Hepatic protein kinase Cbeta deficiency mitigates late-onset obesity.

Although aging is associated with progressive adiposity and a decline in liver function, the underlying molecular mechanisms and metabolic interplay are incompletely understood. Here, we demonstrate that aging induces hepatic protein kinase Cbeta (PKCß) expression, while hepatocyte PKCß deficiency (PKCßHep-/-) in mice significantly attenuates obesity in aged mice fed a high-fat diet. Compared with control PKCßfl/fl mice, PKCßHep-/- mice showed elevated energy expenditure with augmentation of oxygen consumption and carbon dioxide production which was dependent on ß3-adrenergic receptor signaling, thereby favoring negative energy balance. This effect was accompanied by induction of thermogenic genes in brown adipose tissue (BAT) and increased BAT respiratory capacity, as well as a shift to oxidative muscle fiber type with an improved mitochondrial function, thereby enhancing oxidative capacity of thermogenic tissues. Furthermore, in PKCßHep-/- mice, we determined that PKCß overexpression in the liver mitigated elevated expression of thermogenic genes in BAT. In conclusion, our study thus establishes hepatocyte PKCß induction as a critical component of pathophysiological energy metabolism by promoting progressive hepatic and extrahepatic metabolic derangements in energy homeostasis, contributing to late-onset obesity. These findings have potential implications for augmenting thermogenesis as a means of combating aging-induced obesity.

Role of hepatic PKCß in nutritional regulation of hepatic glycogen synthesis.

The signaling mechanisms by which dietary fat and cholesterol signals regulate central pathways of glucose homeostasis are not completely understood. By using a hepatocyte-specific PKCß-deficient (PKCßHep-/-) mouse model, we demonstrated the role of hepatic PKCß in slowing disposal of glucose overload by suppressing glycogenesis and increasing hepatic glucose output. PKCßHep-/- mice exhibited lower plasma glucose under the fed condition, modestly improved systemic glucose tolerance and mildly suppressed gluconeogenesis, increased hepatic glycogen accumulation and synthesis due to elevated glucokinase expression and activated glycogen synthase (GS), and suppressed glucose-6-phosphatase expression compared with controls. These events were independent of hepatic AKT/GSK-3α/ß signaling and were accompanied by increased HNF-4α transactivation, reduced FoxO1 protein abundance, and elevated expression of GS targeting protein phosphatase 1 regulatory subunit 3C in the PKCßHep-/- liver compared with controls. The above data strongly imply that hepatic PKCß deficiency causes hypoglycemia postprandially by promoting glucose phosphorylation via upregulating glucokinase and subsequently redirecting more glucose-6-phosphate to glycogen via activating GS. In summary, hepatic PKCß has a unique and essential ability to induce a coordinated response that negatively affects glycogenesis at multiple levels under physiological postprandial conditions, thereby integrating nutritional fat intake with dysregulation of glucose homeostasis.

Hepatocyte-specific PKCß deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis.

OBJECTIVE: Nonalcoholic hepatic steatosis, also known as fatty liver, is a uniform response of the liver to hyperlipidic-hypercaloric diet intake. However, the post-ingestive signals and mechanistic processes driving hepatic steatosis are not well understood. Emerging data demonstrate that protein kinase C beta (PKCß), a lipid-sensitive kinase, plays a critical role in energy metabolism and adaptation to environmental and nutritional stimuli. Despite its powerful effect on glucose and lipid metabolism, knowledge of the physiological roles of hepatic PKCß in energy homeostasis is limited. METHODS: The floxed-PKCß and hepatocyte-specific PKCß-deficient mouse models were generated to study the in vivo role of hepatocyte PKCß on diet-induced hepatic steatosis, lipid metabolism, and mitochondrial function. RESULTS: We report that hepatocyte-specific PKCß deficiency protects mice from development of hepatic steatosis induced by high-fat diet, without affecting body weight gain. This protection is associated with attenuation of SREBP-1c transactivation and improved hepatic mitochondrial respiratory chain. Lipidomic analysis identified significant increases in the critical mitochondrial inner membrane lipid, cardiolipin, in PKCß-deficient livers compared to control. Moreover, hepatocyte PKCß deficiency had no significant effect on either hepatic or whole-body insulin sensitivity supporting dissociation between hepatic steatosis and insulin resistance. CONCLUSIONS: The above data indicate that hepatocyte PKCß is a key focus of dietary lipid perception and is essential for efficient storage of dietary lipids in liver largely through coordinating energy utilization and lipogenesis during post-prandial period. These results highlight the importance of hepatic PKCß as a drug target for obesity-associated nonalcoholic hepatic steatosis.

