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PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells Jixin Ding, Satomi Kato, Keyong Du⁎ Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111, USA
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
TRB3 is a pseudokinase whose expression is regulated during stress response and changing
Received 22 October 2007
of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression
Revised version received
is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3
12 January 2008
expression and Akt activation. We find that insulin induces TRB3 expression in cell type
Accepted 31 January 2008
dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle
Available online 11 February 2008
cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles
Keywords:
of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of
P13K
TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3
TRB3
expression whereas inhibition of PKCζ expression impairs TRB3 expression induced by
Insulin gene expression
insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3
Akt atypical PKC
expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced. © 2008 Elsevier Inc. All rights reserved.
Introduction TRB3 belongs to a newly identified family of pseudo-kinases consisting of three mammalian isoforms: TRB1, TRB2 and TRB3. TRBs are closely related to Drosophila tribbles, a negative regulator of cell growth [1,2]. Members of TRB family are characterized by containing a domain that shares high homology with protein kinases [3]. However, this domain of TRBs contains variants in key amino acid residues that are essential for protein kinase catalytic activity and lacks ATP binding domain. As a result, TRBs have no detectable kinase catalytic activity, thereby, termed as pseudo-kinases [4]. However, the kinase homology domain of TRBs possesses a substrate-binding domain that may function as protein interacting module. Consistently, TRBs are found to associate with numerous proteins including transcription factors,
protein kinase as well as ubiquitin E3 ligase. By interacting with different factors, TRBs regulate numerous biological processes including cell growth, differentiation and metabolism. TRB1 interacts with MAPK and modulate MAPK kinase activity [5]. By modulating MAPK signaling, TRB1 has been implicated in smooth muscle cell proliferation [6]. TRB2 has been shown to interact with C/EBPβ, a leucine zipper transcription factor. By modulating C/EBP activity, TRB2 has been implicated in adipocytes differentiation [7] and acute myelogenous leukemia [8]. TRB3 interacts with Akt and inhibits activation of Akt. In both liver [9–11] and muscle [12], decrease in TRB3 expression enhances insulin sensitivity, thereby increasing glucose tolerance. Indeed, a human polymorphism of TRB3 (Q84R), which has higher capacity of inhibiting Akt activation, was found to be associated with insulin resistance and related cardiovascular risk [13]. TRB3
⁎ Corresponding author. Keyong Du Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington St, Boston, MA 02111, USA. Fax: +1 617 636 6217. E-mail address: [email protected] (K. Du). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.01.026
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also directly interacts with E3 ligase COP1 and targets COP1 to acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis[14]. By linking COP1 and ACC1, TRB3 facilitates the degradation of ACC and regulates lipid metabolism [14]. Furthermore, TRB3 interacts with and inhibits ATF4 [15,16], the key factor that regulates amino acid metabolism during integrated stress response [17]. By interacting with ATF4, TRB3 inhibits ATF4 mediated gene expression and may protect ATF4 induced cell cytotoxity [18]. Finally, TRB3 appears also playing important role in cell differentiation as overexpression of TRB3 blocks differentiation of adipocytes [19], muscle [20] and osteoblast [21]. TRB3 was first cloned as an inducible gene in the liver undergoing fatty degeneration [22] and in neuronal cells undergoing apoptosis following NGF withdrawal [23], an indication that TRB3 expression is subjected to regulation. Subsequent studies demonstrated that TRB3 is an inducible gene whose expression is induced by a variety of signals including ER stress [24,25], change in nutritious status [26] and insulin [27]. Since TRB3 inhibits insulin-induced Akt activation, a further examination of such a paradoxical effect is merited. In current study, we examined the dynamic relationship between TRB3 expression and Akt activation. Our studies demonstrate that induction of TRB3 expression provides a checking mechanism for proper Akt activation. TRB3 expression by insulin may reflect the balance of insulin action.
Materials and methods Reagents Insulin, dexamethasone (Dex) and 3-Isobutyl-1-methylxanthine (IBMX) are from Sigma. MG132, thapsigargin and all kinase inhibitors are from Calbiochem. Anti-Akt1 and Akt2 rabbit antibodies, anti-phospho-Akt at Ser473 rabbit antibody are from Upstate Biotech; anti-HA monoclonal antibody is from Covance; Anti-flag antibodies, anti-HA antibodies and anti-beta-tubulin are from (Sigma). The anti-TRB3 rabbit antibody has been described [9]. All of protein kinase inhibitors are from Calbiochem.
Plasmids TRB3 shRNA has been described [20]. PKCζ shRNA was purchased from Addgene (Cambridge MA, Plasmid 10803). To generate Myr-Akt expressing pMIGR1, Myr-Akt was released from pCMV6-Myr-Akt with BglII and EcoRI and cloned into pMIGR1 retroviral vectors. PKCζ expression vectors are gifts of Dr. RV Farese of University of South Florida College of Medicine [28]. TRB3 promoter luciferase construct is generated as described [29].
Cell culture, transient transfection and luciferase assay Fao cells were grown in RPMI1640 supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin,
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and 100 μg/ml streptomycin (Invitrogen). For viral infection of Fao cells, 40% confluent Fao cells were incubated with TRB3 shRNA lentivirus plus 6 μg/ml of polybrene. 3T3 L1 preadipocytes were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The adipocyte differentiation of 3T3 L1 preadipocytes is as follows. Briefly, 3T3 L1 preadipocytes were cultured for two additional days after reaching 100% confluence and treated with differentiation medium (DMEM-high glucose containing 10% FBS, 2.5 μg/ml insulin, 0.5 mM IBMX and 2.5 mM Dex, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 4 days. Then, the medium was changed to regular medium. After 7 days differentiation, the adipocytes were used for experiments. Wildtype, Akt1−/−, and Akt2−/− mouse embryonic Fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).
Western blotting After indicated treatments, cells were washed twice with phosphate-buffered saline and extracted with cell lysate buffer (20 mM Tris pH = 7.6, 250 mM NaCl, 0,5 mM EDTA, 0,5 mM DTT, 10 mM β-glycerophosphate, 10% glycerol and protease inhibitors). Equal amounts of protein were subjected to SDS PAGE electrophoresis and transferred to nitrocellulose membranes (Biorad). After blocking in 5% dry milk, the membranes were incubated with each primary antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. The protein bands were visualized using the ECL detection system (Amersham Biosciences). In all cases, blots were not re-probed. Instead, the artifact introduced by stripping the blots was avoided by running parallel blots.
RNA and RT-PCR The total RNA were prepared by using Qiagen Rneasy Kit according to manufacturer's instruction. RT-PCRs were carried by using RT-PCR kit from Ambion with primers for TRB3: 5′ATGCGAGCTACACCTCTGGC-3 and 5′-TAAGGCCCCAGTCGAGTTGC-3′ and 36B4: forward: 5-ATGATTATCCAAAAT GCTTCATTG-3 and reverse 5-AACAGCA TATCCCGAATCTCA-3. Quantification of all western blots are quantified with Genetool.
