Overexpression of latent transforming growth factor-ß binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts

Belen Santiago-Josefat1, Sonia Mulero-Navarro1, Sarah L. Dallas2 and Pedro M. Fernandez-Salguero1,*

1 Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06071-Badajoz, Spain
2 Department of Oral Biology, School of Dentistry, University of Missouri at Kansas City, Missouri, USA

* Author for correspondence (e-mail: pmfersal{at}unex.es)

Accepted 9 October 2003


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The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR–/–) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-ß binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR–/– MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-ß(TGF-ß) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR–/– conditioned medium had higher levels of active and total TGF-ß activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-ß concentrations. TGF-ß did not appear to directly regulate Ltbp-1 given that addition of TGFß neutralizing antibody or TGFß protein to AhR–/– MEF had no effect on Ltbp-1 expression. AhR–/– MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFß activity and cell proliferation.

Key words: Dioxin receptor, LTBP-1, TGF-ß, Matrix metalloproteinases, Differential display


    Introduction
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aryl hydrocarbon (dioxin) receptor (AhR) has essential roles in mediating xenobiotic-induced toxicity and carcinogenesis (Fernandez-Salguero et al., 1996Go; Pohjanvirta and Tuomisto, 1994Go; Shimizu et al., 2000Go). Mechanistically, the AhR functions as a transcription factor that enters the cell nucleus and binds to conserved regulatory sequences (dioxin responsive elements, DREs) located upstream in the promoter of target genes. This process usually results in increased rates of gene expression (Denison and Whitlock, 1995Go; Gonzalez and Fernandez-Salguero, 1998Go; Swanson and Bradfield, 1993Go; Whitlock et al., 1996Go). Although a large number of genes have putative DREs in their promoter region (Lai et al., 1996Go), most of the AhR-regulated genes code for xenobiotic metabolizing enzymes such as the well-known cytochromes P4501A1, 1A2 and 1B1, UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (Hankinson, 1995Go; Nebert et al., 1990Go; Nebert et al., 2000Go; Schmidt and Bradfield, 1996Go). Recently, a new dioxin-inducible cytochrome P450 (CYP450), CYP2S1, has been isolated in mouse Hepa-1 cells by representational difference analysis (Rivera et al., 2002Go).

The high degree of conservation and wide expression of the AhR across different species, together with the many phenotypic changes observed in AhR-null mice (Abbott et al., 1999Go; Benedict et al., 2000Go; Fernandez-Salguero et al., 1995Go; Fernandez-Salguero et al., 1996Go; Fernandez-Salguero et al., 1997Go; Lahvis et al., 2000Go; Mimura et al., 1997Go; Schmidt et al., 1996Go), provide strong support for the existence of endogenous roles for the AhR not related to xenobiotic metabolism. In this regard, recent studies have shown that the AhR is involved in regulating the expression of constitutive proteins such as the DNA polymerase kappa (Ogi et al., 2001Go), N-myristoyltransferase 2 (Kolluri et al., 2001Go), p27Kip1 (Kolluri et al., 1999Go) and Bax (Matikainen et al., 2001Go). Interestingly, inhibitory regulatory elements (iDRE) have been also identified in estrogen responsive genes such as pS2 and cathepsin D (Safe et al., 1998Go), and in the T-cadherin promoter (Niermann et al., 2003Go). Thus, it appears plausible that the AhR could regulate the expression of genes involved in maintaining normal cell physiology.

Previous studies have shown that AhR-null primary hepatocytes and mouse embryo fibroblasts (MEFs), in the absence of xenobiotics, had increased levels of transforming growth factor ß (TGFß) and decreased proliferation, suggesting a role for the AhR in TGFß signaling and cell cycle (Elizondo et al., 2000Go; Zaher et al., 1998Go). TGFß is a dimeric protein that participates in cell homeostasis and development by triggering a complex intracellular transduction pathway following its interaction with membrane-bound serine-threonine receptors (reviewed by Massague, 1998Go). The TGFß dimer is secreted to the extracellular medium forming a latent complex with its propeptide LAP (TGFß latency-associated protein). In most cells, however, the latent form of TGFß is associated before secretion to a third protein called latent TGFß binding protein (LTBP). This process leads to the formation of the large latent TGFß complex (a monomer of LTBP plus a dimer of LAP-TGFß), which will be the more abundant form for TGFß secretion (Koli et al., 2001Go; Lawrence, 2001Go; Saharinen et al., 1999Go). Within the large complex, the carboxylic region of LTBP binds latent TGFß by forming a disulphide bond between one of its 8-Cys repeats and LAP. The amino terminus of LTBP, however, anchors the large complex to the fibers of the extracellular matrix. Different proteases such as plasmin, elastase, chymase and metalloproteinases have been involved in the release of latent TGFß from the extracellular matrix via cleavage of LTBP, and some of these can also activate latent TGFß (reviewed by Koli et al., 2001Go; Lawrence, 2001Go; Taipale and Keski-Oja, 1997Go). Although traditionally considered as a localization protein to establish a reservoir of latent TGFß, recent studies have suggested that LTBP also participates in the assembly, secretion and activation of this cytokine (Annes et al., 2003Go; Dallas et al., 2002Go; Koli et al., 2001Go; Lawrence, 2001Go; Oklu and Hesketh, 2000Go; Taipale and Keski-Oja, 1997Go). Four isoforms of LTBP have been cloned from humans and rodents that differ in their gene structure, alternative splicing, usage of alternative promoters and cell-specific patterns of expression (Giltay et al., 1997Go; Koli et al., 2001Go; Koski et al., 1999Go; Moren et al., 1994Go; Saharinen et al., 1998Go; Tsuji et al., 1990Go; Yin et al., 1995Go). The phenotypic alterations found in the organs of mice lacking expression of different LTBPs strongly support a role for these proteins in TGFß bioavailability and activation (Dabovic et al., 2002Go; Sterner-Kock et al., 2002Go). The AhR and TGFß appear to be related, as primary cultures of hepatocytes and embryonic fibroblasts from AhR–/– mice produced increased levels of this cytokine. Moreover, the lower proliferation and higher apoptotic rates found in those cells could be due, at least in part, to TGFß overexpression (Elizondo et al., 2000Go; Zaher et al., 1998Go). In vivo, the periportal area of AhR–/– liver had increased collagen deposition that appeared to be associated with a certain degree of fibrosis (Peterson et al., 2000Go). Interestingly, this organ also presented higher expression of TGFß protein and elevated apoptotic numbers (Zaher et al., 1998Go).

