MAP Kinase Pathways in the Control of Hepatocyte Growth, Metabolism and Survival

MAP Kinase Pathways

in the Control of Hepatocyte Growth, Metabolism and Survival

Paul Dent

19

19.1

Introduction

In the past 15 years, multiple new signal transduction pathways within cells have been discovered. Many of these pathways belong to what is now termed “the mitogen-activated protein kinase (MAPK) super- family.” The roles of each MAPK pathway in the control of hepatocyte growth, metabolism and sur- vival are still under investigation. It is believed that exposure of hepatocytes to growth factors induces simultaneous activation of multiple MAPK path- ways that act in a coordinated manner to control cell growth, survival and metabolism. In addition, bile acids have more recently been shown to activate growth factor and death receptors in hepatocytes, which can interact with growth factor-induced sig- naling to generate a further layer of complexity in the activation of MAPK pathways. The expression and release of growth factors such as tumor necro- sis factor- α (TNF-α) from Kupffer cells during liver regeneration also has the potential to modulate the responses of MAPK pathways in hepatocytes. Thus the ability of growth factors and bile acids to acti- vate MAPK signaling pathways in hepatocytes may depend on the expression of multiple growth factor receptors, the synthesis of paracrine factors and bile acid levels. This review will describe the enzymes within four major MAPK signaling pathways and discuss their activation and roles in hepatic prolif- erative, survival and metabolic responses.

19.2

The "Classical" Mitogen-Activated Protein Kinase/Extracellular Regulated Kinase Pathway

"Mitogen-activated protein-2 kinase" was first re- ported by the laboratory of Dr. Thomas Sturgill in 1986 [131]. This protein kinase was originally described as a 42-kDa insulin-stimulated protein

kinase activity whose tyrosine phosphorylation in- creased after insulin exposure, and which phospho- rylated the cytoskeletal protein MAP-2. Contempo- raneous studies from the laboratory of Dr. Melanie Cobb identified an additional 44-kDa isoform of this enzyme, termed extracellular signal-regulated kinase (ERK1) [9]. Since many growth factors and mitogens could activate these enzymes, the acronym for this enzyme was subsequently changed to denote mitogen-activated protein (MAP) kinase. Addition- al studies demonstrated that the p42 (ERK2) and p44 (ERK1) MAP kinases regulated another protein kinase activity (p90

) [132], and that they were themselves regulated by protein kinase activities designated MKK1/2 (MAPK kinase; MAP2K), also termed MEK1/2 [154, 155]. MKK1 and MKK2 were also regulated by reversible phosphorylation. The protein kinase responsible for catalyzing MKK1/2 activation was initially described as the proto-on- cogene Raf-1 [19, 41]. This was soon followed by an- other MEK1/2-activating kinase, termed MEKK1, which was a mammalian homolog with similarity to the yeast Ste 11 and Byr 2 genes [74]. However, fur- ther studies have shown that the primary function of MEKK1 is to regulate the c-Jun NH2-terminal kinase (JNK)1/2, rather than the ERK1/2, pathway [157].

19.2.1

Growth Factor Receptors, Ras and Raf

Plasma membrane receptors transduce signals through the membrane to its inner leaflet, leading to the recruitment and activation of guanine nucle- otide exchange factors, which increase the amount of GTP bound to membrane-associated GTP-bind- ing proteins, in particular Ras family proteins [97]

(Fig. 19.1). There are four widely recognized isofor-

ms of Ras: Harvey (H), Kirsten (K4A, K4B) and Neu-

roblastoma (N) [114]. Receptor-stimulated guanine

nucleotide exchange of “Ras” to the GTP-bound

form permits Raf proteins and P110 phosphatidyl

inositol (PI) 3-kinase to associate with “Ras”, result- ing in kinase translocation to the plasma membrane environment where activation of these kinases takes place. Ras contains a GTPase activity that converts bound GTP to GDP resulting in inactivation of the Ras molecule. Mutation of Ras results in a loss of GTPase activity, generating a constitutively active Ras molecule that can lead to elevated activity with- in downstream signaling pathways. Approximately one third of human cancers have Ras mutations, pri- marily the K-Ras isoforms; however, Ras mutation is not a common occurrence in hepatoma, whereas it is relatively common in cholangiocarcinoma [136]. Of note, B-Raf mutations have also been shown to play a role in tumorigenesis in thyroid and cholangiocar- cinoma but not in hepatoma [63, 136].

Raf-1 is a member of a family of serine/threonine protein kinases also comprising B-Raf and A-Raf [104]. All “Raf” family members can phosphorylate MKK1/2 and activate the ERK1/2 pathway [140].

Thus the “Raf” kinases act at the level of a MAPK

kinase kinase (MAP3K). The NH

domain of Raf- 1 can reversibly interact with Ras family members in the plasma membrane and the ability of Raf-1 to associate with Ras is dependent upon the Ras mol- ecule being in the GTP-bound state [84, 92, 143].

