Protein kinase C (PKC) represents a family of serine/threonine protein kinases that plays a key role in signal transduction and regulation of gene expression (11,39,40,43). Molecular cloning and biochemical studies have revealed at least 12 PKC subspecies, which can be classified into three groupsclassic (cPKC), novel (nPKC), and atypical (aPKC)-on the basis of their structures. The PKC family comprises a regulatory domain in the amino terminus and a catalytic domain in the carboxyl terminus. The cPKCs, including PKC␣, -␤I, -␤II, and -␥, have two common regions, C1 and C2, in the regulatory domain. The C1 region has two cysteine-rich loops that are the binding site for diacylglycerol (DAG) and phorbol ester (tetradecanoyl phorbol acetate [TPA]). The C2 region binds to calcium. The nPKCs, including PKC␦, -ε, -, and -, lack the C2 region. The aPKCs, including PKC and -/, lack the C2 region and have only one cysteine-rich loop in the C1 region. Key lipid cofactors of PKCs are DAG, phosphatidylserine (PS), and calcium. All of these are required to activate cPKCs, whereas calcium is not required to activate nPKCs. aPKCs are insensitive to both DAG and calcium. Other lipid species, such as cholesterol sulfate, fatty acids, lysophospholipids, and phosphatidylinositols, also modulate the activity of certain PKC isoforms (3,12,24,41). Several PKC family members displaying responsiveness to calcium, DAG, and fatty acids are expressed in hepatic cells (16,26), but their precise functional roles in cholesterol uptake and biosynthesis are not yet clearly defined.Animal cells regulate their cholesterol content by fine-tuning of the supply of exogenous and endogenous cholesterol (5). Both cholesterol and fatty acids have been demonstrated to be important regulators of plasma low-density lipoprotein (LDL) cholesterol levels due to alterations in LDL receptor activity (6,7,20). The mechanism by which sterols modulate LDL receptor gene transcription has been examined in great detail. Involved in this pathway are a family of proteins, designated sterol regulatory element binding proteins (SREBPs), that play an integral role in the feedback pathway by which cholesterol suppresses transcription of LDL receptor gene. The SREBPs are transcription factors that are bound to the endoplasmic reticulum and nuclear envelope by virtue of two membranespanning regions. When these membranes are depleted of sterols, a two-step proteolytic process releases the amino terminal of SREBPs, which then travel to the nucleus and activate LDL receptor transcription via interaction with multiple nuclear factors and coactivators (7,42). When cells are overloaded with sterols, cleavage of SREBPs is inhibited, resulting in suppression of the LDL receptor gene. A similar mechanism has been shown for the regulation of other sterol-responsive genes

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.

ICI182,780,[7][8][9]4,5,5,5, sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17 ␤ -diol; LPDS, lipoproteindeficient serum; MAPK, mitogen-activated protein kinase; p42/44 MAPK , extracellular signal-regulated kinase; PKC, protein kinase C; pp90 RSK , 90 kDa ribosomal S6 kinase; SRE, sterol regulatory element; SREBP, SRE binding protein; WT, wild type.

Our previous observation that induction of low density lipoprotein (LDL) receptor expression by a variety of extracellular signals is blocked by PD98059, a specific mitogen-activated protein kinase kinase inhibitor, led to the suggestion that the growth-responsive p42/44(MAPK) cascade plays a critical role in regulating LDL receptor transcription. To analyze the specific contribution of the p42/44(MAPK) cascade in regulating cell growth and LDL receptor induction, we established a HepG2-derived cell line that stably expresses an inducible form of oncogenic human Raf-1 kinase. Using this system, we provide direct evidence that specific activation of this cascade alone is not only required but is sufficient to fully induce LDL receptor expression. Interestingly, degree of p42/44(MAPK) activation determines the extent of LDL receptor induction. However, activation of p42/44(MAPK) in the above cells led to the inhibition of DNA synthesis, caused growth arrest, decrease in cyclin A and upregulation of p21(Cip) expression in a time-dependent manner. These results suggest that each of these two processes can be regulated independently of each other in response to p42/44(MAPK) activation. Thus, extent of p42/44(MAPK) activation may be important in transducing divergent cellular responses in human cells with implications for altered signaling resulting in hypercholesterolemia.