In this study, using wild-type and AhR-null MEF cells, we have analyzed differential patterns of gene expression in the absence of xenobiotics. With this approach, we have made an attempt to identify AhR-regulated genes potentially relevant in normal cell physiology. We have found that LTBP-1 is negatively regulated at the transcriptional and protein levels in AhR-null MEFs. Overexpression of LTBP-1 was coincident with increased levels of latent and active TGFß and with decreased matrix metalloproteinase-2 activity. These results not only identified LTBP-1 as a new AhR-regulated gene but also provide further support for the implication of this receptor in the control of TGFß activity.


    Materials and Methods
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Chemicals and reagents
Taq DNA polymerase and MMLV reverse transcriptase were from Ecogen and from Ambion, respectively. Fetal bovine serum (FBS) was obtained from BioWhittaker and was heat inactivated before use. Rabbit antibody against mouse ß-actin and anti-rabbit IgG-HRP (horse-radish peroxidase) were from Sigma and Pierce, respectively. Anti-TGFß 1D-11 neutralizing antibody and human TGFß1 recombinant protein were obtained from R&D Systems. The specificity of the anti-LTBP-1 `hinge' antibody has been described previously (Dallas et al., 2000Go). The anti-rabbit-TRICT (tetramethylrhodamine isothiocyanate) secondary antibody was purchased from Sigma. Protein A/G plus agarose was purchased from Santa Cruz Biotechnology. Dulbecco's-modified Eagle's medium (D-MEM), OPTI-MEM and cell media supplements were purchased from Invitrogen Corporation (GIBCOTM). Differential display (DD) was performed using the kit Hieroglyph mRNA profile from Genomix.

Cell culture and treatments
AhR-null and wild-type control mice of the same genetic background (C57BL6/Nx129/sV) were produced as previously reported (Fernandez-Salguero et al., 1995Go). Mice were genotyped by PCR from tail genomic DNA using the primers forward 5'-GGCTAGCGTGCGGGTTTCTC-3' and reverse 5'-CTAGAACGGCACTAGGTAGGTCAGA-3' and the conditions described below. MEF were isolated from 14.5-day post coitum embryos as described previously (Santiago-Josefat et al., 2001Go). Cells were grown in D-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO2 atmosphere. MEF from the first or second passage at around 80% confluence were used in all the experiments. Mv1Lu (mink lung epithelial, CCL64) cells were grown under the same conditions in complete medium supplemented with 1 mM sodium pyruvate. For the analysis of LTBP-1 protein expression and to obtain MEF-conditioned media, cells were maintained at confluence for 72 hours. To determine TGFß levels and matrix metalloproteinase (MMP) activity, MEF were maintained for 72 hours of post-confluence in OPTI-MEM medium. Treatments with actinomycin D and cycloheximide were done for 3 and 6 hours at 1.5 µM and 175 µM, respectively. Human TGFß recombinant protein was used for the indicated times at 10 ng/ml, whereas anti-TGFß antibody was added at 1 µg/ml for 24 hours. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) treatment was done at a concentration 10 nM for 24 hours.

Differential display
DD was used to randomly amplify and compare mRNA expression between AhR+/+ and AhR–/– MEF. The Genomix Hieroglyph kit was used on a Genomix LX apparatus following the instructions provided by the manufacturer. Briefly, total RNA was purified with the RNeasy kit (Qiagen) and 0.1 µg reverse transcribed (RT) at 42°C for 1 hour using MMLV reverse transcriptase and one of the different 3' anchored primers (such as dT12-GA, dT12-GC, dT12-GG) annealing to the poly-A tail of the mRNAs. An aliquot of 2 µl of RT product was then amplified by PCR with the same anchored primer (reverse) and one of the different arbitrary 10-mer forward primers included in the kit. PCR was performed in 20 µl reaction volume containing 67 mM Tris-HCl pH 8.8, 16 mM (NH4)SO4, 0.01% Tween-20, 1.5 mM MgCl2, 5 µM each dNTP, 0.2 µM each primer, 2.5 µCi [{alpha}-33P]dATP (esp. act. 3000 Ci/mmol) and 1 U Taq-polymerase. Amplification conditions were as follows: initial denaturation at 94°C for 2 minutes; four cycles denaturing at 92°C for 15 seconds, annealing at 46°C for 30 seconds and extending at 72°C for 2 minutes; 25 cycles denaturing at 92°C for 15 seconds, annealing at 60°C for 30 seconds and extending at 72°C for 2 minutes. An aliquot of 7 µl from each PCR reaction was denatured and electrophoresed in a 4.5% urea-HR1000 sequencing gel at 800 V for 16 hours according to the manufacturer's indications. The gels were dried and exposed to X-ray film. Bands corresponding to differentially expressed mRNAs were excised from the gel, re-amplified by PCR using the same primers and the resulting products employed as probes in northern blots of total RNA. DD bands confirmed to be differentially expressed by northern blot were sequenced with an ABI Prism automated sequencer using di-deoxiterminator chemistry (Applied Biosystems).