Additional protein serine/threonine and tyrosine phosphorylation(s) are also known to play a role increasing Raf-1 activity when in the plasma mem- brane environment [20]. PKC isoforms, which can be activated by bile acids, have been proposed to be Raf-1-activating kinases [11]. The initial biochemi- cal analyses of purified Raf-1 demonstrated consti- tutive Y340-Y341 phosphorylation when the protein was co-expressed with Src [20, 41]. PI-3 kinase-de- pendent phosphorylation of S338 by PAK family en- zymes may facilitate Raf-1 tyrosine phosphorylation by Src family members, leading to full activation of Raf-1 [64]. PAK enzymes may also phosphorylate MEK1, promoting Raf-1–MEK1 association and thus pathway activation [14, 28]. In this regard, bile acids, compared to epidermal growth factor (EGF), potently enhance tyrosine phosphorylation of Raf-1 [111].

Phosphorylation of Raf-1 at S259 by either Akt or the cAMP-dependent-protein kinase (PKA) can inhibit Raf-1 activity and its activation by upstream stimuli [24]. Phosphorylation of Raf-1 at S43 by PKA inhibits the interaction of Raf-1 with Ras molecules, thereby blocking Raf-1 translocation to the plasma membrane and its Ras-dependent activation [156].

In contrast to Raf-1, the B-Raf isoform does not con- tain an equivalent to S43, but contains multiple sites of Akt (and potentially PKA)-mediated phosphor- ylation in addition to the B-Raf equivalent of S259 [42]. B-Raf can be activated by both Ras and cAMP via the Rap1 GTPase [162]. Thus the regulation of the MEK1/2-ERK1/2 pathway is very complex, and in some cell types may be both inhibited by cAMP/

PKA, through Raf-1, and stimulated by cAMP/Rap1, through B-Raf.

These findings may have considerable impact on catecholamine signaling in hepatocytes during liver regeneration. During hepatocyte proliferation/liver regeneration, the expression of α

-adrenergic re- ceptors declines and the levels of β

-adrenergic re- ceptors increase. In non-proliferating hepatocytes, the α

-adrenergic receptor agonist phenylephrine enhances Raf-1 and ERK1/2 activity in a similar manner to growth factors such as EGF, and gluca- gon inhibits phenylephrine or EGF-induced ERK1/2 activity by blocking Raf-1 activation [129]. In prolif- erating hepatocytes, phenylephrine cannot activate Raf-1/ERK1/2 and furthermore, the β

-adrenergic receptor agonist isoproteranol inhibits EGF-in- duced ERK1/2 activation. As ERK1/2 signaling has been linked to a proliferative response in hepato-

Fig. 19.1. Some of the characterized signal transduction path- ways in mammalian cells. Growth factor receptors, e.g., the ERBB family, insulin receptor can signal down through GTP-binding proteins into multiple intracellular signal transduction path- ways. Predominant among these pathways are the MAP kinase superfamily of cascades (ERK1/2, ERK5, JNK, p38). Growth fac- tor receptors, Ras proteins and downstream pathways are often activated in tumor cells and inhibitors have been developed to block the function of these molecules, thereby slowing cell growth and promoting cell death responses. Multiple inhibi- tors for the ERBB family receptors have been developed, e.g., the ERBB1 inhibitor AG1478. Inhibitors of Ras farnesylation (and geranylgeranylation) are in clinical trials, as are inhibitors of the ERK1/2/5 pathway. It should be noted that MEK1/2 inhibitors PD98059 and U0126 are also capable of inhibiting the “Big” MAP kinase pathway via blocking activation of MEK5

cytes, these findings suggest that catecholamines may initially promote, and then inhibit, rodent liver regeneration.

In gene deletion studies, loss of Raf-1 function was embryonically lethal due to weak placental an- giogenesis and hepatoblast apoptosis [152, 153]: in Raf-1 null hepatocytes, ERK1/2 was activated by B- Raf rather than Raf-1, which suggests: (a) fetal hepa- tocytes may utilize different “Raf” molecules to ac- tivate the ERK1/2 pathway in comparison to adult hepatocytes and (b) deletion of Raf-1 caused hepa- tocytes to survive by recruiting in compensation B- Raf as the MAP3K activator for ERK1/2, which will tend to promote growth arrest over proliferation.

With reference to comment (a), the relative ability of Raf-1 and B-Raf to activate the ERK1/2 pathway in primary hepatocytes and established HepG2 and HuH7 hepatoma cells is also different: in primary hepatocytes and HepG2 cells, ERK1/2 pathway acti- vation by growth factors is dependent on Raf-1 and inhibited by cAMP [3], whereas in HuH7 cells, B-Raf and ERK1/2 activation can be enhanced by cAMP (P.B. Hylemon and P. Dent, unpublished data). This implies primary and fetal hepatocytes, and estab- lished liver-derived cell lines can have very different signaling behavioral characteristics.

19.2.2

Hepatocyte Proliferation and ERK1/2 Signaling

There are multiple studies that link ERK1/2 signal- ing to hepatocyte proliferation and present evidence that inhibition of MEK1/2/5 blunts the induction of proteins essential for G1 phase progression and S phase entry, such as cyclin D1, PCNA and cyclin A [15, 22, 27, 34, 51, 61, 77, 100, 101, 106, 113, 135, 140]. Activation of ERK1/2 by constitutive or induc- ible Raf-1 and MEK1 constructs can enhance cyclin D1 expression in hepatocytes [22, 34, 100, 101]. One in vivo study using PD98059 has shown inhibition of liver regeneration; however, as PD98059 causes acute kidney toxicity, these results may not be solely due to direct effects on liver biology [135]. PD98059 acts as an inhibitor of “Raf”-mediated phosphoryla- tion of MEK1/2/5 molecules but it is not a kinase domain inhibitor of “Raf” proteins or of activated MEK1: in cells PD98059 is a relatively good inhibitor of MEK1 and MEK5 (IC50 ~5 µM) but a much poorer inhibitor of MEK2 (IC50 ~40 µM) [17]. U0126 is re- ported as a more equipotent inhibitor of MEK1/2/5 that blocks both activating phosphorylations on the MEK1/2/5 proteins as well as their kinase domain activity, with IC50 values in the ~0.5 µM range [17].

