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

Affiliations
  • 1Hyonam Kidney Laboratory, Soon Chun Hyang University, Korea. hblee@hkl.ac.kr
  • 2Department of Internal Medicine, Soon Chun Hyang University College of Medicine, Seoul, Korea.

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.


Reference

1. Miyazono K, Hellman U, Wernstedt C, Heldin CH. Latent high molecular weight complex of transforming growth factor-β1. Purification from human platelets and structural characterization. J Biol Chem. 1988. 263:6407–6415.
2. Olofsson A, Miyazono K, Kanzaki T, Colosetti P, Engstrom U, Heldin CH. Transforming growth factor-β1, -β2, and -β3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes. J Biol Chem. 1992. 267:19482–19488.
3. Taipale J, Lohi J, Saarinen J, Kovanen PT, Keski-Oja J. Human mast cell chymase and leukocyte elastase release latent transforming growth factor-β1 from the extracellular matrix of cultured human epithelial and endothelial cells. J Biol Chem. 1995. 270:4689–4696.
Article
4. Wakefield LM, Smith DM, Flanders KC, Sporn MB. Latent transforming growth factor-β from human platelets. A high molecular weight complex containing precursor sequences. J Biol Chem. 1988. 263:7646–7654.
5. Kanzaki T, Olofsson A, Moren A, Wernstedt C, Hellman U, Miyazono K, Claesson-Welsh L, Heldin CH. TGF-β1 binding protein: a component of the large latent complex of TGF-β1 with multiple repeat sequences. Cell. 1990. 61:1051–1061.
Article
6. Tsuji T, Okada F, Yamaguchi K, Nakamura T. Molecular cloning of the large subunit of transforming growth factor type β masking protein and expression of the mRNA in various rat tissues. Proc Natl Acad Sci USA. 1990. 87:8835–8839.
7. Dubois CM, Laprise MH, Blanchette F, Gentry LE, Leduc R. Processing of transforming growth factor-β1 precursor by human furin convertase. J Biol Chem. 1995. 270:618–624.
8. Gentry LE, Lioubin MN, Purchio AF, Marquardt H. Molecular events in the processing of recombinant type I pre-pro-transforming growth factor β to the mature polypeptide. Mol Cell Biol. 1988. 8:4162–4168.
9. Saharinen J, Taipale J, Keski-Oja J. Association of the small latent transforming growth factor-β with an eight cysteine repeat of its binding protein LTBP-1. EMBO J. 1996. 15:245–253.
10. Gleizes PE, Beavis RC, Mazzieri R, Shen B, Rifkin DB. Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-β binding protein-1 that mediates bonding to the latent transforming growth factor-β1. J Biol Chem. 1996. 271:29891–29896.
Article
11. Miyazono K, Olofsson A, Colosetti P, Heldin CH. A role of the latent TGF-β1-binding protein in the assembly and secretion of TGF-β1. EMBO J. 1991. 10:1091–1101.
12. Taipale J, Miyazono K, Heldin CH, Keski-Oja J. Latent transforming growth factor-β associates to fibroblast extracellular matrix via latent TGF-β1 binding protein. J Cell Biol. 1994. 124:171–181.
13. Flaumenhaft R, Abe M, Sato Y, Miyazono K, Harpel J, Heldin CH, Rifkin DB. Role of the latent TGF-β binding protein in the activation of latent TGF-β by co-cultures of endothelial and smooth muscle cells. J Cell Biol. 1993. 120:995–1002.
14. Dallas SL, Miyazono K, Skerry TM, Mundy GR, Bonewald LF. Dual role for the latent transforming growth factor-β binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein. J Cell Biol. 1995. 131:539–549.
15. Moren A, Olofsson A, Stenman G, Sahlin P, Kanzaki T, Clasesson-Welsh L, ten Dijke P, Miyazono K, Heldin CH. Identification and characterization of LTBP-2, a novel latent transforming growth factor-β-binding protein. J Biol Chem. 1994. 269:32469–32478.
16. Olofsson A, Ichijo H, Moren A, ten Dijke P, Miyazono K, Heldin CH. Efficient association of an amino-terminally extended form of human latent transforming growth factor-β binding protein with the extracellular matrix. J Biol Chem. 1995. 270:31294–31297.
Article
17. Koski C, Saharinen J, Keski-Oja J. Independent promoters regulate the expression of two amino terminally distinct forms of latent transforming growth factor-β binding protein-1 (LTBP-1) in a cell type-specific manner. J Biol Chem. 1999. 274:32619–32630.