Northern blot and RT-PCR
Total RNA was isolated with the RNeasy kit from Qiagen. For northern analysis, 10 µg RNA were separated in 6% formaldehyde-agarose gels. Gels were transferred to Gene-Screen Plus nylon membranes and RNA fixed by UV-crosslinking. Blots were prehybridized at 65°C for 3 hours in Rapid-Hyb buffer (Amersham). cDNA probes for mouse Ltbp-1, AhR, Tgf-ß1 and ß-actin were labeled by random priming using [32P]-dCTP (esp. act. 3000 Ci/mmol) and Klenow DNA polymerase. Probes were purified, diluted to 8x105 cpm/ml in Rapid-Hyb buffer and incubated with the membranes for 4 hours at 65°C. Background was reduced by sequential washing in 2xSSC (3 M sodium chloride, 0.3 M sodium citrate, pH 7.5), 0.1% SDS at room temperature for 30 minutes and in 0.1xSSC, 0.1% SDS at 65°C for another 30 minutes. Membranes were exposed and developed using a Molecular Imager FX imaging system (Bio-Rad Labs). For RT-PCR analysis, 1 µg total RNA was reverse transcribed at 42°C for 60 minutes using oligo(dT) priming and MMLV reverse transcriptase (Ambion). Ltbp-1, Ltbp-2, Ltbp-3, Ltbp-4, Mmp-2, Timp-1 (tissue inhibitor of MMP), Timp-2 and ß-actin cDNAs were amplified by PCR using the primers listed in Table 1. Amplification was carried out for 28 cycles (25 for Timps) in 50 µl reaction mixture containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.5 µM each primer, 2.5 units Taq polymerase and 3 µl of each reverse transcription reaction as template. Cycling conditions were: denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute and extension at 72°C for 1 minute (2 minutes for Ltbp-2, 3 and 4). PCR products were visualized in agarose gels stained with ethidium bromide.


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Table 1. Primer sequences used to analyze the expression of LTBPs, TIMPs and MMP-2 in MEF cultures by RT-PCR

 

Western blot and immunoprecipitation
MEF cultures growing at confluence for 72 hours were washed twice with phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenyl-methyl sulfonyl fluoride, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, 10 mM ß-glycerophosphate and 4 µg/ml Complete protease inhibitor cocktail (Roche). Lysates were centrifuged at 15,000 g for 30 minutes at 4°C and protein concentration determined in the supernatant by the Coomassie protein assay system (Pierce) using bovine serum albumin as standard. Fifteen micrograms of protein were mixed with SDS-sample buffer, denatured and electrophoresed in 8% SDS-PAGE gels. Gels were transferred to nitrocellulose membranes that were blocked for 2 hours at room temperature in TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Tween-20) containing 5% nonfat milk. Blots were sequentially incubated with anti-LTBP-1 or anti-ß-actin primary antibodies at a concentration of 0.45 µg/ml and with the HRP-bound secondary antibody. After washing in TBS-T, membranes were developed using the Super-signal substrate (Pierce) and a chemiluminescence imaging screen (Bio-Rad Labs). LTBP-1 was also immunoprecipitated from AhR+/+ and AhR–/– conditioned media. A volume of medium equivalent to the same number of cells of each genotype was incubated with 2 µg/ml anti-LTBP-1 antibody for 16 hours at 4°C. Antibody-treated media were then incubated with 30 µl protein A/G plus agarose beads at 4°C for 1 hour. Beads were washed for three times each in buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% sodium deoxycholate and 1% Triton X-100) and buffer B (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1% Triton X-100). Laemmli's sample buffer was added and SDS-PAGE performed under reducing conditions as indicated above.

Immunocytochemistry
LTBP-1 was immunolocalized in the extracellular matrix of MEF cultures at 72 hours of postconfluence. After removing the medium, plates were washed with PBS, fixed in 95% ethanol for 10 minutes at –20°C and air-dried. Cells were then permeabilized by three 5 minutes washes in PBS solution containing 0.05% Triton X-100. Blocking was performed for 30 minutes at room temperature in PBS containing 2% bovine serum albumin and 10% normal goat serum. The primary anti-LTBP-1 hinge antibody was added at a concentration of 4.5 µg/ml for 16 hours at 4°C. Following three washes in PBS solution containing 0.05% Triton X-100, anti-rabbit-TRICT secondary antibody was added and incubation continued at room temperature for 1 hour. Plates were washed three times in PBS-0.05% Triton X-100 solution and photographed using a Zeiss fluorescence microscope.

Bioassay for TGFß activity
AhR+/+ and AhR–/– MEF were cultured in serum-free OPTI-MEM medium for 72 hours. Conditioned media were removed, centrifuged at 500 g for 15 minutes at 4°C and the supernatants supplemented with 0.1 mg/ml bovine serum albumin and 0.57 mM phenyl-methyl sulfonyl fluoride. Mv1Lu were seeded in 24-well plates at 2.5x104 cells/well and washed twice in OPTI-MEM. Conditioned media from MEF cultures, diluted 1:2 or 1:4 with fresh OPTI-MEM, were added to Mv1Lu plates that were incubated for 24 hours. In some experiments, 1 µg/ml neutralizing anti-TGFß antibody was added to the conditioned media before the incubation with the Mv1Lu cells. To determine total TGFß production by MEF, conditioned media were pre-incubated at 80°C for 8 minutes before dilution and addition to the Mv1Lu plates. Two hours before the end of the 24 hour incubation period, 0.5 µCi [3H]-methyl-thymidine (esp. act. 7 Ci/mmol) were added to the Mv1Lu cultures to label DNA synthesis. Cells were then fixed for 1 hour in 1 ml methanol:acetic acid (3:1), washed in 80% methanol, trypsinized in 0.05% trypsin and solubilized in 1% SDS. The amount of radiactivity incorporated into DNA was measured in a Beckman LS-3801 liquid scintillation counter. To calculate TGFß concentration, a calibration curve was constructed by adding known amounts (0 to 100 pg) of human recombinant TGFß to Mv1Lu cultures and measuring [3H]-thymidine incorporation into DNA as described above. To analyze the contribution of LTBP-1 to TGFß activation, AhR–/– MEF were grown in presence of 1 µg/ml anti LTBP-1 antibody for 48 hours and the conditioned medium obtained used in TGFß bioassays as indicated above. Previous experiments were performed to determine the neutralizing activity of the anti-TGFß antibody. In a bioassay using conditioned medium from AhR–/– MEF, an amount of 0.06 µg/ml antibody was able to produce 50% neutralization. On the basis of this activity, a 1 µg/ml concentration of anti-TGFß antibody was used in all the experiments.