The clinically used MEK1/2 inhibitor PD184352

(CI1040) acts in a similar potent manner to U0126 and at concentrations below 10 µM has shown spe- cificity for MEK1/2 over MEK5 [91, 130]; it may be a useful tool to demarcate between MEK1/2-ERK1/2 and MEK5-ERK5 signaling dependencies.

The livers of mice deleted for insulin growth factor 1-binding protein, which regenerate poorly, have more recently been shown to lack an ERK1/2 activation response after partial hepatectomy as well as expression of the ERK1/2 downstream tar- get C/EBP β [6, 77]. The role of the cyclin-depend- ent kinase (CDK) inhibitor p21

(p21) and other CDK inhibitors in hepatocyte growth has also been extensively investigated [3, 6, 52, 87, 93, 142, 148]. These findings are very similar to those in other cell types. Hepatocytes from older rodents, which have reduced liver regeneration capability, have higher basal expression of the cyclin kinase inhibitor p21 and lower growth factor-induced ac- tivation of ERK1/2 [138]. These findings are also in agreement with data arguing that the livers of p21 – /– mice regenerate more quickly than those of wild- type animals [1]. In hepatocytes ERK1/2 signaling positively regulates the p21 promoter via the tran- scription factors C/EBP β and Ets2, and to a lesser extent C/EBP α [100, 101]. In addition, ERK1/2 sig- naling enhances p21 mRNA stability and increases p21 protein half life (potentially by phosphorylation at T57 and S130) [62]. In hepatocytes that cannot express p21, proliferation is stimulated by intense ERK1/2 signaling that is dependent on C/EBP β and Ets2 [10, 101].

A number of other studies using various exog-

enous agents that cause prolonged intense ERK1/2

pathway activation in hepatocytes and hepatoma

cells have suggested that this signal is antiprolif-

erative. The novel vitamin K analog Cpd5 and vi-

tamin K3 (menadione) potently enhanced ERK1/2

signaling in hepatocytes, which was associated with

growth suppression [57, 145]. In HepG2 cells stably

transfected with the α

-adrenergic receptor, phe-

nylephrine induced prolonged intense ERK1/2 sig-

naling that was responsible for induction of p21 and

growth arrest; antisense mRNA ablation of p21 pro-

tein expression converted prolonged intense ERK1/2

activation into a growth-promoting signal [3, 4],

which was subsequently confirmed [142]. Acute

ethanol exposure of hepatocytes in vitro combined

with growth factor treatment promotes superinduc-

tion of ERK1/2 signaling, and of p21 and p16 expres-

sion, leading to growth arrest, which is in general

agreement with the known antiproliferative effects

of ethanol on liver regeneration [140]. Chronic etha-

nol exposure of animals appears to block liver regen-

eration, not by promoting, but instead by inhibiting,

growth factor-induced activation of ERK1/2 [13] and

by promoting a p38 MAPK-dependent increase in p21 expression [68]. Chronic exposure of animals to xenobiotic chemical agents that block liver regener- ation can also alter the ability of the liver function- ally to activate the ERK1/2 pathway, for example, the carcinogenic drug 2-acetylamino fluorine does not block EGF-induced activation of ERK1/2 but pre- vents nuclear translocation of ERK1/2, blunting en- hanced transcription factor function(s) [127]. Hepa- titis B virus (HBV) X protein enhances or suppress- es p21 expression in a cell type-dependent manner [67, 75, 76, 102, 106]. For example, in some estab-

lished hepatocyte cell types, X protein enhances ERK1/2 and promotes cell proliferation [7, 66], whereas in primary cells it can promote activation of MAPK pathways and enhance expression of p21 and p27

, leading to growth arrest [75, 106]. Genetic differences between primary and established hepa- tocyte cells as well as different expression levels of X protein in cells may explain these discrepancies.

In many tumor cell types, it was initially noted that ERK1/2 activity was elevated compared to non- transformed “normal” tissues. Immunohistochemi- cal analyses have shown that liver tumors have ele- vated levels of phospho-ERK1/2 [51]. A strong corre- lation between reduced hepatoma cell proliferation and the pathway inhibitory actions of MEK1/2/5 in- hibitors has been noted, suggesting that the ERK1/2 pathway could be a useful therapeutic target in the treatment of hepatocellular carcinoma [e.g., 3, 53].

