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Novel non-TGF-ß-binding splice variant of LTBP-4 in human cells and tissues provides means to decrease TGF-ß deposition

Departments of Virology and Pathology, the Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland
^* Author for correspondence (e-mail: katri.koli{at}helsinki.fi

View larger version (35K): [in a new window]   Fig. 1. RT-PCR analysis of LTBP expression in human cell lines. Total RNA isolated from different cell lines was reverse transcribed followed by PCR amplification using two different primer pairs per each LTBP. (A) LTBP-x.1 primers amplify products spanning from the last EGF-repeats to the noncoding region, whereas LTBP-x.2 primers amplify regions around the 3rd 8-Cys repeat (for primer sequences see Table 1). `RPA' and `3rd 8-Cys' probes used in northern blotting and RNase protection assays are indicated under LTBP-4. In B, human lung fibroblast (CCL-137) RNA was used in RT-PCR reaction with the eight different primer pairs. Agarose gel electrophoreses of the amplified fragments are shown. An arrowhead indicates the LTBP-48-Cys3rd form. The molecular weight markers (nt) are shown on the left. (C) A summary of the arbitrary quantitation of the amounts of different LTBPs in the panel of the indicated cell lines. The amounts of specific products were equalized using G3PDH levels in each cell line, and compared with those of CCL-137 cells. These cell lines included human embryonic lung fibroblasts (CCL-137, WI-38), human skin fibroblasts, SV-40 transformed embryonic lung fibroblasts (VA-13; counterpart of WI-38 cells), human fibrosarcoma cells (HT-1080), human umbilical vein endothelial cells (HUVEC) and immortalized human skin keratinocytes (HaCat). The results, obtained from several independent experiments using two different primer pairs of each LTBP, were combined and are expressed arbitrarily from positive (+) to strong positive (++++).

View larger version (29K): [in a new window]   Fig. 2. Identification of a new LTBP-4 splice form. (Top) RT-PCR amplification of RNA from different cell lines using LTBP-4.2 primers. The migration of the expected 486 bp product as well as the novel 147 bp product in an agarose gel are indicated on the left. The cell line designations are indicated on the top. Endothelial denotes human microvascular endothelial cells. Both PCR products were cloned and sequenced. (Bottom) A schematic view of the amplified sequences. The new splice variant, LTBP-48-Cys3rd is formed by splicing of a proline rich region and the 3rd 8-Cys repeat.

View larger version (17K): [in a new window]   Fig. 3. Genomic structure of LTBP-4 around the 3rd 8-Cys repeat. (A) PAC/LTBP-4 clone containing genomic sequences from LTBP-4 was used as a template in PCR amplification using LTBP-4.2 and `3rd 8-Cys' primers. The `3rd 8-Cys' primers amplify a region that is absent from LTBP-48-Cys3rd (for primer sequences see Table 1). The migration of 4.5 kb and 650 bp PCR products in an agarose gel is shown. The molecular weight markers (nt) are shown on the left. (B) Schematic representation of LTBP-4 as well as the exon-intron structure around the 3rd 8-Cys repeat. The sequences obtained from splice-site junctions of introns A, B and C are shown.

View larger version (24K): [in a new window]   Fig. 4. Expression of LTBP-4 in cell lines. (A) RNase protection assay. Total cellular RNA was extracted from HT-1080, WI-38 and VA-13 cells. Total RNA (5 µg) was used in the assay. The probe was radioactively labeled and incubated with or without cellular RNA. After incubation RNA not protected with the probe was digested with RNase. After precipitation the protected fragments were separated in 6% polyacrylamide gels containing 8 M urea. Intact probe was used as a control. The two protected fragments corresponding to LTBP-4 containing the 3rd 8-Cys repeat as well as LTBP-48-Cys3rd are indicated on the right. The mobilities of molecular weight markers (nt) are shown on the left. (B) Northern blot analysis of human lung fibroblast (CCL-137) total RNA (10 µg). The RNA-filter was probed with `RPA' probe detecting both LTBP-4 forms or with `3rd 8-Cys' probe detecting specifically LTBP-4 containing the 3rd 8-Cys repeat. An expected band of 5 kb was detected. In addition, a minor mRNA species of 2 kb was detected by both probes (arrowhead). The migration of RNA markers (nt) is shown on the left.

View larger version (20K): [in a new window]   Fig. 6. RT-PCR from tissue mRNA with `RPA' primers. RT-PCR amplification of polyA+ RNA from heart tissue and HL-60 promyelocytic leukemia cells was performed using LTBP-4 primers (`RPA', see Materials and Methods). The migration of the expected 331 bp product as well as a novel 259 bp product is indicated and presented schematically. The molecular weight markers (nt) are shown on the left. The sequence between nucleotides 3665 and 3697 is also presented. An asterisk indicates a single nucleotide difference in the LTBP-4 sequence obtained from the heart and HL-60 cells.

View larger version (24K): [in a new window]   Fig. 5. RNase protection assay from tissue-derived mRNA. RNase protection assays were performed with 0.5 µg of polyA+ RNA derived from different tissues as illustrated. The probe, `RPA-2', was radioactively labeled and incubated with or without tissue mRNA as in Fig. 4A. Relative values of LTBP-4 mRNAs (see text for details) compared with LTBP-4 levels in the heart (set as 100%). Phosphoimager quantification of two separate experiments is presented with error bars. (Inset) RNase protection assay of heart and lung mRNAs. The protected fragments corresponding to LTBP-4 containing the 3rd 8-Cys repeat, LTBP-48-Cys3rd as well as LTBP-4EGF-13 are indicated on the right. The mobilities of the molecular weight markers (nt) are shown on the left of the inset.

View larger version (56K): [in a new window]   Fig. 7. Expression of LTBP-4 fragments and immunoblotting from cell conditioned medium. Expression constructs pFull, p8-Cys3rd and p24 8-Cys were transfected into 293T cells and serum-free cell conditioned media were collected for 48 hours. Aliquots of the media were separated by 4-15% SDS-PAGE under reducing conditions, transferred to nitrocellulose filters and immunoblotted with the indicated antibodies. -HA antibody recognizes the C-terminal HA-tag of the constructs, whereas #28-3 and #33-4 recognize the 3rd and 4th 8-Cys repeats of LTBP-4, respectively (Saharinen et al., 1998). The mobilities of the molecular weight markers (kDa) are shown on the left.

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