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

Abstract

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-β (PKCβ) in hepatic adaptation to high-fat/cholesterol diets was gained recently through the use of knockout mice. The emerging evidence indicates that PKCβ 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 PKCβ expression and signaling in the intestine and liver, while systemic PKCβ deficiency promotes accumulation of cholesterol in the liver and bile. PKCβ 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 PKCβ signaling converge on liver ERK1/2 to dictate diet-induced cholesterol and bile acid homeostasis. Collectively, PKCβ is an integrator of dietary fat/cholesterol signal and mediates changes to cholesterol homeostasis.

the western diet is an important environmental factor that predisposes to various metabolic diseases, including atherosclerosis and cholesterol gallstone formation (24, 44, 47, 73, 83, 99). Such diet usually consists of complex combinations of lipids (cholesterol, fat, etc.) that might act synergistically with intestinal bile acids to aggravate dysregulation of cholesterol homeostasis (43, 85, 90, 94, 100). Abnormal levels of cholesterol can have serious cellular consequences and can affect onset of the above-mentioned cholesterol-related metabolic diseases (24, 44, 83).

The liver plays an important role in maintaining body’s cholesterol homeostasis by regulating absorption and synthesis to prevent net accumulation of cholesterol in the plasma and tissues (28, 29). The small intestine also has a major impact on cholesterol homeostasis at the level of cholesterol and bile absorption, fecal excretion, and de novo synthesis. Dietary fats have been shown to alter several metabolic pathways in the liver and intestine, the combined effects of which are reflected by elevations in liver cholesterol levels as well as plasma lipid and lipoprotein profiles. Stringent adaptation of cholesterol homeostasis to dietary fat/cholesterol intake is essential, because the Western diet delivers fat and cholesterol simultaneously from animal sources. In addition, recent studies have shown that dietary fat and dietary cholesterol act synergistically to impair liver metabolism, including cholesterol homeostasis (43, 85, 90, 94, 100). Although significant advances have been made in resolving the molecular identity of individual transcription factors sensitive to fat, cholesterol, or bile acids (11, 26, 48, 49), the dietary signals that sense a combined load of dietary fat and cholesterol and fine-tune cholesterol regulatory network to handle such dietary load are only partially understood. In particular, the critical signaling links and the underlying mechanisms in the body during gut and liver adaptations to such diets remain insufficiently explored. Above all, it is also unclear whether body mounts a specific “defense” mechanism to counteract detrimental effects of the combined fat/cholesterol dietary load on hepatic and whole-body cholesterol homeostasis. Assessing the impact of a high-fat/cholesterol load, rather than an individual component alone, on cholesterol homeostasis should be more physiologically relevant.

Liver, intestine, and cross talk between them play a critical role in cholesterol homeostasis via regulatory networks of transcription factors that translate signals evoked by dietary cholesterol into selective gene expression. Cholesterol homeostatic control is achieved by coordinated actions of several transcription factors, and the recent work from several laboratories suggests that the sterol regulatory element-binding protein (SREBP) and liver X receptor (LXR) transcriptional pathways work in a coordinated and reciprocal fashion to maintain cellular and systemic cholesterol homeostasis (Fig. 1) (11, 29, 49). SREBP regulates the biosynthesis and uptake of cholesterol, whereas LXR family is critical for the elimination of excess cholesterol. The SREBP and LXR transcription factors also work together with the farnesoid X receptor (FXR) to integrate cholesterol homeostasis through regulating bile acid metabolism. In the enterohepatic system, FXR plays a major role in determining the expression levels of genes involved in the maintenance of cholesterol, bile acid, and triglyceride homeostasis. FXR-regulated fibroblast growth factor 15/19 (FGF15/19) is reported to regulate cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase gene (Cyp8b1) expression and thereby bile acid biosynthesis and hydrophobicity (75). Cyp7a1 is the rate-limiting enzyme in bile acid biosynthesis, whereas Cyp8b1 is critical in regulating bile hydrophobicity of the bile acid pool by regulating the cholic acid-to-chenodeoxycholic acid ratio (14).

Fig. 1.