Matrix metalloproteinase activity
MMP activity was determined by a gelatinolytic assay using conditioned medium from AhR+/+ and AhR–/– MEF cultures growing in OPTI-MEM for 72 hours. After centrifugation at 10,000 g for 15 minutes at 4°C, a volume of medium equivalent to the same number of cells was mixed with nonreducing Laemmli's sample buffer (62.2 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 0.025% bromophenol blue) and directly applied into an 8% SDS-PAGE gel that was polymerized in the presence of 1% gelatin. After electrophoresis, the gel was washed three times in a solution containing 2.5% Triton X-100 to eliminate the SDS and to allow reconstitution of the protein. MMP activity was obtained by incubating the gel at 37°C for 16 hours in reaction buffer (50 mM Tris-HCl pH 6.8, 150 mM NaCl, 5 mM CaCl2 and 0.05% sodium azide). The position of the MMPs was visualized by staining the gel in Coomassie G-250 solution (25% methanol, 10% acetic acid, 0.05% Coomassie brilliant blue G-250). Conditioned medium from the human fibrosarcoma cell line HT-1080 was used to identify the position of MMP-2. To determine the contribution of LTBP-1 and TGFß to decreased MMP-2 activity, AhR–/– MEF were cultured for 48 hours in the presence of 1 µg/ml anti-LTBP-1 or anti-TGFß antibodies and the resulting conditioned media used in gelatinolytic assays as described above.


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Differential expression of LTBP-1 in AhR-null MEF cells
We have used DD to identify genes that could be regulated through the AhR in the absence of xenobiotics. A representative DD gel for AhR+/+ (+) and AhR–/– (–) MEF, using several combinations of anchored and arbitrary primers, is shown in Fig. 1A. Considering the number of anchored and arbitrary primers used, we have estimated that close to 15% of the mRNAs present in MEF under basal culturing conditions were analyzed in this study. Among the candidate genes that appeared differentially expressed between both genotypes, we focused on one band, of a size close to 700 bp, which had a higher level of expression in AhR–/– with respect to wild-type MEF. Re-amplification by PCR and sequencing of the differentially expressed band allowed us to compare its nucleotide sequence with the database BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). We found a 97% homology in nucleotide sequence between this band and the mouse gene named latent transforming growth factor-ß binding protein 1 (LTBP-1, accession number AF022889). To confirm differential expression of this gene in our MEF cultures, a cDNA probe was synthesized by RT-PCR using the specific primers for Ltbp-1 indicated in Table 1. Northern blot analysis of total RNA revealed a threefold increase in Ltbp-1 expression between AhR-null and AhR+/+ MEF (Fig. 1B). The use of alternative promoters in the mouse Ltbp-1 gene produces mRNAs of 5.4 (Ltbp-1S) and 7.4 (Ltbp-1L) Kbp (Noguera et al., 2003Go; Weiskirchen et al., 2003Go). In our northern blots, only a major band in the range of 7.0 Kbp could be observed (Fig. 1B), whereas no additional bands around 5 Kbp were found (for comparison, the position of the 28S rRNA is shown in Fig. 1B). These data indicated that MEF transcribed the large form of Ltbp-1. Increased expression of this gene in AhR–/– MEF was further supported by RT-PCR amplification with the specific primers listed in Table 1. As can be seen in Fig. 1C, AhR-null MEF expressed higher levels of Ltbp-1 transcripts than AhR+/+ mice, in close agreement to the northern blot results.



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Fig. 1. Ltbp-1 mRNA expression is increased in AhR-null mice. (A) Total RNA from AhR+/+ and AhR–/– MEF was isolated and analyzed by differential display using the Hieroglyph kit and a Genomix LX apparatus following the instructions provided by the manufacturer. A representative DD gel is shown for different combinations of arbitrary (forward) and anchored (reverse) primers. The band corresponding to LTBP-1 is indicated by an arrow. (B) A differentially expressed band from the DD gel was isolated, re-amplified by PCR and sequenced. Comparison of its sequence with the database BLAST revealed a 97% homology with the Ltbp-1 mRNA. Gene-specific primers (Table 1) were synthesized and used to produce a cDNA probe that detected the mouse Ltbp-1 mRNA in northern blots using 10 µg total RNA. (C) Specific primers (Table 1) were also used to amplify Ltbp-1 by RT-PCR in order to verify overexpression in AhR–/– MEF. Relative levels of gene expression were obtained by using the Quantity One software on a Molecular Imager FX system (Bio-Rad Laboratories) as follows: background was subtracted from each band and the resulting expression normalized by that of ß-actin. Expression in AhR+/+ MEF was assigned an arbitrary value of 1.0. The experiments were repeated at least three times using different MEF preparations.