Results Cell type specific induction of TRB3 expression by insulin TRB3 expression was induced in hepatocytes by insulin [27]. To explore this matter further, we analyzed insulin-induced TRB3 expression in Fao rat hepatoma cells, 3T3L1 adipocytes, β-TC3 mouse, a clonal β cells, L6 rat myotubes and 3T3-L1 preadipocytes by western blot after these cells were treated with insulin for 4 h. We observed that TRB3 expression was induced by insulin in Fao cells (Fig. 1a, top panel,) and 3T3-L1 adipocytes (Fig. 1b, top panel,). However, no change of TRB3
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Fig. 1 – Cell type specific TRB3 expression by insulin. a) Western blot analysis of Fao cells total lysates. b) Western blot analysis of 3T3L1 adipocyte total cell lysates. c) Western blot analysis of β-TC3 cell lysates. d) Western blot analysis of rat L6 myotube. e) Western blot analysis of 3T3-L1 preadipocyte lysates. The cells are serum-deprived for overnight. All treatment is for 4 h. The amount of reagents is Insulin (Ins) 1 μg/ml, MG132 (MG) 10 μM. The antibodies used are anti-TRB3 and Akt antibodies. f) The quantitative analysis of TRB3 expression in each cell lines from three independent experiments. In all of cases, the amount of TRB3 expression was normalized to total amount of Akt and set as 1 under unstimulated condition. The error bars represent the standard deviation or standard error.
expression was detected in β-TC3 cells (Fig. 1c), L6 myotubes (Fig. 1d) and 3T3L1 fibroblast following insulin treatment. The inability of insulin to induce TRB3 expression in these cells is unlikely due to the period of insulin treatment as no TRB3 expression was observed after 16 h insulin treatment (data not shown). As a control, we also treated these different cells with 10 μM proteasomal inhibitor MG132 which is known to elevate TRB3 levels due to blocking protein degradation [30]. Consistently, MG132 was effective on inducing TRB3 expression in all types of cells, suggesting that insulin-induced TRB3 expression is cell type specific. In addition, induction of TRB3 expression by insulin in 3T3L1 adipocytes but not preadipocytes suggests that inducibility of TRB3 expression is acquired during differentiation. We also examined the Akt activation responding to each stimulus by assessing Akt phosphorylation. Insulin induced Akt activation in most cell lines. Unexpectedly, MG132 also induced Akt activation in Fao cells, an event not understood at present, but in PI3K dependent manner (data not shown). Regardless, our data demonstrated that TRB3 expression by insulin is regulated in the cell type manner. Because TRB3 expression is specifically induced in Fao cells and 3T3-L1 adipocytes, we further examined the relationship between TRB3 expression and Akt activation. In both Fao cells (Fig. 2a) and 3T3-L1 adipocytes (Fig. 2b), the induction of TRB3 lagged the activation of Akt. Akt remained active for 2 h while the induction of TRB3 commenced at 2 h post-insulin treatment. TRB3 has been shown to modulate Akt activation in the liver and muscle [9,12]. One might wonder what is the impact of TRB3 expression induced by insulin on Akt activation? To address this question, we assessed Akt activation by insulin under TRB3 knockdown in Fao cells. To this end, Fao cells were transduced with shRNA expressing or controlling lentivirus.
Fig. 2 – The relationship between Akt activation and TRB3 expression in Fao cells a) and 3T3-L1 adipocytes b) Fao cells or 3T3-L1 adipocytes were serum-starved for overnight and treated with 100 nM insulin (Ins). The total cell lysates were prepared as indicated time for western blot analysis. The quantitative analysis of Akt phosphorylation and TRB3 expression is shown in left panel of each figure. In this assay, the amount of Akt phosphorylation or TRB3 expression at time zero was set as 1 after normalized to amount of Akt in each lane. These experiments were repeated four times in Fao cell and twice in 3T3 L1 cells.
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We first assessed the efficiency of this shRNA to suppress TRB3 mRNA expression by RT-PCR. Consistent with previous report that TRB3 shRNA inhibits TRB3 gene expression [10,20], expression of TRB3 shRNA in Fao almost completely abolished TRB3 mRNA expression (Fig. 3a lanes 3 and 4), indicating the efficiency of TRB3 shRNA. Next, the virally transduced Fao cells were serum-deprived overnight, followed by a time course of insulin treatment. Total cell lysates were prepared for western blot analysis to assess how TRB3 expression relates to Akt phosphorylation at Ser473. Since TRB3 expression is not induced until 1 h after insulin treatment, our focus of Akt activation was on the later time points of insulin stimulation. As shown in Fig. 3b, in agreement with the view that TRB3 shRNA inhibits TRB3 expression, no induction of TRB3 was observed at the presence of TRB3 shRNA (lanes 7– 12). Quantification of level of Akt phosphorylation indicated that the level of Akt phosphorylation under TRB3 shRNA expression is generally higher (∼ 20%) than that of normal condition (Fig. 3b left panel), in agreement that TRB3 negatively regulates Akt activation. It is known that prolonged-insulin stimulation induces insulin resistance. There-
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fore, we assessed whether TRB3 expression contributes to such process. To this end, we further stimulated 8-hour insulin pretreated Fao cells with insulin for 30 min. Shown in Fig. 3b, we observed that TRB3 expression prevents a premature upregulation of Akt activity (compare lanes 6 and 12). Next we examined whether TRB3′s ability to restrain Akt activation can be extended to other agonists e.g. MG132 as MG132 induces Akt activation and TRB3 expression in Fao cells. Similarly, knockdown of TRB3 also enhances Akt activation in MG132 treated Fao Cells (Fig. 3c). Based on all of information presented here, we suggest that TRB3 expression induced provides a feedback loop to modulate Akt activation in Fao cells.
The negative role of Akt in TRB3 expression Because of the inverse relationship between Akt activation and TRB3 expression, we investigated the role of Akt activation in insulin-induced TRB3 expression. To this end, we treated Fao cells with P3IK inhibitor LY294002 (10 μM) and Akt inhibitor VIII (2 μM, specific to Akt1 and 2) to block Akt activation. Pretreatment of Fao cells with LY294002 (10 μM)
Fig. 3 – Induction of TRB3 expression restrains Akt activation. a) RT-PCR analysis of TRB3 expression in control and TRB3 shRNA expressing Fao cells. Fao cells were transduced with control or TRB3 shRNA expressing lentiviruses. 30 h later, the cells in serum free medium were treated with or without 100 nM insulin (Ins) for 4 h and total RNA were prepared for RT-PCR with primers specific to TRB3 (top panel) and 36B4 (bottom panel), respectively. These experiments were repeated twice with similar results. b) Knockdown of TRB3 in Fao cells enhances Akt activation. Control or TRB3 shRNA (iT3) expressing Fao cells were serum-starved overnight followed by insulin (100 nM) treatment. In addition after 8 h insulin treatment, the cells were re-stimulated with 100 nM for additional 30 min (lane 830). The total cell lysates were prepared at indicated time for western blot analysis of with anti-phospho-Akt at p473, Akt and TRB3 antibodies respectively. These experiments were repeated three times with the similar results. The quantitative analysis of Akt phosphorylation from three independent experiments is shown. The error bar stands for standard deviation. c) Induction of TRB3 expression attenuates Akt activation by MG132. Wild type and TRB3 shRNA expressing Fao cells were serum-deprived for overnight followed by 4 h MG132 (10 μM) treatments. The total cell lysates were prepared at 4 h and subjected to western blot assay with different antibodies. Top: pAkt473. Middle: Akt. Bottom: TRB3. The quantitative analysis of Akt phosphorylation from three different experiments is shown. In all of cases, the amount of Akt phosphorylation was normalized to total amount of Akt and set as 1 under unstimulated condition. The error bars represent the standard deviation or standard error.
