Stimulation of Id1 Expression by Bone Morphogenetic Protein Is Sufficient and Necessary for Bone Morphogenetic Protein–Induced Activation of Endothelial Cells
Background— Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-β family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior.
Methods and Results— Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration.
Conclusions— The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration.
Received April 22, 2002; revision received July 29, 2002; accepted July 29, 2002.
During angiogenesis, the transition from the resolution to the activation phase and vice versa is determined by an intricately regulated balance between positive and negative regulators.1 In adults, angiogenesis is essential for the female reproductive cycles (eg, during menstruation and pregnancy) and for the repair and remodeling of tissues (eg, wound healing, hard tissue repair, and during ischemia). Neovascularization plays a pivotal role in pathological processes such as tumor growth and metastasis, chronic inflammation, and diabetic vasculopathy.1
Bone morphogenetic proteins (BMPs) belong to a large family of structurally and multifunctional proteins, known as the transforming growth factor-β (TGF-β) superfamily, to which TGF-βs and activins also belong.2 Both endothelial cells (ECs) and vascular smooth muscle cells have been found to express BMPs, including BMP-6.3–5⇓⇓ BMPs have been reported to suppress vascular smooth muscle cell proliferation and increase the expression of smooth muscle differentiation markers.4,6⇓ Effects of BMPs on cultured ECs have not been reported.
Like all members of the TGF-β superfamily, BMP elicits its cellular effects by activating specific combinations of type I and type II receptors with intrinsic serine/threonine kinase activity.2,7⇓ Three specific BMP type I receptors (ie, activin receptor-like kinase [ALK] 2, ALK3 and ALK6) and one specific BMP type II receptor (ie, BMPRII) have been identified.7 Type I receptors determine signal specificity in the BMP receptor complex. Activated BMP type I receptors initiate intracellular signaling by phosphorylating specific receptor-regulated R-Smad proteins (ie, Smad1, Smad5, or Smad8), which assemble into heteromeric complexes with the common-partner Smad4. These heteromeric complexes translocate efficiently to the nucleus, where they regulate the transcription of target genes.2,7⇓
That TGF-β superfamily members are indeed of importance in vascular morphogenesis is demonstrated with different knockout mice of TGF-β family signaling components. Mice lacking TGF-β1 or specific TGF-β type receptors die at midgestation due to defects in angiogenesis.8 The BMP-2/4– and BMP-5/7–deficient mice show abnormal development of the heart and the vasculature.8 Mice deficient in the BMP receptors die early in development, except for those with a knockout of ALK6, whose defects are restricted to the appendicular elements.9 Cardiac myocyte-specific conditional deletion of ALK3 reveals an essential role for this receptor in cardiac septation and atrioventricular cushion morphogenesis.10 Smad1 and Smad5 knockout mice die at embryonic week 10.5 to 11.5 due to defects in vascular development that show enlarged blood vessels surrounded by a decreased number of vascular smooth muscle cells.8,11,12⇓⇓
Perturbation of TGF-β family receptor signaling in humans has also been shown to lead to vascular disorders. Hereditary hemorrhagic telangiectasia, an autosomal-dominant disease with vascular dysplasia resulting in arterial malformations and telangiectasia, is genetically linked to mutations in 2 TGF-β receptors, endoglin and ALK1.8 Interestingly, familial primary pulmonary hypertension, which is also an autosomal-dominant disorder, has been linked to mutations in BMPRII.13 The fact that mice deficient in BMPs, their receptors, or their intracellular Smad effector proteins have impaired vascular development and the linkage of PPH with mutations in BMPRII implicates a role of the BMP/Smad signaling cascade in the formation of the cardiovascular system.
We studied the expression and function of BMP receptors and their downstream signaling components in ECs and examined the effect of BMP on EC behavior. Interestingly, BMP was found to be a stimulator of EC migration and tube formation. Our findings are of relevance for the pathophysiology of vascular morphogenesis.