19.2.3

ERK1/2 Signaling, Apoptosis and Bile Acids

Activation of the ERK1/2 pathway in many cell types is associated not only with proliferation but also with protection from toxic stresses. For exam- ple, exposure of hepatoma cells to transforming growth factor beta (TGF β) can increase ERK1/2, JNK1/2 and p38 activity: inhibition of ERK1/2 sig- naling promotes further JNK1/2 activation and en- hances cell death [103]. Serum and insulin have also

Fig. 19.2a, b. Inhibition of ERK1/2 and PI-3 kinase enhances bile acid toxicity in hepatocytes. a Hepatocytes were treated with the indicated bile acid (50 µM) in the presence or absence of either PD98059 (50 µM), U0126 (10 µM) or PD184352 (10 µM) for 6 h. Ap- optosis was determined by morphological examination of slides as noted in [105]. b Hepatocytes were treated with either 1 µM AG1478 (ERBB1 inhibitor) or 1 µM AG1024 (INS-R, IGF-1-R inhibi- tor) in the presence or absence of DCA (50 µM) for 0–120 min.

ERK1/2, AKT and GSK3 activity was determined by phospho-im- munoblotting as described [105, 110]

Fig. 19.3. Inhibition of ERBB1 using AG1478 promotes and in- hibits DCA-induced apoptosis, which is dependent on the con- centration of DCA. Hepatocytes were treated with 1 µM AG1478 (ERBB1 inhibitor) in the presence or absence of DCA (50 µM;

250 µM; 450 µM) for 6 h. Apoptosis was determined by mor- phological examination of slides as noted in [105, 110]. Arrows indicate enhancement, no effect or inhibition of apoptosis by AG1478

been shown to mediate their anti-apoptotic effects in hepatoma cells via ERK1/2 signaling [56, 90]. In some instances, prolonged intense ERK1/2 activa- tion has been shown to promote cell death. The agent Cpd5 can cause prolonged intense ERBB1-ERK1/2 activation in hepatocytes, which is responsible for cell death [144]. Menadione and fatty acids promote intense ERK1/2 activation in established RALA255- 10G hepatocytes: yet inhibition of ERK1/2 signaling promotes menadione toxicity but inhibits fatty acid toxicity (Dr. M. Czaja, personal communication).

In general, it appears that ERK1/2 (and PI-3 kinase) activation is a compensatory survival re- sponse to balance potential toxic signals, such as bile acid exposure. This laboratory has shown that unconjugated forms of deoxycholic acid (DCA), CDCA and ursodeoxycholic acid (UDCA) all acti- vate the ERK1/2 pathway in primary hepatocytes, which was partially dependent on the PI-3 ki- nase pathway, and that inhibition of MEK1/2 with

PD98059, U0126 or PD184352 enhances bile acid toxicity [105, 108–110] (Fig. 19.2a). Similar data for ERK1/2 protection against DCA toxicity have been generated in colon cancer cells [107]. Inhibition of PI-3 kinase with LY294002 is relatively less effec- tive at promoting unconjugated bile acid-induced apoptosis compared to that caused by a MEK1/2 inhibitor [105, 108]. In contrast to these findings, MEK1/2 inhibitors more weakly enhanced the tox- icity of conjugated bile acids, whereas PI-3 kinase inhibition more efficaciously promoted conjugated bile acid toxicity (P. Dent and P.B. Hylemon, unpub- lished observations) [119, 134, 147]. One likely possi- bility is that conjugated and unconjugated bile acids differentially activate alternate cassettes of growth factor receptors, in different subcellular locations, and thus the ERK1/2 and PI-3 kinase signaling path- ways, in a dose-dependent fashion.

Several independent studies have demonstrated that bile acid-induced ERK1/2 signaling is depend-

Fig. 19.4. Possible mechanisms by which ERK1/2 signaling could protect hepatocytes from toxic stresses. Apoptosis occurs following activation of effector caspases (e.g., caspases-3, -6, -7), which can be triggered by either the extrinsic or intrinsic path- ways. The extrinsic pathway is characteristically initiated by liga- tion of the Fas ligand with its receptor, leading to formation of the death-inducing signaling complex (DISC), which permits the Fas-associated death domain (FADD) to cleave and activate pro- caspase-8. Activated caspase-8 can activate effector caspases such as procaspase-3 or initiate mitochondrial injury via BID. The intrinsic, or mitochondrial pathway, becomes engaged follow- ing mitochondrial injury (e.g., loss of mitochondrial membrane potential; and/or release of pro-apoptotic proteins such as cyto- chrome c). Cytochrome c, in association with dATP, promotes the caspase-9-mediated activation of procaspase-3. Considerable

crosstalk exists between the intrinsic and extrinsic pathways.

For example, while caspase-8 directly activates caspase-3, it also cleaves and activates the pro-apoptotic BH3-only domain Bcl-2 family member BID, which then triggers cytochrome c release and results in further procaspase-3 activation. A large and ex- panding group of pro- and anti-apoptotic Bcl-2 family proteins has been described, which may act by modulating BAD/BIM interactions and mitochondrial pore function, or, in the case of IAP proteins, including c-FLIP molecules, by directly inhibiting caspase activation. The relevance of apoptosis for hepatic cell biology is underscored by accumulating evidence that diverse signaling pathways downstream of ERBB receptors, e.g., the ERK1/2 pathway, regulate cell survival and response to bile acids by modulating the apoptotic threshold

ent on the activation of growth factor receptors in hepatocytes, predominantly of the ERBB family, and, furthermore, in hepatoma cells, by paracrine ligands such as TGF α [47, 105, 108, 149, 160]. Oth- ers have shown some bile acids (TLCA) can bind to and directly activate serpentine receptors [60]. Ac- tivation of ERBB1 and the insulin receptor in pri- mary rodent hepatocytes by bile acids is dependent on mitochondria-dependent generation of reactive oxygen/nitrogen species and the inactivation of pro- tein tyrosine phosphatases (PTPase) [33, 105]. The generation and/or modulation of cellular reactive oxygen species (ROS) levels by bile acids has been described in detail by several groups [116, 117, 159].