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blocked TRB3 expression induced by insulin. In sharp contrast, pretreatment of Fao cells with Akt inhibitor VIII enhanced TRB3 expression by 2 folds (Fig. 4a, compare lanes 2 and 4). Akt inhibitor VIII used was effective in these experiments, as it blocked phosphorylation of Akt at Ser473 as well as phosphorylation of GSK3α/β at Ser9/12 (panel ii and iv). As a control, p38 inhibitor SB203580 (SB) had no effect on either TRB3 expression or Akt activation (compare lanes 2 and 5). Increase of TRB3 expression under Akt inhibition implies that Akt may negatively regulate TRB3 expression induced by insulin. To test this notion, we transduced Fao cells with either control (GFP) or different amount of constitutive active Akt (Myr-Akt) expressing pMIGR1 retrovirus and examined the impact of overexpression of constitutive active Akt on TRB3 expression induced by insulin. Shown in Fig. 4b, TRB3 expression is normally induced by insulin in control Fao cells (top panel, lanes 1 and 4). However, the induction of TRB3 expression by insulin is severely impaired at the presence of constitutive active Akt (compare lanes 4, 5 and 6). Furthermore, the amount of TRB3 protein induced by insulin is
inversely correlated with amount of Myr-Akt expressed (compare top and middle panels). As a control, expression of β-tubulin was not affected under any condition (Fig. 4b. bottom panel). To further examine the role of Akt in TRB3 expression, we analyzed TRB3 expression in MEFs that lack either Akt1 or Akt2. As presented in Fig. 4c, the level of TRB3 expression in Akt1−/− and Akt2−/− MEFs is 3 times higher than that of wildtype MEFs. Furthermore, treatment of MEFs with Akt inhibitor IVIII induces TRB3 expression in all of types of MEFs, suggesting that there is an adding effect of Akt1 and Akt2 on inhibition of TRB3 expression. To further examine the regulation of TRB3 expression by Akt, we assessed how Akt modulates TRB3 promoter activity. In transient transfection and luciferase assays in HepG2 cells, TRB3 promoter driven luciferase activity was reduced more than 50% under overexpression of either Akt1 (Fig. 4d) or Akt2 (Fig. 4e), supporting the notion that Akt plays a negative role in TRB3 expression. The inhibition of luciferase reporter activity by Akt is specific to TRB3 promoter as Akt had no inhibitory effect on parental pXP-luciferase reporter activity. Interestingly, the
Fig. 4 – Akt negatively regulates TRB3 expression. a) blocking Akt activation enhances TRB3 expression. Fao cells were serum-deprived for overnight and treated with indicated inhibitors: 10 μM LY294002 (LY), 2.5 μM Akt inhibitor VIII (AI) and 2.5 μM SB29308 (SB) for 45 min followed by 4-hour insulin treatment. After these treatments, the total cell lysates were prepared for western blot analysis. The antibodies used are anti-TRB3 (i), anti-phospho-Akt at Ser473 (ii), anti-Akt (iii), anti-phospho-GSK3α/β and anti-GSK3α/β antibodies, respectively. The quantitative analysis of TRB3 is shown from three different experiments. The error bars represent standard deviation. “*” indicates P N 0.003 from Student's T test. In this assay, amount of TRB3 expression normalized the total amount of Akt and is set as 1 in untreated cells. b) Overexpression of Myr-Akt impairs insulin-induced TRB3 expression. Fao cells were transduced with either GFP or HA-Myr-Akt expressing pMIGR1 retroviruses. 48 h posttransduction, the cells treated with or without insulin for 4 h. The total cell lysates were prepared for western blot analysis with anti-TRB3, anti-HA and anti-β-tubulin, respectively. The quantitative analysis was shown in left panel from two independent experiments. c) TRB3 expression is elevated in Akt null MEFs. Wildtype, Akt1 and Akt2 null MEFs The total cells lysates were prepared for western blot analysis with anti-TRB3, anti-Akt1, Anti-Akt2 and anti-β-tubulin (tub) antibodies, respectively. These experiments were repeated twice with MEFs from different Akt knockout mice and similar results were obtained. The quantification of TRB3 expression from these two experiments is shown in the top. The level of TRB3 expression in control wildtype MEFS was set as 1. The error bars stands standard error. d) Akt1 inhibits TRB3 promoter activity in HepG2 cells. TRB3 promoter luciferase construct (100 ng) were co-transfected with indicated amount of Akt1 expression plasmids. 30 h later, the total cells lysates were prepared for luciferase assays. The experiments were carried out as triplicate and repeated twice. The finally luciferase activity was normalized to β-galactosidase activity co-transfected with reporter and effector plasmids. TRB-luc: TRB3 promoter luciferase reporter. pXP2: parental luciferase reporter without TRB3 promoter e) The same as d) except Akt2 expression vector used. The error bar represents standard deviation. The * indicates P b 0.02 from one way ANOVA comparing with control cells.
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ability of Akt to inhibit TRB3 promoter activity depends on the dose of Akt expression as increase amount of Akt expression showed less inhibition.
Atypical PKCζ positively regulates TRB3 expression induced by insulin To further investigate putative kinase(s) that is required for TRB3 expression induced by insulin, we examined the impact of a panel protein kinase inhibitors including CamK inhibitor NK-93, MAPK inhibitors PD98059 and U0126, JNK inhibitor SP600125, PKC inhibitors GF109203x and RO32-0432 and p38 inhibitor SB203580, on TRB3 expression induced by insulin. As shown in Fig. 5a, other than RO32-0432, none of these inhibitors affected TRB3 expression induced by insulin, suggesting that an RO32-0432 sensitive kinase activated by PI3K, mediates insulin-induced TRB3 expression. Both GF109203x and RO32-0432 inhibit PKCs. GF109203x mainly inhibits conventional and novel PKCs while RO32-0432 inhibits atypical PKCs including PKCζ. Inhibition of TRB3 expression by R032-0432 implies that atypical PKC may be the kinase that is required for insulin-induced TRB3 expression. To address this question, we examined whether PCKz modulates TRB3 promoter activity. In transient transfection
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and luciferase assay (Fig. 5b), 8-hour insulin treatment led to a 2.5-fold increase of TRB3 promoter-mediated luciferase activity, demonstrating that TRB3-luciferase reporter is insulin responsive. Co-transfection of a PKCz expression vector with TRB3-luciferase reporter led to 3-fold increase of basal and 4fold increase of insulin stimulated TRB3 promoter mediated luciferase activity while co-transfection of the dominant negative (K281W) PKCz and PKCz shRNA reduced the basal level of TRB3 promoter activity and impaired insulin-induced TRB3 promoter activity. To further confirm this result, we examined the impact of PKCz on TRB3 expression in HepG2 cells. As shown in Fig. 5c, TRB3 expression is elevated by insulin treatment (compare lanes 1 and 2) in HepG2 cells. Expression of PKCz significantly increases TRB3 expression (2 folds) while dominant negative PKCz reduces TRB3 protein expression. Taken together our data support the notion that TRB3 expression is positively regulated by atypical PKCζ. In addition, we also examined the effect of dominant negative PKCζ on TRB3 promoter activity induced by MG132 (Fig. 5b). Consistent with the notion that MG132 is a stronger inducer of TRB3 expression, MG132 exhibited a higher ability to enhance TRB3 promoter activity (4 folds) (Fig. 4b). Expression of DN PKCζ reduced MG132-induced TRB3 promoter activity by 30%. This likely reflects the view that MG132 activated multiple
Fig. 5 – Atypical PKCζ activates TRB3 promoters. a) Western blot analysis of Fao cell lysates pretreated with different kinase inhibitors followed by 4 h insulin treatment. LY: LY294002-10 μM, RO: RO32-0432-2 μM, GF: GF109203x-2 μM, PD: PD9805-10 μM UO: U0126-1 μM, SP: SP600125-5 μM and NK: NK-93-10 μM. The amount of TRB3 expression is normalized to the amount of beta-tubulin in corresponding lane and was set as 1 in unstimulated condition. The * indicates F b 0.06 from one way AVONA F-test. b) Transient transfection and luciferase assays of TRB3 promoter in HepG2 cell. HepG2 cells in 12-well dish were transfected with TRB3-promoter reporter (100 ng of per well) plus (100 ng) empty vector pcDNA3 (EV) or pcDNA3-HA-tagged wildtype PKCζ (WT) or dominant negative PKCζ (DN), iRNA: PKCζ shRNA. 30 h later, the cells were treated with or without 100 nM insulin for 8 h. The total cell lysates were prepared for luciferase assays. The experiments were carried out as triplicate and repeated twice. The finally luciferase activity was normalized to β-galactosidase activity co-transfected with reporter and effector plasmids. The error bars represents standard deviation from three experiments. The * indicates F b 0.02 from one way AVONA assay. c) Western blot analysis to show that PKCζ promotes TRB3 expression in HepG2 cells. HepG2 cells in 6-well dished were transfected with pcDNA3 (EV) or pcDNA3-HA-PKCζ (WT) or pcDNA-HA-KA-PKCζ (DN) (300 ng per well). 30 h post-transfection, the cells were treated with (Ins+) or without (Ins−) insulin for 8 h. The total cell lysates were prepared for western blot analysis with anti-TRB3 (top), anti-HA (middle) and anti-beta-tubulin (bottom) antibodies, respectively. In left panel, the amount of TRB from three different experiments is normalized to beta-tubulin and set as 1 in unstimulated control cells. The * indicates p b 0.043 from a Student's T test. The ** indicates p b 0.051.
