Vitamin D₃ metabolites regulate LTBP1 and latent TGF-β1 expression and latent TGF-β1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-β1 in latent form (LTGF-β1) and store it in the extracellular matrix via LTGF-β1 binding protein (LTBP1). 1,25-(OH)₂D₃ (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)₂D₃ (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1-mediated storage of TGF-β1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-β1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-β1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10^-7-10^{- 9} M 24,25 and 10^-8-10^-10 M 1,25, respectively. LTBP1 and TGF-β1 mRNA levels were measured by in situ hybridization; production of LTGF-β1, LTGF- β2, and LTBP1 protein in the conditioned 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-β1 in the media and matrix of the cultures. Matrix-bound LTGF-β1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37°C. LTBP1 and TGF-β1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-β1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-β1, LTGF-β2, and LTBP1. GC cells also produce LTGF-β2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-β1 protein found in the matrix, significant at 10^-8 and 10^-9 M, and a corresponding decrease in TGF-β1 in the media. 24,25 had no effect on the level of TGF-β1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-β1 content of the media is reduced through the formation of latent TGF-β1-LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-β1 incorporation into the matrix is not regulated by this vitamin D₃ metabolite. Thus, vitamin D₃ metabolites may play a role in regulating the availability of TGF-β1 by modulating LTBP1 production.