PPARγ Response Element Research Articles

Identification of a peroxisome-proliferator-activated-receptor response element in the apolipoprotein E gene control region

Apolipoprotein E (apoE) is a protein involved in reverse cholesterol transport. Among other tissues, apoE is expressed in macrophages where its expression increases when macrophages develop into foam cells. It has been recently shown that peroxisome-proliferator-activated receptor gamma (PPARγ) is involved in this conversion. Northern-blot analysis was carried out in the macrophage cell line THP1 to determine whether apoE mRNA levels were regulated by ciglitazone, a PPARγ inducer. The results indicated that treatment with ciglitazone doubled the levels of apoE mRNA. To identify a possible PPARγ response element (PPRE), several portions of apoE gene control region were used to construct luciferase reporter plasmids. In U-87 MG cells, a 185bp fragment located in the apoE/apoCI intergenic region was sufficient to induce a 10-fold increase in the luciferase activity of the extract of cells co-transfected with a PPARγ expression plasmid. Subsequent analysis revealed the presence of a sequence with a high level of sequence similarity to the consensus PPRE. Mutations in this sequence resulted in a lack of functionality both in transient transfection and in electrophoretic-mobility-shift assays. These results demonstrated the presence of a functional PPRE in the apoE/apoCI intergenic region. These results have implications for the regulation of apoE gene expression and could be relevant for understanding the anti-atherogenic effect of thiazolidinediones.

PPARγ Expression and Function in Mesangial Cells: Implication for Renal Disease

7 Dominant negative mutations which inactivate the peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor in man are associated with severe insulin resistance, diabetes and hypertension (Barroso I et al. Nature, 1999;402:880-883). Using RNase protection assay, Western blot analysis and immunostaining of cultured primary rat mesangial cells, we identified the presence of PPARγ message and protein. Electrophoretic mobility of a labeled PPARγ response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARγ is functional in these cells. Addition of unlabelled PPRE efficiently competed away the PPARγ-PPRE protein complex, confirming specificity of binding of the PPARγ to the PPRE. PPARγ ligands, rosiglitazone (10 μM) and troglitazone (10 μM), both completely inhibited platelet derived growth factor (PDGF)-induced DNA synthesis measured as bromodeoxyuridine (BrdU) incorporation (p

PPARγ Expression and Function in Mesangial Cells: Implication for Renal Disease

7 Dominant negative mutations which inactivate the peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor in man are associated with severe insulin resistance, diabetes and hypertension (Barroso I et al. Nature, 1999;402:880-883). Using RNase protection assay, Western blot analysis and immunostaining of cultured primary rat mesangial cells, we identified the presence of PPARγ message and protein. Electrophoretic mobility of a labeled PPARγ response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARγ is functional in these cells. Addition of unlabelled PPRE efficiently competed away the PPARγ-PPRE protein complex, confirming specificity of binding of the PPARγ to the PPRE. PPARγ ligands, rosiglitazone (10 μM) and troglitazone (10 μM), both completely inhibited platelet derived growth factor (PDGF)-induced DNA synthesis measured as bromodeoxyuridine (BrdU) incorporation (p<0.05). When administered in anti-diabetic doses to streptozotocin-induced diabetic rats, troglitazone substantially normalized albumin excretion at 4 months (from 687.1 to 137.6 μg urinary albumin/mg creatinine, p<0.05), but did not affect hyperglycemia or hypertension in this model. These data suggest PPARγ activation may have direct effects to attenuate diabetic glomerular disease, possibly by inhibiting mesangial growth, which occurs early in the process of diabetic nephropathy. These results have therapeutic implications for diabetic glomerulosclerosis as well as proliferative mesangial diseases of the kidney.

Advanced Glycation End Product-Induced Peroxisome Proliferator-Activated Receptor γ Gene Expression in the Cultured Mesangial Cells

We identified the AGEs-induced expression of peroxisome proliferator-activated γ (PPAR γ) in the cultured mesangial cells using reverse transcription-polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and Western immunoblotting. Administration of AGEs-BSA into the cultured mesangial cells resulted in an increase in the levels of mRNA and proteins for PPAR γ in a dose-dependent manner. Specific bands which indicate the protein binding to PPAR γ responsive element (PPRE) in the nuclear extracts were also detected in AGEs-BSA-treated mesangial cells, but not found in BSA-treated cells by EMSA. Antioxidants, NAC, PDTC, and aminoguanidine, attenuated the gene expression and activity of PPAR γ induced by AGEs. These results indicate that PPAR γ was induced and activated by the oxidative signal(s) evoked by AGEs-ligand-receptor interactions. AGEs-induced gene expression of PPAR γ and the signal intensity of PPAR γ and PPRE complex were attenuated furthermore by protein kinase C inhibitors, calphostin C and staurospolin, but not abolished completely, indicating that both signal transduction pathways through the induction of PKC activation and independent of PKC activation were involved in the AGEs-mediated expression and activation process of PPAR γ. AGEs also increased the gene expression of smooth muscle α-actin, which is a marker for phenotypic change in mesangial cells. It is suggested therefore that AGEs-induced transcription factor as the oxidative stress may have a role in the differentiation of mesangial cells.

Expression of macrophage scavenger receptors in various human tissues and atherosclerotic lesions

Expression of CD36 scavenger receptors on macrophages is involved in oxidized low-density lipoprotein (OxLDL) uptake and foam cell formation during atherosclerotic lesion development. It has been shown previously in vitro and in vivo that the atherosclerotic risk factor homocysteine (Hcy) stimulates macrophage CD36 expression and OxLDL uptake. We now examined the effects of aged garlic extract (AGE), a garlic preparation enriched in water-soluble organic sulfur-containing compounds, on Hcy-induced CD36 expression and foam cell formation in human monocytes/macrophages.Incubation with Hcy (200 μM for 72 h) in THP-1-derived macrophages and primary human macrophages caused a 37.8 ± 5.2% and 60.7 ± 4.2% increase in CD36 expression compared to control, respectively. Coincubation with AGE (5 mg/ml) significantly suppressed CD36 expression in THP-1 derived macrophages by 48.6 ± 9.0% compared to Hcy-incubated cells only. AGE (1–5 mg/ml) dose dependently inhibited Hcy-induced CD36 expression in primary human macrophages, and decreased binding of nuclear proteins to a PPARγ response element. Preincubation with AGE significantly inhibited DiI-labeled OxLDL uptake.These data indicate that AGE inhibits CD36 expression and OxLDL uptake in human macrophages by modulating the PPARγ pathway, and suggest that the extract could be useful for the prevention of atherosclerotic lesions.