Antibodies and Ligands
Polyclonal antisera against BMP receptors and Smads have been described previously.14–18⇓⇓⇓⇓ Recombinant BMP-6, activin, and TGF-β3 were generously provided by Drs Kuber Sampatrh, (Creative Biomolecules, Inc, Hopkinton, Mass), Y. Eto (Ajinomoto Co, Kawasaki, Japan), and K. Iwata (OSI Pharmaceuticals, Inc, Farmingdale, NY).
Cell Culture and Adenoviral Infections
Bovine aortic ECs (BAEC)s and mouse embryonic ECs (MEEC)s were cultured as described previously.17 BAECs were generously provided by Dr Haivitz-Friedman. ECs were infected with adenoviruses expressing LacZ or hemagglutinin (HA)-tagged constitutively active (ca) ALK2, caALK3, caALK6 (a gift from K. Miyazono, Cancer Institute, Tokyo, Japan), Id1, or Id3 (a gift from Dr T. Taga, Kumamoto University, Kumamoto, Japan) using a multiplication of infection of 500. After 16 hours, cells were washed and allowed to recover for 24 hours before use in proliferation, migration, or tube formation assays.
Immunoprecipitation, Western Blotting, and Immunohistochemistry
RNA Isolation and Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from BAECs or MEECs with RNeasy columns (Qiagen) according to the manufacturer’s instructions. cDNA synthesis and reverse transcription–polymerase chain reaction (RT-PCR) were performed as previously described.17 Details of PCR primers used are available on request. PCR products were loaded on an agarose gel and stained with ethidium bromide. Identity of the PCR products was confirmed by DNA sequencing.
Transient transfections, transcriptional reporter assays, [32P]ortho-phosphate labeling, loading of ECs with oligonucleotides, EC migration, and a scratch assay were performed as previously described.17 For the Matrigel-based tube formation assay, wells of a chilled 24-well plate were coated with Matrigel (Becton Dickinson) and incubated at 37°C for 30 minutes to form a gel. Adenoviral-infected BAECs were serum-starved overnight, trypsinized, and added to the well. After 24 hours of incubation in the absence or presence of BMP, the BAECs were fixed and photographed.
Expression of BMP Receptors
Immunohistochemistry on sections from human and mouse tissue revealed the expression of BMP type I and type II receptors in ECs. ALK3, ALK6, and BMPRII, but not ALK2, were expressed in ECs in the human colon (Figure 1A). The staining results were confirmed with antibodies raised against different epitopes (purchased from SantaCruz Biotechnology) for ALK3, ALK6, or BMPRII (data not shown). ECs that expressed BMP receptors stained positive with antibodies raised against PECAM/CD31 and endothelin (purchased from Pharmingen; data not shown). The underlying parenchyma in arterioles stained positively with ALK3 antibody. ALK6 immunoreactivity was found in muscle and fibroblast layers in arterioles, whereas BMPRII immunoreactivity was restricted to the endothelial layer. In the mouse heart, BMPRII and ALK3 were expressed in both large vessels and capillaries. ALK2 and, to lesser extent, ALK6 were expressed only in capillaries. Diffuse immunoreactivity for ALK6 was detected in stroma (Figure 1B). In the mouse aorta, ALK3, ALK6, and BMPRII, but not ALK2, were expressed in ECs lining the aorta (Figure 1C). Phosphorylated Smad1, Smad5, and/or Smad8 were detected in these ECs using a phospho-Smad1 (PS1) antibody that specifically recognizes phosphorylated Smad1, Smad5, and/or Smad8,16,17⇓ suggesting that these cells received BMP stimulation. Smad4 was also expressed there, which together with the activated R-Smad, is capable of transducing the BMP receptor signal into a nuclear transcriptional response. Thus, the BMP signaling cascade is present in ECs in vivo.