An overview of key signaling kinases (PKCs and ERK1/2) targeting transcription factors involved critical for cholesterol homeostasis. Cholesterol homeostasis is governed by 3 key transcription factors: SREBP-2, LXR, and FXR. SREBP-2 upregulates target genes involved in cholesterol biosynthesis and uptake in response to low-cellular cholesterol, whereas cholesterol catabolism and efflux are promoted through LXR in response to excess cholesterol. Bile acids activate FXR, which reduces conversion of cholesterol to bile acids by downregulating the expression of enzymes involved in bile acid synthesis, such as Cyp7a1 and Cyp8b1. It also promotes fatty acid synthesis via induction of SREBP-1c and its targets. Secretion of triglyceride-rich very low-density lipoproteins by the liver transports lipids to peripheral tissues. Bile acid-sensitive FXR reduces conversion of cholesterol to bile acids by downregulating expression of Cyp7a1 and Cyp8b1 genes. FXR also promotes the transport of bile acids to the gallbladder via bile salt export pump, multidrug resistance proteins 2 and 3. Within the intestine, FXR reduces the bile acid absorption via downregulation of the apical sodium-dependent bile acid transporter, promotes bile acid movement across the enterocytes via ileal bile acid binding protein, and promotes recycling of bile acids to the liver. FXR also promotes the release of FGF15 in mice or FGF19 in humans from the intestine. FGF15/19 travels to the liver, acting on FGF4 receptor to reduce Cyp7a1 expression and thus repress bile acid synthesis. FXR reduces lipogenesis via inhibition of SREBP-1c. Red and orange arrows indicate ERK1/2- and PKC-dependent metabolic steps, respectively. HDL, High-density lipoprotein; HDLR, HDL receptor; LDL, Low-density lipoprotein; LDLR, LDL receptor; VLDL, Very low density lipoprotein.

In addition to the above classical regulators of cholesterol and bile metabolism, endogenous bile acids can also activate the pregnane X receptor and constitutive androstane receptor, which are known to regulate genes responsible for the detoxification and elimination of a broad spectrum of potentially toxic endogenous and exogenous compounds (21, 84, 92). Likewise, vitamin D receptor (VDR) is also an intestinal sensor for secondary bile acids such as lithocholic acid (62). Altered functions of these nuclear receptors are involved in both pathogenesis and adaptation to cholestatic liver diseases (93). The above nuclear receptors utilize distinct combinations of transcriptional cofactors to effectively regulate their target genes at the transcriptional level (77, 86).

Accumulating evidence supports that above transcription factors are subject to extensive transcriptional, posttranscriptional, and posttranslational regulation (Table 1). While posttranslational regulatory mechanisms (such as phosphorylation and acetylation) control activity and subcellular localization for rapid changes in activity, transcriptional mechanisms account for intermediate and long-term changes in expression. The coordinated transcriptional and posttranscriptional regulation of transcription factors and cofactors enables the liver to rapidly respond to changes in cholesterol and bile acid homeostasis (55).

Table 1.

Summary of the reported effects of PKCs and ERK1/2 on proteins involved in cholesterol homeostasis

Kinase	Protein and Effect of Phosphorylation on Function	Known Role of Phosphorylated Protein	References
PKCα	LXR↓	Cholesterol transport and modulation; reverse cholesterol transport; cholesterol uptake; bile acid metabolism intestinal absorption and excretion	16
PKCα/βΙ	FXR↑	Bile acid synthesis, secretion, transport and detoxification	87
PKC	ABCA1↑	Cholesterol efflux	90, 92
PKCβ	VDR↑	Bile acid metabolism	32
PKC	PXR↓	Bile acid metabolism	18
PKC	RXR↑	Bile acid metabolism	94
ERK-1/2	SREBP-2↑	Cholesterol biosynthesis	3, 47
ERK-1/2	SHP↓	Bile acid metabolism	60
GSK-3β	SREBPs	Cholesterol and fatty acid biosynthesis	87

Up arrow indicates activation, and down arrow indicates repression.

Besides classical transcriptional regulators of cholesterol metabolism, recent studies have highlighted the importance of noncoding RNAs, termed microRNAs (miRNAs), as important posttranscriptional regulators of cholesterol homeostasis (22, 67). In particular, microRNA-33 (miR-33) has been shown to downregulate expression of ABCA1 and ABCG1 to reduce cholesterol efflux and high-density lipoprotein biogenesis (80, 81). Other miRNAs, such as miR-122, miR-370, miR-378, miR-125a, miR-27, and miR-355, have also been shown to regulate cholesterol homeostasis (22).

