Phorbol 12-myristate 13-acetate promotes nuclear translocation of hepatic steroid response element binding protein-2

The International Journal of Biochemistry & Cell Biology 75 (2016) 1–10

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Phorbol 12-myristate 13-acetate promotes nuclear translocation of hepatic steroid response element binding protein-2 Tsz Yan Wong a,1 , Yan Qin Tan a , Shu-mei Lin c , Lai K. Leung a,b,∗ a b c

Food and Nutritional Sciences Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, Hong Kong Biochemistry Programmes, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, Hong Kong Dept. of Food Science, National Chiayi University, Chiayi City, Taiwan, ROC

a r t i c l e

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Article history: Received 31 October 2015 Received in revised form 16 March 2016 Accepted 21 March 2016 Available online 23 March 2016 This work is dedicated to Daisy Lai Ping Leung. Keywords: Phorbol ester PKC SREBP-2 Liver

a b s t r a c t Sterol regulatory element-binding protein (SREBP)-2 is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid profiles. Phorbol 12-myristate 13-acetate (PMA), which is a common protein kinase C (PKC) activator, was shown to promote the post-translational processing and nuclear translocation of SREBP-2 in hepatic cells in the current study. Following SREBP-2 translocation, the transcripts of its target genes HMGCR and LDLR were upregulated as demonstrated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Electrophoretic mobility shift assays (EMSA) also demonstrated an induced DNA-binding activity on the sterol response element (SRE) domain under PMA treatment. The increase of activated Srebp-2 without the concurrent induced mRNA expression was also observed in an animal model. As the expression of SREBP-2 was not increased by PMA, the activation of PKC was the focus of investigation. Specific PKC isozyme inhibition and overexpression supported that PKC␤ was responsible for the promoting effect. Further studies showed that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not 5 adenosine monophosphate-activated protein kinase (AMPK), were the possible downstream signaling proteins of PKC␤. In conclusion, this study illustrated that PKC␤ increased SREBP-2 nuclear translocation in a pathway mediated by MEK/ERK and JNK, rather than the one dictated by AMPK. These results revealed a novel signaling target of PKC␤ in the liver cells. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Cholesterol homeostasis is a highly controlled series of events in humans, and sterol-regulatory element binding protein (SREBP) plays a central role in these events. SREBPs are transcriptional factors that regulate lipid synthesis (Horton et al., 2002). Involvement in fatty acid and glucose metabolism has been documented for

Abbreviations: CVD, cardiovascular disease; RT-PCR, reverse transcriptasepolymerase chain reaction; SCAP, SREBP-cleavage activating protein; COPII, coatamer protein II; S1P, Site-1 protease; S2P, Site-2 protease; EMSA, electrophoretic mobility shift assay; SRE, sterol response element; INSIG-1,-2, insulin-induced proteins; SREBP, sterol regulatory element-binding proteins; MAPK, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; AMPK, 5 ádenosine monophosphate-activated protein kinase. ∗ Corresponding author at: Food and Nutritional Sciences/Biochemistry Programme, School of Life Sciences, The Chinese University of Hong Kong, Rm.507C MMW Bldg., Shatin, N.T., Hong Kong. E-mail addresses: [email protected], [email protected] (L.K. Leung). 1 Present address of TY Wong: Department of Chemistry, Faculty of Science, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong. http://dx.doi.org/10.1016/j.biocel.2016.03.010 1357-2725/© 2016 Elsevier Ltd. All rights reserved.

the subtypes -1a and -1c, whereas the -1a and -2 subtypes are for regulating cholesterol homeostasis. HMG-CoA reductase (HMGCR) catalyses the rate-limiting reaction of cholesterol synthesis, and is an target enzyme for treating hypercholesterolemia. The gene encoding for HMGCR can be regulated by SREBP-2. The LDL receptor (LDLR) that mediates the uptake of blood LDL is another target gene of the transcription factor. The precursor form of SREBP-2 (125 kDa) is encoded by the SREBF2 gene and is truncated and activated by SREBP-cleavage activating protein (SCAP) in a post-translational processing event. When sterol concentrations are low, the SCAP-SREBP-2 complex binds to coatamer protein II (COPII) and migrates from the ER to the Golgi. In the organelle, Site-1 protease (S1P) and Site-2 protease (S2P) truncate the SREBP-2 precursor into the active transcriptional factor. The activated SREBP-2 (approximately 68 kDa) then translocates to the nucleus and binds to the Sterol Responsive Element (SRE) of target genes. At high sterol concentrations, cholesterol binds to the sterol-sensing domain of SCAP. The cholesterol-bound SCAP interacts with insulin-induced gene 1 and 2 proteins (INSIG1,-2) and the SCAP-SREBP-2 complex stays at the ER. As a result,

