Human Molecular Genetics, 2000, Vol. 9, No. 8 1227-1237
© 2000 Oxford University Press
Analysis of ALK-1 and endoglin in newborns from families with hereditary hemorrhagic telangiectasia type 2
Cancer and Blood Research Programme, The Hospital for Sick Children, and Department of Immunology, University of Toronto, Toronto M5G 1X8, Canada and 1Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain
Received 28 January 2000; Revised and Accepted 5 March 2000.
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ABSTRACT |
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ALK-1 (activin receptor-like kinase-1), a type I receptor of the transforming growth factor (TGF)-ß superfamily, is the gene mutated in hereditary hemorrhagic telangiectasia type 2 (HHT2) while endoglin is mutated in HHT1. Using a novel polyclonal antibody to ALK-1, we measured ALK-1 expression on human umbilical vein endothelial cells (HUVEC) of newborns from HHT families whose affected members had normal endoglin levels. ALK-1 levels were specifically reduced in three HUVEC with ALK-1 missense mutant codons, and normal in two newborns not carrying the missense mutations present in the clinically affected relatives. Levels were also normal in a HUVEC with deletion of S232 in the ATP binding site of ALK-1. Thus HHT2 appears to be associated with a loss of function of the mutant allele due to a reduction in either protein level or activity. We also report three new ALK-1 missense mutations leading to G48E/A49P, C344Y and E407D substitutions. In COS-1 transfected cells, ALK-1 was found in the TGF-ß1 and -ß3 receptor complexes in association with endoglin and TßRII, but not in activin receptor complexes containing endoglin. In HUVEC, ALK-1 was not detectable in the TGF-ß1 or -ß3 receptor complexes. However, in the absence of ligand, ALK-1 and endoglin interactions were observed by immunoprecipitation/western blot in HUVEC from normal as well as HHT1 and HHT2 patients. Our data suggest a transient association between these two proteins of the TGF-ß superfamily, both required at a critical level to ensure vessel wall integrity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia (1). Molecular heterogeneity of the disease has been revealed by genetic linkage studies and identification of two distinct loci. The first locus was mapped to chromosome 9q33 (2,3) and endoglin was recognized as the affected gene in HHT1 (4). The second locus was mapped to chromosome 12q13 (5,6) and ALK-1 (activin receptor-like kinase-1) was identified as the gene mutated in HHT2 (7). The clinical features of the disorder include recurrent nosebleeds, mucocutaneous telangiectases and arteriovenous malformations (AVMs) in lung, brain and liver (8–11). Families with HHT1 have a higher incidence of pulmonary involvement while HHT2 families tend to have a later onset and milder manifestations of the disease (12–14).
Both endoglin and ALK-1 gene products are highly expressed on endothelial cells (15,16) and are associated with the transforming growth factor (TGF)-ß superfamily. TGF-ß and related factors signal through a heteromeric complex of related Ser/Thr kinase receptors, type I (also known as activin-like kinases, ALKs) and type II (17). ALK-1 was shown to bind TGF-ß or activin in COS cells when co-expressed with the corresponding type II receptors (16,18,19) but the natural ALK-1 ligand in vivo remains unknown. For TGF-ß, the type II receptor (TßRII) binds ligand, recruits, transphosphorylates and activates the type I receptor (TßRI/ALK-5) (20,21). The type I receptors act on a family of downstream effector proteins known as Smads (22,23). Bone morphogenetic proteins (BMPs) generally signal through Smads 1 and 5 (24,25), whereas activin and TGF-ß signal through Smads 2 and 3 (26). In a recent study, a constitutively active form of ALK-1 activated Smad 1 and 5 but not Smad 2 pathways (27), suggesting that ALK-1 functions in regulating BMP signaling. Endoglin, a homodimeric membrane glycoprotein was shown to interact with TGF-ß1, TGF-ß3, activin-A, BMP-2 and BMP-7, when co-expressed in COS-1 cells with the respective ligand binding kinase receptors (28).
ALK-1 shares a high degree of similarity with other type I receptors in the Ser/Thr kinase subdomains, Gly/Ser (GS) rich domain and short C-terminal tail (16,29). There are 10 Cys residues in the extracellular domain, the spacing of which is conserved among type I receptors (17) and is likely to be critical for ligand binding. The intracellular region consists almost entirely of the kinase domain that contains 12 subdomains with conserved amino acids (30). To date, 18 mutations have been reported in the coding region of the ALK-1 gene (7,31,32). These mutations are found in the extracellular, transmembrane and kinase domains of ALK-1 and include small deletions, insertions and nonsense mutations leading to truncated proteins, as well as missense mutations.
