Receptor Activin Receptor-like Kinase Research Articles

Benzo[a]pyrene and a high-fat diet induce aortic injury and promote low-density lipoprotein accumulation in the endothelium

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant which mainly exposed though diet. High-fat diet (HFD) can induce atherosclerosis, as can BaP. Unhealthy dietary habits lead to high intake of both BaP and lipids. However, the combined effect of BaP and HFD on atherosclerosis and lipid accumulation in the arterial wall, the initial stage of atherosclerosis, is unclear. In this study, C57BL/6 J mice were subchronically exposed to BaP and a HFD, and the mechanism of lipid accumulation was investigated in EA.hy926 and HEK293 cells. Results showed that BaP and HFD increased blood lipids and damaged aortic wall synergistically. Meanwhile, LDL enhanced the toxicity of BaP, and BaP promoted the production of reactive oxygen species and malonaldehyde in EA.hy926 cells, which aggravated LDL-induced cell injury. Moreover, BaP and HFD/LDL induced LDL accumulation in the aortic wall of C57BL/6 J mice/EA.hy926, and the mechanism was by activating AHR/ARNT heterodimer to combine with the scavenger receptor BⅠ (SR-BⅠ) and activin receptor-like kinase 1 (ALK1) promoter regions to transcriptional upregulate its expression, which enhanced the uptake of LDL, and promoting the production of AGEs to inhibit reverse cholesterol transport by SR-BI. BaP and lipid synergistically promoted aortic and endothelial damage, and the health risk of their combined intake should be paid attention to.

Thrombosis in hereditary hemorrhagic telangiectasia

Thrombosis in hereditary hemorrhagic telangiectasia

Activin A promotes hyaluronan production and upregulates versican expression in human granulosa cells†.

Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

ALK1 signaling is required for the homeostasis of Kupffer cells and prevention of bacterial infection.

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. The BMP receptor activin receptor–like kinase 1 (ALK1) is well known for its role in blood vessel formation; however, its role within the immune system has never been revealed to our knowledge. Here, we found that BMP9/BMP10/ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen, and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages showed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

BMP9 attenuates occurrence of venous malformation by maintaining endothelial quiescence and strengthening vessel walls via SMAD1/5/ID1/α-SMA pathway

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9−/− zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

A Signaling Crosstalk between BMP9 and HGF/c-Met Regulates Mouse Adult Liver Progenitor Cell Survival.

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-β type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Bone Morphogenetic Protein 9 Is a Paracrine Factor Controlling Liver Sinusoidal Endothelial Cell Fenestration and Protecting Against Hepatic Fibrosis.

Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.

Advances in the molecular regulation of endothelial BMP9 signalling complexes and implications for cardiovascular disease.

Bone morphogenetic protein 9 (BMP9), a member of the transforming growth factor β (TGFβ) superfamily, is a circulating vascular quiescence and endothelial protective factor, accounting for the majority of BMP activities in plasma. BMP9 and BMP10 bind preferentially to the high-affinity type I receptor activin receptor-like kinase 1 on vascular endothelial cells. Recently, many reports have highlighted the important roles of BMP9 in cardiovascular disease, particularly pulmonary arterial hypertension. In vivo, BMP9 activity and specificity are determined by tightly regulated protein-protein recognition with cognate receptors and a co-receptor, and may also be influenced by other proteins present on the endothelial cell surface (such as low-affinity receptors) and in circulation (such as TGFβ family ligands competing for the same receptors). In this review, we summarise recent findings on the role and therapeutic potential of BMP9 in cardiovascular disease and review the current understanding of how the extracellular protein-protein interaction milieu could play a role in regulating endothelial BMP9 signalling specificity and activity.

Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER+ breast cancer.

Despite the success of approved systemic therapies for estrogen receptor α (ER)-positive breast cancer, drug resistance remains common. We hypothesized that secreted factors from the human tumor microenvironment could modulate drug resistance. We previously screened a library of 297 recombinant-secreted microenvironmental proteins for the ability to confer resistance to the anti-estrogen fulvestrant in 2 ER+ breast cancer cell lines. Herein, we considered whether factors that enhanced drug sensitivity could be repurposed as therapeutics and provide leads for drug development. Screening data revealed bone morphogenic protein (BMP)4 as a factor that inhibited cell growth and synergized with approved anti-estrogens and cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). BMP4-mediated growth inhibition was dependent on type I receptor activin receptor-like kinase (ALK)3-dependent phosphorylation (P) of mothers against decapentaplegic homolog (SMAD/P-SMAD)1 and 5, which could be reversed by BMP receptor inhibitors and ALK3 knockdown. The primary effect of BMP4 on cell fate was cell-cycle arrest, in which RNA sequencing, immunoblot analysis, and RNA interference revealed to be dependent on p21WAF1/Cip1 upregulation. BMP4 also enhanced sensitivity to approved inhibitors of mammalian target of rapamycin complex 1 and CDK4/6 via ALK3-mediated P-SMAD1/5 and p21 upregulation in anti-estrogen-resistant cells. Patients bearing primary ER+ breast tumors, exhibiting a transcriptomic signature of BMP4 signaling, had improved disease outcome following adjuvant treatment with anti-estrogen therapy, independently of age, tumor grade, and tumor stage. Furthermore, a transcriptomic signature of BMP4 signaling was predictive of an improved biologic response to the CDK4/6i palbociclib, in combination with an aromatase inhibitor in primary tumors. These findings highlight BMP4 and its downstream pathway activation as a therapeutic opportunity in ER+ breast cancer.-Shee, K., Jiang, A., Varn, F. S., Liu, S., Traphagen, N. A., Owens, P., Ma, C. X., Hoog, J., Cheng, C., Golub, T. R., Straussman, R., Miller, T. W. Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER+ breast cancer.

