Rachel J. Davies

Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

Mutations in transforming growth factor-β (TGF-β) receptor superfamily members underlie conditions characterized by vascular dysplasia. Mutations in endoglin and activin-like kinase receptor 1 (ALK1) cause hereditary hemorrhagic telangiectasia, whereas bone morphogenetic protein type II receptor (BMPR-II) mutations underlie familial pulmonary arterial hypertension. To understand the functional roles of these receptors, we examined their relative contributions to BMP signaling in human pulmonary artery endothelial cells (HPAECs). BMP9 potently and selectively induced Smad1/5 phosphorylation and Id gene expression in HPAECs. Contrary to expectations, BMP9 also stimulated Smad2 activation. Furthermore, BMP9 induced the expression of interleukin 8 and E-selectin. Using small interfering RNA, we demonstrate that the type I receptor, ALK1, is essential for these responses. However, small interfering RNA and inhibitor studies showed no involvement of ALK5 or endoglin. We further demonstrate that, of the candidate type II receptors, BMPR-II predominantly mediated IL-8 and E-selectin induction and mitogenic inhibition by BMP9. Conversely, activin receptor type II (ActR-II) contributed more to BMP9-mediated Smad2 activation. Only abolition of both type II receptors significantly reduced the Smad1/5 and Id responses. Both ALK1 and BMPR-II contributed to growth inhibition of HPAECs, whereas ActR-II was not involved. Taken together, our findings demonstrate the critical role of type II receptors in balancing BMP9 signaling via ALK1 and emphasize the essential role for BMPR-II in a subset of BMP9 responses (interleukin 8, E-selectin, and proliferation). This differential signaling may contribute to the contrasting pathologies of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Mutations in Bone Morphogenetic Protein Type II Receptor Cause Dysregulation of Id Gene Expression in Pulmonary Artery Smooth Muscle Cells Implications for Familial Pulmonary Arterial Hypertension

Heterozygous germ line mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor occur in more than 80% of patients with familial pulmonary arterial hypertension. Because inhibitors of DNA binding (Id) genes are major targets of BMP/Smad signaling, we studied the regulation of these transcription factors in pulmonary artery smooth muscle cells harboring mutations in BMP type II receptor and control cells. Mutant cells demonstrated a marked deficiency in BMP4-stimulated Id1 and Id2 gene and protein expression compared with control cells. Mutant cells were deficient in Smad1/5 signaling in response to BMPs but also in extracellular signal-regulated kinase (ERK)1/2 activation. We provide evidence for an important interaction between Smad1/5 and ERK1/2 signaling in the regulation of Id gene expression. Thus, BMP4-induced Id1 expression was negatively regulated by ERK1/2 activation. The mechanism involves ERK1/2-dependent phosphorylation of the Smad1 linker region (serine 206), which limits C-terminal serine 463/465 phosphorylation and inhibits Smad nuclear accumulation. Furthermore, activation of ERK1/2 by platelet-derived growth factor BB also caused Smad1 linker region phosphorylation and inhibited BMP4-induced Id1 gene expression. In contrast, Id2 expression was positively regulated by ERK1/2. Moreover, we show that both BMP type II receptor mutation and Id1 knockdown leads to loss of growth suppression by BMPs. Taken together, these findings indicate an important interaction between ERK1/2 and Smad1/5 in the regulation of Id genes. Platelet-derived growth factor, via ERK1/2, further impairs the deficiency in Smad signaling found in BMP type II receptor mutant cells. The integration of these signals at the level of Id gene expression may contribute to the pathogenesis of familial pulmonary arterial hypertension.

BMP type II receptor deficiency confers resistance to growth inhibition by TGF-β in pulmonary artery smooth muscle cells: role of proinflammatory cytokines

Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (HPAH) and a significant proportion of sporadic cases. Pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) not only exhibit attenuated growth suppression by BMPs, but an abnormal mitogenic response to transforming growth factor (TGF)-β1. We sought to define the mechanism underlying this loss of the antiproliferative effects of TGF-β1 in BMPR-II-deficient PASMCs. The effect of TGF-β1 on PASMC proliferation was characterized in three different models of BMPR-II dysfunction: 1) HPAH PASMCs, 2) Bmpr2(+/-) mouse PASMCs, and 3) control human PASMCs transfected with BMPR-II small interfering RNA. BMPR-II reduction consistently conferred insensitivity to growth inhibition by TGF-β1. This was not associated with altered canonical TGF-β1/Smad signaling but was associated with a secreted factor. Microarray analysis revealed that the transcriptional responses to TGF-β1 differed between control and HPAH PASMCs, particularly regarding genes associated with interleukins and inflammation. HPAH PASMCs exhibited enhanced IL-6 and IL-8 induction by TGF-β1, an effect reversed by NF-κB inhibition. Moreover, neutralizing antibodies to IL-6 or IL-8 restored the antiproliferative effect of TGF-β1 in HPAH PASMCs. This study establishes that BMPR-II deficiency leads to failed growth suppression by TGF-β1 in PASMCs. This effect is Smad-independent but is associated with inappropriately altered NF-κB signaling and enhanced induction of IL-6 and IL-8 expression. Our study provides a rationale to test anti-interleukin therapies as an intervention to neutralize this inappropriate response and restore the antiproliferative response to TGF-β1.