In this study, we describe three new missense mutations in the coding region of the ALK-1 gene and also identify in newborns, mutations previously published in their affected family members. A novel polyclonal antibody to ALK-1 was used to measure protein levels on seven human umbilical vein endothelial cells (HUVEC) of newborns from HHT2 families. This allowed us to establish that some mutations in the ALK-1 gene, which alter important structural residues, led to reduction or absence of expression of the mutant proteins. However, one mutation in the ATP binding site did not alter the level of protein expression but likely inactivated the enzyme. We also demonstrate the association of ALK-1 and endoglin in endothelial cells of normal and HHT newborns.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of a polyclonal antibody to ALK-1
To analyze the expression of ALK-1 protein, we developed a novel polyclonal antibody (PAb
ALK-1) by infecting rabbits with a recombinant vaccinia virus expressing ALK-1 (VV-ALK-1). This poxvirus was selected for in vitro and in vivo infections because it can produce relatively high levels of the corresponding recombinant protein (33). Expression of HA-tagged recombinant ALK-1 as a result of an in vitro infection of BSC-40 African monkey cells was observed by western blot analysis with monoclonal antibodies (MAb) to HA (Fig. 1A). A broad band of 55–65 kDa was present in the VV-ALK-1 infected cells, but absent in the control lanes. In addition, infection of rabbits with VV-ALK-1 virus resulted in the in vivo expression of recombinant ALK-1, which in turn led to production of anti-ALK-1 antibodies in the sera of the infected animals (Fig. 1B).
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The specificity of the PAb
ALK-1 was further analyzed in COS-1 cells transfected with ALK-1/HA, ALK-2/HA (activin type I receptor) or ALK-5/HA (TGF-ß type I receptor) (Fig. 1C). Specific reactivity with ALK-1 was observed with the PAb ALK-1, as a band of expected size was present in the ALK-1 transfectant and absent from the pCMV5 empty vector and from samples immunoprecipitated with non-immune (NI) sera (lanes 1–6). No reactivity was observed with ALK-2 (lanes 7 and 9) or ALK-5 (lanes 10 and 12), indicating no cross-reaction with these related type I receptors. The HA immunoprecipitates confirmed the efficiency of transfection and the identity of the respective type I receptors (lanes 5, 8 and 11). Endoglin transfectants showed reactivity with MAb P3D1 but not with PAb ALK-1 (lanes 13–15) indicating that this antiserum did not cross-react with endoglin.
Quantitation of ALK-1 and endoglin protein levels in HHT families
We previously reported that mutations in endoglin result in reduced levels of expression of the corresponding protein at the surface of HUVEC and activated monocytes from HHT1 affected individuals (34,35). We therefore measured the levels of expression of endoglin on activated monocytes obtained from peripheral blood of clinically affected relatives of the newborns from six HHT families using metabolic labeling and immunoprecipitation, and flow cytometry. Expression of normal levels of endoglin, relative to age-matched controls, was found in all individuals (Table 1), which made them possible HHT2 candidates. We then analyzed seven HUVEC from newborns in these families and also observed normal levels of endoglin relative to the control HUVEC (C1–C5) analyzed in each experiment (Fig. 2A and Table 1).
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Since ALK-1 can only be measured on HUVEC as it is not expressed on peripheral blood mononuclear cells, we tested whether PAb
ALK-1 reacts with ALK-1 protein on endothelial cells by metabolic labeling and immunoprecipitation of HUVEC derived from control (C) newborns. A distinct band estimated at 63 kDa was observed with ALK-1 but not with the NI sera, as shown for C1 and C2 in Figure 2B and C3–C5 in Figure 2C. ALK-1 protein levels were then quantified in HUVEC of the seven newborns from potential HHT2 families using metabolic labeling and immunoprecipitation. Reduced ALK-1 levels were found in HUVEC samples from patients H358, H360 and H69, relative to normal HUVEC analyzed in the same experiment (Fig. 2B and Table 1). The remaining four HUVEC samples (patients H241, H242, H143 and H48) expressed normal levels of ALK-1 (Fig. 2C and Table 1).
Using flow cytometry analysis, >90% of HUVEC were found to express ALK-1 relative to the NI sera used as a negative control (Fig. 3). This confirmed the specificity of the PAb ALK-1, which was then used to measure the steady state expression of ALK-1 at the cell surface of seven HUVEC from potential HHT2 families. Percentage of positive cells and mean fluorescence intensity were determined for each sample relative to matched control. The expression of ALK-1 was specifically reduced on HUVEC from newborns H358, H360 and H69, but was normal on HUVEC from newborns H241, H242, H143 and H48 (Fig. 3 and Table 1). Levels of endoglin, 5ß1 integrin and CD31 were unchanged on these HUVEC (Fig. 3), confirming the specific reduction of ALK-1 in three of seven HUVEC, and in agreement with the metabolic labeling studies (Table 1).
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Mutation analysis in six HHT2 families
To establish the HHT2 genotype of the families involved in the current study, DNA samples from clinically affected family members and newborns were screened for mutations in the ALK-1 gene. We confirm that all six families have HHT2 and report new missense mutations in the coding region of the ALK-1 gene in families 109, 48 and 64, respectively (Fig. 4 and Table 2).
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In family 109, a complex mutation was observed in exon 3: a G143A substitution, deletion of G145 and insertion of T147 (Fig. 4A and Table 2). This mutation was found in DNA samples obtained from HUVEC and placenta of newborn H358, and peripheral blood lymphocytes from the clinically affected mother H357, but not from the father H386. The mutation converts adjacent amino acids G48 to E and A49 to P in the extracellular domain of ALK-1.
In family 48, the new mutation is a G to A substitution at position 1031 of exon 7. DNA obtained from both HUVEC and placenta of the newborn H143 and from peripheral blood lymphocytes of the mother H142, who had an uncertain clinical diagnosis of HHT, did not exhibit this mutation. It was found in the DNA from a clinically affected uncle H167 of the newborn H143 (Fig. 4B and Table 2). DNA from the non-affected father was normal. This mutation converts C344 to Y.
