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Correspondence to Katri Koli: katri.koli{at}helsinki.fi
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Abstract |
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Introduction |
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High molecular mass latent TGF-ß complexes contain the 25-kD mature TGF-ß associated noncovalently with its propeptide (latency-associated peptide) and a latent TGF-ß binding protein (LTBP; for review see Saharinen et al., 1999). Recent evidence indicates that LTBPs play important roles not only in the secretion of TGF-ßs (Miyazono et al., 1991) but also in the targeting of the complexes to specific extracellular sites and in the activation process (for reviews see Taipale et al., 1998; Annes et al., 2003). These complexes are activated through various mechanisms. Thrombospondin- and integrin-mediated mechanisms are probably among the most significant (Crawford et al., 1998; Munger et al., 1999). Activation by proteolytic enzymes also appears to be important, for example, during invasive processes (Mignatti and Rifkin, 1993).
LTBPs, together with fibrillins, constitute a family of extracellular glycoproteins, which are mainly composed of repeated domain structures that are important for the function of LTBPs (for reviews see Oklu and Hesketh, 2000; Koli et al., 2001a). For example, the LTBPs contain EGF-like repeats and 8-Cys repeats, which are involved in proteinprotein interactions. Proline-rich regions in the molecule provide flexibility to the protein, are sensitive to proteolytic cleavage, and also contain heparin-binding sequences that may be important for cell recognition and matrix association (Annes et al., 2004).
Three mammalian fibrillins have been identified, of which fibrillins-1 and -2 are major constituents of the 10-nm microfibrils (for review see Ramirez and Pereira, 1999). Of the four LTBP isoforms (Kanzaki et al., 1990; Moren et al., 1994; Saharinen et al., 1998; Penttinen et al., 2002), three associate with the small latent TGF-ß. The third 8-Cys repeats of LTBP-1, -3, and -4 are responsible for the covalent association with latency-associated peptide (Saharinen and Keski-Oja, 2000). Although LTBP-1 and -3 bind all three isoforms of TGF-ß, LTBP-4 associates with TGF-ß1 only (Saharinen and Keski-Oja, 2000). LTBP-2 does not associate with the small latent TGF-ß, but has functions in cell adhesion and migration (Hyytiäinen and Keski-Oja, 2003; Vehviläinen et al., 2003). Although there is considerable overlap in the expression patterns of LTBPs, studies in knockout mice suggest that each isoform has a tissue-specific function. For example, LTBP-2 null mice die early in development, probably due to defective implantation (Shipley et al., 2000), LTBP-3 null mice develop skeletal abnormalities (Dabovic et al., 2002), and LTBP-4 hypomorphic mice develop severe pulmonary aplasia/emphysema and colorectal cancer (Sterner-Kock et al., 2002).
Tissue-specific abnormalities in mice expressing nearly null alleles of LTBP-4 (/) are associated with defects in elastic fiber structure and reduced levels of TGF-ß signaling (Sterner-Kock et al., 2002). However, although defective TGF-ß signaling causes cancer in both humans and mice, and is consistent with the development of colorectal cancers in the absence of LTBP-4, its contribution to abnormal lung development is still unclear. To better understand the molecular changes associated with reduced LTBP-4 expression, we have isolated lung fibroblasts from LTBP-4 / mice and compared them with wild-type (wt) fibroblasts. The most prominent changes observed were decreased production of active TGF-ß and increased accumulation of fibronectin-rich ECM. The decrease in active TGF-ß was associated with increased secretion of latent forms of TGF-ß, which suggests defects in activation but not in secretion. Lack of LTBP-4mediated TGF-ß1 activation was followed by enhanced secretion of latent TGF-ß2 and -ß3 and the activation of the bone morphogenic protein (BMP)4 signaling pathway both in vitro and in vivo. Increased accumulation of ECM in / fibroblasts was attributed to the activation of the BMP-4 pathway. Our results indicate that LTBP-4 modulates the BMP-4 signaling pathway by decreasing TGF-ß activity, which in turn leads to reduced expression of the BMP inhibitor gremlin. These changes may explain in part the pulmonary aplasia/emphysema phenotype, which is a consequence of the lack of LTBP-4 protein in the / mice.