Article
18. Mangasser-Stephan K, Gartung C, Lahme B, Gressner AM. Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts. Liver. 2001. 21:105–113.
19. Weikkolainen K, Keski-Oja J, Koli K. Expression of latent TGF-beta binding protein LTBP-1 is hormonally regulated in normal and transformed human lung fibroblasts. Growth Factors. 2003. 21:51–60.
20. Wada T, Hamakawa S, Hori Y, Kaname S, Shimizu S, Kurokawa K, Katoh T. Immunohistochemical localization of latent transforming growth factor-β binding protein in IgA nephropathy. Kidney Int. 1997. 52:S182–S184.
21. Kinnman N, Andersson U, Hultcrantz R. In situ expression of transforming growth factor-beta1-3, latent transforming growth factor-beta binding protein and tumor necrosis factor-alpha in liver tissue from patients with chronic hepatitis C. Scand J Gastroenterol. 2000. 35:1294–1300.
22. Khalil N, Parekh TV, O'Connor R, Antman N, Kepron W, Yehaulaeshet T, Xu YD, Gold LI. Regulation of the effects of TGF-beta 1 by activation of latent TGF-beta 1 and differential expression of TGF-beta receptors (T beta R-I and T beta R-II) in idiopathic pulmonary fibrosis. Thorax. 2001. 56:907–915.
23. Saika S, Miyamoto T, Tanaka T, Ishida I, Ohnishi Y, Ooshima A. Latent TGF-beta binding protein-1 and fibrillin-1 in human capsular opacification and in cultured lens epithelial cells. Br J Ophthalmol. 2001. 85:1362–1366.
Article
24. Higashi T, Sasagawa T, Inoue M, Oka R, Shuangying L, Saijoh K. Overexpression of latent transforming growth factor-beta 1 (TGF-beta 1) binding protein 1 (LTBP-1) in association with TGF-beta 1 in ovarian carcinoma. Jpn J Cancer Res. 2001. 92:506–515.
25. Border WA, Noble NA. Transforming growth factor β in tissue fibrosis. N Engl J Med. 1994. 331:1286–1292.
Article
26. Lee LK, Meyer TW, Pollock AS, Lovett DH. Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney. J Clin Invest. 1995. 96:953–964.
Article
27. Lee HB, Cha MK, Song KI, Kim JH, Lee EA, Kim SI, Kim J, Yoo MH. Pathogenic role of advanced glycosylation end products in diabetic nephropathy. Kidney Int. 1997. 60:S60–S65.
28. Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, Border WA. Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci USA. 1993. 90:1814–1818.
Article
29. Oh JH, Ha H, Yu MR, Lee HB. Sequential effects of high glucose on mesangial cell transforming growth factor-β1 and fibronectin synthesis. Kidney Int. 1998. 54:1872–1878.
Article
30. van Det NF, Verhagen NA, Tamsma JT, Berden JH, Bruijn JA, Daha MR, van der Woude FJ. Regulation of glomerular epithelial cell production of fibronectin and transforming growth factor-β by high glucose, not by angiotensin II. Diabetes. 1997. 46:834–840.
Article
31. Rocco M, Chen Y, Goldfarb S, Ziyadeh FN. Elevated glucose stimulates TGF-β gene expression and bioactivity in proximal tubule. Kidney Int. 1992. 41:107–114.
Article
32. Green DF, Hwang KH, Ryan US, Bourgoignie JJ. Culture of endothelial cells from baboon and human glomeruli. Kidney Int. 1992. 41:1506–1516.
Article
33. Park S, Ahn H, Kim SW, Lee JD, Park JS. Culture of human glomerular endothelial cells. Korean J Nephrol. 1997. 16:221–229.
34. Lee HB, Yu MR, Yang Y, Jiang Z, Ha H. Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol. 2003. 14:S241–S245.
Article
35. Roth-Eichhorn S, Heitmann B, Flemming P, Kubicka S, Trautwein C. Evidence for the decreased expression of the latent TGF-beta binding protein and its splice form in human liver tumors. Scand J Gastroenterol. 2001. 36:1204–1210.
36. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 1999. 13:9–22.
Article
37. Kobayashi T, Liu X, Wen FQ, Fang Q, Abe S, Wang XQ, Hashimoto M, Shen L, Kawasaki S, Kim HJ, Kohyama T, Rennard Si. Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts. Biochem Biophys Res Commun. 2005. 327:393–398.
38. Yamaguchi K, Nishimura Y, Shigematsu S, Takeuchi Y, Nakamura J, Aizawa T, Hashizume K. Vascular endothelial cell growth factor attenuates actions of transforming growth factor-beta in human endothelial cells. J Biol Chem. 2004. 279:55104–55108.
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