In family 64, a G to T substitution at position 1221 was identified in exon 8, which corresponds to a region in the ALK-1 kinase domain. It was absent from DNA of the fraternal twins H241 and H242, but present in DNA of the clinically affected mother H210 and grandfather. This mutation converts E407 to D (Fig. 4C and Table 2).
In family 110, a G150T substitution in exon 3 was identified in the DNA samples obtained from HUVEC and placenta of newborn H360 and from peripheral blood lymphocytes of the clinically affected mother (Table 2), but not in control DNA from the father. This mutation, which was previously reported in two apparently unrelated families (7,32), converts W50 to C in the extracellular domain. We could not confirm any relation of family 110 to the previously described families.
We also identified mutations in DNA samples of newborns from HHT2 families 16 and 22 (Table 2). Mutations in these families were previously published (6,7). In family 16, a CTC deletion at position 696–698, observed in DNA samples from newborn H48, results in the loss of S232 in the kinase domain of the ALK-1 gene (6). In family 22, a G998 to T substitution in exon 7, which results in conversion of S333 to I, was detected in DNA from newborn H69 (Table 2) (7). We have also confirmed these two mutations by sequencing the cDNA, indicating expression of these mutants at the mRNA level.
ALK-1 associates with endoglin in TGF-ß1 and -ß3 but not in activin-A receptor complexes in COS-1 transfected cells
Since ALK-1 was previously reported to bind TGF-ß1 and activin when overexpressed in COS-1 cells, we tested whether ALK-1 and endoglin could be found in receptor complexes for TGF-ß1, -ß3 and activin-A. Affinity labeling with [125I]TGF-ß1 or -ß3 was performed in COS-1 cells transiently transfected with endoglin or ALK-1 alone, endoglin together with ALK-1 and TßRII. No binding was observed when endoglin and ALK-1 were co-expressed (data not shown). However, cotransfection of TßRII with endoglin and ALK-1 led to binding of [125I]TGF-ß1 and -ß3 to both endoglin and ALK-1, as observed from the analysis of total lysates (Fig. 5A, lanes 1, 5 and 9). Specific immunoprecipitation revealed the position of radiolabeled complexes containing endoglin (compare lanes 3 and 7) and ALK-1 (compare lanes 4 and 8). PAb C16 (TßRII) co-immunoprecipitated [125I]TGF-ß1 or -ß3 cross-linked to endoglin/TßRII (lane 2) and to ALK-1/TßRII complexes (lane 6). When ALK-1 was co-expressed with endoglin and TßRII, both PAb C16 (TßRII) and MAb P3D1 (END) revealed ALK-1 co-precipitation (lanes 10 and 11). MAb 12CA5 (HA) immunoprecipitated the type I receptor ALK-1/HA (lane 12) but not the associated TßRII and endoglin.
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We next tested binding of activin-A to ALK-1 in COS-1 transfectants and examined its association with endoglin in activin-A receptor complexes. COS-1 cells were transfected with endoglin and ALK-1 alone or in combination with ActRII and were then affinity labeled with activin-A (Fig. 5B). SDS–PAGE analysis of total lysates showed activin-A binding to ActRII alone (lane 1), ActRII and endoglin (lane 3), ActRII and ALK-1 (lanes 4 and 5), whereas ALK-1 and endoglin did not bind ligand (lane 2). Immunoprecipitation with MAb P3D1 (
END) revealed co-precipitation of endoglin with ActRII in activin-A complexes, but not when cotransfected with ALK-1 (lanes 6 and 7). However, ALK-1 did co-precipitate with ActRII as seen using MAb to HA but endoglin was excluded from this complex (lanes 8 and 9). When similar experiments were conducted with ALK-2, a type I receptor that binds activin-A, a complex containing ActRII, endoglin and ALK-2 could be precipitated (Fig. 5C). Again, total lysates showed binding of activin-A to ActRII and ALK-2 only when ActRII was expressed (lanes 1–5), whereas activin-A binding to endoglin was only seen by immunoprecipitation with a MAb to endoglin (lanes 6 and 7) which also showed ActRII association. The high molecular weight bands seen, particularly in lane 7 (Fig. 5C), likely represent a cross-linked complex of endoglin and ActRII observed under reducing conditions. When ALK-2 was co-expressed with ActRII and endoglin, ALK-2 also associated with the complex containing endoglin, ActRII and radiolabeled activin-A (lanes 8 and 9). These results suggest that endoglin interacts with specific activin-A complexes containing ActRII and ALK-2 but not ActRII and ALK-1.
Endogenous ALK-1 is not detectable in TGF-ß1 or -ß3 receptor complexes in HUVEC
Using the characterized PAb ALK-1, we next tested whether endogenous ALK-1 could be detected in the TGF-ß receptor complex in HUVEC, as these cells have relatively high levels of ALK-1 (Fig. 6A). HUVEC were incubated with radiolabeled TGF-ß1, chemically cross-linked and solubilized with digitonin. This detergent is milder than Triton X-100 (36) and can preserve protein–protein interactions as previously shown for endoglin–TßRII interactions (28). Immunoprecipitation analysis revealed TGF-ß1 binding and effective co-precipitation of endoglin, TßRII and a trace of a type I receptor, likely to be ALK-5 (lanes 1, 4, 5 and 8), as previously reported (28). We could not detect binding of TGF-ß1 to ALK-1, as no product was immunoprecipitated by PAB ALK-1 (lanes 3 and 7).