 

To determine if increased mRNA levels were followed by an increase in LTBP-1 protein, immunoblots were performed in total cell extracts from AhR+/+ and AhR–/– MEF cultures (Fig. 2). LTBP-1 protein expression in MEF lacking AhR was increased between 2.5 and 3-fold with respect to control fibroblasts (Fig. 2A). Additionally, a major protein band with a molecular weight close to 190 kDa was detected in MEF. The putative short form of the protein, with a reported molecular mass of 140-150 kDa (Noguera et al., 2003Go; Weiskirchen et al., 2003Go), was undetectable in these mouse cultures (Fig. 2A). A fraction of the extracellular LTBP-1 was found in the conditioned medium of MEF cells, as could be observed by immunoprecipitation (Fig. 2B). Although AhR-null MEF also had elevated levels of this form of LTBP-1 (about 1.8-fold with respect to AhR+/+ MEF), the increase was less pronounced than that observed at the level of intracellular protein (Fig. 2A). LTBP-1 is not only a targeting molecule for TGFß but also appears to be a component of the extracellular matrix (Koli et al., 2001Go). To determine if increased LTBP-1 protein expression could also be associated with its accumulation in the extracellular matrix, postconfluent AhR+/+ and AhR–/– MEFs were analyzed by immunocytochemistry (Fig. 2C). AhR-null fibroblasts showed a large increase in LTBP-1 deposition in the extracellular matrix, a situation that could be due, at least in part, to higher levels of protein expression. Three additional members of the LTBP family have been identified in the mouse. To determine if the AhR genotype could also influence the expression of these other isoforms, RT-PCR analysis was performed on total RNA from wild-type and AhR-null MEF (Fig. 3). Contrary to the results obtained with Ltbp-1, Ltbp-2 (Fig. 3A), Ltbp-3 (Fig. 3B) and Ltbp-4 (Fig. 3C) mRNA levels did not change significantly in the absence of AhR. Northern blot analysis for Ltbp-2, -3 and -4 revealed a similar result to that obtained by RT-PCR (data not shown). These results indicate that the AhR could be involved in the control of Ltbp-1 expression through isoform-specific mechanisms.



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Fig. 2. LTBP-1 protein is overexpressed in AhR–/– MEF. (A) LTBP-1 protein expression was analyzed using cellular extracts from wild-type and AhR-null MEF. A total of 15 µg protein were resolved in SDS-PAGE gels and used for immunoblotting with a specific anti-LTBP-1 `hinge' antibody. The position of the 190 kDa LTBP-1 band is indicated. (B) LTBP-1 was immunoprecipitated from conditioned medium from MEF of both genotypes and subjected to immunoblot analysis as indicated above. The position of the 190 kDa LTBP-1 band is indicated. (C) LTBP-1 overexpression was also detected in the extracellular matrix of AhR–/– MEF by immunocytochemistry using the `hinge' antibody. A significant increase in matrix deposition could be observed in MEF lacking AhR (arrowheads). Quantitations of protein expression were done as indicated in Fig. 1, except for B, in which ß-actin normalization could not be applied. The experiment was performed in three different MEF cultures for each genotype with similar results.

 


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Fig. 3. mRNA expression of Ltbp-2, -3 and -4 in AhR–/– MEF. RNA expression for additional members of the LTBP family was performed by RT-PCR using total RNA and the gene-specific primers listed in Table 1. No significant differences were found in the expression of Ltbp-2 (A), Ltbp-3 (B) or Ltbp-4 (C) between AhR+/+ and AhR–/–. The expression of ß-actin was used to check for RNA quantitation and integrity. The experiment was performed by duplicate in at least two MEF preparations.

 

The high steady-state level of Ltbp-1 mRNA found in the absence of AhR required active transcription as addition of the transcriptional inhibitor actinomycin D (Act D) markedly decreased Ltbp-1 overexpression (Fig. 4A). Interestingly, in the presence of the protein synthesis inhibitor cycloheximide (CHX) the increase in Ltbp1 expression was maintained above basal levels in AhR–/– fibroblasts (Fig. 4A), indicating that newly synthesized co-repressor (s) could cooperate to maintain transcriptional control of this gene in MEF. Additional data involving the AhR in negative regulation of Ltbp-1 came from experiments showing that activation of this receptor by the exogenous ligand TCDD (10–9 M) produced a decrease in steady-state Ltbp-1 mRNA levels (Fig. 4B). The role of the AhR in LTBP regulation was further supported by experiments treating AhR+/+ MEF cultures with recombinant TGFß, which is known to decrease AhR expression and activity in A549 human lung carcinoma cells (Dohr et al., 1997Go; Wolff et al., 2001Go). Treatment of MEF with TGFß for 3 hours decreased AhR mRNA to very low levels. In parallel, a significant increase in Ltbp-1 mRNA expression could be observed (Fig. 4C). Moreover, when MEF cells lacking AhR were treated with TGFß under the same conditions, no effect on Ltbp-1 mRNA expression was found (Fig. 4D). Finally, the addition to AhR-null MEF of a neutralizing antibody against TGFß activity had no effect on Ltbp-1 expression (Fig. 4E). Therefore, these data indicated that the AhR was involved in the negative control of LTBP-1 expression in MEF cells.



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Fig. 4. Regulation of Ltbp-1 expression by the AhR. (A) AhR–/– MEFs were treated for 3 and 6 hours with 1.5 µM actinomycin D (Act D) or 175 µM cycloheximide (CHX). Ltbp-1 and ß-actin mRNA expression were analyzed by northern blot using 10 µg total RNA. (B) AhR+/+ MEF were treated with the solvent DMSO (–) or 10 nM of the AhR ligand TCDD (+) and Ltbp-1 expression analyzed by RT-PCR. (C) AhR+/+ MEF were treated with 10 ng/ml recombinant TGFß protein for 1.5 or 3 hours. AhR and Ltbp-1 expression were determined by northern blot using 10 µg total RNA. (D) AhR–/– MEF were treated with 10 ng/ml recombinant TGFß protein for the indicated times and Ltbp-1 expression measured by northern blot using 10 µg total RNA. (E) AhR–/– MEF were treated for 24 hours with 1 µg/ml neutralizing anti-TGFß antibody and Ltbp-1 expression analyzed by northern blot using 10 µg total RNA. The levels of the 28S rRNA shown in panels C, D and E were obtained from ethidium bromide-stained agarose gels and were used to confirm equal loading. The experiments were done in duplicate in two MEF cultures.