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Results |
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LTBP-4 / fibroblasts produce increased amounts of ECM
TGF-ß1 is a potent regulator of ECM production and degradation that generally causes an accumulation of ECM. Therefore, we analyzed whether or not the lack of LTBP-4mediated TGF-ß1 targeting would reduce ECM production. Cells were metabolically labeled for 2 d, after which extracellular matrices were prepared and analyzed by SDS-PAGE. Unexpectedly, the amount of sodium deoxycholateresistant matrix was considerably higher in / fibroblasts (Fig. 2 A), and the amount of the 250-kD fibronectin polypeptide was particularly increased. Immunofluorescence analyses of 5-d-old fibroblast cultures indicated that in wt cells, fibronectin was localized into fibrillar structures in the matrices, whereas in / cells fibronectin was more abundant and formed a uniform sheetlike structure around the cells (Fig. 2 B). Because the altered ECM structure may change the sodium deoxycholate solubility of the ECM-associated fibronectin, the massive increase seen in Fig. 2 A is plausibly due to both increased protein deposition and decreased solubility. Neither fibronectin mRNA levels in vitro (Fig. 2 D) or in vivo (not depicted) nor secreted protein levels in / fibroblasts (not depicted) were up-regulated, suggesting increased deposition and/or decreased turnover. The cell surface expression levels of the fibronectin receptor 5ß1 integrin were not changed in / fibroblasts (unpublished data). Accordingly, immunohistochemical analysis of lung tissues indicated that fibronectin levels were highly up-regulated in the / mice (Fig. 2 C). LTBP-4 binds to fibrillin-1 (Isogai et al., 2003) and loss of fibrillin fibers in mice leads to changes in the levels of active TGF-ß (Neptune et al., 2003). Therefore, fibrillin-1 distribution was evaluated, but no significant changes were observed (Fig. 2 C).
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Inhibition of TGF-ß signaling does not reverse the / fibroblast phenotype
Although the amount of active TGF-ß in the conditioned medium was decreased, the / fibroblasts exhibited a phenotype similar to TGF-ßtreated wt cells. TGF-ß induces the production of ECM and enhances the expression of PAI-1 and CTGF in wt cells (unpublished data). The enhanced secretion of latent forms of TGF-ß led us to speculate whether or not TGF-ß might be activated by a distinct mechanism. For example, direct activation of TGF-ß and subsequent binding to cell surface receptors may occur without release into the medium. To address this possibility, TGF-ß signaling was inhibited by stable expression of a dominant-negative TGF-ß type II receptor (RII) in / fibroblasts (see Materials and methods). This form of the receptor lacks the kinase domaincontaining cytoplasmic tail, and is therefore unable to transduce TGF-ß signals (Wieser et al., 1993). To verify that the transgene prevented TGF-ß signaling, the cells were transiently transfected with a TGF-ß responsive promoter ((CAGA)12-luc; Dennler et al., 1998), followed by overnight treatment with TGF-ß1. In / fibroblasts, TGF-ß1 (100 pg/ml) induced the activity of (CAGA)12-luc promoter by
30-fold, whereas in cells expressing
RII the response was almost completely lost (Fig. 3 A). Analyses of ECM production revealed that the inhibition of TGF-ß signaling in / fibroblasts did not lead to reversal of the phenotype (Fig. 3 B and see Fig. 7), which suggested that enhanced TGF-ß activity is not involved in the induction of this phenotype.