As PTPases are ~100-fold more active as enzymes than protein kinases, a small reduction in cellu- lar PTPase activity will result in an increase in the steady-state tyrosine phosphorylation of the pro- tein kinases, e.g., receptor tyrosine kinases, whose phosphorylation they suppress. Hence, inhibition of DCA-stimulated ERBB1 or the insulin receptor sign- aling significantly blunts DCA-induced ERK1/2 ac- tivation (Fig. 19.2b). In some cell types, the insulin receptor can promote activation of ERBB1 via release of TGF α, as has been observed for bile acid-induced activation of ERBB1 in HuH7 hepatoma cells [149].

Treatment of primary hepatocytes with 50 µM DCA causes almost no induction of apoptosis within 6 h;

however, DCA toxicity is significantly enhanced by AG1478, an ERBB1 inhibitor (Fig. 19.3) [105]. Treat- ment of hepatocytes with either 250 µM or 450 µM DCA causes a considerably greater increase in hepa- tocyte apoptosis within 6 h: AG1478 does not poten- tiate 250 µM DCA-induced killing, despite signifi- cantly blocking PI-3 kinase and ERK1/2 activation [47, 105], and reduces 450 µM DCA-induced hepa- tocyte death. These findings may explain the con- flicting findings reported by different groups using lower and higher concentrations of toxic bile acids, e.g., [105, 112]. Hence bile acid-induced activation of growth factor receptors may promote both protec- tive and toxic cellular responses that are dependent on the concentration, and presumably duration, of bile acid exposure. It should be noted that all of the above findings in bile acid-treated hepatocytes have similarities to observations made in carcinoma cells exposed to UV and ionizing radiation [21].

The mechanisms by which ERK1/2 signaling protects hepatocytes from toxic stresses are likely to be found at multiple levels within the cell (Fig. 19.4).

First, ERK1/2 signaling regulates the activities of transcription factors that are known to elicit cy- toprotective responses, e.g., CREB, C/EBP β [101, 108]. These factors in turn can alter the transcrip- tion of diverse pro- and anti-apoptotic genes such as PUMA, c-FLIP isoforms, Mcl-1 and Bcl-

[8, 55,

98, 110]. Second, in various cells ERK1/2 and p90

may directly phosphorylate and inhibit the func- tions of pro-apoptotic proteins, e.g., BAD, BIM, pro- caspase 9 [2, 32, 69, 78, 79, 105, 110, 121, 147]. Third, ERK1/2 pathway signaling can promote or inhibit the expression of both protective and toxic growth factors, e.g., TNF- α, EGF [49]. Finally, bile acid-in- duced ERK1/2 signaling could potentially regulate both the expression of MDM2, the E3 ligase that can ubiquinate the tumor suppressor p53; as well as ex- pression of p19

, which blocks the ubiquination of p53 by MDM2. In a variety of cell types the bal- ance of MDM2 versus p19 expression can determine p53 levels, and those of p21, as well as the apoptotic threshold [115]. While expression of p21 in many cell systems has been associated with a protection of toxic stresses, p21 can enhance bile acid toxicity in hepatocytes [72, 109]. Thus ERK1/2 signaling may have a dual protective and toxic nature in hepato- cytes; short-term activation of ERK1/2 promotes cell survival after a toxic insult, whereas prolonged ERK1/2 signaling (>~24 h) may enhance cell killing [144].

19.2.4

ERK1/2 Signaling and Metabolism

Activation of ERK1/2 can enhance plasma mem- brane translocation of the glucose transporter GLUT4 in adipocytes and myotubes [120]. In some cell types, ERK1/2 signaling can also control the activity of glycogen synthase kinase 3 (GSK3) and that of serine/threonine protein phosphatase 1, co- localized on the glycogen particles, which could in- fluence the rate of glycogen synthesis [30, 122]. Bile acids regulate glycogen synthase activity in hepato- cytes via the insulin-R/PI-3 kinase/GSK3 pathway to the same degree as exposure to 50 nM insulin [47]. In hepatocytes and hepatoma cells, growth fac- tor and bile acid-induced ERK1/2 signaling controls the expression of the LDL receptor and plays a role in stimulating the excretion of bile salts leading to increased canalicular transport capacity [70, 111].

19.3

The JNK Pathway

c-Jun NH

-terminal kinase 1 and 2 were initially de-

scribed biochemically to be stress-induced protein

kinase activities that phosphorylated the NH

-ter-

minus of the transcription factor c-Jun; hence the

pathway is often called the stress-activated protein

kinase (SAPK) pathway [23, 48]. Multiple stresses

increase JNK1/2 (and the subsequently discovered JNK3) activity including UV- and γ-irradiation, cy- totoxic drugs, bile acids and ROS (e.g., H

O

). Phos- phorylation of the NH

-terminal sites Ser63 and Ser73 in c-Jun increases its ability to trans-activate AP-1 enhancer elements in the promoters of many genes. It has been suggested that JNK can phos- phorylate the NH2-terminus of c-Myc, potentially playing a role in both proliferative and apoptotic signaling [96]. In a similar manner to the previously described ERK1/2 MAPK pathway, JNK1/2 activities were regulated by dual threonine and tyrosine phos- phorylation, which were found to be catalyzed by a protein kinase analogous to MKK1/2, termed stress- activated extracellular regulated kinase 1 (SEK1), also called MKK4 [23, 141]. An additional isoform of MKK4, termed MKK7, was subsequently discovered [141]. As in the case of MKK1 and MKK2, MKK4 and MKK7 were regulated by dual serine phosphoryla- tion.