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pathways including inhibiting protein degradation and PI3K pathway to induce TRB3 expression.
Discussion In this report, we have examined the dynamic relationship between TRB3 expression and Akt activation. We found that while insulin induces TRB3 expression, it only does so in certain cells, namely hepatocytes and adipocytes, but not others, such as beta cell and muscle type cells. Inability of insulin to induce TRB3 expression in beta cells and muscle cells is not due to that TRB3 gene is inactive as MG132 increases TRB3 expression in all of type cells. In both Fao cells and adipocytes, the induction of TRB3 expression and Akt activation by insulin are inversely related, suggesting that there is functional relationship between Akt activation and TRB3 expression. To determine the functional relationship between Akt and TRB3 expression, we have examined how suppression of TRB3 expression by TRB3 shRNA impacts on Akt activation. Our data demonstrated TRB3 expression sustains Akt activation, consistent with the early finding [31] and renders Akt refractory to further activation. Role of TRB3 in Akt activation has been studied in different contents. In both mouse muscle and liver, increased TRB3 expression is associated with impaired Akt activation [9,11,12] and in primary hepatocytes [11] with some conflicts [32]. In our studies, we found that expression of TRB3 sustained Akt activation, but not abolished Akt activation. In addition, our data indicated that induction of TRB3 expression by insulin renders Akt refractory to further activation. In this regard, the role of TRB3 in Akt activation is to fine-tune Akt activation, instead of abolishing Akt activation. Therefore, it is tempting to believe that TRB3 expression is more important in Akt signaling where Akt activity requires carefully balanced. It is noteworthy that recent studies of TRB3 null mice indicated that inaction of TRB3 in mice had no apparent effect on glucose and lipid metabolism under normal condition [33]. This is inconsistent with the notion that TRB3 negative regulates Akt activation. However, it is possible that under pathological conditions such as obesity, TRB3 null mice may behave differently as the author proposed. In an attempt to get insight into the regulation of TRB3 expression by insulin, we have examined the role of PI3K and its downstream kinases, namely Akt and PCKζ in TRB3 expression. In agreement with the early studies [26,27], insulin induces TRB3 expression in PI3K dependent manner. However, completely unexpected, Akt negatively regulates TRB3 expression by insulin evidenced by a) blocking Akt activation enhances TRB3 expression; b) overexpression of Akt inhibits TRB3 expression (Fig. 4b) and c) Akt1 and 2 null MEFs have significant higher TRB3 expression than wildtype MEFs (Fig. 4c). In this regard, the level of TRB3 expression induced by insulin, in part, depends on relative level of Akt activation. Lower level of Akt activation will lead to higher TRB3 expression and vice versa. This may explain several observations of regarding TRB3 expression. TRB3 expression is induced during fasting and elevated in insulin resistant state [10,11]. Though TRB3 expression during fasting could be due to increase in the level of Glucogan, expression of TRB3 by Dex/
Forsklin has not been consistent [27,34]. In addition, glucose deprivation has been shown to induce TRB3 expression [35]. It has been speculated that lower glucose in blood induces TRB3 expression. However, this could not be the reason that TRB3 expression is elevated in insulin resistant condition because blood glucose level is high under insulin resistance state. Based on our hypothesis, we believe that increase of TRB3 expression during fasting could be due to decrease of Akt activation, the result of drop of insulin level, therefore, TRB3 expression is increased. Furthermore, in insulin resistance, Akt activation is reduced. Accordingly TRB3 expression is elevated. Recently, it is reported that expression of constitutive active FoxO activates Akt and inhibits TRB3 expression in hepatocytes [27,36]. It was further shown that FoxO1 inhibited TRB3 expression independent of FoxO1 DNA binding activity. The possible explanation for this could be that FoxO1 selectively enhances Akt activation, thereby inhibiting TRB3 expression. It is noteworthy that, in these studies, that expression of Myr-Akt via adenoviral transfer was found to enhance TRB3 mRNA expression in T-antigen immortalized hepatocytes. The reason for the discrepancy between our and those studies was not clear. However, it could be due to either cell type differences (Fao cell in our study vs antigen immortalized hepatocytes) or alternatively the different ways to express Myr-Akt (retrovirus vs adenovirus). In spite of this discrepancy, our findings that MEFs that are either Akt1-null or Akt2-null have a higher level of TRB3 expression argue that Akt negatively regulates TRB3 expression. Because insulin-induced TRB3 expression depends on PI3K kinase and Akt plays a negative role in TRB3 expression, we have sought to investigate the positive signal activated by PI3K for insulin mediated TRB3 expression. Our data suggest that atypical PKCζ could be the kinase that mediates TRB3 expression as atypical PKC inhibitor R032-0432 specifically abolished insulin-induced TRB3 expression This was further demonstrated that overexpression of PKCζ enhanced whereas that of dominant negative PKCz and PKC shRNA suppressed TRB3 promoter activity and TRB3 protein expression. At present, it is not clear how PKCζ enhances TRB3 expression. One of interesting recent finding is that PCKζ can regulate SP1 transcription activity [37]. There are several putative SP1 binding sites in TRB3 promoter. It will be greatly interesting in the future studies to determine whether PKCζ mediates TRB3 expression via phosphorylation of SP1. In spite of this, the potential role of PCKζ in TRB3 expression may provide the insight into cell type specific TRB3 expression. PKCζ is mainly activated via PI3K-IRS-2 pathway [38,39]. IRS-2 is the major isoform of IRS protein family in hepatocytes and adipocytes. Thus, it is likely that PKCζ is specifically activated in hepatocytes and adipocytes, thereby, activating TRB3 expression. PKCζ blunted insulin signaling via interacting with Akt [40] or phosphorylating IRS-1 [41]. Interestingly, inhibition of Akt by PKCζ depends on PI3K [41,42]. As TRB3 negatively regulates Akt activation, mediation of TRB3 expression induced by insulin by PCKζ may provide an additional means for PKCζ to negatively regulate insulin signaling in hepatic cells. In any signal transduction pathway, protein kinase activation needs to be balanced as improper kinase activity is often associated with different pathological conditions. Here we have demonstrated that insulin transmits both negative and positive signals to regulate TRB3 expression. We suggest that
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the level of TRB3 expression induced by insulin depends on relative level Akt activation and PKCζ activation. Thus, TRB3 expression may represent a balanced action of insulin. [14]
Acknowledgments We thank Dr. PN Tsichlis for providing mouse embryonic fibroblasts, Dr. RV Farese for providing PKCζ expression vectors and Dr. R. Cunard for TRB3 promoter constructs. KD is the recipient of Thomas R Lee career development award from The American Diabetes Association.