Molecular Mechanisms of Pulmonary Arterial Hypertension: Role of Mutations in the Bone Morphogenetic Protein Type II Receptor

Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of small, peripheral resistance vessels in the lung involving proliferation and migration of vascular smooth muscle, endothelial cell and fibroblasts. The increase in pulmonary vascular resistance leads to right heart failure, and, without treatment, death occurs within 3 years of diagnosis. The etiology of PAH is multifactorial. In some patients, there is a major genetic predisposition in the form of heterozygous germline mutations in a transforming growth factor-beta superfamily receptor, the bone morphogenetic type II receptor (BMPR-II). In addition, it is likely that additional factors, such as inflammation, are important to manifest disease. The currently available treatments for PAH were developed mainly as vasodilators, and although they improve symptoms they have limited impact on survival. This review examines the role of the BMPR-II signaling pathway in the process of pulmonary vascular remodeling. We discuss the ways in which manipulation of BMPR-II signaling might be targeted with the aim of preventing or reversing vascular remodeling and improving survival in patients with PAH.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

Mutations in the gene for the transforming growth factor (TGF)-beta superfamily receptor, bone morphogenetic protein receptor II, underlie heritable forms of pulmonary arterial hypertension (PAH). Aberrant signaling via TGF-beta receptor I/activin receptor-like kinase 5 may be important for both the development and progression of PAH. We investigated the therapeutic potential of a well-characterized and potent activin receptor-like kinase 5 inhibitor, SB525334 [6-(2-tert-butyl-5-{6-methyl-pyridin-2-yl}-1H-imidazol-4-yl)-quinoxaline] for the treatment of PAH. In this study, we demonstrate that pulmonary artery smooth muscle cells from patients with familial forms of idiopathic PAH exhibit heightened sensitivity to TGF-beta1 in vitro, which can be attenuated after the administration of SB525334. We further demonstrate that SB525334 significantly reverses pulmonary arterial pressure and inhibits right ventricular hypertrophy in a rat model of PAH. Immunohistochemical studies confirmed a significant reduction in pulmonary arteriole muscularization induced by monocrotaline (used experimentally to induce PAH) after treatment of rats with SB525334. Collectively, these data are consistent with a role for the activin receptor-like kinase 5 in the progression of idiopathic PAH and imply that strategies to inhibit activin receptor-like kinase 5 signaling may have therapeutic benefit.

BMP-9 Induced Endothelial Cell Tubule Formation and Inhibition of Migration Involves Smad1 Driven Endothelin-1 Production

Background Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof. Methodology/Principal Finding Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation. Conclusions/Significance Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.

Transforming Growth Factor-β1 Represses Bone Morphogenetic Protein–Mediated Smad Signaling in Pulmonary Artery Smooth Muscle Cells via Smad3

Previous studies of pulmonary arterial hypertension (PAH) have implicated excessive transforming growth factor (TGF)-β1 signaling and reduced bone morphogenetic protein (BMP) signaling in the disease pathogenesis. Reduced BMP signaling in pulmonary artery smooth muscle cells (PASMCs) from patients with heritable PAH is a consequence of germline mutations in the BMP type II receptor (BMPR-II). We sought to establish whether the TGF-β1 and BMP4 pathways interact in PASMCs, and if this is altered in cells with BMPR-II mutations. Control PASMCs or from patients with PAH harboring BMPR-II mutations were treated with BMP4, TGF-β1, or cotreated with both ligands. Signaling was assessed by examination of Smad phosphorylation, luciferase reporters, and the transcription of BMP4 or TGF-β1-responsive genes. TGF-β1 attenuated BMP4-mediated inhibitors of differentiation 1/2 induction and abolished the response in BMPR-II mutant PASMCs, whereas BMP4 did not alter TGF-β1-mediated transcription. Activin-like kinase 5 inhibition blocked this effect, whereas cycloheximide or pharmacological inhibitors of TGF-β-activated kinase 1, extracellular signal-regulated kinase 1/2, or p38 mitogen-activated protein kinase were ineffective. BMP4 and TGF-β1 cotreatment did not alter the activation or nuclear translocation of their respective Smad signaling proteins. Small interfering RNA for Smad3, but not Smad2, Smad6, or Smad7, reversed the inhibition by TGF-β1. In addition, TGF-β-activated kinase 1 inhibition blocked Smad3 phosphorylation, implying that C-terminal Smad3 phosphorylation is not required for the inhibition of BMP4 signaling by TGF-β1. TGF-β1 reduces BMP4 signaling in PASMCs, a response that is exacerbated on the background of reduced BMP responsiveness due to BMPR-II mutations. These data provide a rationale for therapeutic inhibition of TGF-β1 signaling in PAH.