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Similarly, when HUVEC were affinity labeled with TGF-ß3 (Fig. 6B), analysis of total cell lysates revealed specific cross-linking to TßRII, endoglin dimers (180 kDa) and oligomers (>200 kDa; lanes 1–3). Endoglin dimers were immunoprecipitated under non-reducing conditions by MAb P4A4 (
END, lane 4) while monomers were seen under reducing conditions (lane 8). Anti-TßRII co-precipitated endoglin dimers and oligomers (lane 5) and monomers (lane 9), confirming that endoglin and TßRII form a complex with TGF-ß3. No traces of ALK-1 were observed (lanes 6 and 7, 10 and 11). We were thus unable to identify endogenous ALK-1 in the TGF-ß1 or -ß3 receptor complexes on HUVEC.
ALK-1/endoglin interactions in HUVEC of normal and HHT newborns
Since endoglin can associate with TßRII in the absence of exogenous ligand (28), we investigated its ability to interact with TßRII and ALK-1 in HUVEC of normal and HHT newborns using western blot analysis of specific immunoprecipitates. Polyclonal antibodies to endoglin, TßRII and ALK-1 were used and the immunoprecipitates were then probed with MAb P4A4 to endoglin (Fig. 7A–C). PAb END specifically immunoprecipitated endoglin in all analyzed samples, as illustrated for controls C6 and C7, and for HUVEC from non-affected newborn H241 (lanes 1, END). Low reactivity was observed with the NI sera (NI lanes). PAb C16 to TßRII co-precipitated TßRII and endoglin in HUVEC from control (C6) as well as from HHT1 (H344) and HHT2 (H48) newborns (Fig. 7A, lanes 3, 5 and 7, respectively), confirming that ligand-independent association of these two proteins was still occurring in endothelial cells from HHT newborns.
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We then tested whether an association between ALK-1 and endoglin could be detected in HUVEC obtained from normal and HHT newborns (Fig. 7B). When extracts were immunoprecipitated with PAb
ALK-1 and probed with MAb P4A4 under non-reducing conditions, endoglin was found to be associated with ALK-1 in control HUVEC (lane 3). This interaction was also detected in HUVEC from an HHT1 patient (H344; lane 5) and in three HHT2 HUVEC analyzed (H69, H358 and H360; lanes 7, 9 and 11, respectively).
Because it is known that ALK-1 and endoglin are expressed in placenta, we tested whether their interaction could be demonstrated in extracts of placental membrane fractions obtained from control and HHT newborns. Figure 7C shows that PAb ALK-1 co-precipitated endoglin and ALK-1 in samples from non-affected newborn H241 from HHT2 family 64, HHT1 affected newborns H129, H136 (34) and HHT2 affected newborn H48 (Fig. 7C, lanes 3, 5, 7 and 9, respectively). These results suggest that an interaction between endoglin and ALK-1 can still be detected, in absence of ligand, in HUVEC and placenta of HHT1 and HHT2 newborns.
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DISCUSSION |
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In this paper, we describe a novel polyclonal antibody that specifically detects ALK-1 and we were able to quantify its expression on seven HUVEC from six HHT2 families. ALK-1 protein levels were reduced in three of the seven HUVEC and ALK-1 missense mutations (G48E/A49P, W50C and S333I) were found in all three cases, revealing that these newborns were affected with HHT2. It is likely that these mutations lead to structural alterations that result in protein misfolding and intracellular degradation, explaining the lack of surface expression of the mutant proteins. The first two mutations occur between Cys46 and Cys51, while the third replaces S333. These residues are highly conserved among the type I receptors (17,30) and are likely to be critical for proper folding of the protein. In the case of the S333I substitution, mutant mRNA could be sequenced, confirming expression of the message and suggesting instability of the mutant protein.
Three other HUVEC, two of which were from fraternal twins, showed normal levels of ALK-1 protein. Mutation analysis confirmed that these newborns were not affected because they did not express the disease-related mutation (C344Y or E407D) found in other affected family members. Thus, in six of seven families analyzed, levels of ALK-1 correlated with the presence or absence of a mutant protein.
However, HUVEC from newborn H48 expressed relatively normal levels of ALK-1 protein, as measured by metabolic labeling (89%) and flow cytometry (85%), while carrying a 3 bp deletion (CTC 696–698) that was previously reported for other affected family members (6). This mutation deletes S232, which is one of two adjacent Ser residues in the ATP binding site of the kinase domain of ALK-1. These Ser residues are conserved in most of the type I receptors of the TGF-ß superfamily (30). This mutation is thus likely to yield a non-functional enzyme. Our results support the hypothesis that HHT2, like HHT1, is associated with haploinsufficiency as previously suggested (7). A loss of ALK-1 expression due to structural mutations that lead to misfolded and unstable proteins (e.g. as in families 22, 109 and 110), or a loss of function of the mutated allele (as predicted for family 16) are likely to be the most common mechanisms responsible for HHT2.
We report three novel missense mutations (G48E/A49P, C344Y and E407D) in the coding region of the ALK-1 gene. The first, seen in family 109, consists of substitution, followed by deletion and insertion of single base pairs in exon 3 of ALK-1. The net effect of the mutation is the conversion of adjacent amino acids G48 to E and A49 to P in the extracellular domain. In family 48, C344 is replaced by Y, while in family 64 the mutation is E407 to D substitution. These two mutations occur in exons 7 and 8 of the kinase domain, respectively.