[15]
[16]
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[30] N. Ohoka, S. Yoshii, T. Hattori, K. Onozaki, H. Hayashi, TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death, EMBO J. 24 (2005) 1243–1255. [31] L. He, F.A. Simmen, H.M. Mehendale, M.J. Ronis, T.M. Badger, Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane. Role of TRB3 in inhibition of Akt/protein kinase B activation, J. Biol. Chem. 281 (2006) 11126–11134. [32] P.B. Iynedjian, Lack of evidence for a role of TRB3/NIPK as an inhibitor of PKB-mediated insulin signalling in primary hepatocytes, Biochem. J. 386 (2005) 113–118. [33] H. Okamoto, E. Latres, R. Liu, K. Thabet, A. Murphy, D. Valenzeula, G.D. Yancopoulos, T.N. Stitt, D.J. Glass, M.W. Sleeman, Genetic deletion of Trb3, the mammalian Drosophila tribbles homolog, displays normal hepatic insulin signaling and glucose homeostasis, Diabetes 56 (2007) 1350–1356. [34] S.Z. Yacoub Wasef, K.A. Robinson, M.N. Berkaw, M.G. Buse, Glucose, dexamethasone and the unfolded protein response regulate TRB3 mRNA expression in 3T3-L1 adipocytes and L6 myotubes, Am. J. Physiol.: Endocrinol. Metab. 291 (2006) E1274–E1280. [35] R. Schwarzer, S. Dames, D. Tondera, A. Klippel, J. Kaufmann, TRB3 is a PI 3-kinase dependent indicator for nutrient starvation, Cell Signal 18 (2006) 899–909. [36] M. Naimi, N. Gautier, C. Chaussade, A.M. Valverde, D. Accili, E. Van Obberghen, Nuclear Foxo1 controls and integrates key signaling pathways in hepatocytes, Endocrinology 148 (2007) 2424–2434.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells Jixin Ding, Satomi Kato, Keyong Du⁎ Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111, USA
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
TRB3 is a pseudokinase whose expression is regulated during stress response and changing
Received 22 October 2007
of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression
Revised version received
is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3
12 January 2008
expression and Akt activation. We find that insulin induces TRB3 expression in cell type
Accepted 31 January 2008
dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle
Available online 11 February 2008
cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles
Keywords:
of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of
P13K
TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3
TRB3
expression whereas inhibition of PKCζ expression impairs TRB3 expression induced by
Insulin gene expression
insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3
Akt atypical PKC
expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced. © 2008 Elsevier Inc. All rights reserved.
Introduction TRB3 belongs to a newly identified family of pseudo-kinases consisting of three mammalian isoforms: TRB1, TRB2 and TRB3. TRBs are closely related to Drosophila tribbles, a negative regulator of cell growth [1,2]. Members of TRB family are characterized by containing a domain that shares high homology with protein kinases [3]. However, this domain of TRBs contains variants in key amino acid residues that are essential for protein kinase catalytic activity and lacks ATP binding domain. As a result, TRBs have no detectable kinase catalytic activity, thereby, termed as pseudo-kinases [4]. However, the kinase homology domain of TRBs possesses a substrate-binding domain that may function as protein interacting module. Consistently, TRBs are found to associate with numerous proteins including transcription factors,
protein kinase as well as ubiquitin E3 ligase. By interacting with different factors, TRBs regulate numerous biological processes including cell growth, differentiation and metabolism. TRB1 interacts with MAPK and modulate MAPK kinase activity [5]. By modulating MAPK signaling, TRB1 has been implicated in smooth muscle cell proliferation [6]. TRB2 has been shown to interact with C/EBPβ, a leucine zipper transcription factor. By modulating C/EBP activity, TRB2 has been implicated in adipocytes differentiation [7] and acute myelogenous leukemia [8]. TRB3 interacts with Akt and inhibits activation of Akt. In both liver [9–11] and muscle [12], decrease in TRB3 expression enhances insulin sensitivity, thereby increasing glucose tolerance. Indeed, a human polymorphism of TRB3 (Q84R), which has higher capacity of inhibiting Akt activation, was found to be associated with insulin resistance and related cardiovascular risk [13]. TRB3
⁎ Corresponding author. Keyong Du Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington St, Boston, MA 02111, USA. Fax: +1 617 636 6217. E-mail address: [email protected] (K. Du). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.01.026
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also directly interacts with E3 ligase COP1 and targets COP1 to acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis[14]. By linking COP1 and ACC1, TRB3 facilitates the degradation of ACC and regulates lipid metabolism [14]. Furthermore, TRB3 interacts with and inhibits ATF4 [15,16], the key factor that regulates amino acid metabolism during integrated stress response [17]. By interacting with ATF4, TRB3 inhibits ATF4 mediated gene expression and may protect ATF4 induced cell cytotoxity [18]. Finally, TRB3 appears also playing important role in cell differentiation as overexpression of TRB3 blocks differentiation of adipocytes [19], muscle [20] and osteoblast [21]. TRB3 was first cloned as an inducible gene in the liver undergoing fatty degeneration [22] and in neuronal cells undergoing apoptosis following NGF withdrawal [23], an indication that TRB3 expression is subjected to regulation. Subsequent studies demonstrated that TRB3 is an inducible gene whose expression is induced by a variety of signals including ER stress [24,25], change in nutritious status [26] and insulin [27]. Since TRB3 inhibits insulin-induced Akt activation, a further examination of such a paradoxical effect is merited. In current study, we examined the dynamic relationship between TRB3 expression and Akt activation. Our studies demonstrate that induction of TRB3 expression provides a checking mechanism for proper Akt activation. TRB3 expression by insulin may reflect the balance of insulin action.
Materials and methods Reagents Insulin, dexamethasone (Dex) and 3-Isobutyl-1-methylxanthine (IBMX) are from Sigma. MG132, thapsigargin and all kinase inhibitors are from Calbiochem. Anti-Akt1 and Akt2 rabbit antibodies, anti-phospho-Akt at Ser473 rabbit antibody are from Upstate Biotech; anti-HA monoclonal antibody is from Covance; Anti-flag antibodies, anti-HA antibodies and anti-beta-tubulin are from (Sigma). The anti-TRB3 rabbit antibody has been described [9]. All of protein kinase inhibitors are from Calbiochem.
Plasmids TRB3 shRNA has been described [20]. PKCζ shRNA was purchased from Addgene (Cambridge MA, Plasmid 10803). To generate Myr-Akt expressing pMIGR1, Myr-Akt was released from pCMV6-Myr-Akt with BglII and EcoRI and cloned into pMIGR1 retroviral vectors. PKCζ expression vectors are gifts of Dr. RV Farese of University of South Florida College of Medicine [28]. TRB3 promoter luciferase construct is generated as described [29].