In contrast to the ERK1/2 pathway, however, which appears primarily to utilize the three protein kinases of the Raf family to activate MKK1/2, at least ten protein kinases are known to phosphorylate and activate MKK4/7, including the Ste 11/Byr2-ho- mologs MKKK1–4, as well as proteins such as TAK-1 and Tpl-2 [74, 123, 124, 157]. Cleavage of MEKK1 by caspase molecules into a constitutively active mol- ecule may play an amplifying role in the execution of apoptotic processes [124, 151].

Upstream of the MAP3K enzymes are another layer of JNK pathway protein kinases, e.g., Ste20-ho- mologs and low molecular weight GTP-binding pro- teins of the Rho family, in particular Cdc42 and Rac1 (Fig. 19.1) [12, 36, 163]. It is not clear how growth factor receptors, e.g., ERBB1, activate the Rho fam- ily low molecular weight GTP-binding proteins; one mechanism may be via the Ras proto-oncogene, whereas others have suggested via PI-3 kinase and/

or protein kinase C isoforms [139]. In addition, other groups have shown that agonists acting through the TNF- α and FAS receptors, via sphingomyelinase en- zymes generating the lipid second-messenger cera- mide, can activate the JNK pathway by mechanisms that may act through Rho family GTPases.

19.3.1

Hepatocytes and JNK1/2 Signaling

c-Jun NH

-terminal kinase signaling plays an essen- tial role in hepatocyte proliferation. Genomic dele- tion of c-Jun was shown to be embryonically lethal due to a lack of liver development, and in a more recently published study, deletion of MKK4 (SEK1) has also been found to block liver development in

vivo [37]. The in utero lethality of c-Jun –/– and MKK4 –/– animals was enhanced in c-Jun –/– MKK4 –/– embryos, suggesting that the JNK pathway sig- naling regulates transcription factors in addition to c-Jun to promote liver growth and survival [29, 37, 95, 146, 150]. In contrast, the embryonic lethality of MEK1 and Raf-1 deletions has been shown to be dependent on a lack of vasculature development and to a lesser extent on hepatoblast survival [39].

In vitro studies examining the role of JNK pathway signaling in the control of hepatocyte DNA synthe- sis have largely agreed with the embryonic lethality studies. Interruption of JNK pathway signaling by use of dominant negative MKK4, JNK1 and c-Jun blocks growth factor-induced activation of the AP- 1 transcription factor complex and hepatocyte DNA synthesis in vitro [5, 26, 150]. Studies using high doses of the JNK1/2 inhibitor SP600125 have also suggested JNK signaling controls expression of cy- clin D1 and liver regeneration in vivo; however, at the concentration used, SP600125 may also have had non-specific inhibitory effects on other MAPK path- ways [125]. Liver regeneration/hepatocyte prolifera- tion is controlled by many growth factors, some of which potently activate the JNK pathway, includ- ing TNF- α, insulin and to a lesser extent EGF and hepatocyte growth factor (HGF). TNF- α, primarily generated by Kupffer cells [126], can stimulate the proliferation of hepatocytes. TNF- α signaling also activates the transcription factor NF- κB, which acts in a coordinated fashion with JNK1/2 and ERK1/2 to promote hepatocyte growth and protect hepato- cytes from TNF- α toxicity [65, 118].

19.3.2

JNK1/2 Signaling, Apoptosis and Bile Acids

In hepatocytes both pro- and anti-apoptotic actions of JNK signaling have been observed and appear to be stimulus-dependent [5, 25, 38, 81, 83, 89]. Lig- and-induced activation of the TNF- α receptor in hepatocytes is important in proliferative responses, while at the same time, many groups have argued that TNF- α-induced JNK signaling is toxic, par- ticularly when combined with oxidative stress [83].

Others have argued that TNF- α and ceramide/PKC zeta-induced JNK signaling plays a protective role, and that loss of both JNK and NF- κB induction pro- mote TNF- α toxicity [81], including that induced by chemotherapeutic agents [85, 86]. Activation of MEKK1, either by upstream stimuli or by caspase- dependent cleavage has been linked to many of the above observations [151].

Bile acids can activate the JNK pathway in hepa-

tocytes. DCA increases ceramide production in

hepatocytes and DCA- and TCA-induced JNK acti- vation is abolished in FAS-R –/– and acidic sphin- gomyelinase –/– mouse hepatocytes, whereas the ERBB1 inhibitor or ROS/RNS-quenching agent N- acetyl cysteine has no inhibitory effect on JNK ac- tivity [43, 44]. In FAS-R –/– cells, TNF- α can still activate the JNK pathway and generate ceramide, and DCA can still activate the ERK1/2 pathway, ar- guing that bile acids do not functionally activate the TNF- α receptor towards the JNK pathway and that ERBB1 signaling is intact in FAS-R –/– cells. This is of note because ceramide has been linked to altered Raf-1–RAS interactions in some cell systems. Treat- ment of FAS-R –/– hepatocytes with exogenous neu- tral or acidic sphingomyelinase does not activate the JNK pathway, suggesting ceramide generation is upstream of the initial bile acid target (possibly acidic sphingomyelinase) and the FAS-R.