Recent data showed that introducing the W50C mutation into the extracellular domain of the ALK-1/TßRI chimera led to abrogation of signaling activity (37). As this mutation is associated here with reduced expression of ALK-1 in HUVEC of newborn H360, the reported loss of signaling activity in COS-1 cells was likely to be due to low levels of expression of the unstable chimeric receptor.
As previously reported by Attisano et al. (16), ALK-1 can be found in the TGF-ß1 receptor complex when co-transfected in COS-1 cells. We now also demonstrate that in COS-1 cells it can be present in the TGF-ß3 receptor complex. This supports recent data demonstrating that chimeric ALK-1/TßRI receptors can signal for TGF-ß1 and -ß3, and therefore are capable of binding both of these ligands (37). We also observed that the PAb C16 to TßRII is the most efficient at bringing down the TGF-ß receptor complex containing endoglin and ALK-5 (28) and/or ALK-1 (current study). Our results, that ALK-1 and endoglin are not found in the activin receptor complex in COS-1 cells despite the fact that ALK-1 can interact with ActRII, are in keeping with the observations that the chimeric ALK-1/ActRI receptor does not signal for activin-A (37). In our experiments with HUVEC, binding of TGF-ß1 and -ß3 to endogenous ALK-1 could not be demonstrated. In addition, in the absence of ligand, we were unable to demonstrate ALK-1 interaction with either TßRII or endoglin by surface labeling (data not shown). However, using immunoprecipitation/western blot analysis, we detected an interaction between ALK-1 and endoglin in HUVEC and placenta in the absence of ligand. This interaction was also observed in samples obtained from HHT1 and HHT2 newborns.
Since the true physiological ligand for ALK-1 is not known, and the type II receptor(s) it interacts with is unclear, it is difficult to establish which ligand binding complexes involve both endoglin and ALK-1. Endoglin functions in signaling receptor complexes of multiple members of the TGF-ß superfamily (28). It binds TGF-ß1/-ß3 via its association with TßRII, and activin and BMP-7 via ActRII/ActRIIB, regardless of the co-expressed type I receptor. Endoglin also binds BMP-2 via its association with the type I receptors ALK-3 and ALK-6. ALK-1 has now been implicated in TGF-ß1/-ß3 receptor complexes as well as that of an unknown ligand present in serum (37). A constitutively active form of ALK-1 was shown to act on Smads 1, 5 and 8, which are involved in BMP signaling (27,38). One of the critical questions that remains to be answered is whether HHT is associated with modified responses to TGF-ß1/-ß3 or to an as yet unknown ligand. Endoglin null mice die at mid-gestation due to impaired vascular development (39,40) and ALK-1 null embryos appear to have a similar phenotype (as quoted in 37). Mice deficient in TGF-ß1 or TßRII are also embryonic-lethal due to defects in yolk sac vasculogenesis (41,42). These studies suggest that endoglin and ALK-1 mediate the effects of TGF-ß1 in vascular development. Furthermore, as a loss of function of the mutated allele of either endoglin or ALK-1 leads to the vascular disorder HHT, we hypothesize that both endoglin and ALK-1 function in a non-classical TGF-ß1 pathway that is critical for vascular integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical samples
Blood samples and medical histories were obtained with informed consent from all individuals participating in the study. In the case of umbilical cord and placenta samples, consent was obtained from the parents in families with a clinical diagnosis of HHT. The procedures were reviewed and approved by the Research Ethics Board of the Research Institute of The Hospital for Sick Children, Toronto, Canada. All patients and families were given a number and clinically diagnosed patients or newborns from HHT families are referred to with a prefix H (for HHT). In total, samples from six families with HHT2 were included in the study. In the case of families 16 and 22, for which mutations were previously reported (7,31), only samples from newborns were available. Samples obtained from newborns H129, H136 and H344, all expressing reduced levels of endoglin and with known mutations in endoglin gene (34), were also analyzed. Mutation in H129 is a 1-bp (A) insertion at position 1470, in exon 11, causing a frameshift that leads to a truncated protein terminating at L490. Newborn H136 carries a nonsense mutation in exon 5 of endoglin gene created by a C to T substitution at position 587, which converts codon 196 into a stop. The mutation in newborn H344 is an insertion of G in exon 8 that caused a frameshift and led to a stop at position 1112.
HUVEC were derived from umbilical cords of newborns from HHT families and controls from local deliveries (15,34,35). In all assays, experimental and control HUVEC at equivalent cell densities and passage numbers were employed. The mononuclear cells, derived from venous blood of clinically affected HHT patients and known non-affected relatives or unrelated volunteers, were used for measurement of endoglin levels as described previously (34,35,43).
Mammalian expression constructs and transient transfection
The pCMV5 expression constructs containing cDNAs for endoglin, TßRII, ActRII, ALK-1/HA (tagged at the C-terminus with the influenza hemagglutinin epitope, HA), ALK-2/HA and ALK-5/HA have all been described previously (16,20,22,44–46). All constructs for Ser/Thr kinase receptors were provided by J. L. Wrana and L. Attisano. COS-1 cells were maintained and transiently transfected with expression constructs using the DEAE–dextran–chloroquine method as reported (22,24). Assays were performed 2 days post-transfection.
