The triterpenoid CDDO inhibits expression of matrix metalloproteinase-1, matrix metalloproteinase-13 and Bcl-3 in primary human chondrocytes

Abstract

A synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), has been reported to have anti-inflammatory properties and to decrease the interleukin-1 (IL-1)-induced expression of matrix metalloproteinase-1 (MMP-1) and MMP-13. We have shown previously that IL-1 induces expression of the inhibitor of NF-kappaB (IkappaB) family member Bcl-3, and that this contributes to MMP-1 expression. To quantify the effects of CDDO on IL-1-induced MMP-1, MMP-13 and Bcl-3 expression, we stimulated the chondrosarcoma cell line SW-1353 and human primary chondrocytes with IL-1, in the presence or absence of CDDO. Harvested RNA was subjected to quantitative real-time reverse-transcriptase polymerase chain reaction. In SW-1353 cells, 300 nM CDDO significantly decreased the induction of MMP-1 and MMP-13 by IL-1. In human primary chondrocytes, 300 nM CDDO inhibited the induction of these genes by IL-1 to an even greater extent. In both cell types, inhibition of MMP-1 required 24 hours of pretreatment with CDDO, whereas MMP-13 could be inhibited when CDDO and IL-1 were added simultaneously to culture. In human primary chondrocytes, IL-1-induced Bcl-3 expression was inhibited when cells were pretreated with CDDO. To determine whether the inhibitory effect of CDDO on MMP worked through inhibition of Bcl-3 gene expression, SW-1353 cells stably transfected with a Bcl-3 expression plasmid were treated with IL-1 and/or CDDO, and MMP gene expression was assayed. Overexpression of Bcl-3 increased MMP-1, but not MMP-13, mRNA levels. Furthermore, overexpressed Bcl-3 could sustain the CDDO-dependent inhibition of IL-1-induced MMP-1 expression. Our data demonstrate that CDDO inhibits IL-1-induced MMP-1 and MMP-13 expression in human chondrocytes. CDDO also inhibits the expression of Bcl-3, an IL-1-responsive gene that preferentially contributes to MMP-1 gene expression.

Figure 1

IL-1-induced MMP-1 and MMP-13 expression is inhibited in SW-1353 cells preincubated with CDDO for 24 hours. SW-1353 cells were treated for 24 hours with 300 nM CDDO before the addition of 10 ng/ml IL-1β. Alternatively, CDDO was added simultaneously with the cytokine. After a further 18 hours of culture, RNA was harvested, reverse transcribed and subjected to quantitative real-time PCR for MMP-1 (a) and MMP-13 (b). Results were normalized to GAPDH, and are means of culture triplicates. They are expressed as molecules of MMP per molecule of GAPDH. Two-tailed Student's t-tests were performed on the data, and significance is indicated as follows: *P < 0.1; **0.01 <P < 0.05; ***P < 0.01.

Figure 2

Expression of MMP-1, MMP-13, and Bcl-3 is inhibited in human chondrocytes pretreated with CDDO for 24 hours. Human primary chondrocytes (passage 1) were treated for 24 hours with 300 nM CDDO before the addition of 10 ng/ml IL-1. Alternatively, CDDO was added simultaneously with the cytokine. After a further 18 hours of culture, RNA was harvested, reverse transcribed and subjected to quantitative real-time PCR for MMP-1 (a), MMP-13 (b), and Bcl-3 (c). Results were normalized to GAPDH, and are means of culture triplicates. They are expressed as molecules per molecule of GAPDH. Two-tailed Student's t-tests were performed on the data, and significance is indicated as follows: *P < 0.1; **0.01 <P < 0.05; ***P < 0.01.

Figure 3

Increasing doses of CDDO inhibit the expression of MMP-1, MMP-13, and Bcl-3 in human primary chondrocytes. Human primary chondrocytes (passage 1) were treated for 24 hours with 300 nM to 5 μM CDDO before the addition of 10 ng/ml IL-1. After a further 18 hours of culture, RNA was harvested, reverse transcribed and subjected to quantitative real-time PCR for MMP-1 (a), MMP-13 (b), and Bcl-3 (c). Results were normalized to GAPDH, and are means of culture triplicates. They are expressed as molecules per molecule of GAPDH. Two-tailed Student's t-tests were performed on the data, and significance is indicated as follows: *P < 0.1; **0.01 <P < 0.05; ***P < 0.01.

Figure 4

Overexpression of Bcl-3 sustains MMP-1 expression against inhibition by CDDO in SW-1353 cells. SW-1353 cells, stably transfected with a pBkRSV vector with or without a Bcl-3 insert, were incubated for 24 hours with 300 nM CDDO before the addition of 10 ng/ml IL-1. After a further 18 hours of culture, RNA was harvested, reverse transcribed and subjected to quantitative real-time PCR for MMP-1 (a) and MMP-13 (b). Results were normalized to GAPDH, and are means of culture triplicates. They are expressed as molecules per molecule of GAPDH. Two-tailed Student's t-tests were performed on the data, and significance is indicated as follows: *P < 0.1; **0.01 <P < 0.05; ***P < 0.01.