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Gene expression profiling of fibroblasts
To identify candidate genes involved in the development of the / phenotype, we analyzed differentially expressed genes by oligonucleotide microarrays for over 12,000 mouse genes comparing RNA from / and wt lung fibroblasts (see Materials and methods). A total of 119 genes or ESTs were either up- or down-regulated greater than threefold in / lung fibroblasts (see Online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200403067/DC1). The expression of LTBP-4, which was lost, served as an internal control for the analyses. LTBP-1 and -3 were expressed at comparable levels in wt and / fibroblasts, whereas LTBP-2 expression levels were slightly enhanced in / fibroblasts (unpublished data). Special attention was paid to genes regulated greater than fivefold, genes known to be involved in the TGF-ß response pathway, genes involved in the regulation of the ECM, and genes of growth factors or their receptors (Table I). The expression of FGF-7 was found to be down-regulated. Although FGF-7 plays a role in lung development, knockout animals have no obvious defects in the lung (Guo et al., 1996). In addition, several ECM components or proteases were either up- or down-regulated in LTBP-4 / cells, probably contributing to the enhanced matrix phenotype. One of the most prominent changes was down-regulation of the expression of thrombospondin-2, which is a matricellular protein involved in the regulation of cellmatrix interactions (for review see Bornstein, 2001).
A prominent observation in the / fibroblasts was a fivefold increase in the expression of BMP-4, in parallel with almost complete loss of expression of the BMP inhibitor gremlin. Northern analyses confirmed these observations (Fig. 5 A). Immunoblotting of cell-conditioned medium revealed increased levels of BMP-4 protein in the medium of / fibroblasts (Fig. 5 B). In addition, the BMP-4 target gene inhibitor of differentiation (Id)2 was induced in the gene array. Northern analyses of Id2 as well as Id1 indicated 510-fold increases in expression levels (Fig. 5 A). The expression levels of the BMP-4 receptors BMPR-IA (ALK-3), BMPR-IB (ALK-6), and BMPR-II were comparable in wt and / fibroblasts (gene array analyses; unpublished data). These results suggest that BMP-4 signaling is considerably increased in LTBP-4 / fibroblasts.
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Discussion |
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The excessive accumulation of fibronectin and the gene expression profile of the / fibroblasts were very similar in TGF-ßtreated wt cells. However, neutralization of TGF-ß signaling did not rescue the phenotype and ruled out the involvement of enhanced levels of TGF-ß in the induction of this phenotype. In accordance with our previous in vivo findings (Sterner-Kock et al., 2002), we observed that the amount of active TGF-ß was significantly decreased in the / fibroblasts. Interestingly, the / fibroblasts produced increased amounts of latent forms of TGF-ß. The expression and secretion of TGF-ß2 and -ß3 increased considerably in / fibroblasts, but the activation of TGF-ß complexes was compromised. TGF-ß1 down-regulates the expression of TGF-ß2 and -ß3 (Bascom et al., 1989), and the lack of LTBP-4mediated activation of TGF-ß1 might contribute to the observed increase in the levels of the other isoforms. Interestingly, BMP-4 regulated TGF-ß3 levels in wt fibroblasts, so increased secretion of BMP-4 may also regulate TGF-ß3 mRNA expression in / fibroblasts. Because both LTBP-1 and -3 are expressed in the mouse lung (Yin et al., 1995; Noguera et al., 2003), they may compensate for the loss of LTBP-4 in the secretion of the small latent complexes. However, the pericellular locations and activation mechanisms of various complexes are likely to be different. This finding is supported by the observation that overexpression of LTBP-1 in / fibroblasts could not substitute for the loss of LTBP-4 in the reversal of the phenotype. Similarly, isoform-specific function of LTBP-1 in TGF-ß1 activation cannot be substituted by LTBP-3 in vß6 integrinmediated activation model (Annes et al., 2004).
The microarray analyses revealed that BMP-4 expression was induced in / fibroblasts in parallel with the loss of expression of its inhibitor gremlin. Activation of the BMP pathway was also reflected by the up-regulation of BMP-4 target genes Id1 and -2. In addition, CTGF, which was highly up-regulated in / fibroblasts, is a direct target of both BMP-4 and TGF-ß1 (Abreu et al., 2002). Inhibition of the BMP-4 pathway partially rescued the / phenotype, further indicating the importance of this signaling cascade for the development of the LTBP-4 / phenotype. The observations were further supported by in vivo data because immunohistochemical staining of / mouse lung tissue revealed increased BMP-4 levels in parallel with decreased gremlin levels and increased phosphorylation of the BMP-specific Smad protein.