The roles of each JNK isoform appear to be dif- ferent in hepatocyte survival responses. Activation of JNK2 enhances the apoptotic response of mouse hepatocytes exposed to DCA, whereas inhibition of JNK1 function by expression of dominant nega- tive JNK1 reduces the apoptotic response of mouse hepatocytes exposed to DCA [110]. Treatment of hepatocytes with Brefeldin A, which blocks the bile acid-induced translocation of FAS-R from intrac- ellular stores to the plasma membrane [128], does not block bile acid-induced JNK activation [43]. The above findings are of particular note as ligand-inde- pendent activation of the FAS-R by bile acids, and to a lesser extent TRAIL receptor family members, has been causally linked to bile acid-stimulated apopto- sis [35, 49, 82, 105]. Thus bile acids activation of the JNK pathway in hepatocytes, via the activation of the FAS-R at an intracellular site, presumably is in a dynamic regulatory balance for cell survival with FAS-R-stimulated activation of pro-caspases. Pre- sumably, the dependence of bile acid-induced JNK activation on ceramide generation argues that clus- tering of FAS-R molecules into raft-like structures is required for downstream signal transduction, since ceramide generation causes the clustering of death receptors into rafts, which activate JNK [16, 99].

19.3.3

JNK1/2 Signaling and Metabolism

JNK1/2 signaling in non-hepatic cells has gener- ally not been linked to altered metabolic processes in cells. However, in hepatocytes the rapid activa- tion of JNK/AP-1 signaling has been linked to the downregulation of cholesterol 7 alpha hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis [44]. More recent

studies have suggested that prolonged activation of the sterol-regulated transcription factor FXR can promote the expression of the growth factor FGF-19, leading to activation of the FGF receptor 4 and downstream JNK pathway, thereby controlling CYP7A1 expression [50].

19.4

The p38 MAPK Pathway

The p38 MAPK pathway was originally described as a mammalian homolog to a yeast osmolarity sens- ing pathway [45]. It was soon discovered that many cellular stresses activated the p38 MAPK pathway, in a manner not dissimilar to that described for the JNK pathway. Rho family GTPases appear to play an important role as upstream activators of the p38 MAPK pathway and via several MAP3K enzymes, e.g., the PAK family, regulate the MAP2K enzymes MKK3 and MKK6 [46]. At least four isoforms of p38 MAPK exist, termed p38 α, β, γ and δ [73]. There are several protein kinases downstream of p38 MAPK enzymes that are activated following phosphoryla- tion by p38 isoforms, including p90

and MSK1/2 [18]. P90

phosphorylates and acti- vates HSP27, while MSK1/2 can phosphorylate/acti- vate transcription factors that regulate e.g., CREB.

Bile acid-induced activation of CREB in primary hepatocytes appears dependent on the ERK1/2 path- way, rather than p38 signaling [110].

The role of p38 MAPK signaling in cellular re- sponses is diverse, depending on the cell type and stimulus. For example, p38 MAPK signaling has been shown to promote cell death as well as to en- hance cell growth and survival [83, 161]. The ability of bile acids to regulate p38 MAPK activity in cells derived from the liver appears to be highly variable, with different groups reporting either no activation, weak activation or strong activation [40, 110, 111], which may be due to differences between primary and established cells. This is in contrast to the clas- sical MAPK–ERK1/2 pathway where bile acid-in- duced activation has been consistently observed by many groups.

19.4.1

Hepatocyte Proliferation and p38 Signaling

A positive role for p38 signaling in hepatocyte prolif-

eration was first noted in 1997, and subsequently by

a variety of groups [13, 129] (see Fig. 19.5). Ethanol-

induced impairment of liver regeneration has been

linked to both a reduction in stimulated p38 phos- phorylation and sustained p38 activity, which was correlated to high basal expression of p21 [13, 68].

The growth-promoting role of p38 may be stimulus specific as activation of G-protein-coupled recep- tors, including those for prostaglandin F(2alpha), vasopressin and norepinephrine, does not appear to stimulate hepatocyte growth via either the ERK1/2 or p38 pathways [94]. Expression of HBV X protein has been shown to activate the p38 pathway, which may be responsible for activation of STAT3 and in- creased cyclin D1 levels [137]: however, other studies have argued that STAT3 and p38 signaling can sup- press cyclin D1 levels in a differentiation-specific manner in liver-derived cells [88].

19.4.2

P38 Signaling, Apoptosis and Metabolism

Activation of the p38 pathway has been linked to the induction of apoptosis: TGF β-induced apoptosis has been shown to utilize p38 signaling as a pro-ap- optotic signal [80, 103]. However, hepatitis C virus core protein has also been shown to inhibit FAS-de- pendent p38 signaling and apoptosis [158]. In HuH7 cells, bile acid-induced apoptosis also recruits p38 as a pro-apoptotic stimulus that is inhibited by c- FLIP-L [40]; of note, c-FLIP-S expression is PI-3 kinase and ERK1/2 dependent in DCA-treated pri- mary rodent hepatocytes [105, 110]. Hepatocellular carcinoma cells have been noted to have lower levels of apoptosis and p38 activity [54]. P38 signaling has also been linked to choloretic efflux of bile acids, by regulating the distribution of the translocation to the plasma membrane of the bile salt export pump [71].

