The role of acetylation in Timp-1 regulation

Introduction  Aberrant matrix turnover, mediated by a family of proteases known as matrix metalloproteinases (MMPs), is involved in a number of pathologies including rheumatoid arthritis and osteoarthritis, tumour invasion and metastasis, and liver fibrosis. The active forms of all of the MMPs are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As inhibition represents a major level of control of MMP activity, a detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important in developing therapies for these diseases. Histone acetyl transferases (HATs) play a crucial role in gene regulation via acetylation of both histones (to loosen nucleosomal structure and to recruit accessory factors) and transcription factors. Here, we describe the effects of a histone deacetylase inhibitor (Trichostatin A – TSA) on the TGF-β1 and Phorbol ester (PMA) induction of Timp-1.

Materials and methods  RNA isolation, TaqMan^® real-time RT-PCR, cell culture, transient transfections and reporter gene assays along with electrophoretic mobility shift assays (EMSA) were performed essentially as described (Young et al. 2002).

Results  In murine C3H10T1/2 cells both TGF-β1 and PMA induce Timp-1 expression in a protein synthesis-dependent manner as measured by Northern blotting and TaqMan^® real-time RT-PCR. TSA superinduces PMA-induced Timp-1 expression but represses TGF-β-induced Timp-1 expression. A TSA dose–response experiment further demonstrates that TGF-β and PMA stimulate Timp-1 gene expression via different mechanisms. The effects of TGF-β, PMA and TSA on Timp-1 expression can be reiterated in transient transfection studies with Timp-1 promoter containing reporter constructs. Deletion of the proximal AP-1 motif from the promoter demonstrates that this sequence is critical for TGF-β1-induced reporter expression, while PMA appears to act via other sequences/mechanisms.

Discussion  Repression of deacetylation by TSA differentially affects the TGF-β or PMA induction of Timp-1. These results will help elucidate the different molecular mechanisms by which these factors regulate the expression of Timp-1 and other genes. Furthermore, the effects of acetylation on TGF-β induction of Timp-1 may act as a paradigm for other cytokine-induced genes.

This work was funded and supported by the Arthritis Research Campaign (ARC) and the Dunhill Medical Trust.