Cell culture, transient transfection and luciferase assay Fao cells were grown in RPMI1640 supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin,
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and 100 μg/ml streptomycin (Invitrogen). For viral infection of Fao cells, 40% confluent Fao cells were incubated with TRB3 shRNA lentivirus plus 6 μg/ml of polybrene. 3T3 L1 preadipocytes were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The adipocyte differentiation of 3T3 L1 preadipocytes is as follows. Briefly, 3T3 L1 preadipocytes were cultured for two additional days after reaching 100% confluence and treated with differentiation medium (DMEM-high glucose containing 10% FBS, 2.5 μg/ml insulin, 0.5 mM IBMX and 2.5 mM Dex, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 4 days. Then, the medium was changed to regular medium. After 7 days differentiation, the adipocytes were used for experiments. Wildtype, Akt1−/−, and Akt2−/− mouse embryonic Fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).
Western blotting After indicated treatments, cells were washed twice with phosphate-buffered saline and extracted with cell lysate buffer (20 mM Tris pH = 7.6, 250 mM NaCl, 0,5 mM EDTA, 0,5 mM DTT, 10 mM β-glycerophosphate, 10% glycerol and protease inhibitors). Equal amounts of protein were subjected to SDS PAGE electrophoresis and transferred to nitrocellulose membranes (Biorad). After blocking in 5% dry milk, the membranes were incubated with each primary antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. The protein bands were visualized using the ECL detection system (Amersham Biosciences). In all cases, blots were not re-probed. Instead, the artifact introduced by stripping the blots was avoided by running parallel blots.
RNA and RT-PCR The total RNA were prepared by using Qiagen Rneasy Kit according to manufacturer's instruction. RT-PCRs were carried by using RT-PCR kit from Ambion with primers for TRB3: 5′ATGCGAGCTACACCTCTGGC-3 and 5′-TAAGGCCCCAGTCGAGTTGC-3′ and 36B4: forward: 5-ATGATTATCCAAAAT GCTTCATTG-3 and reverse 5-AACAGCA TATCCCGAATCTCA-3. Quantification of all western blots are quantified with Genetool.
Results Cell type specific induction of TRB3 expression by insulin TRB3 expression was induced in hepatocytes by insulin [27]. To explore this matter further, we analyzed insulin-induced TRB3 expression in Fao rat hepatoma cells, 3T3L1 adipocytes, β-TC3 mouse, a clonal β cells, L6 rat myotubes and 3T3-L1 preadipocytes by western blot after these cells were treated with insulin for 4 h. We observed that TRB3 expression was induced by insulin in Fao cells (Fig. 1a, top panel,) and 3T3-L1 adipocytes (Fig. 1b, top panel,). However, no change of TRB3
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Fig. 1 – Cell type specific TRB3 expression by insulin. a) Western blot analysis of Fao cells total lysates. b) Western blot analysis of 3T3L1 adipocyte total cell lysates. c) Western blot analysis of β-TC3 cell lysates. d) Western blot analysis of rat L6 myotube. e) Western blot analysis of 3T3-L1 preadipocyte lysates. The cells are serum-deprived for overnight. All treatment is for 4 h. The amount of reagents is Insulin (Ins) 1 μg/ml, MG132 (MG) 10 μM. The antibodies used are anti-TRB3 and Akt antibodies. f) The quantitative analysis of TRB3 expression in each cell lines from three independent experiments. In all of cases, the amount of TRB3 expression was normalized to total amount of Akt and set as 1 under unstimulated condition. The error bars represent the standard deviation or standard error.
expression was detected in β-TC3 cells (Fig. 1c), L6 myotubes (Fig. 1d) and 3T3L1 fibroblast following insulin treatment. The inability of insulin to induce TRB3 expression in these cells is unlikely due to the period of insulin treatment as no TRB3 expression was observed after 16 h insulin treatment (data not shown). As a control, we also treated these different cells with 10 μM proteasomal inhibitor MG132 which is known to elevate TRB3 levels due to blocking protein degradation [30]. Consistently, MG132 was effective on inducing TRB3 expression in all types of cells, suggesting that insulin-induced TRB3 expression is cell type specific. In addition, induction of TRB3 expression by insulin in 3T3L1 adipocytes but not preadipocytes suggests that inducibility of TRB3 expression is acquired during differentiation. We also examined the Akt activation responding to each stimulus by assessing Akt phosphorylation. Insulin induced Akt activation in most cell lines. Unexpectedly, MG132 also induced Akt activation in Fao cells, an event not understood at present, but in PI3K dependent manner (data not shown). Regardless, our data demonstrated that TRB3 expression by insulin is regulated in the cell type manner. Because TRB3 expression is specifically induced in Fao cells and 3T3-L1 adipocytes, we further examined the relationship between TRB3 expression and Akt activation. In both Fao cells (Fig. 2a) and 3T3-L1 adipocytes (Fig. 2b), the induction of TRB3 lagged the activation of Akt. Akt remained active for 2 h while the induction of TRB3 commenced at 2 h post-insulin treatment. TRB3 has been shown to modulate Akt activation in the liver and muscle [9,12]. One might wonder what is the impact of TRB3 expression induced by insulin on Akt activation? To address this question, we assessed Akt activation by insulin under TRB3 knockdown in Fao cells. To this end, Fao cells were transduced with shRNA expressing or controlling lentivirus.
Fig. 2 – The relationship between Akt activation and TRB3 expression in Fao cells a) and 3T3-L1 adipocytes b) Fao cells or 3T3-L1 adipocytes were serum-starved for overnight and treated with 100 nM insulin (Ins). The total cell lysates were prepared as indicated time for western blot analysis. The quantitative analysis of Akt phosphorylation and TRB3 expression is shown in left panel of each figure. In this assay, the amount of Akt phosphorylation or TRB3 expression at time zero was set as 1 after normalized to amount of Akt in each lane. These experiments were repeated four times in Fao cell and twice in 3T3 L1 cells.
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We first assessed the efficiency of this shRNA to suppress TRB3 mRNA expression by RT-PCR. Consistent with previous report that TRB3 shRNA inhibits TRB3 gene expression [10,20], expression of TRB3 shRNA in Fao almost completely abolished TRB3 mRNA expression (Fig. 3a lanes 3 and 4), indicating the efficiency of TRB3 shRNA. Next, the virally transduced Fao cells were serum-deprived overnight, followed by a time course of insulin treatment. Total cell lysates were prepared for western blot analysis to assess how TRB3 expression relates to Akt phosphorylation at Ser473. Since TRB3 expression is not induced until 1 h after insulin treatment, our focus of Akt activation was on the later time points of insulin stimulation. As shown in Fig. 3b, in agreement with the view that TRB3 shRNA inhibits TRB3 expression, no induction of TRB3 was observed at the presence of TRB3 shRNA (lanes 7– 12). Quantification of level of Akt phosphorylation indicated that the level of Akt phosphorylation under TRB3 shRNA expression is generally higher (∼ 20%) than that of normal condition (Fig. 3b left panel), in agreement that TRB3 negatively regulates Akt activation. It is known that prolonged-insulin stimulation induces insulin resistance. There-
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fore, we assessed whether TRB3 expression contributes to such process. To this end, we further stimulated 8-hour insulin pretreated Fao cells with insulin for 30 min. Shown in Fig. 3b, we observed that TRB3 expression prevents a premature upregulation of Akt activity (compare lanes 6 and 12). Next we examined whether TRB3′s ability to restrain Akt activation can be extended to other agonists e.g. MG132 as MG132 induces Akt activation and TRB3 expression in Fao cells. Similarly, knockdown of TRB3 also enhances Akt activation in MG132 treated Fao Cells (Fig. 3c). Based on all of information presented here, we suggest that TRB3 expression induced provides a feedback loop to modulate Akt activation in Fao cells.