 

Overexpression of LTBP-1 was related to increased TGFß levels in AhR-null MEF
LTBP-1 is involved in localizing TGFß to the extracellular matrix. To study if LTBP-1 overexpression could be related to differences in TGFß activity between AhR+/+ and AhR–/–, we used a Mv1Lu-based bioassay to measure the amount of TGFß released to the medium by MEF of both genotypes (Fig. 5A, left). The addition of conditioned medium from wild-type MEF (+/+) inhibited Mv1Lu proliferation, suggesting the presence of inhibitory molecules. A neutralizing anti-TGFß antibody restored cell proliferation, indicating that such inhibitory molecules could be TGFß. As previously reported (Elizondo et al., 2000Go), conditioned medium from AhR-null MEF (–/–) strongly inhibited Mv1Lu proliferation and such an effect could be also recovered by anti-TGFß antibody. Heating the conditioned medium from AhR+/+ and AhR–/– activated the fraction of latent TGFß and further inhibited Mv1Lu proliferation. The inhibitory effect of heat-activated medium could be partially recovered by anti-TGFß antibody. Therefore, AhR+/+ and AhR–/– MEFs secreted active (inhibited Mv1Lu proliferation without additional processing) and latent (required activation by heating) TGFß protein into the culture medium. By constructing a calibration curve using known amounts of recombinant TGFß protein (Fig. 5A, right), we could quantify the levels of active and total (active plus latent) TGFß present in the culture media (Table 2). AhR-null produced about twofold more total TGFß than wild-type MEF (314±56 vs 147±42). More significant is the ratio for active TGFß between both genotypes, given that AhR–/– secretion exceeded AhR+/+ by over fourfold (190±34 vs 44±0.8). Additionally, although active TGFß represented 30% of the total amount of this cytokine in wild-type MEF, this fraction increased to 60% in AhR-null cells. Thus, MEF lacking AhR expression secreted increased levels of active and total TGFß, and the fraction active/total was higher with respect to control MEF. Increased TGFß activity in AhR–/– MEF was not the consequence of increased expression of the gene coding for this cytokine in the absence of AhR since Northern blot analysis for Tgf-ß1 revealed no significant differences in mRNA levels between both genotypes (Fig. 5B).



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Fig. 5. TGFß levels are increased in AhR–/– MEF. (A) The amount of TGFß secreted by MEF was analyzed in conditioned medium from AhR+/+ and AhR–/– fibroblasts. The inhibitory effect of TGFß on the proliferation of Mv1Lu mink lung epithelial cells was determined by measuring the rate of [3H]-thymidine incorporation during DNA synthesis. Conditioned medium was obtained after 72 hours of culture in OPTI-MEM medium. In some experiments, 1 µg/ml neutralizing anti-TGFß antibody was added during incubation of the Mv1Lu cells with conditioned medium. To determine total TGFß activity, conditioned media were heated at 80°C for 8 minutes before addition to Mv1Lu cultures. To transform [3H]-thymidine incorporation into TGFß concentration, a standard curve was constructed with known amounts of recombinant TGFß protein. Experiments as the one shown were used to calculate the concentration of active and total TGFß presented in Table 2. (B) Tgfß mRNA expression was determined in AhR+/+ and AhR–/– MEF by northern blot using 10 µg total RNA. The expression of ß-actin was used to normalize for equal loading. The experiments were done in triplicate in at least two different MEF cultures.

 

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Table 2. TGFß levels in conditioned media from AhR+/+ and AhR–/– MEF cultures

 

Matrix metalloproteinase MMP-2 activity was decreased in AhR-null MEF
TGFß has been reported to downregulate MMP-2 activity in cultured mesanglial cells (Wang and Hirschberg, 2003Go) and in vivo in mouse models of rheumatoid arthritis (Chernajovsky et al., 1997Go). We have shown that AhR-null mice had a moderate level of liver fibrosis resulting from extracellular matrix accumulation in the periportal areas of this organ (Peterson et al., 2000Go). To determine whether increased levels of active TGFß in AhR–/– could be related to lower MMP-2 activity, we have performed gelatinolytic assays using conditioned medium from wild-type and AhR-null MEF (Fig. 6). AhR–/– MEF secreted significantly reduced levels of MMP-2 protein, whether in its precursor (72 kDa), intermediate (64 kDa) or active (62 kDa) forms (Fig. 5A). Decreased MMP-2 activity in AhR–/– conditioned medium could not be attributed to lower expression of the gene as RT-PCR analysis revealed no significant difference in Mmp-2 mRNA between both genotypes (Fig. 6B). To analyze whether decreased MMP-2 activity could be the result of increased levels of the MMP inhibitors Timp-1 and Timp-2, we analyzed their expression by RT-PCR. As can be seen in Fig. 6C, Timp-1 and Timp-2 mRNA levels were very similar in AhR+/+ and AhR–/– MEFs. These data indicated that lower MMP-2 activity was not due to changes in Mmp-2, Timp-1 or Timp-2 expression.



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Fig. 6. MMP-2 is downregulated in AhR–/– MEF. (A) MMP-2 activity was analyzed in conditioned media from AhR+/+ and AhR–/– growing for 72 hours in OPTI-MEM. A gelatinolytic assay was employed as indicated in Materials and Methods. The positions of the pro-(72 kDa), intermediate (64 kDa) and active (62 kDa) MMP-2 are indicated by arrows. Gelatinolytic activity in conditioned media from HT1080 cells was used as control. (B) Mmp-2 mRNA expression was determined by RT-PCR in AhR+/+ and AhR–/– MEF cultures using the primers listed in Table 1. (C) mRNA expression of the MMP inhibitors Timp-1 and Timp-2 was also analyzed by RT-PCR in AhR+/+ and AhR–/– MEF cultures with the primers indicated in Table 1. In panels B and C, ß-actin levels were also analyzed. The experiments were done in duplicate in two different MEF cultures.

