Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2002) 26, 971–983 (Printed in Great Britain)
EXPRESSION AND REGULATION OF SPARC, FIBRONECTIN, AND COLLAGEN IV BY DEXAMETHASONE IN LENS EPITHELIAL CELLS
Rajinder S. Sawhneyf1
Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263, U.S.A.


Abstract

Cataract formation is a deleterious side effect of some hormone therapies, thus, it is important to understand how hormones regulate lens basement structure and function. We have examined the effects of dexamethasone (DEX) on the regulation of Secreted Protein Acidic and Rich in Cysteines (SPARC), fibronectin (FN), and collagen IV (CN IV). To radiolabel newly synthesized proteins, cultured monolayers of bovine anterior lens capsule epithelial (ALCE) cells were pulsed with [3H] proline. To identify proteins, an immunofluorescent technique, immunoprecipitation with specific antibodies, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used. DEX increased production of total proteins, whereas it specifically decreased synthesis of FN and CN IV. A decrease in FN and CN IV synthesis by DEX in ALCE cells may decrease adhesion of lens epithelium to the basement membrane (BM), which may in turn cause pathogenesis. Messenger RNAs were identified by Northern blot analyses using specific DNA probes. Treatment of lens epithelial cells with DEX causes a 100–150% up-regulation of SPARC mRNA in a concentration-dependent fashion. The increase in the expression of FN mRNA by DEX was in a dose-response fashion and varied from 50–600%. A 24-hour treatment with DEX (10−6M) increased CN IV mRNA levels to 386% over baseline levels. Thus showing a differential upregulation by DEX of mRNAs of SPARC, FN, and CN IV. Results of nuclear run-on transcription assays indicate that regulation of RNAs by DEX may occur, in part, at the transcriptional level. The aberrant expression of lens basement membrane proteins by DEX may contribute to abnormal lens cell function and ultimately to anterior subcapsular cataract.


Keywords: basement membrane, cataractogenesis, epithelial cells, lens capsule, gene regulation..

f1To whom correspondence should be addressed: Tel.: +1 (716) 8455874; Fax: +1 (716) 845-8857; E-mail:rajinder.sawhney@roswellpark.org


References

1 Babizhayev, MA, Zhukotskii, AV and Sologub, AA (1992) Image analysis of the lens opacities induced in developing chick embryo by glucocorticoids. Exp Eye Res 55, 521-37

2 Brekken, RA and Sage, EH (2000) SPARC, a matricellular protein: at the crossroads of cell-matrix communication. Matrix Biol 19, 816-27

3 Grant, ME, Harwood, R and Williams, IF (1976) Increased synthesis of glomerular basement membrane collagen in streptozotocin diabetes. J Physiol 257, 56-7

4 Gronowicz, GA and McCarthy, MB (1995) Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease beta 1-integrin levels. Endocrinology 136, 598-608

5 Jobling, AI and Augusteyn, RC (2001) Is there a glucocorticoids receptor in the bovine lens? Exp Eye Res 72, 687-94

6 Lane, TF and Sage, EH (1994) The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J 8, 163-73

7 Lash, GE, Fitzpatrick, TE and Graham, CH (2001) Effect of hypoxia on cellular adhesion to vitronectin and fibronectin. Biochem & Biophys Res Comm 287, 622-9

8 Mason, IJ, Taylor, A, Williams, JG, Sage, H and Hogan, BLM (1986) Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell ‘culture shock’ glycoprotein of Mr 43000. EMBO J 5, 1465-72

9 Miller, D, Tijerina, ML and Mayman, C (1979) In vitro production of steroid cataract in bovine lens. Acta Opthalmol 57, 1101-6

10 Mohan, PS and Spiro, RG (1986) Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures. J Biol Chem 261, 4238-336

11 Ng, KW, Manji, SS, Young, DF and Findlay, DM (1989) Opposing influences of glucocorticoids and retinoic acid transcriptional control in preosteoblasts. Mol Endocrinol 3, 2079-85

12 Oikarinen, A, Salo, T, Ala-kokko, L and Tryggvason, K (1987) Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming-fibrosarcoma (HT-1080) cells. Biochem J 245, 235-41

13 Pao, C-I, Farmer, PK, Begovic, S, Goldstein, S, Wu, G-J and Phillips, LS (1992) Expression of hepatic insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 genes is transcriptionally regulated in streptozotocin-diabetic rats. Mol Endocrinol 6, 969-77

14 Reynolds, GE, Schmitz, JA, Mattson, DE, Pielstick, L and Luedke, AJ (1985) “White-eye calf” syndrome in Oregon associated with bluetongue and epizootic hemorrhagic disease viruses. Prog Clin Biol Res 178, 67-9

15 Rüdiger, JJ, Roth, M, Bihl, MP, Cornelius, BC, Johnson, M, Ziesche, R and Block, L-H (2002) Interaction of C/EBPa and the glucocorticoid receptor in vivo and in nontransformed human cells. FASEB 16, 177-84