Several genes important for the development of the lung were dysregulated in LTBP-4 / fibroblasts. FGF-7, BMP-4, and TGF-ß13 have specific expression patterns in the developing lung (for review see Warburton et al., 2000). FGF-7 promotes epithelial proliferation and functions as a differentiation factor for the developing lung. However, FGF-7 null animals do not show any detectable lung phenotype (Guo et al., 1996), which may be due to overlapping functions of lung morphogens. BMP-4 is an important lung morphogen and its overexpression under the surfactant protein promoter in the lung could severely perturb lung development (Bellusci et al., 1996). The correct targeting and expression levels of these factors are crucial for lung morphogenesis. The up-regulation of TGF-ß activation in fibrillin-1deficient mice led to emphysema, which was rescued by inhibition of TGF-ß1 and -ß2 activities with neutralizing antibodies (Neptune et al., 2003). Interestingly, the down-regulation of TGF-ß activation in LTBP-4 / and ß6 integrin null mice could also lead to emphysema-like conditions in the lung (Sterner-Kock et al., 2002; Morris et al., 2003). Although the mechanisms differ, the correct balance of growth factor activation appears crucial for the development of the lung.
TGF-ß regulates the expression of gremlin in mesangial cells (McMahon et al., 2000). Gremlin expression levels are drastically down-regulated in / fibroblasts, which probably allows BMP-4 signaling and accumulation of the fibronectin-rich ECM. Restoring wt levels of TGF-ß activity to / fibroblasts reverts gremlin expression and reduces ECM accumulation, suggesting that TGF-ß is a strong regulator of gremlin expression and BMP-4 signaling also in lung fibroblasts. LTBP-4 affects the targeting and activation of TGF-ß1 in an isoform-specific manner. Although the accumulation of matrix components is reduced in TGF-ß1 / mouse embryo fibroblasts, mRNA levels remain unchanged for TGF-ß2 and -ß3 (Sudarshan et al., 1998). These differences may reflect the differentiation status of the cells. A contribution of LTBP functions that are independent of TGF-ß in the induction of the / phenotype cannot be ruled out. Our findings suggest that TGF-ß1 targeting as well as LTBP-4dependent modulation of growth factor pathways are important for the development of the lung. Because BMP-4 is essential for pulmonary morphogenesis, and its transgenic overexpression produces abnormal lungs (Bellusci et al., 1996), it is likely that the pulmonary aplasia/emphysema in LTBP-4 / mice results at least in part from the switch from TGF-ß to BMP-4 signaling.
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Materials and methods |
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cDNA constructs
Full-length human LTBP-4S (Saharinen et al., 1998) was cloned into pEF-IRES-P vector (Hobbs et al., 1998) as an XhoIEcoRI fragment generating a construct hLTBP-4S/pEF-IRES. The dominant-negative TGF-ß receptor (RII) cDNA insert was removed from kdTßRII (construct was provided by M.G. Brattain, Roswell Park Cancer Institute, Buffalo, NY; Ko et al., 1998) as an EcoRIXbaI fragment and cloned into pEF-IRES-P generating a
RII/pEF-IRES construct. mGremlin/pEF-IRES was constructed by RT-PCR amplification of the coding sequence of mouse gremlin followed by cloning into pEF-IRES-P as an XhoIEcoRI fragment. The primer sequences used for the amplification were 5'-CCGCTCGAGATGAATCGCACCGCA-3' and 5'-GGAATTCTGACAGGCTGAATGTGC-3'. Construct pLTBP-1 has been described previously (Saharinen and Keski-Oja, 2000).
Cell culture and transfections
Lung fibroblast cultures from adult wt and LTBP-4 / mice were established according to standard protocols. In brief, the lung tissues were finely chopped, and primary explant cultures were allowed to attach and produce outgrowth for 10 d. This was followed by trypsinization of the cells, removal of the primary explants, and culturing the cells as adherent monolayer cultures until confluency. During the following 1012 wk in culture the cells went through a "crisis" and spontaneous immortalization produced continuous cell lines. Two separate cell lines of both wt and / lung fibroblasts were used in the studies and they produced similar results. Mv1Lu mink lung epithelial cells (CCL64) were obtained from American Type Culture Collection. Cells were cultured in DME supplemented with 10% FCS (Life Technologies), 100 IU/ml penicillin, and 50 µg/ml streptomycin.