Dietary fat/cholesterol-sensitive PKCß-RB signaling: Potential role in NASH/HCC axis.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis, and environmental factors significantly contribute to the risk. Despite knowledge that a Western-style diet is a risk factor in the development of nonalcoholic steatohepatitis (NASH) and subsequent progression to HCC, diet-induced signaling changes are not well understood. Understanding molecular mechanisms altered by diet is crucial for developing preventive and therapeutic strategies. We have previously shown that diets enriched with high-fat and high-cholesterol, shown to produce NASH and HCC, induce hepatic protein kinase C beta (PKCß) expression in mice, and a systemic loss of PKCß promotes hepatic cholesterol accumulation in response to this diet. Here, we sought to determine how PKCß and diet functionally interact during the pathogenesis of NASH and how it may promote hepatic carcinogenesis. We found that diet-induced hepatic PKCß expression is accompanied by an increase in phosphorylation of Ser780 of retinoblastoma (RB) protein. Intriguingly, PKCß-/- livers exhibited reduced RB protein levels despite increased transcription of the RB gene. It is also accompanied by reduced RBL-1 with no significant effect on RBL-2 protein levels. We also found reduced expression of the PKCß in HCC compared to non-tumorous liver in human patients. These results raise an interesting possibility that diet-induced PKCß activation represents an important mediator in the functional wiring of cholesterol metabolism and tumorigenesis through modulating stability of cell cycle-associated proteins. The potential role of PKCß in the suppression of tumorigenesis is discussed.

PKCß: Expanding role in hepatic adaptation of cholesterol homeostasis to dietary fat/cholesterol.

Cholesterol homeostasis relies on an intricate network of cellular processes whose deregulation in response to Western type high-fat/cholesterol diets can lead to several life-threatening pathologies. Significant advances have been made in resolving the molecular identity and regulatory function of transcription factors sensitive to fat, cholesterol, or bile acids, but whether body senses the presence of both fat and cholesterol simultaneously is not known. Assessing the impact of a high-fat/cholesterol load, rather than an individual component alone, on cholesterol homeostasis is more physiologically relevant because Western diets deliver both fat and cholesterol at the same time. Moreover, dietary fat and dietary cholesterol are reported to act synergistically to impair liver cholesterol homeostasis. A key insight into the role of protein kinase C-ß (PKCß) in hepatic adaptation to high-fat/cholesterol diets was gained recently through the use of knockout mice. The emerging evidence indicates that PKCß is an important regulator of cholesterol homeostasis that ensures normal adaptation to high-fat/cholesterol intake. Consistent with this function, high-fat/cholesterol diets induce PKCß expression and signaling in the intestine and liver, while systemic PKCß deficiency promotes accumulation of cholesterol in the liver and bile. PKCß disruption results in profound dysregulation of hepatic cholesterol and bile homeostasis and imparts sensitivity to cholesterol gallstone formation. The available results support involvement of a two-pronged mechanism by which intestine and liver PKCß signaling converge on liver ERK1/2 to dictate diet-induced cholesterol and bile acid homeostasis. Collectively, PKCß is an integrator of dietary fat/cholesterol signal and mediates changes to cholesterol homeostasis.

Modulation of Hepatic Protein Kinase Cß Expression in Metabolic Adaptation to a Lithogenic Diet.

