First published online 3 February 2004
doi: 10.1242/jcs.00932
Journal of Cell Science 117, 849-859 (2004)
Published by The Company of Biologists 2004
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
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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.
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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.
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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.
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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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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.
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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.
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© The Company of Biologists Ltd 2004