For stable transfection, the cells were cultured overnight in P60 culture dishes. The transfection was performed the next day using 3 µg of plasmid DNA and 9 µl of FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions. After transfection, the cells were washed and incubated overnight with serum containing growth medium. The antibiotic selection was started a day after transfection with 2 µg/ml of puromycin (Sigma-Aldrich). A gradual increase of puromycin concentration was performed if higher expression levels were needed.
Cells to be transiently transfected were plated in 6-well plates. The next day, the cells were cotransfected with a total of 2 µg of (CAGA)12-luc or (BRE)2-luc promoter constructs (provided by P. ten Dijke) together with pRL-TK (Renilla luciferase control; Promega) plasmid using FuGENE 6 transfection reagent (Roche). After 24 h, the cells were changed to serum-free medium, and TGF-ß1 or BMP-4 were added at the indicated concentrations. Subsequently, 48 h after transfection, the cells were lysed and subjected to luciferase activity measurements by a Dual Luciferase Kit (Promega) and Digene DCR-1 luminometer (MGM Instruments).
TGF-ß activity assay
Mink lung epithelial cells stably transfected with a fragment of PAI-1 promoter fused to luciferase gene (TMLC) were provided by D.B. Rifkin (New York University School of Medicine, New York, NY). These cells produce luciferase in response to TGF-ß. TGF-ß standards and medium samples to be assayed for TGF-ß activity were analyzed as described previously (Abe et al., 1994). The assays were performed at least three times. The results have been presented as relative value of TGF-ß activity (wt medium set as one). The specificity was controlled by adding neutralizing pan-specific TGF-ß antibodies where indicated (AB-100-NA; R&D Systems).
Isolation of extracellular matrices
Cells were cultured in the presence of 35S-labeled amino acids (Easy Tag Express-[35S]; NEN Life Sciences Products) for 2 d. Extracellular matrices were prepared as described previously (Hedman et al., 1979; Taipale et al., 1992). In brief, cell cultures were washed once with PBS, and then treated three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer, pH 8.0, at 0°C for 10 min. The plates were then washed again with PBS, allowed briefly to dry, and the ECM samples were collected by extraction with Laemmli sample buffer and heat treated at 95°C for 5 min. Aliquots of matrix samples were separated by SDS-PAGE and the radiolabeled proteins were visualized by fluorography.
SDS-PAGE and immunoblotting
SDS-PAGE was performed using 7.5% or 415% gradient Tris-HCl polyacrylamide gels (Bio-Rad Laboratories). Conditioned cell culture medium, equalized according to cell number, was concentrated 10-fold using Microcon YM-10 or YM-30 centrifugal filters (Millipore) where indicated. Electrophoretically separated proteins were transferred to nitrocellulose membranes using semidry blotting system (Bio-Rad Laboratories). Immunodetection was performed as described previously (Penttinen et al., 2002).
Immunofluorescence analysis and microscopy
Cells were grown on glass coverslips for 5 d. Coverslips were washed three times with PBS and the cells fixed in ice-cold methanol at 20°C for 15 min. After washing three times with PBS, the cells were incubated in Dulbecco's PBS containing 3% BSA to prevent nonspecific binding of the antibodies. The cells were incubated with the primary antibody in Dulbecco's PBS for 1 h. The binding of the antibodies was detected using FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The coverslips were finally washed in water, mounted on glass slides using Vectashield anti-fading reagent (Vector Laboratories), and examined under the imaging microscope (model Axioplan 2; Carl Zeiss MicroImaging, Inc.) using a 40x objective. Images were acquired with a camera (model AxioCamHRc; Carl Zeiss MicroImaging, Inc.) and software (AxioVision3.1; Carl Zeiss MicroImaging, Inc.).
