Selective role of sterol regulatory element binding protein isoforms in aggregated LDL-induced vascular low density lipoprotein receptor-related protein-1 expression.

Low density lipoprotein receptor-related protein (LRP1) is upregulated in vascular smooth muscle cells by intravascular aggregated LDL (agLDL) - LDL trapped in the arterial intima and systemic LDL. LRP1 upregulation in hypercholesterolemic aortas is concomitant with SREBP downregulation. However, the specific role of SREBP isoforms in LRP1 transcription and LDL-induced LRP1 upregulation in human vascular smooth muscle cells (VSMC) is unknown. In the present study we report that specific silencing of either SREBP-1 or SREBP-2 enhanced LRP1 whereas overexpression of the active SREBP isoforms decreased LRP1 expression. Gel mobility shift and ChIP assays demonstrated that SREBP-1a, SREBP-1c and SREBP-2 were able to bind to three putative SRE sequences; SRE-A (-1042 to -1028), SRE-B (-115 to -101) and SRE-C (+226 to +234). ChIP assays demonstrated that agLDL (100µg/mL, 24h) significantly and specifically decreased SREBP-2 binding to the LRP1 promoter. Luciferase assays demonstrated that agLDL increased the transcriptional activity of A/B or A/C double mutants but failed to increase that of the double B/C mutant. Our results show that both SREBP-1 and SREBP-2 negatively modulated LRP1 transcription. Furthermore, agLDL exerted an upregulatory effect on LRP1 expression by decreasing SREBP-2 binding to LRP1 promoter. Two SRE-like sequences control the response of LRP1 to agLDL.

Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide.

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alteration of the CYP apoprotein profile that after 24 h of culture is quite different from that found in the liver of uninduced rats. This process is reverted by the early and efficient inhibition of NO synthesis, which allows for (1) maintenance of total P450 holoenzyme content, (2) preservation of the initial constitutive CYP pattern in culture and (3) the early expression of the normal inducibility in response to model inducers.

Involvement of peroxynitrite on the early loss of p450 in short-term hepatocyte cultures.

The biological chemistry of nitric oxide (NO) in the oxygenated cellular environment is extremely complex. It involves the direct interaction of NO with specific biomolecules and the so-called indirect effects, due to secondary more potent oxidant species derived from NO which are also able to react with DNA, lipids, thiols and transition metals (Wink et al., 1996; Nathan, 1992). In addition to its regulatory role as a signalling molecule (Nathan, 1992; Moncada and Palmer, 1991) it has become evident that NO (or NO-derived species) is a critical factor involved in various toxicological mechanisms (Wink et al., 1996; Wang et al., 1998; Estevez et al., 1999; Wink et al., 1999). Some controversy exists however about the damaging vs. protective actions of NO on oxidative injury, whose biological significance in living cells and tissues remains still ill defined. Research in this laboratory (López-García, 1998; López-García and Sanz-Gonzalez, 2000) has shown that NO synthesis is significantly activated in hepatocytes from control rats following isolation by the classical collagenase-based procedure. NO overproduction appears to be due to the very early activation of liver constitutive Ca2+-dependent NO synthase (cNOS). Previous results have also provided first experimental evidence for the direct involvement of endogenously generated NO as a causal factor responsible for important phenotypic changes commonly observed in short-term cultured hepatocytes, which includes the early impairment of hepatocyte mitochondrial function--i.e., transient cell energy depletion--and glucose metabolism, and the well-known quick and irreversible loss of P450 content (López et al. 1987; López-García, 1998). This study aims to further characterise the mechanisms underlying this phenomenon. Results show that the hepatocyte isolation procedure (the commonly employed collagenase-based two step liver perfusion method) induces strong oxidative stress that lasts for at least 4 h in culture and involves both oxygen-derived (ROS) and nitrogen-derived (RNS) reactive species. On the basis of the combined use of dihydrorhodamine 123 (DHR) as a probe and L-NAME (N(G)-nitro-L-arginine methyl ester) to efficiently block NO synthesis, the analysis of the amount, the time-course pattern, and the nature of the species involved support the view that peroxynitrite* (PN) is readily formed within the early culture hours. Immunodetection of protein bound 3-nitrotyrosine provides direct evidence for PN generation upon hepatocyte isolation: several nitrated protein bands--most already present after only 30 min of liver perfusion and quantitatively increasing for the first 2 hours in culture--have been identified as preferential PN protein targets in the different cellular compartments. Since the early inhibition of NO synthesis is enough to provide full maintenance of the hepatocyte initial P450 content, results support the view that PN--while not affecting cell viability and monolayer development--is the main species likely responsible for the early loss of P450 in short-term cultured hepatocytes.