Studies Implicating PKC in the Regulation of Diet-Induced Cholesterol and Bile Acid Homeostasis

Protein kinase C (PKC) family plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis (70). The PKC family comprises a family of lipid-activated enzymes that are structurally and functionally similar and are categorized into conventional (α, βI, βII, and γ; require diacylglycerol and calcium for activation), novel (δ, ε, η, and θ; require only diacylglycerol for activation), and atypical (ζ and λ; require neither diacylglycerol nor calcium for activation) isoforms (71). The general structure of a PKC molecule consists of a catalytic and a regulatory domain found at the COOH- and NH₂-terminus, respectively. In the inactive state, the regulatory region (composed of a conserved C1 and C2 domain) is bound to the catalytic region and inhibits the activity of the enzyme. Dissociation of this intramolecular inhibitory interaction results in activation of the enzyme. Although functional role of individual PKC isoenzymes, many of which are coexpressed in the same cell, are poorly studied, emerging studies support that different PKC isoforms display highly distinct functions in vivo (87). The functional differences are due in part to the differential expression profiles of isoforms and partially caused by the various biochemical properties responsible for differential integration of the PKC isoforms into signaling networks and transcriptional complexes.

Several previous studies have indirectly suggested roles of the PKC family in modulating the cholesterol and bile acid homeostasis and are briefly summarized here: First, dietary constituents (bile acids, cholesterol, and to some extent fatty acids) as well as diacylglycerol are reported to activate PKCs including PKCβ, and according to many reports do so in a synergistic manner (4, 17, 27, 33, 46, 56, 60, 69, 74, 78). Accordingly, uptake of various lipoproteins is associated with the induction of PKCβ expression in various cell culture models (6, 10, 72, 82). PKC isoforms have also been implicated in the regulation of lipoprotein uptake (2, 10, 37, 54), and reverse cholesterol transport, such as modulating Abca1 stability and ApoAI-dependent efflux (98, 101, 104). Furthermore, tauroursodeoxycholic acid used for the treatment of cholestatic liver disease stimulates excretion of bile acids through Ca²⁺-dependent PKC activation (7). Lastly, PKCβ (Chr 7, 117.5 Mb, 65.7 cM) can be a positional candidate for Lith 22 gallstone susceptibility loci detected on chromosome 7, with peak linkage at 65 cM (51, 61). This linkage has a mechanistic basis since we found that PKCβ^−/− mice, and not PKCδ^−/− mice, show sensitivity to diet-induced gallstone formation (39). Second, PKCs have been shown to phosphorylate and regulate the activity of several nuclear receptors (Table 1). For example, VDR DNA binding and transactivation is inhibited by PKCβ (36), which is involved in regulating ileum fibroblast growth factor 15 (FGF15) expression and thereby bile acid synthesis (88); PKC phosphorylation of nuclear receptors, such as retinoid X receptor-α (RXRα), is shown to promote its cytoplasmic localization in cell culture models (95); Both PKCα and β are shown to phosphorylate and activate transactivation activity of farnesoid X receptor (FXR) (25), whereas PKCα is reported to inactivate LXRα transactivation (18); and human retinoid A receptor-α (RARα) can be phosphorylated by PKC resulting in a decrease in DNA binding activity, dimerization with RXRα, and transactivation (19). Recently, treatment by PKC activator phorbol 12-myristate 13-acetate (TPA) was shown to induce mitochondrial localization of orphan nuclear receptor Nurr77 and RXRα (9, 12, 31), as well as promote degradation of RXRα (103). Third, PKCβ can also regulate cholesterol homeostasis by modulating insulin signaling in the liver, as PKCβ is reported to disrupt insulin signaling cascade (45, 58, 59, 79). PKCβ is also shown to mediate insulin-induced liver SREBP-1c expression (102), and downstream mitogen/extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) is reported to modulate SREBP-2 transactivation (3, 52) (Table 1) and cholesterol-7α-hydroxylase (Cyp7a1) expression via small heterodimer partner modification (65). Moreover, PKCs are shown to inhibit glycogen synthase kinase-3β (30), which is shown to modulate SREBP degradation (96); Finally, ERK1/2 inhibition promotes very low-density lipoprotein (VLDL) assembly/secretion (97), whereas we have reported earlier that ERK1/2 also controls hepatic low-density lipoprotein (LDL) receptor expression levels (20, 50, 53, 54, 91). Despite the implications of PKCs in cellular mechanisms critically important to regulation of cholesterol and bile acid homeostasis by in vitro studies and cell culture models, no in vivo evidence existed to suggest a physiologically relevant role for specific PKC isoforms in this process. Understanding the individual role of specific PKC isoforms has become more important with the development of several successful drugs that target this cell signaling pathway (16, 66).

