J Korean Med Sci.  2005 Aug;20(4):628-635. 10.3346/jkms.2005.20.4.628.

Expression and Regulation of Latent TGF-beta Binding Protein-1 Transcripts and Their Splice Variants in Human Glomerular Endothelial Cells

• Kwak JH
• Woo JS
• Shin K
• Kim HJ
• Jeong HS
• Han DC
• Kim SI
• Park CS

• KMID: 1712745
• DOI: http://doi.org/10.3346/jkms.2005.20.4.628

Abstract

Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H2O2, and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1S delta 53. TGF-beta1, but not high glucose, H2O2 or VEGF, tended to increase LTBP-1S delta 53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H2O2, and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S delta 53. Modification of LTBP-1S delta 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.

Keyword

Latent TGF-beta binding protein; Splice Variant of LTBP-1; Endothelial Cells; Trnasforming growth factor beta1; Blood Glucose; Hydrogen Peroxide; Vascular Endothelial Growth Factor

MeSH Terms

*Alternative Splicing
Amino Acid Sequence
Cell Line
Cells, Cultured
Cloning, Molecular
Comparative Study
Endothelial Cells/drug effects/*metabolism
*Gene Expression Regulation
Glucose/pharmacology
Humans
Hydrogen Peroxide/pharmacology
Intracellular Signaling Peptides and Proteins/*genetics
Kidney Glomerulus/cytology
Protein Isoforms/genetics
RNA, Messenger/genetics/metabolism
Research Support, Non-U.S. Gov't
Reverse Transcriptase Polymerase Chain Reaction
*Transcription, Genetic
Transfection
Transforming Growth Factor beta/pharmacology
Vascular Endothelial Growth Factor A/pharmacology

Figure

• 

  Fig. 1 Structural features of LTBP-1S and LTBP-1SΔ53. Simple structural features of LTBP-1S, primer sets, the size of PCR products and the deleted region in LTBP-1SΔ53 are shown. A putative heparin-binding site is shown by amino acid sequence (HRRRPIHHHVGK) and other typical motifs, repeats and sites are illustrated with various symbols.

• 

  Fig. 2 Estimation of basal LTBP-1 expression in HGEC. Northern blot analysis for LTBP-1 expression was carried out with RNA from HGEC and WI-38. The results of three independent experiments are presented. 28S and 18S served as internal controls (A). (B) Transient transfection assay for LTBP-1 expression was carried out with L-1S PROM, a reporter construct covering the 1,751 bp upstream region of LTBP-1S gene. The reporter construct was introduced with pRL-CMV into HGEC and WI-38 and cell lysate of each culture was prepared after 24 hr of transfection. The mean value of the activity from three independent experiments is shown with the value of WI-38 as 100%.

• 

  Fig. 3 Expression patterns of LTBP-1 in HGEC cultured under different conditions. The first stranded cDNA was synthesized with 3 µg of total RNA from HGEC and then PCR with primers (LSF3 and LSR1) was carried out 40 cycles. For standardization of the amount of the respective RNA applied, β-actin was amplified with the same cDNA prepared. One fifth of PCR volume was applied on the 1.5% agarose gel electrophoresis. The results of five independent experiments are shown as fold change relative to control (B). Upper panel (A) is a representative bands of PCR products from 5 experiments. C: control; HG: high glucose (30 mM); H2O2: 100 µM H2O2; TGF-β1: 2.5 ng/mL of TGF-β1; VEGF: 10 ng/mL of VEGF; M: molecular size marker; *p<0.05 compared to 12 hr control.

• 

  Fig. 4 Identification of LTBP-1S and LTBP-1SΔ5 by restriction mapping. Restriction mapping with Sca I (Promega) was carried out to screen the candidate clones for LTBP-1S. Two Sca I sites are in LTBP-1S (lane 12), but only one site in LTBP-1SΔ53 (lane 1-11).

• 

  Fig. 5 Alternative spliced-form of LTBP-1 RNA in HGEC and WI-38. The first stranded cDNA was synthesized with 3 µg of total RNA from HGEC and WI-38. PCR with internal primers (LSF2 and LSR2) was carried out 30 cycles for WI-38 (lane 1) and 40 cycles for HGEC (lane 2 and 3). For standardization of the amount of respective RNA applied, β-actin was amplified with 30 cycles of PCR with the same cDNA prepared. One fifth of PCR volume was applied on the 1.5% agarose gel electrophoresis.

• 

  Fig. 6 Changes of the splicing pattern in HGEC cultured under different conditions. RT-PCR was done with the same method described in Fig. 5. Upper panel (A) is a representative bands for LTBP-1S and LTBP-1SΔ53 under different stimuli from PCR products from 5 independent experiments. Lower panel (B) shows the mean±SE of LTBP-1SΔ53/LTBP-1S ratio from 5 experiments. C: control; HG: high (30 mM) glucose; H2O2: 100 µM H2O2; TGF-β1: 2.5 ng/mL of TGF-β1; VEGF: 10 ng/mL of VEGF; M: molecular size marker.

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