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control various cellular and physiological processes, such as gene expression, cell proliferation, and secretion. The family consists of 12 isozymes, and they are classified into three groups according to their differential activation characteristics. Classical PKCs containing ␣, ␤I, ␤II, and ␥ isoforms are responsive to diacylglycerol (DAG), phosphatidylserine (PPS) and calcium (Ca). Novel PKCs, which consist of ␦, ␧, ␩, and ␪ isozymes, are activated by DAG and PPS. The atypical PKC ␨, ␫, ␭ and PKM␨ on the other hand, are stimulated by PPS only (Nitti et al., 2008). A previous study has demonstrated that PKC␤ activates SREBP1c but not SREBP-1a (Yamamoto et al., 2010). In the current study, we hypothesized that PKC was capable of controlling the posttranslation processing of SREBP-2. 2. Materials and methods 2.1. Chemicals Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma Chemical Company (St Louis, MO, USA). Kinase inhibitors like SB203580, H-89, Compound C, Bisindolylmaleimide I, SP600125, PD98059, pAKT inhibitor and U0126 were purchased from Calbiochem (San Diego, CA, USA). LY333531 and HBDDE were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals, if not stated, were acquired from Sigma Chemicals. PKC␤I and ␤II pcDNA3.1 expression plasmids were gifts from Professor Lau Kwok Fai (School of Life Sciences, the Chinese University of Hong Kong, HKSAR, China). 2.2. Cell culture studies HepG2, L-02, Bel-7404 and WRL cells (American Type Culture Collection, Rockville, VA, USA) were cultured in RPMI—1640 phenol red-free media (Sigma Chemicals) with 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD) and incubated at 37 ◦ C and 5% carbon dioxide. These cells were routinely subcultured when reaching 80% of confluency. Three days before the experiment, the cultures were switched to RPMI—1640 phenol red free media (Sigma Chemicals) and 5% charcoal-dextran treated fetal bovine serum (Hyclone, Utah, USA). Sub-confluent cell cultures were treated with various concentrations of PMA with DMSO as the carrier solvent. The final concentration of the solvent was 0.1% v/v, and the control cultures received DMSO only. The cell density in each experiment was maintained at 5 × 102 cells/mm2 . Fig. 1. Serum lipid parameters in PMA-treated mice. ICR mice were tail-injected with 0, 1, 2, 4 ␮g PMA. Blood samples were collected 24 h after the injection. Serum total cholesterol (TC)(Fig. 1A), HDL-C (Fig. 1B) and non-HDL-C (Fig. 1C) levels were determined. Values of lipids are means ± SEM, n = 3. Means labeled with (*) are significantly different from the control.

less SREBP-2 will be processed and activated (Lewis et al., 2011; Edwards et al., 2000; Horton et al., 2002). Regulation at the transcriptional and post-translational levels has been described for SREBP-2. Some signal transduction pathways can also be a part of the activation or deactivation process. Phosphatidylinositol 3-kinase and Akt stimulate the transport of SREBP-2 to the Golgi for post-translational processing (Du et al., 2006), while AMPK phosphorylates the factor and prevents it from processing into its truncated active form (Li et al., 2011). In addition, ERK-1/2 increase the activity of SREBP-2 by phosphorylation (Kotzka et al., 2004), whereas phosphorylation mediated by GSK3 promotes transcriptional factor degradation (Hirano et al., 2001). Protein kinase Cs (PKCs) are one of the most studied signaling protein families. As reviewed by Mackay and Twelves (2007), they