BACKGROUND & AIMS: Dietary factors are likely an important determinant of gallstone development, and difficulty in adapting to lithogenic diets may predispose individuals to gallstone formation. Identification of the critical early diet-dependent metabolic markers of adaptability is urgently needed to prevent gallstone development. We focus on the interaction between diet and genes, and the resulting potential to influence gallstone risk by dietary modification. METHODS: Expression levels of hepatic protein kinase C (PKC) isoforms were determined in lithogenic diet-fed mice, and the relationship of hepatic cholesterol content and PKCß expression and the effect of hepatic PKCß overexpression on intracellular signaling pathways were analyzed. RESULTS: Lithogenic diet feeding resulted in a striking induction of hepatic PKCß and PKCδ mRNA and protein levels, which preceded the appearance of biliary cholesterol crystals. Unlike PKCß deficiency, global PKCδ deficiency did not influence lithogenic diet-induced gallstone formation. Interestingly, a deficiency of apolipoprotein E abrogated the diet-induced hepatic PKCß expression, whereas a deficiency of liver X receptor-α further potentiated the induction, suggesting a potential link between the degree of hepatic PKCß induction and the intracellular cholesterol content. Furthermore, our results suggest that PKCß is a physiologic repressor of ileum basal fibroblast growth factor 15 (FGF15) expression and activity of hepatic proto-oncogene serine/threonine-protein kinase Raf-1/mitogen-activated protein (MAP) kinase kinase/extracellular signal-regulated kinases 1/2 (Raf-1/MEK/ERK1/2) cascade proteins, and the complex interactions between these pathways may determine the degree of hepatic ERK1/2 activation, a potent suppressor of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase expression. We found that PKCß regulated Raf-1 activity by modulating the inhibitory Raf-1Ser259 phosphorylation. CONCLUSIONS: Our results demonstrate a novel interaction between the hepatic PKCß/Raf-1 regulatory axis and ileum PKCß/FGF15/ERK axis, which could modulate the bile lithogenecity of dietary lipids. The data presented are consistent with a two-pronged mechanism by which intestine and liver PKCß signaling converges on the liver ERK1/2 pathway to control the hepatic adaptive response to a lithogenic diet. Elucidating the impact and the underlying mechanism(s) of PKCß action could help us understand how different types of dietary fat modify the risk of gallstone formation, information that could help to identify novel targets for therapeutic approaches to combat this disease.

Protein kinase C-beta: An emerging connection between nutrient excess and obesity.

There is currently a global epidemic of obesity as a result of recent changes in lifestyle. Excess body fat deposition is caused by an imbalance between energy intake and energy expenditure due to interactions between genetic and environmental factors. The signals and biological mechanisms that trigger fat accumulation by disrupting energy homeostasis are not well understood. There is considerable evidence now supporting a possible role of protein kinase C beta (PKCß) in energy homeostasis. This review highlights recent findings on the role of PKCß activation in the genesis and progression of obesity, and of PKCß repression in mediating the beneficial effects of physical exercise. Available data support a model in which adipose PKCß activation is among the initiating events that disrupt mitochondrial function through interaction with p66shc and amplify fat accumulation and adipose dysfunction, with systemic consequences. Manipulation of PKCß levels, activity, or signaling could provide a therapeutic approach to combat obesity and associated metabolic disorders.

Emerging role of protein kinase C in energy homeostasis: A brief overview.

Protein kinase C-ß (PKCß), a member of the lipid-activated serine/threonine PKC family, has been implicated in a wide range of important cellular processes. Very recently, the novel role of PKCß in the regulation of triglyceride homeostasis via regulating mitochondrial function has been explored. In this review, I aim to provide an overview of PKCß regarding regulation by lipids and recently gained knowledge on its role in energy homeostasis. Alterations in adipose PKCß expression have been shown to be crucial for diet-induced obesity and related metabolic abnormalities. High-fat diet is shown to induce PKCß expression in white adipose tissue in an isoform- and tissue-specific manner. Genetically manipulated mice devoid of PKCß are lean with increased oxygen consumption and are resistant to high-fat diet-induced obesity and hepatic steatosis with improved insulin sensitivity. Available data support the model in which PKCß functions as a "diet-sensitive" metabolic sensor whose induction in adipose tissue by high-fat diet is among the initiating event disrupting mitochondrial homeostasis via intersecting with p66(Shc) signaling to amplify adipose dysfunction and have systemic consequences. Alterations in PKCß expression and/or function may have important implications in health and disease and warrants a detailed investigation into the downstream target genes and the underlying mechanisms involved. Development of drugs that target the PKCß pathway and identification of miRs specifically controlling PKCß expression may lead to novel therapeutic options for treating age-related metabolic disease including fatty liver, obesity and type 2 diabetes.

