Lisa Patel

Resistin is expressed in human macrophages and directly regulated by PPARγ activators

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

Tumor suppressor and anti-inflammatory actions of PPARγ agonists are mediated via upregulation of PTEN

The PTEN tumor suppressor gene modulates several cellular functions, including cell migration, survival, and proliferation [1] by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling cascades. Mechanisms by which the expression of PTEN is regulated are, however, unclear. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) [2] has been shown to regulate differentiation and/or cell growth in a number of cell types [3, 4, 5], which has led to the suggestion that PPARgamma, like PTEN [1, 6], could act as a tumor suppressor. PPARgamma has also been implicated in anti-inflammatory responses [7, 8], although downstream mediators of these effects are not well defined. Here, we show that the activation of PPARgamma by its selective ligand, rosiglitazone, upregulates PTEN expression in human macrophages, Caco2 colorectal cancer cells, and MCF7 breast cancer cells. This upregulation correlated with decreased PI 3-kinase activity as measured by reduced phosphorylation of protein kinase B. One consequence of this was that rosiglitazone treatment reduced the proliferation rate of Caco2 and MCF7 cells. Antisense-mediated disruption of PPARgamma expression prevented the upregulation of PTEN that normally accompanies monocyte differentiation and reduced the proportion of macrophages undergoing apoptosis, while electrophoretic mobility shift assays showed that PPARgamma is able to bind two response elements in the genomic sequence upstream of PTEN. Our results demonstrate a role for PPARgamma in regulating PI 3-kinase signaling by modulating PTEN expression in inflammatory and tumor-derived cells.

Legumain and cathepsin-L expression in human unstable carotid plaque

OBJECTIVE The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. METHODS AND RESULTS Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P<0.001) and activity (P<0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls. CONCLUSION Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.

Chylomicron remnants and oxidised low density lipoprotein have differential effects on the expression of mRNA for genes involved in human macrophage foam cell formation

The effects of chylomicron remnants (non-oxidised or oxidised) and oxidised low density lipoprotein (oxLDL) on the expression of mRNA for a wide range of genes believed to play a role in macrophage foam cell formation were compared using macrophages derived from the human monocyte cell line THP-1. Chylomicron remnant-like particles (CMR-LPs), oxidised CMR-LPs (oxCMR-LPs) and oxLDL were incubated with THP-1 macrophages, and the relative abundance of mRNA transcripts for genes involved in lipoprotein uptake, intracellular lipid metabolism, transport and storage and cholesterol efflux from macrophages was determined. The results show that CMR-LPs and oxLDL differ markedly in their effects on the expression of mRNA for a number of the genes tested. OxLDL increased mRNA levels for the scavenger receptors CD36 (×3.2) and lectin-like oxLDL receptor 1 (×2.1), and peroxisome proliferator-activated receptor γ while CMR-LPs did not. In contrast, the expression of mRNA for the LDL receptor-like protein was raised by CMR-LPs (×1.8) but not oxLDL. Furthermore, down-regulation of mRNA levels for the ATP-binding cassette transporter (ABC) A1 was observed with CMR-LPs (×0.6), compared to the up-regulation found with oxLDL (×4.4). In addition, a number of significant differences were found between the effects of CMR-LPs and oxCMR-LPs, with the oxidised particles causing a striking rise in mRNA expression for the multi-drug resistance 1 gene (×13.7), but otherwise showing pattern more similar to that seen with oxLDL. These findings provide evidence to indicate that chylomicron remnants cause lipid accumulation in macrophages by influencing the expression of genes which regulate lipid metabolism at the transcriptional level, and that the mechanisms involved differ in important respects from those triggered by oxLDL.

Human tribbles homologue 2 is expressed in unstable regions of carotid plaques and regulates macrophage IL-10 in vitro

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.