2.3. Quantitative real time RT-PCR assay Liver cells were seeded in 6-well Costar plates and underwent various treatments. After 24 h, total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad CA, USA). The RNA’s concentration and purity were determined by its absorbance at 260/280 nm. First DNA strands were synthesized from 3 ␮g of total RNA using oligo-dT primers and M-MLV Reverse Transcriptase (USB Corporation, Cleveland, Ohio, USA). Target fragments were quantified by real-time PCR and an ABI prism 7700 Sequence Detection System (Applied Biosystems) was employed for these assays. Taqman® /VIC® MGB probes and primers for SREBF2, HMGCR, LDLR and GAPDH (Assay-on-DemandTM ) and Real-time PCR Taqman Universal PCR Master Mix were all obtained from Applied Biosystems. PCR reactions were set up as described in the manual, which were validated by the company. Signals obtained for GAPDH served as a reference to normalize the amount of RNA amplified in each reaction. Relative gene expression was analyzed using the 2−DC T method (Livak and Schmittgen, 2001).

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Fig. 2. Western blot analysis of SREBP-2 and ERK on PMA-treated mouse liver samples. ICR mice were tail-injected with 0, 1, 2, 4 ␮g PMA. Liver samples were collected 24 h after the injection. Hepatic protein was extracted from the samples, and N-terminal SREBP-2 and p-ERK were determined by Western Blot (Fig. 2A). Optical densities of protein expression (Fig. 2B) are means ± SEM, n = 5-6. Means labeled with (*) are significantly different from the control. The image is a representation of two independent experiments.

2.4. Electromobility shift assay Nuclear protein extract was isolated by using NucBusterTM protein extraction kit (Novagen® , EMD Biosciences, Inc., La Jolla, CA, USA.). In brief, cells were washed, trypsinized, and centrifuged at 500g at 4 ◦ C. Reagent 1 was added to the packed cells. Nuclear extract was isolated from the cell suspension by vortexing and centrifugation. The nuclear protein was stored at −80 ◦ C until assayed. An oligonucleotide mimicking (−205 to −160) HMGCR as shown below was synthesized and labeled by DIG Gel Shift Kit, 2nd Generation (Roche Diagnostics GmbH). HMGCR (−205 to −160) with SREs underlined: 7 × SRE:

5 -GTT GGC CGA GCC CGT GGT GAG AGA TGG TGC GGT GCC TGT TCT TGG -3’ 5 -GTG CGG TGG TGC GGT GGT GCG GTG GTG CGG TGG TGC GGT GGT GCG GTG GTG CGG TG-3

The nuclear protein was incubated with the labeled probe, sonicated salmon sperm DNA, poly(dI-dC), and binding buffer (400 mm KCl, 80 mm HEPES, 2 mm DTT, 0.8 mm EDTA, 80% glycerol, pH8) provided in electrophoretic mobility shift assay accessory kit (Novagen) for 30 min at room temperature. 7 × SRE unlabeled oligonucleotide or SREBP-2 antibody was co-incubated as the competitive control. A non-denaturing, 4–6% agarose gel electrophoresis was used to separate the reaction mix in 0.5 × Trisborate EDTA at 100 V. The labeled oligonucleotide-protein complex was electro-transferred to a Nylon membrane, fixed by uv light, blocked and washed. The shifted oligonucleotide was detected by anti-Digoxigenin-AP conjugate and the chemiluminescent substrate CSPD® provided in the kit.

U.S.A.). 50 ␮g of lysate protein were separated on 10% SDS-PAGE and transferred onto an Immobilon PVDF membrane (Millipore, Bedford, MA, U.S.A.). Primary antibodies of SREBP-2 (Santa Cruz Biotech. Inc., CA), N-terminal SREBP-2 (Abcam plc, Cambridge, UK), HMGCR (Millipore, Bedford, MA), anti-phospho-ERK-1/2, antiphospho-PKC␣/␤II, anti-phospho-PKA (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), and anti-actin primary (Sigma Chemicals) and secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) were used for protein detection. An ECL Detection Kit (Amersham, Arlington Heights, IL, U.S.A.) provided the chemiluminescent substrate for HRP, and the targeted protein was visualized by autochemiluminography. For the nuclear and/or cytosolic protein preparations, the NucBusterTM protein extraction kit (Novagen® ). Histone-4 and ␤actin were used for normalizations in nuclear extracts and cell lysates, respectively. 2.6. Immunoprecipitation Cells were washed twice by PBS (pH 7.4) and harvested into a 1.5 ml microtube with 0.3 ml RIPA lysis buffer supplemented with protease/phosphatase inhibitors. After extracting for 20 min on a rocking platform at 4 ◦ C, lysates were collected and centrifuged at 13000g at 4 ◦ C. 300 ␮g of lysate protein was incubated for 2–6 h at 4 ◦ C with protein A/G plus agarose and p-ERK-1/2 or p-JNK antibody (Santa Cruz). The pelleted beads were washed three times with icecold lysis buffer. The complex was eluted by SDS loading buffer, separated by SDS-PAGE and transferred to PVDF membrane. The interaction was visualized by immunoblotting with anti-SREBP-2. 2.7. Immunocytochemical imaging