Emerging Role of PKCβ in Adaptiveness to High-Fat/Cholesterol Diet

PKCβ belongs to a conventional PKC subfamily whose members require DAG and calcium for activation (70). Despite similarity of stimulatory agonists, it appears that individual PKC isoforms in this subfamily perform unique functions. For example, unlike systemic deletion of either PKCα (57) or PKCγ (1), PKCβ deletion in mice was found to have a significant impact on the body’s lipid and glucose metabolism (5, 37–42, 63). Emerging evidence suggests that PKCβ action is not only limited to triglyceride metabolism; its induction in the liver and intestinal tissues by high-fat/cholesterol diets, with or without cholic acid, enables this kinase to function as a metabolic adaptor of cholesterol homeostasis (39, 42).

Emergence of PKCβ as a specific regulator of cholesterol homeostasis is mainly supported by our observation that high-fat/cholesterol diets induce intestine and liver PKCβ expression, whereas deficiency of PKCβ expression in mice has profound effects on hepatic cholesterol metabolism, including increases in liver and plasma cholesterol, hepatic bile cholesterol saturation index, and hydrophobicity, as well as hypersensitivity to gallstone formation (39, 42). It is also accompanied by ERK1/2 activation and reduced expression of both Cyp7a1 and Cyp8b1 genes in the liver (39).

These results suggest that PKCβ is a critical link in an adaptive response for proper handling of high dietary fat and cholesterol intake by modulating hepatic ERK1/2 activity to coordinately regulate the cellular cholesterol biosynthesis, uptake, and degradation. Our data are consistent with a two-pronged mechanism by which intestinal and liver PKCβ deficiency converges on liver ERK1/2 to modify the expression of genes involved in cholesterol and bile acid homeostasis (Fig. 2). One pathway may cause the stimulation of intestinal FGF15 expression, leading to an increase in ERK1/2 activity in the liver via fibroblast growth factor receptor 4 and coreceptor β-Klotho, resulting in suppression of Cyp7a1 and Cyp8b1 gene transcription and the reduction of cholesterol catabolism (39). The other pathway may rely on the negative regulation of the Raf-1/MEK/ERK signaling axis by PKCβ itself in the liver, resulting in further activation of ERK1/2 to reduce cholesterol catabolism in PKCβ^−/− mice. Interestingly, negative in vivo regulation of the hepatic Raf/MEK/ERK cascade by PKCβ is unexpected, since two earlier in vitro studies using cell culture models suggest that PKCβ is required for ERK1/2 activation (23, 32). Both mechanisms may cooperate to maximize the effectiveness of PKCβ deficiency on hepatic ERK1/2 activation. It is interesting to note that ERK1/2 plays an important role in controlling hepatic LDL receptor expression (20, 50, 53, 54, 91), SREBP-2 expression (52), VLDL assembly (96), and cholesterol efflux (68, 104). The net effect of systemic PKCβ deficiency would be to promote hepatic cholesterol accumulation, as was observed for PKCβ^−/− mice. This model has the potential to explain how PKCβ, either directly or indirectly through ERK1/2, caused differential expression of the genes involved in the cholesterol uptake, biosynthesis, and catabolic reduction of the hepatic cholesterol burden. Establishing PKCβ as the prime kinase involved in fine tuning hepatic ERK1/2 activation in vivo in response to high-fat/cholesterol diet introduces a new model that can be used to investigate the FGF15 regulatory mechanisms within a functional context.

Fig. 2.

Proposed model for the mechanism by which PKCβ acts as a major regulator of cholesterol homeostasis, allowing adaptation to high-fat/cholesterol intake. It appears that PKCβ defends hepatic cholesterol homeostasis by inducing cholesterol excretion pathways, while reducing cholesterol synthesis and uptake pathways. In such a way, PKCβ may modulate bile lipid composition and thus the susceptibility to cholesterol gallstone formation. The underlying mechanism is consistent with a 2-pronged mechanism by which PKCβ deficiency contributes to dysfunctional cholesterol homeostasis through disturbing PKCβ/FGF15/ERK and Raf-1/MEK/ERK regulatory axes critical for hepatic and systemic cholesterol homeostasis. Emerging evidence indicates that these nuclear receptors have essential roles not only in the regulation of cholesterol and bile acid metabolism, but also in the integration of sterol, fatty acid, and glucose metabolism. CSI, bile cholesterol saturation index; HI, bile hydrophobicity index.