Protein kinase C Beta in the tumor microenvironment promotes mammary tumorigenesis.

Protein kinase C beta (PKCß) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCß is pro-tumorigenic in this disease is still unclear. Interestingly, while it is known that PKCß mediates angiogenesis, immunity, fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCß is functionally relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) induced mammary tumorigenesis in the presence and absence of PKCß. We utilize two model systems: one where PKCß is deleted in both the epithelial and stromal compartments to test the global requirement for PKCß on tumor formation, and second, where PKCß is deleted only in the stromal compartment to test its role in the TME. MMTV-PyMT mice globally lacking PKCß live longer and develop smaller tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when PKCß is null exclusively in the stroma, PyMT-driven B6 cells form smaller tumors with diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCß in MMTV-PyMT tumorigenesis. In corroboration with these results, PKCß mRNA (Prkcb) is increased in fibroblasts isolated from MMTV-PyMT tumors. These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCß in the mammary TME is necessary to elucidate how to effectively target this signaling pathway in breast cancer.

PRKCB/protein kinase C, beta and the mitochondrial axis as key regulators of autophagy.

Autophagy is the major intracellular system of degradation, and it plays an essential role in various biological events. Recent observations indicate that autophagy is modulated in response to the energy status of the mitochondrial compartment. However, the exact signaling mechanism that controls autophagy under these conditions remains unclear. In this study, we report that the activation of protein kinase C ß (PRKCB), a member of the classical PRKCs, negatively modulates the mitochondrial energy status and inhibits autophagy. Furthermore, cells treated with a pharmacological PRKCB inhibitor, and prkcb knockout MEFs showed an increase in autophagy both in vitro and in vivo, as well as an increased mitochondrial membrane potential (Ψm), suggesting a strong involvement of mitochondrial energy in the modulation of the autophagy machinery. Finally, we show that factors that increase the Ψm oppose the PRKCB-dependent inhibition of autophagy. Altogether, these data underscore the importance of PRKCB in the regulation of autophagy; moreover, the finding that a pharmacological modulation of the Ψm modifies autophagy levels may be useful in fighting pathologies (including various types of cancer and neurodegenerative disorders) that are characterized by reduced levels of autophagy.

Protein kinase Cß deficiency attenuates obesity syndrome of ob/ob mice by promoting white adipose tissue remodeling.

To explore the role of leptin in PKCß action and to determine the protective potential of PKCß deficiency on profound obesity, double knockout (DBKO) mice lacking PKCß and ob genes were created, and key parameters of metabolism and body composition were studied. DBKO mice had similar caloric intake as ob/ob mice but showed significantly reduced body fat content, improved glucose metabolism, and elevated body temperature. DBKO mice were resistant to high-fat diet-induced obesity. Moreover, PKCß deficiency increased ß-adrenergic signaling by inducing expression of ß1- and ß3-adrenergic receptors (ß-ARs) in white adipose tissue (WAT) of ob/ob mice. Accordingly, p38(MAPK) activation and expression of PGC-1α and UCP-1 were increased in WAT of DBKO mice. Consistent with results of in vivo studies, inhibition of PKCß in WAT explants from ob/ob mice also increased expression of above ß-ARs. In contrast, induction of PGC-1α and UCP-1 expression in brown adipose tissue of DBKO mice was not accompanied by changes in the expression of these ß-ARs. Collectively, these findings suggest that PKCß deficiency may prevent genetic obesity, in part, by remodeling the catabolic function of adipose tissues through ß-ARs dependent and independent mechanisms.

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism.

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cß (PKCß), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCß deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCß(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCß(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCß(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCß in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCß as a major physiological regulator of both triglyceride and cholesterol homeostasis.

Loss of protein kinase Cbeta function protects mice against diet-induced obesity and development of hepatic steatosis and insulin resistance.