19.5

The MEK5-ERK5 "Big MAP Kinase" Pathway

The "big MAP kinase" pathway was first described in 1995 [164]. The term "big" derives from the fact that, whereas the molecular masses of ERK1/2 and JNK1/2 are 42/44 kDa and 46/54 kDa, respectively, ERK5 has a mass of ~90 kDa. The upstream activa- tors of ERK5, the MEK5 isoforms, have a similar molecular mass to other MAP2K molecules [31] and display different subcellular locations. The response of the MEK5-ERK5 pathway to growth factors such as EGF is similar to that of the MEK1/2-ERK1/2 pathway, including in many, but not all cell types, a dependency on Ras signaling [31, 58, 59]. Fur- thermore, MEK5 is also partially inhibited by the

previously described MEK1/2 inhibitors PD98059 and U0126. It appears that PD184352 is much more specific at lower concentrations (<10–20 µM) for MEK1/2 than MEK5 [91]. In this regard, we have found that PD184352 potentiated DCA-induced ap- optosis in primary hepatocytes to the same extent as U0126 and dominant negative MEK1 (S217A), but not dominant negative MEK1 (K97M), suggest- ing MEK5 does not signal to survival after DCA ex- posure (Fig. 19.2a) [105] (L. Qiao and P. Dent, un- published findings). The MAP3K enzymes recently shown to phosphorylate MEK5, MEKK2 and MEKK3 have been previously linked to signaling through the JNK pathway [133]. ERK5 has been proposed to phosphorylate and activate the transcription factors MEF2C, Sap 1a and also c-Myc. In a similar man- ner to the ERK1/2 pathway, the ERK5 cascade has been proposed to play a key role in growth factor- stimulated cell growth and cell survival processes (Fig. 19.1). The activation of MEK5-ERK5 in hepa- tocytes is unknown, although preliminary studies suggest it can be activated by EGF and conjugated/

unconjugated bile acids in primary hepatocytes (C.

Mitchell and P. Dent, unpublished observations).

Based on the fact that EGFR/Ras signaling can pro- mote MEK5-ERK5 activation, and bile acids also ac- tivate these molecules, it is probable that this path-

Fig. 19.5. Inhibition of p38 alpha function blunts hepatocyte DNA synthesis. Rats were subjected to either partial hepatecto- my or sham operation. Twenty-four hours after surgery, primary hepatocytes were isolated and plated in vitro. Hepatocytes were plated in the presence of vehicle (DMSO), the p38 inhibi- tor SB203580 (5 µM), its inactive analog SKF106978 (5 µM) or the MEK1/2/5 inhibitor PD98059 (50 µM). Cells were incubated with 10 µCi [³H]thymidine and DNA synthesis was determined 48 h after plating by [³H]thymidine incorporation into DNA [129]. As indicated by the solid lines, DNA synthesis is strongly inhibited by SB203580 but only weakly inhibited by the MEK1/2/5 inhibi- tor PD98059

way will be stimulated following exposure of cells to many growth factors.

19.6 Conclusions

Multiple MAPK pathways play critical roles in hepa- tocyte proliferation, survival and metabolism con- trol. Although much has been discovered using iso- lated hepatocytes in vitro, significantly more work needs to be performed to understand how MAPK pathway signaling within the intact liver is control- led.

Acknowledgments

P.D. was supported in part by National Institute of Health grants DK52875, CA72955, CA88906, Depart- ment of Defense grants BC98-0148 and BC02-0335, the Jim Valvano "V Foundation" and the Universal Leaf Corporation Professorship in Signal Transduc- tion Research.

Selected Reading

Sturgill TW, Ray LB. Muscle proteins related to microtubule as- sociated protein-2 are substrates for an insulin-stimulatable kinase. Biochem Biophys Res Commun 1986;134:565–571.

Sturgill TW, Ray LB, Erikson E, Maller JL. Insulin-stimulated MAP- 2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 1988;334:715–718.

Derijard B, Raingeaud J, Barrett T et al. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 1995;267:682–685.

Qiao L, Studer E, Leach K et al. Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes:

inhibition of EGFR/mitogen-activated protein kinase-signal- ing module enhances DCA-induced apoptosis. Mol Biol Cell 2001;12:2629–2645.

In Sturgill and Ray, the discovery of "classical" insu- lin-stimulated MAP kinase (now termed ERK2) was reported. In Sturgill et al., the ability of this MAP kinase to phosphorylate and activate ribosomal p90 S6 kinase was noted: these findings represent the first report of a cascade of insulin-stimulated pro- tein kinases, and ultimately led to the elucidation of the component parts of the "insulin black box."

Studies in Derijard et al. describe the discovery of MAP kinase family pathways parallel to the initial classical ERK/MAP kinase cascade. As an example of the complex interplay between MAP kinase path- ways in the liver, Qiao et al. present evidence as to how bile acids, via activation of receptor tyrosine kinases such as the EGF, HER2 and insulin recep- tors, and other receptors such as the FAS receptor, promote simultaneous activation of multiple MAP kinase pathways in hepatocytes.

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