S38 TGF-Beta1 Negatively Regulates BMP4 Signalling in Human Pulmonary Artery Smooth Muscle Cells Via A Smad3-Dependent Mechanism

Introduction BMP4 signals via the Smad pathway to induce the expression of the ID dominant-negative basic helix-loop-helix transcription factors (ID1–4) that regulate cell differentiation. We have shown that ID induction is blunted in human pulmonary artery smooth muscle cells (HPASMCs) from pulmonary arterial hypertension (PAH) patients with mutations in the bone morphogenetic type-II receptor (BMPR-II). TGFβ1 is implicated in the pathogenesis of PAH. We therefore examined whether TGFβ1 and BMP4 signalling directly interact in HPASMCs. Methods Explant-derived HPASMCs from unaffected donors or PAH patients with identified BMPR-II mutations were studied. The transcriptional responses of ID1, ID2, PAI1 and CTGF to BMP4, TGFβ1 or co-treatment were examined by qPCR In some experiments, cells were pre-incubated with cycloheximide or pharmacological inhibitors of ALK5 (SD208), MAP kinase (U0126), TAK1 or p38 MAP kinase (SB203580). The roles of Smad2, Smad3, Smad6 and Smad7 were investigated using siRNAs. Smad-dependent transcription was examined using the BMP-responsive luciferase reporter (BRE-luc) and the TGF-responsive luciferase reporter, CAGA12-luc. Protein lysates collected at 1 and 4hrs were immunoblotted for phosphorylated and total Smads and candidate kinases. In some experiments, nuclear and cytoplasmic fractions were prepared and immunoblotted. Results BMP4 induced ID1 and ID2 expression at 1, 4 and 24h whereas TGFβ1 induced ID1/2 at 1h and repressed them at 4h and 24h. TGFβ1, but not BMP4, induced PAI1 and CTGF expression. BMP4 did not alter TGFβ1-mediated transcriptional responses. In contrast, TGFβ1 attenuated BMP4-mediated ID1/2 induction and the BRE-luc response in donor HPASMCs. Moreover, TGFβ1 abolished BMP4 responses in PAH PASMCs with BMPR-II mutations. This was reversed by the ALK5 inhibitor, SD208, but not by cycloheximide or a TAK1 inhibitor. BMP-Smad phosphorylation and nuclear translocation did not differ between co-treatment and treatment with individual ligands. Smad3 siRNA, but not Smad2, Smad6 or Smad7, reversed the inhibitory effect of TGFβ1. Conclusions TGFβ1 negatively regulates BMP4-mediated ID genes transcription by interfering with BMP-Smad signalling via Smad3, abolishing the BMP signals in cells with BMPR-II mutations. These data reinforce the notion that TGFβ1 plays a pathogenic role in PAH, via direct inhibition of the attenuated BMP response caused by BMPR-II insufficiency.

TGF-β/BMP Signaling in Pulmonary Vascular Disease

The transforming growth factor β (TGF-β) superfamily is a group of multifunctional proteins with over 35 distinct members, including TGF-β, activins, bone morphogenetic proteins (BMPs), and growth differentiation factors.1 They all have profound effects on developmental processes ranging from soft tissue and skeletal development to vasculogenesis.2 The effects are not limited to embryogenesis alone as these molecules are also known to play significant roles in the maintenance and control of adult tissues.2, 3, 4 Although TGF-β is the prototypic member of the family, the largest group of cytokines within the TGF-β superfamily comprises the BMPs. They were originally identified as molecules regulating growth and differentiation of bone and cartilage. However, BMPs are now known to regulate growth, differentiation, and apoptosis in a diverse number of cells lines, including mesenchymal and epithelial cells.2, 3, 4