23 The suppression of lipoprotein lipase expression in J774.2 macrophages by EFN-γ and TNF-α is mediated at the transcriptional level

Abstract

Lipoprotein lipase (LPL) plays a crucial role in lipid metabolism by catalysing the hydrolysis of the triacylglycerol core of chylomicrons and very low density lipoproteins (VLDL), thereby providing non-esterified fatty acids and 2monoacylglycerol for tissue utilisation [ 1-31, LPL is expressed by several tissuedcell types, including macrophages and macrophage-derived cell lines [4-53. Macrophage LPL has generated continuing interest because of its possible role in the pathogenesis of atherosclerosis [6-71. For example, LPL is expressed by macrophage-derived foam cells in atherosclerotic plaques [8] and inbred murine strains, with susceptibility to atherosclerosis, contain an unusually high level of macrophage LPL expression and secretion [9]. In addition, studies in vitro has shown that LPL can modify low density lipoproteins (LDL) to forms that are taken up readily by both macrophage and smooth muscle cells [lo]. Several growth factors and cytokines are known to regulate the varied cellular hnction of the vascular wall [ 1 1-12]. The changes in macrophage LPL expression by such mediators may, therefore, make a major contribution to the progression of atherosclerosis. Unfortunately, studies on the regulation of macrophage LPL by cytokines are limited [2-31. We have, however, recently examined the action of six cytokines on the expression of LPL using the murine J774.2 cell line as a model system [13]. We showed that exposure of the cells to interleukin-1 (IL-l), interleukin-1 1 (IL-I I), interferon gamma (IFN-y) and tumour necrosis factor alpha (TNF-a) produced a dose-dependent decrease in LPL-enzymatic activity, M A levels, and -protein content. By contrast, interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) had no effect. The maximum suppression of LPL activity produced by IL-I, IL-I 1 and TNF-a (77-86%) was higher than that by IFN-y (60%). Because more than 80% of the decrease in LPL enzymatic activity observed when the cells are stimulated with IL-1, IL1 1, IFN-y and TNF-a could be accounted for by a corresponding reduction in LPL mRNA levels, it can be concluded that the responses occur at the level of mRNA metabolism, by either decreasing the LPL mRNA transcription or stability. To explore these possibilities further, we have examined here whether IFNy or TNF-a have any effect on the stability of LPL mRNA. 5774.2 cells were treated with or without cytokines (IFN-y or TNF-a) in the presence of 5@ml actinomycin D to block transcription. The dose of cytokine used (500U/ml TNF-a, 1000U/ml IFN-y) corresponded to that which produced a maximum reduction of LPL activity in a previous study [13J RNA was then prepared from cells at timed intervals and subjected to northern blot analysis. The experiment was restricted to a maximum treatment for 6h because it was observed that longer incubations (10h) produced cell death. Membranes for the northern blot were first probed with radiolabelled LPL cDNA insert, and following autoradiography, “stripped” and reprobed with the p-actin cDNA, assigning the LPL:P-actin ratio in control cells as 100%. As shown in Figure 1, the decay of LPL mRNA produced in the presence of cytokines was not greater than that for control unstimulated cells. It can, therefore, be concluded that the IFN-yor TNF-amediated suppression of LPL expression is mediated primarily at the level of decreased LPL gene transcription ::: r o m y c i n ~ D ~