2.5. Western blot analysis Cells were washed once by PBS (pH 7.4) and harvested into a 1.5 ml microtube with 0.5 ml lysis buffer (PBS, 1%NP40, 0.5% sodium deoxycholate, 0.1% SDS). The lysis buffer contained protease inhibitors (40 mg/L PMSF, 0.5 mg/L aprotinin, 0.5 mg/L leupeptin, 1.1 mmol/L EDTA and 0.7 mg/L pepstatin) and phosphatase inhibitor cocktail (PhosphoSTOP tablet, Roche). The harvested cells were then lysed with a cell disruptor (Branson Ultrasonics Corp., Danbury, CT, U.S.A.) on ice for 30 s. The protein concentration of cell lysate was determined by Dc protein assay (BioRad, Richmond, CA,

HepG2-68 cells were grown on 35 mm glass bottom dishes, and were treated with 3 ␮M PMA at 40–50% confluence for 24 h. After the treatment period, the cells were fixed with 4% paraformaldehyde in PBS with 0.2% (v/v) Tween 20 for 5 min, followed by blocking in 3% BSA PBS for 30 min at room temperature. The dishes were washed, and incubated with anti-SREBP-2 and anti-golgin-97 primary antibody (1:100 dilution in PBS) for 3 h. Subsequently, 1-h incubation of Alexa Fluor 488-labelled (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568-labelled (Molecular Probes, Eugene, OR, USA) secondary antibodies was carried out. Dishes were stained

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with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) and the cells were examined by confocal microscopy. 2.8. Animal experimentation The protocol of this experiment was described by Yamamoto et al. (2010). Twenty male ICR mice aged around 7 weeks were acquired from the Animal Facility of the Chinese University of Hong Kong. Overnight-fasted mice were randomly divided into 4 groups (0, 1, 2, 4 ␮g PMA/mouse, n = 6). PMA was injected in the tail vein, followed by euthanasia 4 h later. Plasma was collected and liver was removed and stored at −80 ◦ C. The protocol adopted in the present study had been approved by Animal Experimentation Ethics Committee of Chinese University of Hong Kong. 2.9. Plasma cholesterol measurements Blood samples obtained were centrifuged at 5000 rpm for 5 min to collect plasma. Plasma total cholesterol was measured using Infinity Total Cholesterol Assay kit (Waltham, MA, USA). For the quantification of plasma HDL, a solution of phosphotungstic acid and magnesium chloride was added to precipitate VLDL and LDL. HDL in the supernatant was measured as that for total cholesterol. Non-HDL was calculated by deducting HDL from TC. 2.10. Statistical methods A Prism® 5.0 (GraphPad Software, Inc., CA, USA) software package was utilized for statistical analysis. Whenever applicable, results were analyzed by One way ANOVA and significant level was set at p < 0.05. 3. Results 3.1. PMA enhanced hepatic N-terminal SREBP-2 protein in vivo The regulation of SREBP-2 by PMA was examined in mice. 4 h after tail-injection, the serum non-HDL-cholesterol (nHDL-C) levels significantly (P < 0.05) increased in mice received 4 ␮g PMA (Fig. 1C) though no changes were observed in TC (Fig. 1A) or HDL (Fig. 1B). Western blot analysis revealed that hepatic N-terminal SREBP-2 was increased with a concurrent increase in p-ERK in mice receiving 4 ␮g PMA injection (Fig. 2A). The optical density data (Fig. 2B) indicated that the increases were significant (P < 0.05). Since Srebf2 mRNA was not induced, the increased amount of N-terminal SREBP2 should be coming from post-translational processing (Fig. 3A). This result was consistent to those observed in the cell culture studies. As downstream genes of SREBP-2, hmgcr and ldlr mRNA expression in livers was determined. Hepatic ldlr mRNA levels were up-regulated (P < 0.05) in mice treated with 4 ␮g PMA (Fig. 3C). A dose-dependent increase in hmgcr mRNA expression was also seen, although the increase did not achieve statistical significance (Fig. 3B). Since an increase in p-ERK was observed (Fig. 2), this signaling pathway would be investigated in the subsequent cell study. 3.2. PMA increased HMGCR and LDLR mRNA expression in liver cells The mRNA transcripts of HMGCR and LDLR displayed a dosedependent increase in HepG2 cells following PMA administration for 24 h (Fig. 4A). The increases of HMGCR and LDLR mRNA were significant at 24 h after treatment with 3 ␮M PMA (Fig. 4B). Nevertheless, the response of LDLR appeared to be more sensitive than that of SREBP-2. These two genes are regulated by multiple pathways, and PMA might activate other signaling molecules which

