VITAMIN-D-3 METABOLITES REGULATE LTBP1 AND LATENT TGF-BETA-1 EXPRESSION AND LATENT TGF-BETA-1 INCORPORATION IN THE EXTRACELLULAR-MATRIX OF CHONDROCYTES

Citation
Ha. Pedrozo et al., VITAMIN-D-3 METABOLITES REGULATE LTBP1 AND LATENT TGF-BETA-1 EXPRESSION AND LATENT TGF-BETA-1 INCORPORATION IN THE EXTRACELLULAR-MATRIX OF CHONDROCYTES, Journal of cellular biochemistry, 72(1), 1999, pp. 151-165
Citations number
45
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Cell Biology
ISSN journal
0730-2312
Volume
72
Issue
1
Year of publication
1999
Pages
151 - 165
Database
ISI
SICI code
0730-2312(1999)72:1<151:VMRLAL>2.0.ZU;2-W
Abstract
Growth plate chondrocytes make TCF-beta 1 in latent form (LTGF-beta 1) and store it in the extracellular matrix via LTGF-beta 1 binding prot ein (LTBP1). 1,25-(OH)(2)D-3 (1,25) regulates matrix protein productio n in growth zone (CC) chondrocyte cultures, whereas 24,25-(OH)(2)D-3 ( 24,25) does so in resting zone (RC) cell cultures. The aim of this stu dy was to determine if 24,25 and 1,25 regulate LTBP1 expression as wel l as the LTBP1-mediated storage of TGF-beta 1 in the extracellular mat rix of RC and CC cells. Expression of LTBP1 and TGF-beta 1 in the grow th plate and in cultured RC and CC cells was determined by in situ hyb ridization using sense and antisense oligonucleotide probes based on t he published rat LTBP1 and TGF-beta 1 cDNA sequences. Fourth passage m ale rat costochondral RC and CC chondrocytes were treated for 24 h wit h 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta 1 mRNA levels were measured by in situ hybridization; pr oduction of LTGF-beta 1, LTGF-beta 2, and LTBP1 protein in the conditi oned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24 ,25 on the level of TGF-beta 1 in the media and matrix of the cultures . Matrix-bound LTGF-beta 1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta 1 mRN As are co-expressed throughout the growth plate, except in the lower h ypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta 1 mRNAs than RC cells. FPLC purification of the conditioned media confir med that RC cells produce LTGF-beta 1, LTGF-beta 2, and LTBP1. GC cell s also produce LTGF-beta 2, but at lower concentrations. 1,25 dose-dep endently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronou nced, effect on RC cells. 1,25 also caused a dose-dependent increase i n the amount of TGF-beta 1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta 1 in th e media. 24,25 had no effect on the level of TGF-beta 1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the pr oduction of LTBP1 by GC cells and suggests that the TGF-beta 1 content of the media is reduced through the formation of latent TGF-beta 1-LT BP1 complexes which mediates storage in the matrix. Although 24,25 ind uced the expression of LTBP1 by RCs, TGF-beta 1 incorporation into the matrix is not regulated by this vitamin D-3 metabolite. Thus, vitamin D-3 metabolites may play a role in regulating the availability of TGF -beta 1 by modulating LTBP1 production. J. Cell. Biochem. 72:151-165, 1999. (C) 1999 Wiley-Liss, Inc.