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

Hugo A. Pedrozo, Zvi Schwartz, Tatyana Mokeyev, Asher Ornoy, Wang Xin-Sheng, Lynda Bonewald, David D. Dean, Barbara D. Boyan

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)151-165
Number of pages15
JournalJournal of Cellular Biochemistry
Volume72
Issue number1
DOIs
StatePublished - Jan 1 1999
Externally publishedYes

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Cholecalciferol
Chondrocytes
Metabolites
Extracellular Matrix
Conditioned Culture Medium
Messenger RNA
Growth Plate
Rats
In Situ Hybridization
Proteins
Oligonucleotide Probes
Antisense Oligonucleotides
Fibrinolysin
Cell culture
Purification
Assays
Carrier Proteins
Complementary DNA
Availability
Immunoassay

Keywords

  • Latent TGF-β, 1,25-(OH)D, 24,25-(OH)D
  • Vitamin D, LTBP1, TGF-β, chondrocytes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Vitamin D3 metabolites regulate LTBP1 and latent TGF-β1 expression and latent TGF-β1 incorporation in the extracellular matrix of chondrocytes. / Pedrozo, Hugo A.; Schwartz, Zvi; Mokeyev, Tatyana; Ornoy, Asher; Xin-Sheng, Wang; Bonewald, Lynda; Dean, David D.; Boyan, Barbara D.

In: Journal of Cellular Biochemistry, Vol. 72, No. 1, 01.01.1999, p. 151-165.

Research output: Contribution to journalArticle

Pedrozo, Hugo A. ; Schwartz, Zvi ; Mokeyev, Tatyana ; Ornoy, Asher ; Xin-Sheng, Wang ; Bonewald, Lynda ; Dean, David D. ; Boyan, Barbara D. / Vitamin D3 metabolites regulate LTBP1 and latent TGF-β1 expression and latent TGF-β1 incorporation in the extracellular matrix of chondrocytes. In: Journal of Cellular Biochemistry. 1999 ; Vol. 72, No. 1. pp. 151-165.
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T1 - Vitamin D3 metabolites regulate LTBP1 and latent TGF-β1 expression and latent TGF-β1 incorporation in the extracellular matrix of chondrocytes

AU - Pedrozo, Hugo A.

AU - Schwartz, Zvi

AU - Mokeyev, Tatyana

AU - Ornoy, Asher

AU - Xin-Sheng, Wang

AU - Bonewald, Lynda

AU - Dean, David D.

AU - Boyan, Barbara D.

PY - 1999/1/1

Y1 - 1999/1/1

N2 - 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.

AB - 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.

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