Obesity is an energy balance disorder in which intake is greater than expenditure, with most excess calories stored as triglyceride (TG). We previously reported that mice lacking the beta-isoform of protein kinase C (PKCbeta), a diacylglycerol- and phospholipid-dependent kinase, exhibit marked reduction in the whole body TG content, including white adipose tissue (WAT) mass. To investigate the role of this signaling kinase in metabolic adaptations to severe dietary stress, we studied the impact of a high-fat diet (HFD) on PKCbeta expression and the effect of PKCbeta deficiency on profound weight gain. We report herein that HFD selectively increased PKCbeta expression in obesity-prone C57BL/6J mice, specifically in WAT; the expression levels were little or unchanged in the liver, muscle, kidney, and heart. Basal PKCbeta expression was also found to be elevated in WAT of obese ob/ob mice. Remarkably, mice lacking PKCbeta were resistant to HFD-induced obesity, showing significantly reduced WAT and slightly higher core body temperatures. Unlike lean lipodystrophic mouse models, these mice did not have fatty livers, nor did they exhibit insulin resistance. Moreover, PKCbeta(-/-) mice exhibited changes in lipid metabolism gene expression, and such alterations were accompanied by significant changes in serum adipokines. These observations suggest that PKCbeta deficiency induced a unique metabolic state congruous with obesity resistance, thus raising the possibility that dysregulation of PKCbeta expression could contribute to dietary fat-induced obesity and related disorders.

AICAR positively regulate glycogen synthase activity and LDL receptor expression through Raf-1/MEK/p42/44MAPK/p90RSK/GSK-3 signaling cascade.

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is a commonly used pharmacological agent to study physiological effects which are similar to those of exercise. However, signal transduction pathways by which AICAR elicits downstream effects in liver are poorly understood. We report here that AICAR not only activated AMPK but also phosphorylated/deactivated glycogen synthase kinase-3 alpha/beta (GSK-3alpha/beta) and dephophorylated/activated glycogen synthase (GS) in a time-dependent manner in human hepatoma HepG2 cells. The signal connection between AICAR and GSK-3 is indirect and involves activation of Raf-1/MEK/p42/44(MAPK)/p90(RSK) signaling cascade as pharmacologic inhibition of MEK significantly reduced phosphorylation/deactivation of GSK-3 and consequent dephosphorylation/activation of GS. Moreover, silencing the expression of p90(RSK), a substrate of p42/44(MAPK), attenuated AICAR-dependent GSK-3 phosphorylation, implicating this kinase as a key mediator of AICAR signaling to GSK-3. Furthermore, consistent with the involvement of Raf-1 kinase cascade, AICAR-induced low-density lipoprotein (LDL) receptor expression in a p42/44(MAPK)-dependent manner. Finally, AICAR requires AMPK-alpha2-dependent and -independent pathways to activate Raf-1 kinase cascade as suppression of AMPKalpha2 activity, and not of AMPKalpha1, partially blocked AICAR-dependent p42/44(MAPK) activation and GSK-3 phosphorylation/deactivation. Collectively, these results highlight Raf-1 signaling cascade as the critical mediator of AICAR action on glucose and lipid metabolism in HepG2 cells.

Protein kinase C deficiency increases fatty acid oxidation and reduces fat storage.

Metabolic syndrome is common in the general population, but there is little information available on the underlying signaling mechanisms regulating triglyceride (TG) content in the body. In the current study, we have uncovered a role for protein kinase Cbeta (PKCbeta) in TG homeostasis by studying the consequences of a targeted disruption of this kinase. PKCbeta(-/-) mutant mice were considerably leaner and the size of white fat depots was markedly decreased compared with wild-type littermates. TG content in the liver and skeletal muscle of PKCbeta(-/-) mice was also significantly low. Interestingly, mutant animals were hyperphagic and exhibited higher food intake and reduced feed efficiency versus wild type. The protection from obesity involves elevated oxygen consumption/energy expenditure and increased fatty acid oxidation in adipose tissue with concurrent increased mitochondria genesis, up-regulation of PGC-1alpha and UCP-2, and down-regulation of perilipin. The ability of PKCbeta deficiency to promote fat burning in adipocytes may suggest novel therapeutic strategies for obesity and obesity-related disorders.

Selective repression of low-density lipoprotein receptor expression by SP600125: coupling of histone H3-Ser10 phosphorylation and Sp1 occupancy.