Fig. 3. Hepatic hmgcr and ldlr mRNA expression in PMA-treated mice. ICR mice were tail-injected with 0, 1, 2, 4 ␮g PMA. Liver samples were collected 24 h after the injection. Total RNA was extracted and real-time RT-PCR was used to determine the mRNA expression. Messenger RNA expression of Srebf2 (Fig. 3A), hmgcr (Fig. 3B) and ldlr (Fig. 3C) was determined. Values of mRNA expression are means ± SEM, n = 3. Means labeled with (*) are significantly different from the control.

could differentiate the response time of the genes. Since the optimal time of treatment appeared to be 24 h, all experiments were carried out using this exposure time. Similar results in HMGCR and LDLR mRNA expression were also observed in the other hepatic cell lines WRL-68 (Supplementary data Fig. 1A), L-02 (Supplementary data Fig. 1B) and Bel7404 (Supplementary data Fig. 1C) after PMA treatment for 24 h. 3.3. PMA activated post-translational processing of SREBP-2 Because SREBP-2 is a key regulator in cholesterol metabolism, we examined the effect of PMA on the regulation of SREBP-2. PMA increased the N- and C-terminal SREBP-2 protein in HepG2 cells in

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Fig. 4. PMA upregulated HMGCR and LDLR mRNA levels in Hepatic cells in a time and concentration dependent manner. HepG2 cells were treated with PMA. Messenger RNA was extracted and assayed for HMGCR and LDLR mRNA expression after 24 h of treatment (Fig. 4A). In Fig. 4B, the RNA was extracted at time points from 2 to 48 h. Values of mRNA expression are means ± SEM, n = 3. Means labeled with (*) are significantly different from the control (0 ␮M or 0 h).

a concentration-dependent manner after treating for 24 h (Fig. 5A). However, we did not see any significant changes in the transcription of SREBF2 (Fig. 5C). These results indicated that the enhanced post-translational processing was solely responsible for the activation of SREBP-2. The increased processing appeared to occur at around 12 h of treatment (Fig. 5B). Similar results were also found in WRL-68 (Supplementary data Fig. 2A), L-02 (Supplementary data Fig. 2B) and Bel7404 cells (Supplementary data Fig. 2C). 3.4. Immunocytochemical staining of SREBP-2 protein The distribution of the SREBP-2 protein in HepG2 cells was further analyzed by confocal microscopy. In the control samples, the Alexa-488-labeled SREBP-2 was mainly localized in the cytoplasm. In contrast, SREBP-2 was mainly found in the DAPI-labeled nuclei (Fig. 6 Merge image) after treating with 3 ␮M PMA (Fig. 6). These observations suggested that PMA induced nuclear translocation of SREBP-2. 3.5. SRE-binding EMSA assay SREBP-2 transactivation represents the most common regulation for HMGCR expression. In view of the enhancement of PMA on SREBP-2 translocation, the DNA binding on the downstream gene promoter was evaluated. EMSA assay was carried out for investigating the interaction between the N-SREBP-2 and SRE motifs (Fig. 7). The position of the interacting band was revealed by co-incubating with 7 × SRE unlabeled oligonucleotide fragment or anti-N-SREBP2. The band was competed out under either treatment. Our data

showed that the interaction intensified when PMA was administered to the HepG2 cells.