Immunohistochemistry
Paraffin sections of mouse tissues harvested at the age of 6 wk were prepared and stained using standard histology procedures. For immunostainings (immunoperoxidase), the deparaffinized and rehydrated tissue slides were first treated for 30 min with 3% H2O2 to inactivate the endogenous peroxidases. After rinsing in ddH2O and soaking in PBS for 510 min, the slides were treated with 10% (wt/vol) BSA in PBS to saturate nonspecific protein binding sites. The slides were exposed to the specific antibodies at 4°C overnight. After removing excess antibody, the slides were treated with appropriate biotin-labeled secondary antibodies (Dianova) at 37°C for 60 min, and finally with HRP-labeled streptavidin (Zymed Laboratories) at 37°C for 20 min. After washing, the slides were incubated in diaminobenzidine (Sigma-Aldrich) at RT for 5 min. The slides were finally examined under a conventional microscope (Carl Zeiss MicroImaging, Inc.) after removing the excess substrate in ddH2O.
RNA isolation and RT-PCR
Total cellular RNA was isolated using RNeasy Mini kit (QIAGEN) according to manufacturer's instructions. RNA concentrations and purities were determined spectrophotometrically (Ultrospec 3000; Amersham Biosciences) as well as by agarose gel electrophoresis followed by ethidium bromide staining.
Reverse transcription was performed with Random primers (Life Technologies) and Superscript II reverse transcriptase (Life Technologies) using 2.5 µg of total RNA according to manufacturer's instructions. The cDNAs were amplified using AmpliTaq Gold (PerkinElmer) in a 30-cycle PCR reaction as described previously (Koli et al., 2001b). The primer sequences used for the amplification of the LTBP-4 fragment were 5'-CCCGCTCCGTTTATACAATG-3' and 5'-AGGAAACCGTCCGGAC-3'. The amplified products were analyzed by agarose gel electrophoresis.
Northern hybridization analyses
10 µg of total cellular RNA was fractionated on 1.2% agarose gels containing formaldehyde and transferred to Hybond-N nylon membranes (Amersham Biosciences) by capillary transfer. Prehybridization and hybridization were performed at 68°C in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.). cDNA probes for genes of interest and a control gene glyceraldehyde-3-phosphate dehydrogenase (GADPH) were labeled with [32P]-dCTP (>3,000 Ci/mmol; Amersham Biosciences) using a Ready-To-Go DNA Labeling Kit (Amersham Biosciences). Radioactivity levels were quantified with a bio-imaging analyzer (model BAS-2500; Fuji) where indicated, and the results expressed as mRNA expression levels relative to the GADPH levels.
Oligonucleotide microarray analyses
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. To assure that the gene expression measured by microarray assay was not affected by degradation of the RNA extracted from the lung fibroblast cells, we used the Bioanalyzer-system (Agilent Technologies) to evaluate the quality of the RNA. The protocol for the sample preparation and microarray processing was according to instructions (Affymetrix, Inc.). 10 µg of fragmented, biotinylated cRNA was hybridized to a MGU74Av2 microarray (Affymetrix, Inc.) for 16 h at 45°C with constant rotation at 60 rpm. After hybridization, the microarray was washed and stained on a fluidics station (Affymetrix, Inc.) and scanned with an argon-ion confocal laser, with a 488-nm emission and detection at 570 nm. GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Data analysis was performed with the GeneSpring software version 4.2 (Silicon Genetics Inc.). To eliminate changes within the range of background noise and to select the most differentially expressed genes, the restrictions used to classify genes as up- or down-regulated included a change in expression of greater than threefold and evidence that the genes were "present" according to the data analysis (Affymetrix, Inc.).
Online supplemental material
The complete data from the gene array analyses (see Gene array analyses) is available at http://www.jcb.org/cgi/content/full/jcb200403067/DC1.
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Acknowledgments |
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This work was supported by grants from the Academy of Finland, the Finnish Cancer Foundation, the Sigrid Juselius Foundation, Biocentrum Helsinki, National Technology Agency (TEKES), Novo Nordisk Foundation, Biomedicum Foundation, Helsinki University Hospital Fund, and the University of Helsinki to J. Keski-Oja and by grants from the Deutsche Forschungsgemeinschaft to H. von Melchner.
Submitted: 10 March 2004
Accepted: 4 August 2004
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