Generation of a polyclonal antibody to ALK-1 using a recombinant vaccinia virus construct
BSC-40 African green monkey kidney cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FCS, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a 5% CO2 atmosphere at 37°C. Culture of the Western reserve strain vaccinia virus and isolation of recombinant virus were performed as described (47). The plasmid pCMV5-ALK-1/HA (16) was digested with HindIII and BamHI, and the resulting 1.6 kb fragment was treated with DNA polymerase I (Klenow fragment) and inserted into the SmaI site of vaccinia virus plasmid pSC11. The resulting plasmid vector co-expressed the ALK-1 gene inserted downstream of the vaccinia 7.5 kDa protein promoter, together with the Escherichia coli ß-galactosidase gene under the control of the vaccinia 11 kDa protein late promoter; both genes were flanked by viral thymidine kinase (TK) sequences. Thus, upon homologous recombination, both genes were inserted into the TK locus of the vaccinia virus genome yielding the VV-ALK-1 virus. Control recombinant TK– vaccinia virus (VV-TK–) was constructed as described for the other recombinant viruses, using the pSC11 plasmid. New Zealand rabbits were immunized twice with a 4-week interval with 109 p.f.u. of either VV-ALK-1 or VV-TK– in PBS. Two weeks after the last injection, sera were collected and reactivity with ALK-1 confirmed by western blot.
Antibodies
MAb P3D1 and P4A4 which recognize different epitopes of the extracellular domain of human endoglin have been described previously (48). A PAb to human endoglin was generated using recombinant vaccinia virus as described previously (49). MAb 5.6E to CD31 (PECAM-1) was purchased from AMAC and MAb JBS5 to 5ß1 integrin was a gift from Dr J. A. Wilkins (University of Manitoba, Winnipeg, Canada). Murine NI IgG1 (Coulter Electronics, Hialeah, FL) was used as a negative control. PAb C16 to peptide 550–565 of the highly conserved C-terminal of human TßRII was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). MAb 12CA5 (Boehringer Mannheim, Laval, Canada) to influenza hemagglutinin (HA) was used for immunoprecipitation of HA-tagged TGF-ß superfamily receptors.
Flow cytometry
HUVEC were released with trypsin–EDTA and washed in Ca2+Mg2+-free PBS. Cells (5 x 105) were incubated with the respective MAb or an IgG1 isotype control and FITC-conjugated F(ab')2 goat anti-mouse IgG (Tago Inc., Burlingame, CA). For NI and immune anti-ALK-1 sera, FITC-conjugated F(ab')2 goat anti-rabbit IgG was used. The HUVEC population was defined by forward light scatter versus side scatter, and live cells were selected with propidium iodide. Percent positive cells within these defined gates and mean fluorescence intensities were determined relative to the respective negative control. In the case of ALK-1, reactivity with NI serum was used to define the negative gate. Patient HUVEC samples were run in parallel with control HUVEC and their relative mean fluorescence intensity estimated from at least two experiments. All samples were analyzed on the FACScan® (Becton Dickinson, Mountain View, CA).
Metabolic labeling and immunoprecipitation
Control and patient HUVEC at sub-confluence (~90%) were metabolically labeled as described (34,35,43). Cells were incubated with 100 µCi/ml [35S]methionine (met) (Trans35S-label; ICN Pharmaceuticals Ltd, Montreal, Canada) in met-free DMEM (low glucose; Life Technologies, Rockville, MD) for 3.5 h and solubilized in lysis solution containing 1% Triton X-100 or 1% digitonin (Wako, Richmond, VA). After overnight preclearing with NI sera and Protein A–Sepharose" CL-4B (Pharmacia Biotech Inc., Baie d’Urfe, Canada) samples were divided equally and immunoprecipitated with saturating amounts of anti-endoglin and anti-ALK-1 antibodies, followed by elution in non-reducing solution (60 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue).
To quantitate ALK-1 and endoglin expression and to correct for the differences in yield between samples and respectively matched controls, we measured total protein by TCA precipitation in aliquots of total lysates. Equivalent c.p.m. were fractionated on 4–12% SDS–PAGE gradient gels (Novex Experimental Technology, San Diego, CA) which were then fixed, dried and exposed to BioMax MS films in the BioMax TransScreen LE cassettes (Eastman Kodak, Rochester, NY). Radioactivity in the bands corresponding to ALK-1, run under reducing conditions, or the fully processed form of endoglin (E) was quantified using a PhosphorImager and ImageQuant Software (Molecular Dynamics, Sunnyvale, CA). The patient to control pixel value ratios were calculated for each immunoprecipitate and the mean ± SD determined from at least four values. Transfected COS-1 cells and activated monocytes, obtained from peripheral blood of controls and patients, were processed as described above.
DNA preparation
Genomic DNA was isolated from HUVEC, placenta and peripheral blood lymphocytes using DNAZOL® reagent (Gibco BRL, Gaithersburg, MD) or Puregene® DNA Isolation Kit (Gentra Systems, Minneapolis, MN) according to manufacturers’ protocols.