 

Antibodies against LTBP-1 and TGFß decreased the inhibitory potential of AhR–/– conditioned medium
To analyze whether overexpression of LTBP-1 could be contributing to higher levels of active TGFß, we have cultured AhR–/– MEF for 48 hours in the presence of antibodies against LTBP-1 or TGFß (Fig. 7). Addition of anti-LTBP-1 antibody during culturing of AhR–/– MEF resulted in a conditioned medium that was less effective in blocking the proliferative potential of Mv1Lu cells as compared with medium from nontreated MEF cultures. Conditioned medium from anti-TGFß antibody-treated AhR–/– MEF was even less able to inhibit Mv1Lu proliferation (Fig. 7A). Blocking LTBP-1 or TGFß also affected MMP-2 activity. As shown in Fig. 7B, conditioned medium from AhR–/– MEF, growing in the presence of anti-LTBP-1 or anti-TGFß antibodies, had a partial recovery in MMP-2 activity as compared with untreated conditioned medium. Thus, LTBP-1 overexpression could be contributing to the growth inhibitory potential and the lower MMP-2 activity observed in AhR–/– MEF.



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Fig. 7. Antibodies against LTBP-1 and TGFß decreased the inhibitory properties of conditioned medium from AhR–/– MEF. AhR–/– MEF were grown for 48 hours in the presence of 1 µg/ml anti-LTBP-1 (hinge) or 1 µg/ml anti-TGFß (1D-11) antibodies and conditioned medium prepared as indicated in Materials and Methods. (A) Conditioned media from cultures left untreated (No Ab), treated with anti-LTBP-1 (anti-LTBP) or anti-TGFß (anti-TGFß) were added to Mv1Lu cells and their inhibitory potential measured as indicated in Materials and Methods. (B) Using the same conditioned media; MMP-2 activity was determined by using a gelatinolytic assay. The experiments were performed in triplicate (A) or duplicate (B) in two MEF cultures.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A large majority of the published studies on AhR function have been focused on its role as a xenobiotic-interacting receptor that regulates the expression of xenobiotic-metabolizing enzymes, which will be the final effectors for clearance of toxic and carcinogenic molecules from the body. Nevertheless, this important role, recent experimental evidence strongly suggests that the AhR could also participate in the control of cellular functions unrelated to xenobiotic metabolism. In vitro studies have shown that the AhR interacts with proteins controlling the cell cycle such as retinoblastoma (Elferink et al., 2001Go; Ge and Elferink, 1998Go; Puga et al., 2000Go) and p300/CBP (Tohkin et al., 2000Go) and nuclear hormone receptors such as the estrogen receptor alpha (Porter et al., 2001Go; Wormke et al., 2003Go). The production and characterization of mouse lines lacking AhR expression has also provided relevant information for the contribution of this receptor to normal cell function (Alexander et al., 1998Go; Andreola et al., 1997Go; Fernandez-Salguero et al., 1995Go; Fernandez-Salguero et al., 1997Go; Lahvis et al., 2000Go; Schmidt et al., 1996Go; Staples et al., 1998Go; Thurmond et al., 1999Go; Thurmond et al., 2000Go). Therefore, one of the important issues in order to define endogenous roles for the AhR is to identify its target genes under normal cellular conditions. In this context, recent work has found that p27 (Kolluri et al., 1999Go), Bax (Matikainen et al., 2001Go), T-cadherin (Niermann et al., 2003Go) and N-myristoyltranferase-2 (Kolluri et al., 2001Go) constitute novel target genes regulated through the AhR pathway.

In this study, using MEF from wild-type and AhR-null mice, we have made an attempt to identify novel target genes for the AhR in the absence of xenobiotics. Using a DD technique, differential patterns of gene expression were compared between both genotypes. We found that Ltbp-1 mRNA was significantly increased in AhR–/– MEF, suggesting that the AhR could be a negative regulator of the endogenous expression of this gene. A higher Ltbp-1 mRNA content in AhR–/– MEF was not the consequence of RNA stabilization but rather of active transcription, as it could be blocked by actinomycin D treatment. The observation that Ltbp-1 expression remained elevated in AhR–/– cells suggested that additional proteins might participate as co-repressors, together with the AhR, in maintaining endogenous Ltbp-1 expression in AhR+/+ MEF. Additional data supported the role of the AhR in a negative regulatory mechanism controlling Ltbp-1 expression. First, the well-characterized AhR ligand TCDD inhibited Ltbp-1 mRNA expression, a consistent observation if this receptor was contributing to the transcriptional repression of this gene. Second, it was shown that TGFß downregulated AhR mRNA expression and activity in A549 human lung carcinoma cells (Dohr et al., 1997Go; Wolff et al., 2001Go). TGFß addition also decreased AhR expression in MEF and such an effect was synchronized with Ltbp-1 upregulation. Finally, Ltbp-1 expression in AhR–/– MEF was insensitive not only to TGFß addition but also to neutralizing antibodies against this protein. These data suggest that the AhR negatively regulate Ltbp-1 expression in MEF in the absence of xenobiotics, and therefore, underline the role of the AhR as a transcriptional repressor. Negative regulation of T-cadherin expression by the AhR has been previously suggested (Niermann et al., 2003Go). Thus, Ltbp-1 represents a new AhR-regulated gene not involved in xenobiotic metabolism. However, from the current data, it cannot be discriminated if the AhR directly drives transcription of the LTBP-1 gene. Current studies are under way to study whether or not the AhR regulates LTBP-1 expression by direct binding to the mouse Ltbp-1 promoter. The expression of three additional LTBPs cloned to date, Ltbp-2, -3 and -4, was detectable in MEF cultures but unaffected by the AhR genotype, suggesting the existence of gene-specific regulatory mechanisms for Ltbp-1.