The negative role of Akt in TRB3 expression Because of the inverse relationship between Akt activation and TRB3 expression, we investigated the role of Akt activation in insulin-induced TRB3 expression. To this end, we treated Fao cells with P3IK inhibitor LY294002 (10 μM) and Akt inhibitor VIII (2 μM, specific to Akt1 and 2) to block Akt activation. Pretreatment of Fao cells with LY294002 (10 μM)
Fig. 3 – Induction of TRB3 expression restrains Akt activation. a) RT-PCR analysis of TRB3 expression in control and TRB3 shRNA expressing Fao cells. Fao cells were transduced with control or TRB3 shRNA expressing lentiviruses. 30 h later, the cells in serum free medium were treated with or without 100 nM insulin (Ins) for 4 h and total RNA were prepared for RT-PCR with primers specific to TRB3 (top panel) and 36B4 (bottom panel), respectively. These experiments were repeated twice with similar results. b) Knockdown of TRB3 in Fao cells enhances Akt activation. Control or TRB3 shRNA (iT3) expressing Fao cells were serum-starved overnight followed by insulin (100 nM) treatment. In addition after 8 h insulin treatment, the cells were re-stimulated with 100 nM for additional 30 min (lane 830). The total cell lysates were prepared at indicated time for western blot analysis of with anti-phospho-Akt at p473, Akt and TRB3 antibodies respectively. These experiments were repeated three times with the similar results. The quantitative analysis of Akt phosphorylation from three independent experiments is shown. The error bar stands for standard deviation. c) Induction of TRB3 expression attenuates Akt activation by MG132. Wild type and TRB3 shRNA expressing Fao cells were serum-deprived for overnight followed by 4 h MG132 (10 μM) treatments. The total cell lysates were prepared at 4 h and subjected to western blot assay with different antibodies. Top: pAkt473. Middle: Akt. Bottom: TRB3. The quantitative analysis of Akt phosphorylation from three different experiments is shown. In all of cases, the amount of Akt phosphorylation was normalized to total amount of Akt and set as 1 under unstimulated condition. The error bars represent the standard deviation or standard error.
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blocked TRB3 expression induced by insulin. In sharp contrast, pretreatment of Fao cells with Akt inhibitor VIII enhanced TRB3 expression by 2 folds (Fig. 4a, compare lanes 2 and 4). Akt inhibitor VIII used was effective in these experiments, as it blocked phosphorylation of Akt at Ser473 as well as phosphorylation of GSK3α/β at Ser9/12 (panel ii and iv). As a control, p38 inhibitor SB203580 (SB) had no effect on either TRB3 expression or Akt activation (compare lanes 2 and 5). Increase of TRB3 expression under Akt inhibition implies that Akt may negatively regulate TRB3 expression induced by insulin. To test this notion, we transduced Fao cells with either control (GFP) or different amount of constitutive active Akt (Myr-Akt) expressing pMIGR1 retrovirus and examined the impact of overexpression of constitutive active Akt on TRB3 expression induced by insulin. Shown in Fig. 4b, TRB3 expression is normally induced by insulin in control Fao cells (top panel, lanes 1 and 4). However, the induction of TRB3 expression by insulin is severely impaired at the presence of constitutive active Akt (compare lanes 4, 5 and 6). Furthermore, the amount of TRB3 protein induced by insulin is
inversely correlated with amount of Myr-Akt expressed (compare top and middle panels). As a control, expression of β-tubulin was not affected under any condition (Fig. 4b. bottom panel). To further examine the role of Akt in TRB3 expression, we analyzed TRB3 expression in MEFs that lack either Akt1 or Akt2. As presented in Fig. 4c, the level of TRB3 expression in Akt1−/− and Akt2−/− MEFs is 3 times higher than that of wildtype MEFs. Furthermore, treatment of MEFs with Akt inhibitor IVIII induces TRB3 expression in all of types of MEFs, suggesting that there is an adding effect of Akt1 and Akt2 on inhibition of TRB3 expression. To further examine the regulation of TRB3 expression by Akt, we assessed how Akt modulates TRB3 promoter activity. In transient transfection and luciferase assays in HepG2 cells, TRB3 promoter driven luciferase activity was reduced more than 50% under overexpression of either Akt1 (Fig. 4d) or Akt2 (Fig. 4e), supporting the notion that Akt plays a negative role in TRB3 expression. The inhibition of luciferase reporter activity by Akt is specific to TRB3 promoter as Akt had no inhibitory effect on parental pXP-luciferase reporter activity. Interestingly, the
Fig. 4 – Akt negatively regulates TRB3 expression. a) blocking Akt activation enhances TRB3 expression. Fao cells were serum-deprived for overnight and treated with indicated inhibitors: 10 μM LY294002 (LY), 2.5 μM Akt inhibitor VIII (AI) and 2.5 μM SB29308 (SB) for 45 min followed by 4-hour insulin treatment. After these treatments, the total cell lysates were prepared for western blot analysis. The antibodies used are anti-TRB3 (i), anti-phospho-Akt at Ser473 (ii), anti-Akt (iii), anti-phospho-GSK3α/β and anti-GSK3α/β antibodies, respectively. The quantitative analysis of TRB3 is shown from three different experiments. The error bars represent standard deviation. “*” indicates P N 0.003 from Student's T test. In this assay, amount of TRB3 expression normalized the total amount of Akt and is set as 1 in untreated cells. b) Overexpression of Myr-Akt impairs insulin-induced TRB3 expression. Fao cells were transduced with either GFP or HA-Myr-Akt expressing pMIGR1 retroviruses. 48 h posttransduction, the cells treated with or without insulin for 4 h. The total cell lysates were prepared for western blot analysis with anti-TRB3, anti-HA and anti-β-tubulin, respectively. The quantitative analysis was shown in left panel from two independent experiments. c) TRB3 expression is elevated in Akt null MEFs. Wildtype, Akt1 and Akt2 null MEFs The total cells lysates were prepared for western blot analysis with anti-TRB3, anti-Akt1, Anti-Akt2 and anti-β-tubulin (tub) antibodies, respectively. These experiments were repeated twice with MEFs from different Akt knockout mice and similar results were obtained. The quantification of TRB3 expression from these two experiments is shown in the top. The level of TRB3 expression in control wildtype MEFS was set as 1. The error bars stands standard error. d) Akt1 inhibits TRB3 promoter activity in HepG2 cells. TRB3 promoter luciferase construct (100 ng) were co-transfected with indicated amount of Akt1 expression plasmids. 30 h later, the total cells lysates were prepared for luciferase assays. The experiments were carried out as triplicate and repeated twice. The finally luciferase activity was normalized to β-galactosidase activity co-transfected with reporter and effector plasmids. TRB-luc: TRB3 promoter luciferase reporter. pXP2: parental luciferase reporter without TRB3 promoter e) The same as d) except Akt2 expression vector used. The error bar represents standard deviation. The * indicates P b 0.02 from one way ANOVA comparing with control cells.
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ability of Akt to inhibit TRB3 promoter activity depends on the dose of Akt expression as increase amount of Akt expression showed less inhibition.