In this study, we show that exposure of human hepatocellular HepG2 cells to SP600125 rapidly and dramatically reduced global histone H3-Ser10 phosphorylation, without significantly affecting the global acetylation of neighboring lysines. The loss of phosphorylation is not due to changes in cell cycle distribution and/or apoptosis and is mediated independent of either p46/54(JNK) or MSK-1/2 inhibition. Moreover, SP600125 repressed the basal expression of the endogenous LDL receptor in a gene-specific manner, whereas the expression of squalene synthase, sterol response element-binding protein-1, and beta-actin was not altered by SP600125. Finally, chromatin immunoprecipitation and in vivo footprinting assays provided direct evidence that localized histone H3-Ser10 dephosphorylation at the low-density lipoprotein receptor promoter was associated with a significant decrease in the occupancy of the Sp1 binding site, with a slight reduction in the occupancy of RNA polymerase II. Together, our findings show that SP600125 is an efficient inhibitor of histone H3-Ser10 phosphorylation in vivo, and our results led us to hypothesize that this modification plays a novel role in regulating transcriptional control by modulating promoter accessibility to maintain basal expression in a gene-specific manner.

Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: potential role of protein phosphatase 2A-protein kinase CbetaII complex.

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.

Phorbol ester promotes histone H3-Ser10 phosphorylation at the LDL receptor promoter in a protein kinase C-dependent manner.

Histone modification is emerging as a major regulatory mechanism for modulating gene expression by altering the accessibility of transcription factors to DNA. This study unravels the relationship between histone H3 modifications and LDL receptor induction, focusing also on routes by which phosphorylation is mediated in human hepatoma HepG2 cells. We show that while histone H3 is constitutively acetylated at LDL receptor chromatin, 12-O-tetradecanoylphorbol-13-acetate (TPA) causes rapid hyperphosphorylation of histone H3 on serine 10 (histone H3-Ser10), despite global reduction in its phosphorylation levels. Ser10 hyperphosphorylation precedes LDL receptor induction and is independent of the p42/44MAPK, p38MAPK, pp90RSK, or MSK-1 cascade. Interestingly, inhibition of protein kinase C (PKC) blocks Ser10 hyperphosphorylation and also compromises LDL receptor induction by TPA. Consistent with its role, recombinant purified PKC phosphorylate purified histone H3-Ser10. Collectively, our findings highlight a novel role for PKC in regulating histone H3-Ser10 phosphorylation and suggest that histone modification provides numerous regulatory opportunities to set the overall range of control attainable for LDL receptor gene induction.

pp90RSK- and protein kinase C-dependent pathway regulates p42/44MAPK-induced LDL receptor transcription in HepG2 cells.

We have previously shown that different extracellular stimuli require signaling through the Raf/MEK/p42/44MAPK cascade to induce LDL receptor expression. The present studies were designed to delineate the molecular mechanisms underlying p42/44MAPK-induced LDL receptor transcription in HepG2-Delta Raf-1:ER cells, a modified HepG2 cell line in which the Raf-1/MEK/p42/44MAPK cascade can be specifically activated by anti-estradiol ICI182,780 in an agonist-specific manner. Using these cells, we show that: a) LDL receptor induction was reduced in reporter constructs containing mutation in either Sp1 or sterol-regulatory element-1 (SRE-1) sites, whereas inactivation of both sites abolished the induction; b) E1A, which inhibits CREB binding protein (CBP), a common activator of SRE-1 binding protein and Sp1, strongly repressed the induction; c) intracellular inhibition of the 90 kDa ribosomal S6 kinase (pp90RSK) cascade reduced LDL receptor induction; d) highly selective protein kinase C (PKC) inhibitors effectively abrogated the induction without affecting activation of pp90RSK; and e) overexpression of PKC beta significantly induced LDL receptor promoter activity. Taken together, these results demonstrate that pp90RSK and PKC beta are downstream effectors and Sp1, SRE-1 binding protein, and CBP are part of the transcriptional complex resulting in induction of LDL receptor expression in response to activation of the Raf/MEK/p42/44MAPK cascade. These findings identify for the first time a role for PKC beta in determining the specificity of p42/44MAPK signaling by participating with pp90RSK in regulating gene expression.

Activation of Raf-1/MEK-1/2/p42/44(MAPK) cascade alone is sufficient to uncouple LDL receptor expression from cell growth.

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.