3.6. Dissecting the participation of protein kinases in cells treated with PMA 3.6.1. PKC Since PMA is the activator of PKC, specific PKC inhibitors were employed to investigate the signal transduction pathway. Pre-treatment of the general PKC inhibitor bisindolymateimide I prevented the processing of SREBP-2 increased by PMA; the PKC␤specific inhibitor LY333531 was also effective whereas the PKC␣ inhibitor HBDDE did not affect the cleavage (Fig. 8A). Because these inhibitors are specific to the respective ␤ and ␣ isoforms, PKC␤ appeared to be the major regulator for the protein processing. Consistent with the inhibitory pattern of SREBP-2, HMGCR and LDLR mRNA expression induced by PMA in HepG2 cells was reduced by bisindolymateimide I and LY333531 (Fig. 8B). Since the results in Fig. 8A and B indicated that PKC␤ was the key signaling protein involved, PKC␤I and PKC␤II were ectopically over-expressed and examined in HepG2 cells. Immunoblot assays verified the expression. These cells overexpressing PKC␤ displayed an increased SREBP-2 cleavage and higher amounts of N-terminal SREBP-2 and HMGCR protein than the control cells (Fig. 8C). These results suggested that PMA promoted SREBP-2 processing through a PKC␤-dependent pathway.

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Fig. 5. PMA induced SREBP-2 cleavage in liver cells. (A) HepG2 cells were treated with various concentrations of PMA and protein lysates were extracted from cells. PMA promoted SREBP-2 protein processing in HepG2 cells in a dose- (Fig. 5A) and time- (Fig. 5B) dependent manner. The mRNA expression of SREBF2 in samples treated with PMA is shown in Fig. 5C. Values of mRNA expression are means ± SEM, n = 3.

Fig. 6. Increased nuclear translocation of SREBP-2 in PMA-treated HepG2 cells. The hepatic cells HepG2 were seeded in 6-well culture plates and treated with PMA at 3 ␮M. After 24 h of treatment, the cells were fixed and incubated with Golgi-specific and SREBP-2 primary antibodies and fluorophore (Alexa 568 and 488)-labeled secondary antibodies. The nuclei were counterstained with DAPI. The organelles were visualized by confocal microscopy. The image is a representation of two independent experiments.

3.6.2. MAPKs Since MAPKs can be downstream signaling proteins of PKC, the phosphorylated forms of MAPKs were determined by immunoblot-

ting. PMA induced the activation of MEK1/2, ERK1/2, P38 and JNK expression in a dose-dependent manner (Fig. 9A). The PMAinduced SREBP-2 processing in HepG2 cells was abrogated by

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pretreatment with the MEK1/2 inhibitor PD98059 and the JNK inhibitor SP600125 (Fig. 9B). Pre-treating the cells with the P38 inhibitor SB203580 did not reverse the induced SREBP-2 processing. This indicated that MEK/ERK and JNK but not P38 were the possible intermediate signaling molecules in the regulation of SREBP-2 processing. Consistently, the PMA-induced mRNA expression of HMGCR and LDLR were reduced by PD98059 or SP600125 (Fig. 9C). An immunoprecipitation assay was performed and NSREBP-2 was co-precipitated with p-JNK or p-ERK. The result showed that treatment of PMA might enhance the interaction between SREBP-2 and p-JNK or p-ERK (Fig. 9D). Taken together, these results suggest that PMA elevated p-PKC␤ and promoted SREBP-2 processing through the activation of MEK/ERK and JNK.