16 Sage, EH (2001) Regulation of interactions between cells and extracellular matrix: a command performance on several stages. J Clin Invest 107, 781-3

17 Saika, S, Kawashima, Y, Miyamoto, T, Okada, Y, Tanaka, SI, Ohmi, S et al. (1998) Immunolocalization of prolyl 4-hydroxylase subunits, α-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants. Exp Eye Res 66, 283-94

18 Sawhney, RS (1995) Identification of SPARC in the anterior lens capsule and its expression by lens epithelial cells. Exp Eye Res 61, 645-8

19 Sawhney, RS (1993) Expression of type I and type III procollagen by lens epithelial cells. Invest Ophthalmol Vis Sci 34, 2195-202

20 Sawhney, RS and Bone, RC (1993) Endotoxin alters the expression of extracellular matrix proteins by cultured endothelial cells. Cell Mol Biol Res 39, 589-99

21 Sawhney, RS and Dixit, SN (1985) Biosynthesis and in vitro translation of type IV procollagens. Eur J Biochem 151, 11-6

22 Sawhney, RS, Hering, TM and Sandell, LJ (1991) Biosynthesis of small proteoglycan II (Decorin) by chondrocytes and evidence for a procore protein. J Biol Chem 266, 9231-40

23 Sawhney, RS, Krishna, M and Bone, RC (1992) . Recent Advances in Cellular and Molecular Biology, Vol. 5 173-80

24 Sawhney, RS, Wood, LS and Vogeli, G (1997) Molecular cloning of the bovine α1 (IV) procollagen gene (Col 4A1) and use in investigating the regulation of expression of type IV procollagen by retinoic acid in bovine lens epithelial cells. Cell Biol Intl 21, 501-10

25 Shanker, G, Olson, O, Bone, R and Sawhney, RS (1999) Regulation of extracellular matrix proteins by transforming growth factor β1 in cultured pulmonary endothelial cells. Cell Biol Intl 23, 61-72

26 Shanker, G and Sawhney, RS (1996) Retinoic acid: identification of specific receptors through which it may mediate transcrip-tional regulation of fibronectin gene in bovine lens epithelial cells. Cell Biol Intl 20, 613-9

27 Southren, AL, Gordon, GG, Yeh, HS, Dunn, MW and Weinstein, BI (1978) Receptors for glucocorticoids in the lens epithelium of the calf. Science 200, 1177-8

28 Steely, HT, Browder, SL, Julian, MB, Miggans, ST, Wilson, KL and Clark, AF (1992) The effects of dexamethasone on fibronectin expression in cultured human trabecular meshwork cells. Invest Ophth Vis Sci 33, 2242-50

29 Stokes, J, Noble, J, Brett, L, Phillips, C, Seckl, JR, O'Brien, C and Andrew, R (2000) Distribution of glucocorticoid and mineralocorticoid receptors and 11 beta-hydroxysteroid dehydrogenases in human and rat ocular tissues. Invest Ophth Vis Sci 41, 1629-38

30 Suzuki, T, Sasano, H, Kaneko, C, Ogawa, S, Darnel, Ad and Krozowski, ZS (2001) Immunohistochemical distribution of 11 beta-hydroxysteroid dehydrogenase in human eye. Mol & Cell Endocrinol 173, 121-5

31 Weiner, FR, Czaja, MJ, Giambrone, M-A, Takahashi, S, Biempica, L and Zern, MA (1987) Transcriptional and posttranscriptional effects of dexamethasone on albumin and procollagen messenger RNAs in murine schistosomiasis. Biochem 26, 1557-62

32 Wenk, EJ, Hernandez, MR, Weinstein, BI, Gordon, GG, Dunn, MW and Southren, AL (1982) Glucocorticoid receptor binding in bovine lens. Invest Ophth Vis Sci 22, 599-605

33 Wewer, UM, Albrechtsen, R, Fisher, LW, Young, MF and Termine, JD (1988) Osteonectin/SPARC/BM-40 in human decidua and carcinoma, tissues characterized by De Novo formation of basement membrane. Am J Pathol 132, 345-55

34 Yanoff, M (1975) Pathology of cataract. Cataract and Abnormalities of the Lens 155-89

35 Yao, J, Bone, RC and Sawhney, RS (1995) Differential effects of tumor necrosis factor-alpha on the expression of fibronectin and collagen genes in cultured bovine endothelial cells. Cell Mol Biol Res 41, 17-28

36 Young, MF, Bolander, ME, Day, AA, Ramis, CI, Gehron Robey, P, Yamada, Y and Termine, JD (1986) Osteonectin mRNA: distribution in normal and transformed cells. Nucleic Acid Res 14, 4483-97


Received 5 December 2001; accepted 23 July 2002

doi:10.1006/cbir.2002.0955


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)