Mutation analysis
Two sets of primers were designed for amplification and subsequent sequencing of each of the nine coding exons of the ALK-1 gene. Each reaction contained 2.5 mM of dNTP, 0.2 µM of oligonucleotide primers, optimal concentration of MgCl2, AmpliTaq polymerase, obtained from Perkin-Elmer (Roche Molecular Systems, Branchburg, NJ) and 300 ng of DNA. Thermocycling conditions were: initial denaturation for 2 min at 94°C followed by 30 cycles of 45 s at 94°C, 45 s annealing at 56°C, 60 s at 72°C and a final extension of 5 min at 72°C. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Mississauga, Canada) and templates were sequenced using a cycle sequencing protocol. The sequencing primers were designed to be at least 5 bp within the amplification primers and were labeled with Cy5.5 fluorescent dye (Visible Genetics Inc., Toronto, Canada). Templates (20–40 ng), 3 pM of Cy5.5 labeled primer, and 4 U of ThermoSequenaseTM enzyme (Amersham Life Sciences, Cleveland, OH) in ThermoSequenaseTM buffer were added to dNTP terminators. Cycle sequencing conditions were: 2 min at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at annealing temperature (56–60°C), 1 min at 70°C and a final extension of 2 min at 72°C. Products were resolved on a MicroGene BlasterTM sequencer at 1300 V and 54°C for 30–40 min and sequences analyzed using the Open GeneTM DNA Analysis Software (Visible Genetics Inc.).
Affinity labeling
TGF-ß1 and TGF-ß3 were from R&D Systems (Minneapolis, MN); recombinant human activin-A was a generous gift from Y. Eto (Ajinomoto Co. Inc., Kawasaki, Japan). Ligands were iodinated with [125I]chloramine-T as previously described (46,50,51). HUVEC were incubated with 200 pM [125I]TGF-ß1, 250 pM [125I]TGF-ß3 or 800 pM [125I]activin-A for 4 h, washed, treated with DSS crosslinker (disuccinimidyl suberate; Pierce, Rockford, IL) and solubilized with lysis buffer containing 1% digitonin or 1% Triton X-100 and protease inhibitors (52). Cell lysates containing equivalent total protein were immunoprecipitated with control NI IgG, MAb P3D1 and P4A4 (-endoglin), 12CA5 (HA), PAb C16 (-TßRII), NI serum and PAb ALK-1. Immune complexes were collected with Protein G– or Protein A– Sepharose", washed with lysis buffer containing either digitonin or Triton X-100 and eluted in 1% SDS. Cross-linked receptors bound to radiolabeled ligands were visualized by separation on 4–12% gradient gels by SDS–PAGE and autoradiography using BioMax MS film and the BioMax TransScreen HE intensifying screen system (Eastman Kodak).
Western blot analysis
Soluble extracts, obtained by lysing uninfected control, infected recombinant TK– vaccinia virus (VV-TK–) and the HA-tagged ALK-1 recombinant vaccinia virus (VV-ALK-1) infected BSC-40 cells were fractionated by 7.5% SDS–PAGE and transferred to nitrocellulose membranes. These were blocked with PBS containing 5% milk powder for 1 h, and probed with MAb 12CA5 (anti-HA), followed by peroxidase-conjugated goat anti-mouse IgG and detected by ECL. Anti-HA immunoprecipitates were subjected to SDS–PAGE, transferred to nitrocellulose and probed with the serum from a rabbit infected with VV-ALK-1, followed by peroxidase-conjugated goat anti-rabbit Ig and chemiluminescence detection.
HUVEC at subconfluence were lysed with 1% Triton X-100 and immunoprecipitated with PAb ALK-1, NI sera and PAb END prior to SDS–PAGE on 4–12% gels under non-reducing conditions. The total amount of protein was estimated and the amount used for immunoprecipitation with ALK-1 (0.2–0.4 mg) was 10 times more than with END. After electrophoretic transfer to nitrocellulose membranes, the immunoprecipitates were probed with mAb P4A4 followed by goat anti-murine IgG-HRP and detection by ECL. Crude membrane fractions, prepared from the placenta by subsequent centrifugation at 5000 and 100 000 g were solubilized in 1% Triton X-100 plus inhibitors and analyzed using the same procedure.