3.6.3. AMPK Previous studies have shown that AMPK is crucial in regulating the processing of SREBP (Yang et al., 2008; Li et al., 2011). However, we did not observe any changes in p-AMPK in cultures treated with 3 ␮M PMA over 2- and 17-h incubation periods (Fig. 10A, upper panel) or with PMA dosages ranging from 0.03 to 6 ␮M for 24 h (Fig. 10A, lower panel). Compound C is an AMPK-specific inhibitor. Administrating compound C induced SREBP-2 cleavage as shown in the absence of PMA (first lane of Fig. 10B). However, adding compound C could not amplify the PMA-induced cleavage of SREBP-2 (last lane of Fig. 10B). These results illustrated that AMPK was not involved in PMA-induced processing of SREBP-2.

Fig. 7. PMA amplified the SRE-DNA interaction. The hepatic cells HepG2 were seeded in 6-well culture plates and treated with PMA at 0, 0.03, 0.3, 1.5, 3, 6 ␮M. After 24 h, nuclear extracts were obtained from the cells and EMSA assay was performed. ( ) is the SREBP-2-SRE interacting band. 6 + Ab is the sample with 6 ␮M PMA incubating with SREBP-2 antibody; 6 + 7X, is the sample with 6 ␮M PMA incubating with repeats of SRE (7 × SRE) oligonucleotide. The image represents one of three independent experiments.

3.6.4. Insulin A previous study has indicated that PKC-␤ may participate in insulin-induced SREBP-1c processing (Yamamoto et al., 2010). A

Fig. 8. PKC inhibitor reverted PMA-induced SREBP-2 cleavage. HepG2 were seeded in 6-well culture plates. In Fig. 8A & B, cells were pre-treated with 2.37 ␮M bisindolymateimide I (PKC inhibitor), 50 ␮M HBDDE (PKC␣-specific inhibitor), or 1 ␮M LY333531(PKC␤-specific inhibitor), followed by treatment with 3 ␮M PMA. In Fig. 5C, cells were transfected with PKC␤ I or II expression plasmids. After 24 h, the cells were lysed and protein or RNA extracts were obtained. The autochemiluminograms shown in Fig. 8A & C are representations of three independent experiments. Messenger RNA values in Fig. 8B are means ± SEM, n = 3. Means labeled with (*) are significantly different from samples treated with PMA only.

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Fig. 9. Involvement of MAPK signaling pathway in PMA-induced SREBP-2 processing and the downstream gene expression. HepG2 cells were treated with PMA (0–6 ␮M), cell lysates were collected and assayed for MAPKs at 24 h (Fig. 9A). Pre-treatment of PD98059 (18.7 ␮M), SB203580 (10 ␮M) or SP600125 (50 ␮M) was administered and SREBP-2 processing was analyzed in Fig. 9B. HMGCR and LDLR mRNA were determined at 24 h in cells pre-treated with PD98059, SB203580 or SP600125 in Fig. 9C. Values are means ± SEM, n = 3. Means marked (*) are significantly lower than the cultures treated with PMA alone (p < 0.05). Fig. 9D is the immunoblot result of N-terminal SREBP-2 from the immunprecipitation with anti-pERK and p-JNK.

Fig. 10. SREBP-2 processing promoted by PMA was independent to AMPK. HepG2 cells were cultured and were treated with PMA and co-treated with the AMPK inhibitor Compound C (10 ␮M). The cell lysates were immunoblotted for AMPK and SREBP-2. Images for total- (t-) and/or phospho- (p-)AMPK with respect to time (upper panel) and concentration (lower panel) are shown in Fig. 10A. Immunoblot of SREBP-2 from samples co-treated with PMA and Compound C is displayed in Fig. 10B.

comparable result was observed for SREBP-2 processing induced by 0.1 ␮M insulin in HepG2 cells in the current study. The PKC-␤ inhibitor LY333531 counteracted the action of insulin. In addition, MEK/ERK and/or JNK were likely the intermediately signals as pretreating with PD98059 or SP600125 could prevent the transcription factor processing (Fig. 11). 4. Discussion In the present study, we demonstrated that PMA activated the post-translational processing machinery of SREBP-2 and enhanced the expression of the cholesterol metabolic enzymes in vitro and in vivo. The nuclear translocation of SREBP-2 was independent of AMPK, while JNK and MEK/ERK could serve as the intermediate