A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10 provides most BMP biological activity in plasma

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

Inhibition of TGF-β Signaling in Tumor Cells by Small Molecule Src Family Kinase Inhibitors.

In a series of studies carried out over the last couple of years in various cell types, it was observed that the experimentally used Src family kinase inhibitors PP1 and PP2 and the clinically used Src/Abl inhibitors AZM475271 and dasatinib are potent inhibitors of TGF-β mediated cellular responses such as Smad and p38 mitogen-activated protein kinase phosphorylation, Smad-dependent transcriptional activation, growth inhibition, epithelial-mesenchymal transition (EMT), and cell motility. While for PP1/PP2 it was demonstrated that these agents directly inhibit the kinase activity of the TGF-β type I receptor activin receptor-like kinase 5, the mechanism of the anti-TGF-β effect of AZM475271 and dasatinib is less clear. In contrast, the anti-TGF-β effect of yet another Src/Abl inhibitor, bosutinib, is more variable with respect to the type of the TGF-β response and the cell type affected, and lacks a clear dose-dependency. In the light of their strong anti-activin receptor-like kinase 5 kinase effect, PP1 and PP2 should not be used when studying the role of c-Src as downstream mediators in TGF-β/activin receptor-like kinase 5 signaling. On the other hand, based upon in vitro findings, it is conceivable that part of the therapeutic effects of AZM475271 and dasatinib seen in preclinical and clinical studies with solid tumors was caused by inhibition of prometastatic TGF-β rather than Src signaling. If AZM475271 and dasatinib can indeed act as dual Src / TGF-β inhibitors in vivo, this may be beneficial for prevention of metastatic disease in more advanced tumor stages.

Transforming Growth Factor-β1/Activin Receptor-like Kinase 5-Mediated Cell Migration is Dependent on the Protein Proteinase-Activated Receptor 2 but not on Proteinase-Activated Receptor 2-Stimulated Gq-Calcium Signaling.

Transforming growth factor-β (TGF-β), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-β1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-β receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-β signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-β1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-β1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-β1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-β1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-β1 synergy may involve TGF-β1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-β's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.

The Increased Transforming Growth Factor-β Signaling Induced by Diabetes Protects Retinal Vessels

The roles of transforming growth factor (TGF)-β in extracellular matrix production and vascular remodeling, coupled with increased TGF-β expression and signaling in diabetes, suggest TGF-β as an important contributor to the microangiopathy of diabetic retinopathy and nephropathy. To investigate whether increased TGF-β signaling could be a therapeutic target for preventing retinopathy, we used a pharmacologic approach (SM16, a selective inhibitor of the type 1 TGF-β receptor activin receptor-like kinase 5, orally active) to inhibit the increased, but not the basal, Tgf-β signaling in retinal vessels of diabetic rats. At the level of vascular gene expression, 3.5 months' diabetes induced minimal changes. Diabetes + SM16 for 3 weeks caused widespread changes in gene expression poised to enhance vascular inflammation, thrombosis, leakage, and wall instability; these changes were not observed in control ratsgiven SM16. The synergy of diabetes and SM16 in altering gene expression was not observed in the lung. At the level of vascular network morphology, 7 months' diabetes induced no detectable changes. Diabetes + SM16 for 3 weeks caused instead distorted morphology and decreased density. Thus, indiabetes, retinal vessels become dependent on a small increase in TGF-β signaling via activin receptor-like kinase 5 to maintain early integrity. The increased TGF-β signaling may protect against rapid retinopathy progression and should not be a target of inhibitory interventions.

Promiscuous signaling of ligands via mutant ALK2 in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disorder characterized by successive heterotopic bone formation, for which at present there is no therapy. Mutations in the bone morphogenetic protein (BMP) type I receptor Activin receptor-like kinase 2 (ACVR1/ALK2) are the main trigger for FOP and inflammation is thought to be the secondary hit. The single nucleotide mutation at position 617 in the cDNA ALK2 sequence, which is found in 98% of FOP patients, results in a R206H change in the intracellular juxtamembrane region of ALK2. Previous studies had revealed that this mutation perturbs the interaction with the negative regulator FKBP12, thereby sensitising cells expressing this mutant receptor to BMPs, which are potent inducers of cartilage and bone formation. Recently, however, a twist in the underlying mechanism of FOP was revealed. Mutant ALK2 was found to respond to Activin-A, whereas wild type ALK2 function is inhibited by Activin-A. The latter cytokine is induced locally upon tissue damage and inflammation. Moreover, therapeutic targeting of Activin-A was found to inhibit heterotopic ossification in a mutant ALK2 knock-in mouse model that is highly reminiscent to human FOP. This review will focus on these latest surprising findings and discuss the implication for treatment of FOP patients.

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5

Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2−/− mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

BackgroundAlthough systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells.Methodology/Principal FindingsHyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes.ConclusionHyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-β1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.