It is possible that greater insulin sensitivity might also play a role in increased ERK1/2 activation in PKCβ^−/− liver of animals fed a high-fat/cholesterol diet. PKCβ has been reported to inhibit several components of the insulin signaling cascade (45, 79). Overexpression of PKCβ in the liver, similar to its overexpression in muscle (35), may result in hepatic insulin resistance, a condition known to affect ERK1/2 activation and the expression of both Cyp7a1 and Cyp8b1 genes. Insulin has been shown to initiate signaling cascades through protein kinase B (Akt) and ERK1/2 signaling pathways and has also been shown to act synergistically with FGF15 to activate these critical signaling pathways (76, 89). Further studies are needed to determine how insulin- and FGF15-specific phosphorylation of ERK1/2 is coordinated with PKCβ-dependent signaling events to respond appropriately to environmental conditions. It is possible the detrimental effect on cholesterol homeostasis exerted by PKCβ deficiency may supersede the potential protective effect offered by insulin sensitivity on gallstone formation (8).

A final consideration is that nutrition plays a major role in determining health of not only the individual, but also of the next generation. There is emerging evidence that, in addition to more traditional regulatory schemes outlined above (Fig. 2), cholesterol homeostasis is also governed by epigenetic mechanisms such as histone phosphorylation and acetylation (34). Interestingly, both PKC and ERK signaling can affect chromatin modifications in multiple ways through phosphorylation of transcription factors, which recruit chromatin-modifying complexes, and/or through direct phosphorylation of histones (13, 15, 37, 38, 64). PKCβ and downstream ERK1/2, being histone kinases, can change gene expression by modifying epigenetic marks and can thus directly link dietary lipids with epigenetic changes.

Concluding Remarks

Frequent metabolic abnormalities such as atherosclerosis and gallstone formation are related to impaired cholesterol homeostasis (24, 44, 73, 99). Understanding of cholesterol homeostasis in response to dietary fat or cholesterol has advanced significantly (11, 26, 48, 49), however much insight is needed into the mechanisms that tend to minimize fluctuations in the amount of cholesterol in the body by combined high-fat/cholesterol load. We provide a brief overview of how studies based on PKCβ^−/− mice may be instrumental in leading to an understanding of the defense mechanism counteracting the deleterious effects of lithogenic stress and some of its implications. Emerging evidence supports the possibility that PKCβ deficiency triggers a cascade of reactions aimed at decreasing hepatic cholesterol to maintain homeostasis. The underlying mechanism appears to involve PKCβ-mediated regulatory loops, leading to the upregulation of genes involved in hepatic bile synthesis, while hepatocellular uptake of cholesterol and cholesterol biosynthesis are inhibited (42). The mechanism through which PKCβ regulates cholesterol homeostasis is incompletely defined, in particular the regulation of ileum FGF15 expression and its physiological significance. Another unanswered question that is of considerable interest is the nature of the signal that initiates activation of PKCβ expression. Is the PKCβ promoter targeted by nuclear receptors and SREBPs? More detailed information is also needed to define the downstream components of the PKCβ signaling pathway regulated by dietary fat/cholesterol intake. For example, is cholesterol efflux regulated by PKCβ and/or ERK (68, 101, 104)? Are there unidentified PKCβ/ERK-dependent targets involved in the effect? Does PKCβ serve as a point of cross talk between signaling pathways by integrating transcriptional inputs on different gene networks? Does PKCβ overexpression in the liver correct the diet-induced dysregulated cholesterol homeostasis? These and many other questions remain to be answered before an understanding of how PKCβ signaling precisely regulate cholesterol and bile acid homeostasis. An important and emergent area, in terms of both physiology and therapeutic exploitation, is the role liver and intestinal PKCβ play in maintaining cholesterol and bile acid homeostasis. It is also anticipated that a greater understanding of the role of PKCβ in diet-induced cholesterol homeostasis may lead to more effective therapeutic strategies for highly prevalent cholesterol-related diseases. Furthermore, if epigenetic events are controllable through dietary interventions via PKCβ, then a new therapeutic approach to atherosclerosis and gallstones is possible. The connections between signaling pathways such as PKCβ to lipid metabolism and epigenetic gene regulation make the explanation of this concept possible in the near future. Finally, even though new insights have been obtained using animal models, more studies are needed to establish a definite relevance of PKCβ to human metabolic diseases.

GRANTS

This review article is based on works supported in part by grants from the Ohio State University Wexner Medical Center and the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

D.M. prepared figures; D.M. drafted manuscript; D.M. and K.D.M. edited and revised manuscript; K.D.M. approved final version of manuscript.