signaling molecules. We further illustrated that PKC␤I & II, but not the ␣ isozyme, activated SREBP-2. PKC␤ could differentially activate SREBP-1 isoforms as previously described (Yamamoto et al., 2010), and our results complemented these findings. Previous studies have shown that PKC␤ could be a regulatory signal in atherosclerosis and lipid metabolism by increasing expression of LDLR (Fan et al., 2014), vascular endothelial growth factor (Amadio et al., 2010) and insulin receptor substrate 2 (Park et al., 2013). Knocking out the pkcˇ gene or administering PKC␤ inhibitor reduces cholesterol accumulation in macrophages (Ma et al., 2006), apoE secretion (Fan et al., 2014), and the progression of atherosclerosis (Harja et al., 2009). The present study indicated that PKC␤ might also participate in cholesterol metabolism through the posttranslational processing of SREBP-2.

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Since PMA is an analogue of the signaling lipid diacylglycerol, the biological activities in the present study might have some physiological implications. Yamamoto et al. (2010) have shown the involvement of PKC-␤ in insulin-induced SREBP-1c processing in hepatocytes. In the present study, PKC-␤ and its downstream MEK/ERK and JNK were possible signaling molecules in SREBP-2 processing induced by insulin. 5. Conclusion In the present study, we demonstrated that PMA promoted SREBP-2 activation at the post-translational level. Further analysis revealed that PKC␤ was responsible for the regulation. Considering that the PKC␤ inhibitor Ruboxistaurin is under regulatory review for the treatment of diabetes, results of the present study may generate new perspectives of the prodrug. Acknowledgements

Fig. 11. Potential physiological application of the signal transduction pathway. HepG2 cells were cultured and pre-treated with inhibitors of JNK (SP600125, 50 ␮M), MEK/ERK (PD98059, 18.7 ␮M), or PKC-␤ (LY333531, 1 ␮M). The cells were treated with insulin (0.1 ␮M) or glucose (33 mM) for 24 h. The cell lysates were immunoblotted for C-terminal SREBP-2, p-PKC-␤, p-ERK-1/2, and p-JNK. The image represents one of two independent experiments with comparable results.

PKC␤ has also been shown to be responsive to dietary intake (Mehta and Mehta, 2014). Consuming a high-fat diet increases pkcˇ expression in brown adipose tissue (Huang et al., 2009), increases hmgcr expression and activates SREBP-2 in the liver (Wu et al., 2013), and increases serum cholesterol (Jiang et al., 2005) in mice. In contrast, high-fat feeding would not increase serum cholesterol in pkcˇ-knockout mice (Huang et al., 2009). Exercise may reduce pkcˇ expression in skeletal muscle and liver (Rao et al., 2013). This study provided a mechanistic insight into the interrelationship of gene expression. Several signal transduction events have been shown to regulate SREBP-2 activation. Phospho-Akt increases SREBP-2 processing by stimulating protein transport from the endoplasmic reticulum to the Golgi (Du et al., 2006; Luu et al., 2012). In contrast, GSK3␤, which can be a substrate of PKC (Goode et al., 1992), reduces SREBP-2 activity by initiating its degradation (Lewis et al., 2011). It is conceivable that PMA, through PKC signaling, could regulate one or multiple regulatory pathways in promoting SREBP-2 nuclear translocation. Results of the current study suggested that ERK and JNK were the possible signal transduction intermediates. As ERK-1/2 can phosphorylate SREBP-2 and increase its transactivity (Kotzka et al., 2004), further studies are needed to determine protein phosphorylation and activation by these kinases. Since the JNK or MEK/ERK inhibitors could reverse PMA-promoted SREBP-2 processing, any intrinsic factors or xenobiotics that potentiate these kinases are possible activators of SREBP-2. AMPK phosphorylates the precursor SREBP-2, and the phosphorylated form will be deterred from processing into its active form (Li et al., 2011). PMA is capable of activating AMPK in monocyte differentiation (Cao et al., 2014) and adhesion (Chang et al., 2012). Studies have also shown that crosstalk exists between PKC and AMPK in blood cells. The collaboration between AMPK and PKC may reduce phosphatidylinositol 4-phosphate 5-kinase activity (van den Bout et al., 2013). However, we did not observe any changes in the AMPK status in the current study. The cell type difference could contribute to this disparity.

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