Differential Roles of Extracellular Signal-regulated Kinase-1/2 and p38MAPK in Interleukin-1β- and Tumor Necrosis Factor-α-induced Low Density Lipoprotein Receptor Expression in HepG2 Cells

Abstract

The inflammatory cytokines interleukin-1␤ (IL-1␤Elevated plasma levels of cytokines, including IL-1␤ and TNF found in various inflammatory, infectious, and malignant diseases, are often associated with hypocholesterolemia (7-11). Systemic infusion of cytokines has been shown to lower serum cholesterol levels in animals and humans (12-14). The concentrations of IL-1␤ and TNF similar to those found following infection induce low density lipoprotein (LDL) receptor expression in human hepatoma (HepG2) cells, and this effect is not part of its mitogenic response, as IL-1␤ did not increase DNA synthesis (15-18). Interestingly, a 1.56-kilobase pair region of the 5Ј-flanking region of the human LDL receptor promoter has been shown to confer IL-1␤-dependent induction to an heterologous gene, suggesting that increased receptor expression results from activation of LDL receptor transcription and is not due to an alteration in LDL receptor mRNA stability (18). These studies have also established that, unlike TNF, IL-1␤-induced LDL receptor transcription does not require protein synthesis and, therefore, likely depends on activation of a preexisting component(s). The initial step in their action is the association of agonist with its cell surface receptors, followed by intracellular protein phosphorylation/dephosphorylation (19 -21). The downstream effectors linking receptor activation with the different cellular responses are still largely to be defined, although several signaling pathways have been proposed (20,21). The signal transduction mechanisms by which these cytokines stimulates LDL receptor expression are poorly characterized. Many extracellular signals elicit specific biological responses through activation of MAPK cascades (22-26). The three major subfamilies of MAPKs in higher eukaryotes include ERK-1/2, 46/54 JNK , and p38 MAPK , all of which are activated by phosphorylation of a tyrosine and a threonine residue catalyzed by a dual specificity MAPK kinase. ERKs are most strongly activated by mitogenic signals such as growth factors or 12-Otetradecanoylphorbol-13-acetate (TPA), whereas p46/54 JNK , and p38MAPK are activated by stressful stimuli such as the inflammatory cytokines, IL-1␤, and TNF, thermal shock, and osmotic shock (23,27). Activation of MAPKs leads to distinct cellular responses mediated by phosphorylation of specific target substrates (25). Recently, we have shown involvement of ERK-1/2 signaling cascade in TPA-induced LDL receptor expression in HepG2 cells (28).In this study, we have investigated the early in vivo signal-

Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

“…Although PD-98059 could have other effects, the concentration used here is consistent with that previously used to inhibit ERK activation (41,68). Moreover, this treatment did not alter the proliferation rate as compared with vehicle treated controls.…”

One-way Cross-talk between p38MAPK and p42/44MAPK

“…Analysis of the signal transduction pathways using the above inhibitors revealed a critical role of p42/44 MAPK activation during induction of low density lipoprotein (LDL) receptor gene expression by a variety of extracellular stimuli (43,44 Cell Culture-Human hepatoma cell line HepG2 and its derivative HepG2-⌬Raf-1:ER cell line that stably expresses the ⌬Raf-1:ER chimera were maintained as monolayer cultures in a humidified 5% CO 2 atmosphere at 37°C in Eagle's minimum essential medium (BioWhittaker) supplemented with 10% fetal bovine serum (Life Technologies), 2 mM L-glutamine, 20 units/ml penicillin, and 20 g/ml streptomycin sulfate.…”

“…Northern Analysis-Total RNA was isolated using TRIzol, and Northern blotting was done essentially as described earlier (43,44). Briefly, 20 g of total cellular RNA was fractionated on 1% formaldehyde agarose gel and transferred to Zeta Probe membrane by capillary blotting.…”

“…Immunoblot Analysis-Proteins were fractionated by SDS-polyacrylamide gel electrophoresis with 10% acrylamide separation gel, and the separated proteins were transferred to nitrocellulose and processed as described previously (43,44). Briefly, membranes were incubated in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.2% (v/v) Tween 20 (Tris/NaCl/ Tween 20) with 5% (w/v) nonfat dried milk for 1 h, washed in Tris/NaCl/ Tween 20 (3 ϫ 5 min), and incubated for 1 h with primary antibody in Tris/NaCl/Tween 20 containing 1% milk at room temperature for nonphosphospecific antibodies and overnight at 4°C for phosphospecific antibodies.…”

Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection

“…50 In summary, it appears that variants in the LDLR gene, which influence the pathogenesis of FH and related diseases, may contribute to variation in severity of fibrosis, inflammation and response to treatment in hepatitis C. There is a complex interaction between IFN-␣ and LDLR activity, which could play an important role in these processes. LDLR activity appears to be increased by IL-1, 51 which is downregulated by its receptor antagonist, IL-1RA. 52 Circulating IL-1RA in turn has been shown to be induced by IFN treatment.…”