LTBP-1 overexpression in AhR-null mice was also observed at the protein level. Increased protein expression was probably responsible for the accumulation of LTBP-1 in the culture medium and the extracellular matrix of MEF. However, although LTBP-1 mRNA and protein were similarly increased in the absence of AhR (e.g. about threefold), the amount of this protein secreted to the medium was elevated by less than twofold, suggesting that a fraction of LTBP could be interacting with the matrix. In agreement, a significant amount of LTBP-1 was detected in the extracellular matrix of AhR-null MEF that could have a structural or growth factor sequestering role, as previously suggested (Dallas et al., 2000Go; Dallas et al., 1995Go; Taipale et al., 1994Go; Taipale et al., 1996Go). LTBP-1 can exist in a short (LTBP-1S) or in a long (LTBP-1L) form depending on the cell-specific activation of alternative promoters (Koski et al., 1999Go; Oklu and Hesketh, 2000Go). Only a transcript of about 7 Kbp and a protein of about 190 kDa, corresponding in size to LTBP-1L, could be detected in our MEF cultures, suggesting that LTBP-1S was not expressed. These results agree with previous data showing that expression of the 1S and 1L forms varies during mouse development, with the 1S undetectable before gestational day 17 (Weiskirchen et al., 2003Go).

LTBP-1 not only localizes TGFß to the extracellular matrix, but also contributes to its release from matrix stores (reviewed by Koli et al., 2001Go; Oklu and Hesketh, 2000Go; Taipale and Keski-Oja, 1997Go). The expression of both proteins was reported to be synchronized in ovarian and breast cancer cells (Higashi et al., 2001Go; Koli and Keski-Oja, 1995Go). Thus, LTBP-1 overexpression in AhR–/– MEF could be related to an increase in TGFß activation. To address this possibility, we have measured the levels of this cytokine in wild-type and AhR-null MEF. As previously reported (Elizondo et al., 2000Go), culture medium from AhR–/– fibroblasts had increased levels of TGFß. In these cells, not only the total level of TGFß was increased by twofold, but more importantly, the fraction of active cytokine increased over fourfold with respect to wild-type MEF. This could be relevant as most primary cells are unable to activate TGFß and secrete this cytokine in its latent form (Koli et al., 2001Go). Although the mechanisms leading to increased TGFß activation in AhR–/– MEF are not yet understood, it could involve alterations in the activity of proteases such as plasmin, elastase or chymase, which are known to release TGFß from its latent form (reviewed by Koli et al., 2001Go; Lawrence, 2001Go; Oklu and Hesketh, 2000Go). Another potential mechanism of activation could be mediated by retinoids, which are known to increase the level of active TGFß (Kojima and Rifkin, 1993Go). Because the liver of AhR–/– mice had increased retinoid content and primary hepatocytes from AhR-null mice secreted high levels of active TGFß (Zaher et al., 1998Go), the possibility exists that AhR–/– MEF could produce high levels of retinoids able to activate TGFß. These alternative mechanisms are currently under consideration. An additional issue relates to how increased LTBP-1 levels could result in higher levels of TGFß. Given that LTBP-1 participates in the assembly and secretion of TGFß (Chen et al., 2002Go; Miyazono et al., 1991Go; Miyazono et al., 1992Go) and the expression of both proteins is corregulated in several cell lines (Koski et al., 1999Go; Miyazono et al., 1991Go; Taipale et al., 1994Go), decreased TGFß degradation or facilitated secretion of the cytokine in the presence of high levels of LTBP-1 could be potential mechanisms involved.

Several studies have reported that TGFß decreases MMP-2 activity not only in cell culture (Wang and Hirschberg, 2003Go) but also in animal models (Chernajovsky et al., 1997Go). MMP-2, however, cleaved matrix-bound and soluble LTBP-1 in primary osteoblast cultures (Dallas et al., 2002Go). MEF lacking AhR had decreased MMP-2 activity compared with wild-type fibroblasts and this effect was not due to decreased expression of the Mmp-2 gene or to increased expression of the MMP inhibitors Timp-1 and Timp-2. Therefore, it is tempting to speculate that increased expression of LTBP-1 could be contributing to higher levels of TGFß and to the inhibition of MMP-2 activity. Indeed, this hypothesis should be taken into account as an antibody against LTBP-1 not only decreased the growth inhibitory properties but also the inhibition of MMP-2 activity present in AhR–/– conditioned medium.

In summary, we have identified mouse Ltbp-1 as a new negatively regulated AhR-target gene not involved in xenobiotic metabolism. LTBP-1 overexpression in the absence of AhR may be related to increased levels of TGFß activation. Increased TGFß activity in AhR-null MEF could contribute to decreased MMP-2 activity. The relationship between AhR, LTBP-1, TGFß and MMP-2 could be relevant to explain some of the phenotypes observed in AhR–/– mouse liver such as decreased organ size (Fernandez-Salguero et al., 1995Go; Lahvis et al., 2000Go), increased fibrosis (Peterson et al., 2000Go) and altered vascular structures (Lahvis et al., 2000Go).


    Acknowledgments
 
We are very grateful to Dr Carlos Lopez-Otin for kindly providing the HT1080 conditioned media. This work has been funded by Grant SAF2002-00034 from the Spanish Ministry of Science and Technology (to P.F.S.). B.S.J. was a recipient of a predoctoral fellowship from the Consejeria de Educacion y Cultura, Junta de Extremadura.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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