that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast to these results, we find no evidence for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations.

Endothelial Cells in Co-culture Enhance Embryonic Stem Cell Differentiation to Pancreatic Progenitors and Insulin-Producing Cells through BMP Signaling

Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9232-z) contains supplementary material, which is available to authorized users.

BMP Signaling Promotes the Growth of Primary Human Colon Carcinomas in vivo

Human colon carcinomas (CCs) represent a growing worldwide problem. One of the pathways that has been negatively implicated in the genesis of CCs is triggered by bone morphogenetic protein (BMP) ligands, which activate BMP receptors leading to the function of SMAD proteins in the nucleus. BMP signaling is altered in familial human polyposis, and mice with compromised BMP signaling in the intestine develop tumors. Here, we have re-evaluated the presence and roles of BMP signaling in advanced sporadic human CCs, using both primary tumors and established cell lines, and directly modulating BMP pathway activity in a cell-autonomous manner using constitutively active and dominant-negative BMP receptor Ib forms. We find evidence for active endogenous BMP signaling in all primary CC samples and for its role in promoting primary CC tumor growth and CC cell survival and proliferation in vivo in xenografts. In vitro, we also document autonomous and non-autonomous effects of enhanced BMP receptor activity on gap closure in culture, suggesting possible roles in invasion. Caution should thus be exerted in trying to augment or restore its activity for therapeutic purposes. In contrast, we raise the possibility that blockade of BMP signaling might have beneficial effects against at least a subset of advanced colon cancers.

Krüppel-like factor 15 regulates BMPER in endothelial cells

Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. Previous studies have shown that BMP endothelial cell precursor-derived regulator (BMPER) plays an important role in endothelial cell function and blood vessel formation. BMPER is a key regulator of BMP4 activity and a prerequisite for BMP pathway activation by BMP4 in endothelial cells. Here, we characterize the BMPER promoter and elucidate mechanisms of BMPER regulation. To investigate transcriptional mechanisms of BMPER expression, the murine BMPER promoter was cloned and characterized. A series of 5' deletions of the BMPER promoter revealed that the proximal promoter contains activating cis-elements. By overexpression or siRNA-based knockdown, we demonstrate that BMPER expression is activated by Krüppel-like factor (KLF) 15. As determined by gelshift analyses, KLF15 binds directly to a predicted KLF-binding element at -284 bp within the BMPER promoter. Co-expression experiments show that Sp1 acts as an antagonist for KLF15-induced promoter activation. Endothelin-1 was identified as a potent inhibitor of KLF15 and BMPER expression in endothelial cells, suggesting that KLF15 is a transducer of endothelin-1 activity on BMPER expression. The selective ET(B) endothelin receptor antagonist BQ788 abolished the downregulation of BMPER expression by endothelin-1. Mechanistically, we found that KLF15 is a strong and direct activator of the BMPER expression. BMPER is downregulated by endothelin-1 in a dose-dependent fashion and in parallel to KLF15. As KLF15 deficiency is accompanied by a vascular phenotype and BMPER is necessary for proper blood vessel formation, we suggest a chain of events in which the effects of endothelin-1 on BMPER are mediated by KLF15.

FRET Reveals Novel Protein-Receptor Interaction of Bone Morphogenetic Proteins Receptors and Adaptor Protein 2 at the Cell Surface

Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 β-isoform and caveolae of the caveolin-1 α/ β-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.

When one skeleton is enough: approaches and strategies for the treatment of fibrodysplasia ossificans progressiva (FOP).

A heterozygous missense mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor, is responsible for fibrodysplasia ossificans progressiva (FOP), the most catastrophic disorder of skeletal metamorphosis in humans. The discovery of the FOP gene establishes a crucial milestone in understanding FOP, reveals a highly conserved target in the BMP signaling pathway for drug development and specifically stimulates therapeutic approaches for the development of inhibitors for ACVR1/ALK2 signaling. Effective therapies for FOP, and possibly for more common conditions of heterotopic ossification, will be based on interventions that selectively block promiscuous ACVR1/ALK2 signaling, and/or themolecular triggers, responding cells and tissue microenvironments that facilitate aberrant skeletal metamorphosis in a permissive genetic background of increased BMP pathway activity.

Conversion of the Nipple to Hair-Bearing Epithelia by Lowering Bone Morphogenetic Protein Pathway Activity at the Dermal-Epidermal Interface

Epithelial appendages, such as mammary glands and hair, arise as a result of epithelial-mesenchymal interactions. Bone morphogenetic proteins (BMPs) are important for hair follicle morphogenesis and cycling and are known to regulate a wide variety of developmental processes. For example, overexpression of BMPs inhibits hair follicle formation. We hypothesized that the down-regulation of the BMP signaling pathway in the basal epidermis expands regions that are competent to form hair follicles and could alter the fate of the epithelium in the mouse nipple to a hair-covered epidermal phenotype. To test our hypothesis, we used a transgenic mouse model in which keratin 14 (KRT14) promoter-mediated overexpression of Noggin, a BMP antagonist, modulates BMP activity. We observed the conversion of nipple epithelium into pilosebaceous units. During normal mammary gland organogenesis, BMPs are likely used by the nipple epithelium to suppress keratinocyte differentiation, thus preventing the formation of pilosebaceous units. In this report, we characterize the morphology and processes that influence the development of hairs within the nipple of the KRT14-Noggin mouse. We demonstrate that Noggin acts, in part, by reducing the BMP signal in the epithelium. Reduction of the BMP signal in turn leads to a reduction in the levels of parathyroid hormone-related protein. We propose that during evolution of the nipple, the BMP pathway was co-opted to suppress hair follicle formation and create a more functional milk delivery apparatus.