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ACKNOWLEDGEMENTS |
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We are grateful to all the patients and their family members who participated in this study and for the support of the HHT Foundation International and to Jamie McDonald from Salt Lake City for providing us with patient information. We thank Drs L. Attisano and J.L. Wrana for the type I Ser/Thr kinase receptor constructs, and Dr J. L. Wrana for the critical review of the manuscript. We are grateful to Ursula Cymerman for technical advice on cycle sequencing, Carmen Langa for excellent technical assistance and Dr J. Dunn from Visible Genetics Inc. for the use of the Microgeneblaster®. This work was supported by grant #NA3434 from the Heart and Stroke Foundation of Ontario, grant #MT6247 from the Medical Research Council of Canada, grant HHT-FY99-677 from March of Dimes (M.L.), and from Comision Interministerial de Ciencia y Tecnologia (CICYT-SAF97-0034) and Comunidad Autonoma de Madrid (CAM) (C.B.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 416 813 6258; Fax: +1 416 813 6255; Email: mablab@sickkids.on.ca
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 S.A. Abdalla, C.J. Gallione, R.J. Barst, E.M. Horn, J.A. Knowles, D.A. Marchuk, M. Letarte, and J.H. Morse Primary pulmonary hypertension in families with hereditary haemorrhagic telangiectasia Eur. Respir. J., March 1, 2004; 23(3): 373 - 377. [Abstract] [Full Text] [PDF]
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R E Harrison, J A Flanagan, M Sankelo, S A Abdalla, J Rowell, R D Machado, C G Elliott, I M Robbins, H Olschewski, V McLaughlin, et al. Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia J. Med. Genet., December 1, 2003; 40(12): 865 - 871. [Abstract] [Full Text] [PDF]
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 C. J. Favre, M. Mancuso, K. Maas, J. W. McLean, P. Baluk, and D. M. McDonald Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1917 - H1938. [Abstract] [Full Text] [PDF]
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T. Seki, J. Yun, and S. P. Oh Arterial Endothelium-Specific Activin Receptor-Like Kinase 1 Expression Suggests Its Role in Arterialization and Vascular Remodeling Circ. Res., October 3, 2003; 93(7): 682 - 689. [Abstract] [Full Text] [PDF]
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J Berg, M Porteous, D Reinhardt, C Gallione, S Holloway, T Umasunthar, A Lux, W McKinnon, D Marchuk, and A Guttmacher Hereditary haemorrhagic telangiectasia: a questionnaire based study to delineate the different phenotypes caused by endoglin and ALK1 mutations J. Med. Genet., August 1, 2003; 40(8): 585 - 590. [Abstract] [Full Text] [PDF]
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S A Abdalla, U W Geisthoff, D Bonneau, H Plauchu, J McDonald, S Kennedy, M E Faughnan, and M Letarte Visceral manifestations in hereditary haemorrhagic telangiectasia type 2 J. Med. Genet., July 1, 2003; 40(7): 494 - 502. [Abstract] [Full Text] [PDF]
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 S. E. DUFF, C. LI, J. M. GARLAND, and S. KUMAR CD105 is important for angiogenesis: evidence and potential applications FASEB J, June 1, 2003; 17(9): 984 - 992. [Abstract] [Full Text] [PDF]
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 D. A. Marchuk, S. Srinivasan, T. L. Squire, and J. S. Zawistowski Vascular morphogenesis: tales of two syndromes Hum. Mol. Genet., April 2, 2003; 12(90001): R97 - 112. [Abstract] [Full Text] [PDF]
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S. van den Driesche, C. L. Mummery, and C. J.J. Westermann Hereditary hemorrhagic telangiectasia: an update on transforming growth factor {beta} signaling in vasculogenesis and angiogenesis Cardiovasc Res, April 1, 2003; 58(1): 20 - 31. [Abstract] [Full Text] [PDF]
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 E. Torsney, R. Charlton, A. G. Diamond, J. Burn, J. V. Soames, and H. M. Arthur Mouse Model for Hereditary Hemorrhagic Telangiectasia Has a Generalized Vascular Abnormality Circulation, April 1, 2003; 107(12): 1653 - 1657. [Abstract] [Full Text] [PDF]
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S. Srinivasan, M. A. Hanes, T. Dickens, M. E. M. Porteous, S. P. Oh, L. P. Hale, and D. A. Marchuk A mouse model for hereditary hemorrhagic telangiectasia (HHT) type 2 Hum. Mol. Genet., March 1, 2003; 12(5): 473 - 482. [Abstract] [Full Text] [PDF]
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 M E Begbie, G M F Wallace, and C L Shovlin Hereditary haemorrhagic telangiectasia (Osler-Weber-Rendu syndrome): a view from the 21st century Postgrad. Med. J., January 1, 2003; 79(927): 18 - 24. [Abstract] [Full Text] [PDF]
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S. Lamouille, C. Mallet, J.-J. Feige, and S. Bailly Activin receptor-like kinase 1 is implicated in the maturation phase of angiogenesis Blood, December 15, 2002; 100(13): 4495 - 4501. [Abstract] [Full Text] [PDF]
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M. Guerrero-Esteo, T. Sanchez-Elsner, A. Letamendia, and C. Bernabeu Extracellular and Cytoplasmic Domains of Endoglin Interact with the Transforming Growth Factor-beta Receptors I and II J. Biol. Chem., August 2, 2002; 277(32): 29197 - 29209. [Abstract] [Full Text] [PDF]
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C Olivieri, E Mira, G Delu, F Pagella, A Zambelli, L Malvezzi, E Buscarini, and C Danesino Identification of 13 new mutations in the ACVRL1 gene in a group of 52 unselected Italian patients affected by hereditary haemorrhagic telangiectasia J. Med. Genet., July 1, 2002; 39(7): e39 - 39. [Full Text] [PDF]
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 R. C. Trembath, J. R. Thomson, R. D. Machado, N. V. Morgan, C. Atkinson, I. Winship, G. Simonneau, N. Galie, J. E. Loyd, M. Humbert, et al. Clinical and Molecular Genetic Features of Pulmonary Hypertension in Patients with Hereditary Hemorrhagic Telangiectasia N. Engl. J. Med., August 2, 2001; 345(5): 325 - 334. [Abstract] [Full Text] [PDF]
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 A. Bourdeau, M. E. Faughnan, M.-L. McDonald, A. D. Paterson, I. R. Wanless, and M. Letarte Potential Role of Modifier Genes Influencing Transforming Growth Factor-{beta}1 Levels in the Development of Vascular Defects in Endoglin Heterozygous Mice with Hereditary Hemorrhagic Telangiectasia Am. J. Pathol., June 1, 2001; 158(6): 2011 - 2020. [Abstract] [Full Text] [PDF]
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