BMP Efficiently Induces Smad5 Phosphorylation and Activates Smad-Dependent Response in ECs
To examine the in vitro effects of BMP, we characterized MEECs and BAECs for BMP receptor expression by performing RT-PCR. BAECs expressed BMPR-II, ALK2, and ALK6, and MEECs expressed BMPR-II, ALK2, and ALK3 (Figure 2A). The lack of PCR products for ALK3 and ALK6 in BAECs and MEECs, respectively, is not due to defective annealing of primers due to species difference (data not shown). By affinity-labeling of BAEC surface proteins with radiolabeled BMP-6, followed by immunoprecipitation of the cell lysates with specific receptor antibodies, we could also show the presence of BMP type I and type II receptors (data not shown). RT-PCR analysis of Smad1, Smad5, Smad8, and Smad4 revealed that their mRNAs are expressed in BAECs and MEECs. Thus, BMP receptors and their effector Smads are expressed in BAECs and MEECs.
To study the activation of endogenous R-Smads in these primary ECs on BMP-6 stimulation, we used PS1 and a phospho-Smad2 antibody (PS2) that specifically detects phosphorylated Smad2.16–18⇓⇓ Consistent with the effects of BMP on other cell types, BMP stimulation specifically induced the phosphorylation of Smad1, Smad5, and/or Smad8 in BAECs (Figure 2B). As previously reported, TGF-β induced both Smad5 and Smad2 phosphorylation in BAECs.17 In contrast to TGF-β and BMP, activin only induced Smad2 phosphorylation (Figure 2B). To verify that BMP stimulation indeed leads to Smad5 phosphorylation, an orthophosphate labeling was performed on MEECs in the absence and presence of BMP-7 (BMP-7 is closely related to BMP-6 and signals using the same BMP receptors), and the lysate was immunoprecipitated with a specific Smad5 antibody. In lysates from BMP-7-treated cells, a [32P]-labeled protein was immunoprecipitated with the expected size of Smad5. The specificity of the antibody is shown by blocking the recognition of Smad5 after adding a cognate peptide (Figure 2C). Interestingly, we observed that in the 35S-methionine/cysteine–labeled cell lysates, the mobility of Smad5 was slightly retarded on BMP-7 stimulation, which is also indicative of BMP-7–induced Smad5 phosphorylation. Identical results were obtained in BAECs (data not shown). Thus, this independent method confirmed that BMP stimulation leads to a strong phosphorylation of Smad5 in ECs (Figure 2C).
Challenging BAECs with different BMP-6 concentrations showed that Smad1, Smad5, and/or Smad8 phosphorylation reaches a peak after stimulation with 25 ng/mL BMP-6 and remains stable up to 500 ng/mL BMP-6 (Figure 2D). To obtain more insight into the kinetics, we examined the BMP-6–induced Smad phosphorylation over a 0- to 24-hour time period. Peak levels of BMP-6–induced Smad phosphorylation were reached between 30 to 60 minutes and were still observed after 24 hours of BMP-6 activation (Figure 2E). On Western blots with a high separating resolution at 50 to 60 kDa that were incubated with PS1 antibody, we observed that BMP-6 induces phosphorylation of 2 Smad proteins, of which the protein with a higher molecular weight is recognized by a Smad5-specific antibody. The lower band may represent phosphorylated Smad1 or Smad8 because PS1 is capable of detecting all these phosphorylated R-Smads.
To study the effect of BMP type I receptors on Smad activation, we infected BAECs with an adenovirus expressing caALK2, caALK3, or caALK6 and analyzed their effect on R-Smad phosphorylation (Figure 2F). As expected, all the caBMP type I receptors induce the phosphorylation of Smad1, Smad5, and/or Smad8. To examine whether BMP-induced Smad phosphorylation can induce transcriptional responses in ECs, the Smad-binding element (SBE)4-luc reporter (capable of responding to BMPs) was transfected in MEEC cells together with caALK3 or stimulated with BMP-6. The (SBE)4-luc reporter was highly induced by both caALK3 and BMP-6 stimulation (Figure 2G). Therefore, the ECs analyzed have all the components needed to transduce the signal from the membrane to the nucleus, resulting in Smad-dependent transcriptional activation.
