Biomedical subjects

G Chinetti

Publications and source records attributed to G Chinetti.

22 records · Page 2Linked to original sources

PubMed1999

Cell culture conditions determine apolipoprotein CIII secretion and regulation by fibrates in human hepatoma HepG2 cells.

Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum. Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs. The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2. First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases. Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them. The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent. Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)alpha activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643. Comparison of the activity of a PPARgamma-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARalpha ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARalpha but not PPARgamma. In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARalpha but not via PPARgamma. However, these effects are only observed in HepG2 cells cultured under appropriate conditions.

Animals↗

PubMed1999

A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity.

The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.

Animals↗

PubMed1998

Activation of proliferator-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages.

Peroxisome proliferator-activated receptors (PPARs) have been implicated in metabolic diseases, such as obesity, diabetes, and atherosclerosis, due to their activity in liver and adipose tissue on genes involved in lipid and glucose homeostasis. Here, we show that the PPARalpha and PPARgamma forms are expressed in differentiated human monocyte-derived macrophages, which participate in inflammation control and atherosclerotic plaque formation. Whereas PPARalpha is already present in undifferentiated monocytes, PPARgamma expression is induced upon differentiation into macrophages. Immunocytochemistry analysis demonstrates that PPARalpha resides constitutively in the cytoplasm, whereas PPARgamma is predominantly nuclear localized. Transient transfection experiments indicate that PPARalpha and PPARgamma are transcriptionally active after ligand stimulation. Ligand activation of PPARgamma, but not of PPARalpha, results in apoptosis induction of unactivated differentiated macrophages as measured by the TUNEL assay and the appearance of the active proteolytic subunits of the cell death protease caspase-3. However, both PPARalpha and PPARgamma ligands induce apoptosis of macrophages activated with tumor necrosis factor alpha/interferon gamma. Finally, PPARgamma inhibits the transcriptional activity of the NFkappaB p65/RelA subunit, suggesting that PPAR activators induce macrophage apoptosis by negatively interfering with the anti-apoptotic NFkappaB signaling pathway. These data demonstrate a novel function of PPAR in human macrophages with likely consequences in inflammation and atherosclerosis.

Apoptosis↗

PubMed1998

Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators.

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARgamma promotes lipid storage by regulating adipocyte differentiation, PPARalpha stimulates the beta-oxidative degradation of fatty acids. PPARalpha-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARalpha is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARalpha ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect. Here we show that PPARalpha is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARalpha ligands, and not PPARgamma ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARalpha repression of NF-kappaB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARalpha inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARalpha in the vascular wall may influence the process of atherosclerosis and re-stenosis.

Acute-Phase Proteins↗