Cholecystokinin Stimulates Extracellular Signal-regulated Kinase through Activation of the Epidermal Growth Factor Receptor, Yes, and Protein Kinase C

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 C␤ (PKC␤), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKC␤ 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 PKC␤ ؊/؊ 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 PKC␤ ؊/؊ mice also displayed decreased expression of hepatic cholesterol-7␣-hydroxylase (CYP7A1) and sterol 12␣-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 PKC␤ ؊/؊ mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKC␤ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKC␤ as a major physiological regulator of both triglyceride and cholesterol homeostasis.

Section: Discussionmentioning

confidence: 99%

Disruption of the Murine Protein Kinase Cβ Gene Promotes Gallstone Formation and Alters Biliary Lipid and Hepatic Cholesterol Metabolism

Huang¹,

Bansode

Rowland

et al. 2011

“…Activation of p47 phox by serine phosphorylation was detected using an anti-phosphoserine antibody (15). The anti-phospho-Src family-Tyr 418 antibody was used to detect activating phosphorylation of Yes (16), and the anti-phosphotyrosine antibody was used to detect tyrosine phosphorylation of EGFR in the respective immunoprecipitates. FADD and caspase 8 association or tyrosine phosphorylation of the immunoprecipitated CD95 samples were detected by Western blot analysis using the respective antibodies (anti-FADD, anti-caspase 8, and anti-phosphotyrosine).…”

Section: Methodsmentioning

confidence: 99%

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Reinehr

Sommerfeld

Keitel

et al. 2008

Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide-and protein kinase C-dependent pathway, which leads to an activating phosphorylation of p47 phox . The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl ؊ ]. Bafilomycin or 4,4-diisothiocyanostilbene-2,2-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47 phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl ؊ ], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction. CD952 (Apo-1/Fas) belongs to the death receptor family and plays an important role in apoptosis induction in many cell types. In hepatocytes, CD95 is mainly located within the cellular interior and can either be activated after ligation with its natural ligand (CD95L) or in a ligand-independent way by hydrophobic bile salts or hyperosmotic cell shrinkage (Refs. 1-4; for review, see Ref. 5). Ligand-dependent and -independent CD95 activation in hepatocytes is a complex process that finally results in CD95 trafficking from the cellular interior to the plasma membrane, subsequent formation of the death-inducing signaling complex (DISC), and eventually apoptosis (1-12).In hepatocytes, proapoptotic stimuli such as CD95 ligand (CD95L), hydrophobic bile salts, or hyperosmotic cell shrinkage induce a rapid oxidative stress (ROS) response (2, 4), which triggers a Yes-dependent but ligand-independent activation of the epiderm...

“…Immunoprecipitates were washed three times with a buffer containing 136 mM NaCl, 20 mM Tris-HCl, 10% (v/v) glycerol, 2 mM EDTA, 50 mM ␤-glycerophosphate, 20 mM sodium pyrophosphate, 0.2 mM Pefabloc, 5 g/ml aprotinin, 5 g/ml leupeptin, 4 mM benzamidine, 1 mM sodium vanadate, and 0.1% (v/v) Triton X-100 and then transferred to Western blot analysis as described above. The anti-phospho-Src family-Y 418 antibody was used to detect activating phosphorylation of Yes or Fyn in the respective immunoprecipitates (20), whereas Yes or Fyn/EGFR association was detected using an anti-EGFR-antibody in the latter samples. EGFR, FADD, and caspase 8 association or tyrosine phosphorylation of the immunoprecipitated CD95 samples were detected by Western blot analysis using the respective antibodies (anti-EGFR, -FADD, -caspase 8, and -phospho-tyrosine).…”

Section: Methodsmentioning

confidence: 99%

The Src Family Kinase Yes Triggers Hyperosmotic Activation of the Epidermal Growth Factor Receptor and CD95

Reinehr

Becker

Höngen

et al. 2004

Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Hä ussinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosM) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.Changes in liver cell hydration are important regulators of hepatic metabolism, gene expression, and transport across the plasma membrane through activation of osmosensing and osmosignaling pathways (1-6). Hypoosmotic cell swelling involves integrin-dependent osmosensing and osmosignaling via Src kinase and mitogen-activated protein kinases (7, 8), resulting in an inhibition of autophagic proteolysis and stimulation of bile acid excretion (7,8).Little is known about osmosensing and osmosignaling in response to hyperosmotic hepatocyte shrinkage. Apart from effects on metabolism and gene expression (1-6), hyperosmotic hepatocyte shrinkage triggers a rapid translocation of intracellular CD95 to the plasma membrane, which is accompanied by DISC 1 formation and sensitizes hepatocytes toward CD95 ligand (CD95L)-induced apoptosis (9, 10). Hyperosmotic membrane targeting and activation of CD95 involves rapid activation of the epidermal growth factor receptor (EGFR) and of c-Jun-N-terminal kinases (JNK) (10), an association of activated EGFR and CD95, and subsequent CD95 tyrosine phosphorylation by the EGFR tyrosine kinase activ...