BMP-6 Stimulates EC Migration and Tube Formation
On demonstrating the presence of the BMP signaling pathway in ECs, we wanted to elucidate the biological effects of BMPs on EC behavior. Therefore, we examined the effects of BMP receptor activation on migration and tube formation. We plated adenovirally-infected cells with caALKs, Smad1, Smad5, or LacZ, with or without BMP-6, in a Boyden chamber assay and measured the chemokinetic response. BAECs challenged with BMP-6, as well as infected with caBMPRIs or Smad1 or Smad5, showed an increased migratory behavior compared with untreated or LacZ-infected BAECs (Figures 3A and 3B and data not shown). A similar stimulatory effect of BMP-6 on EC migration was observed when performing a scratch assay using BAECs (data not shown). To examine the effect of BMP on tube formation, we plated BAECs on Matrigel. BMP-6, as well as infection with caBMPRIs, potently stimulated the formation of cord-like structures (Figure 3C). Taken together, our results implicate that the BMP/Smad pathway induces an activation of the endothelium.
BMP-6 Potently Induces Id1 Expression in ECs
BMP activation in embryonic stem cells is known to stimulate the expression of Id proteins, which act as dominant-negative inhibitors of basic helix loop helix transcription factors.19,20⇓ Northern blot analysis revealed that Id1 mRNA levels are increased after stimulating BAECs with BMP-6 (Figure 4A). In addition, we found that infecting BAECs with an adenovirus expressing the caBMPRIs or stimulating the cells with BMP-6 induced Id1 protein expression (Figure 4B). Consistent with these findings, BMP-6 activated an Id1 promoter-driven luciferase reporter construct (Id1-Luc) in BAECs (Figure 4C). To examine whether Smads are involved in BMP-6-induced transcriptional activation of the Id1 promoter, we cotransfected the Id1-Luc reporter with expression constructs for Smad1, Smad5, dominant-negative Smad4, or inhibitory Smads (ie, Smad6 or Smad7). We found that ectopic expression of Smad1 or Smad5, together with Smad4, promoted basal activation of this reporter, and dominant-negative Smad4 and both inhibitory Smads inhibited BMP-induced luciferase activity (Figure 4D). These findings indicate an involvement of Smad proteins in BMP-induced activation of the Id1 promoter. TGF-β induces Id1 expression in ECs by activating the ALK-1/Smad5 pathway,17 whereas activin, which is capable of phosphorylating Smad2 but not Smad1, Smad5, or Smad8 (Figure 2A), did not activate the Id1 reporter (data not shown). Thus, Id1 is a downstream target of the BMP/Smad pathway in ECs.
Ectopic Expression of Id1 Induces EC Migration and Tube Formation and Inhibiting Id1 Expression Blocks the BMP-Induced Migration
Id proteins affect cell proliferation, migration, invasion, and differentiation and have an essential role in angiogenesis.20 We examined the effects of ectopic Id1 expression on EC migration and tube formation. To analyze chemokinesis, Id1-infected MEECs were plated in a Boyden chamber. Ectopic expression of Id1 promoted EC migration (Figure 4E). Similar results were obtained when BAECs were infected with adenoviral Id1 or Id3 (data not shown). Importantly, BMP-induced migration of MEECs was inhibited when cells were treated with Id1 antisense oligonucleotides but not when challenged with Id1 sense oligonucleotides (Figure 4F). In MEECs treated with antisense-Id1, Id1 expression is selectively inhibited.17 To test the effect of Id1 on tube formation, Id1-infected BAECs were plated on Matrigel. Interestingly, ectopic expression of Id1 induced the formation of cord-like structures in a fashion similar to that of BAECs stimulated with BMP-6 (Figure 4G). Thus, Id1 is an important downstream target of BMP in mediating its stimulatory effect on EC migration and is also likely to have an effect on EC tube formation.
Genetic studies in mice and humans point to an important role for BMPs in cardiovascular processes.8,9,13⇓⇓ Previous in vitro studies on the function of BMPs in the vascular system have been focused on their effects on vascular smooth muscle cells.3–6⇓⇓⇓ In the present study, we show the existence of a BMP signaling pathway in ECs in vivo and in vitro (Figures 1 and 2⇑). We report that BMP receptor/Smad activation stimulates EC migration and tube formation (Figure 3), indicating a role for BMP in regulating the organization and differentiation of the newly formed ECs in a capillary network. The BMP/Smad-induced activation of ECs was found to be critically dependent on the BMP/Smad-induced upregulation of Id1 (Figure 4). Ectopic expression of Id1 (or Id3) was sufficient to induce EC migration and tube formation (Figure 4). Thus, the effect of BMP receptor activation on ECs can be mimicked by the overexpression of its target gene, Id1. Our findings add BMPs to a growing list of potent proangiogenic factors and may provide new insights into mechanisms that underlie cardiovascular phenotypes observed in mice and humans with perturbed BMP/Smad signaling pathways.
We report here the BMP-6-induced phosphorylation of Smad1, Smad5, and/or Smad8 in ECs (Figure 2B). This BMP response, although similar to the effect of BMP on non-ECs,1,7⇓ was distinct from that of TGF-β, which induced phosphorylation of Smad2 and Smad5 via ALK5 and ALK1, respectively,17 and of activin, which induced only Smad2 phosphorylation that was probably mediated via ALK4 (Figure 2B). The kinetics of TGF-β versus BMP-induced Smad5 phosphorylation is distinct; although TGF-β–induced Smad5 phosphorylation is transient,17 BMP-6–induced phosphorylation is very stable (Figure 2E). Because BMP-6 and closely related family members are expressed by cells of the vascular system, including ECs and vascular smooth muscle cells, they may thus stimulate, in an autocrine or paracrine manner, the activation state of endothelium.
The mechanism by which Id1 via BMP stimulates migration and tube formation (Figure 3) is not known. We are currently exploring the possibility that this is caused by changes in transcription factor activity, leading to changes in gene expression for metalloproteinases and integrins. During embryogenesis and tumorigenesis, Id1 and Id3 are abundantly expressed during blood vessel formation.20 It will be of interest to examine whether the BMP/Smad pathway is providing the stimulus for Id expression in these ECs. Id proteins were required for the proliferative and invasive phenotype of ECs during angiogenesis; Id1/Id3 double-null mice show abnormal angiogenesis, forming enlarged, dilated blood vessels, and Id1+/−/Id33−/− double-mutant mice failed to support tumor growth and metastasis from xenografts due to poor vascularization.21 Of note, mice deficient in Smad1 or Smad5,8,11,12⇓⇓ the downstream effectors for BMP receptors in ECs, also show enlarged and dilated blood vessels and are thus reminiscent of Id1/Id3-null mice.
BMPs induce bone formation during embryogenesis and fracture healing by promoting a complex sequential cascade of events, involving chemotaxis, condensation, proliferation, and differentiation of mesenchymal cells. Interestingly, angiogenesis and vascular invasion are key events in endochondral ossification; extensive vascularization precedes bone formation, and interfering with angiogenesis has been shown to block bone formation.22 In this respect, our finding that BMP can directly act on ECs may thus be relevant for the anabolic effect of BMP on bone formation; BMPs may not only stimulate bone formation by acting directly on mesenchymal cells or indirectly on ECs by inducing the expression of angiogenic factors in mesenchymal cells,23 but also by directly activating ECs to stimulate angiogenesis.
This study was supported by grants from the Netherlands Heart Foundation (grant 99-046), Dutch Cancer Society (NKI 2000-22117), Dutch Organization for Scientific Research (MW 902-16-295), EU QLG1-CT-2001-01032, and the Ludwig Institute for Cancer Research.
- ↵Yi SE, LaPolt PS, Yoon BS, et al. The type I BMP receptor BmprIB is essential for female reproductive function. Proc Natl Acad Sci U S A. 2001; 98: 7994–7999.
- ↵Gaussin V, van de Putte T, Mishina Y, et al. Endocardial cushion and myocardial defect after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3. Proc Natl Acad Sci U S A. 2002; 99: 2878–2883.
- ↵Tremblay KD, Dunn NR, Robertson EJ. Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development. 2001; 128: 3609–3621.
- ↵Rosenzweig BL, Imamura T, Okadome T, et al. Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc Natl Acad Sci U S A. 1995; 92: 7632–7636.
- ↵Hollnagel A, Oehlmann V, Heymer J, et al. Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem. 1999; 274: 19838–19845.