Atypical PKCζ positively regulates TRB3 expression induced by insulin To further investigate putative kinase(s) that is required for TRB3 expression induced by insulin, we examined the impact of a panel protein kinase inhibitors including CamK inhibitor NK-93, MAPK inhibitors PD98059 and U0126, JNK inhibitor SP600125, PKC inhibitors GF109203x and RO32-0432 and p38 inhibitor SB203580, on TRB3 expression induced by insulin. As shown in Fig. 5a, other than RO32-0432, none of these inhibitors affected TRB3 expression induced by insulin, suggesting that an RO32-0432 sensitive kinase activated by PI3K, mediates insulin-induced TRB3 expression. Both GF109203x and RO32-0432 inhibit PKCs. GF109203x mainly inhibits conventional and novel PKCs while RO32-0432 inhibits atypical PKCs including PKCζ. Inhibition of TRB3 expression by R032-0432 implies that atypical PKC may be the kinase that is required for insulin-induced TRB3 expression. To address this question, we examined whether PCKz modulates TRB3 promoter activity. In transient transfection
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and luciferase assay (Fig. 5b), 8-hour insulin treatment led to a 2.5-fold increase of TRB3 promoter-mediated luciferase activity, demonstrating that TRB3-luciferase reporter is insulin responsive. Co-transfection of a PKCz expression vector with TRB3-luciferase reporter led to 3-fold increase of basal and 4fold increase of insulin stimulated TRB3 promoter mediated luciferase activity while co-transfection of the dominant negative (K281W) PKCz and PKCz shRNA reduced the basal level of TRB3 promoter activity and impaired insulin-induced TRB3 promoter activity. To further confirm this result, we examined the impact of PKCz on TRB3 expression in HepG2 cells. As shown in Fig. 5c, TRB3 expression is elevated by insulin treatment (compare lanes 1 and 2) in HepG2 cells. Expression of PKCz significantly increases TRB3 expression (2 folds) while dominant negative PKCz reduces TRB3 protein expression. Taken together our data support the notion that TRB3 expression is positively regulated by atypical PKCζ. In addition, we also examined the effect of dominant negative PKCζ on TRB3 promoter activity induced by MG132 (Fig. 5b). Consistent with the notion that MG132 is a stronger inducer of TRB3 expression, MG132 exhibited a higher ability to enhance TRB3 promoter activity (4 folds) (Fig. 4b). Expression of DN PKCζ reduced MG132-induced TRB3 promoter activity by 30%. This likely reflects the view that MG132 activated multiple
Fig. 5 – Atypical PKCζ activates TRB3 promoters. a) Western blot analysis of Fao cell lysates pretreated with different kinase inhibitors followed by 4 h insulin treatment. LY: LY294002-10 μM, RO: RO32-0432-2 μM, GF: GF109203x-2 μM, PD: PD9805-10 μM UO: U0126-1 μM, SP: SP600125-5 μM and NK: NK-93-10 μM. The amount of TRB3 expression is normalized to the amount of beta-tubulin in corresponding lane and was set as 1 in unstimulated condition. The * indicates F b 0.06 from one way AVONA F-test. b) Transient transfection and luciferase assays of TRB3 promoter in HepG2 cell. HepG2 cells in 12-well dish were transfected with TRB3-promoter reporter (100 ng of per well) plus (100 ng) empty vector pcDNA3 (EV) or pcDNA3-HA-tagged wildtype PKCζ (WT) or dominant negative PKCζ (DN), iRNA: PKCζ shRNA. 30 h later, the cells were treated with or without 100 nM insulin for 8 h. The total cell lysates were prepared for luciferase assays. The experiments were carried out as triplicate and repeated twice. The finally luciferase activity was normalized to β-galactosidase activity co-transfected with reporter and effector plasmids. The error bars represents standard deviation from three experiments. The * indicates F b 0.02 from one way AVONA assay. c) Western blot analysis to show that PKCζ promotes TRB3 expression in HepG2 cells. HepG2 cells in 6-well dished were transfected with pcDNA3 (EV) or pcDNA3-HA-PKCζ (WT) or pcDNA-HA-KA-PKCζ (DN) (300 ng per well). 30 h post-transfection, the cells were treated with (Ins+) or without (Ins−) insulin for 8 h. The total cell lysates were prepared for western blot analysis with anti-TRB3 (top), anti-HA (middle) and anti-beta-tubulin (bottom) antibodies, respectively. In left panel, the amount of TRB from three different experiments is normalized to beta-tubulin and set as 1 in unstimulated control cells. The * indicates p b 0.043 from a Student's T test. The ** indicates p b 0.051.
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pathways including inhibiting protein degradation and PI3K pathway to induce TRB3 expression.
Discussion In this report, we have examined the dynamic relationship between TRB3 expression and Akt activation. We found that while insulin induces TRB3 expression, it only does so in certain cells, namely hepatocytes and adipocytes, but not others, such as beta cell and muscle type cells. Inability of insulin to induce TRB3 expression in beta cells and muscle cells is not due to that TRB3 gene is inactive as MG132 increases TRB3 expression in all of type cells. In both Fao cells and adipocytes, the induction of TRB3 expression and Akt activation by insulin are inversely related, suggesting that there is functional relationship between Akt activation and TRB3 expression. To determine the functional relationship between Akt and TRB3 expression, we have examined how suppression of TRB3 expression by TRB3 shRNA impacts on Akt activation. Our data demonstrated TRB3 expression sustains Akt activation, consistent with the early finding [31] and renders Akt refractory to further activation. Role of TRB3 in Akt activation has been studied in different contents. In both mouse muscle and liver, increased TRB3 expression is associated with impaired Akt activation [9,11,12] and in primary hepatocytes [11] with some conflicts [32]. In our studies, we found that expression of TRB3 sustained Akt activation, but not abolished Akt activation. In addition, our data indicated that induction of TRB3 expression by insulin renders Akt refractory to further activation. In this regard, the role of TRB3 in Akt activation is to fine-tune Akt activation, instead of abolishing Akt activation. Therefore, it is tempting to believe that TRB3 expression is more important in Akt signaling where Akt activity requires carefully balanced. It is noteworthy that recent studies of TRB3 null mice indicated that inaction of TRB3 in mice had no apparent effect on glucose and lipid metabolism under normal condition [33]. This is inconsistent with the notion that TRB3 negative regulates Akt activation. However, it is possible that under pathological conditions such as obesity, TRB3 null mice may behave differently as the author proposed. In an attempt to get insight into the regulation of TRB3 expression by insulin, we have examined the role of PI3K and its downstream kinases, namely Akt and PCKζ in TRB3 expression. In agreement with the early studies [26,27], insulin induces TRB3 expression in PI3K dependent manner. However, completely unexpected, Akt negatively regulates TRB3 expression by insulin evidenced by a) blocking Akt activation enhances TRB3 expression; b) overexpression of Akt inhibits TRB3 expression (Fig. 4b) and c) Akt1 and 2 null MEFs have significant higher TRB3 expression than wildtype MEFs (Fig. 4c). In this regard, the level of TRB3 expression induced by insulin, in part, depends on relative level of Akt activation. Lower level of Akt activation will lead to higher TRB3 expression and vice versa. This may explain several observations of regarding TRB3 expression. TRB3 expression is induced during fasting and elevated in insulin resistant state [10,11]. Though TRB3 expression during fasting could be due to increase in the level of Glucogan, expression of TRB3 by Dex/
Forsklin has not been consistent [27,34]. In addition, glucose deprivation has been shown to induce TRB3 expression [35]. It has been speculated that lower glucose in blood induces TRB3 expression. However, this could not be the reason that TRB3 expression is elevated in insulin resistant condition because blood glucose level is high under insulin resistance state. Based on our hypothesis, we believe that increase of TRB3 expression during fasting could be due to decrease of Akt activation, the result of drop of insulin level, therefore, TRB3 expression is increased. Furthermore, in insulin resistance, Akt activation is reduced. Accordingly TRB3 expression is elevated. Recently, it is reported that expression of constitutive active FoxO activates Akt and inhibits TRB3 expression in hepatocytes [27,36]. It was further shown that FoxO1 inhibited TRB3 expression independent of FoxO1 DNA binding activity. The possible explanation for this could be that FoxO1 selectively enhances Akt activation, thereby inhibiting TRB3 expression. It is noteworthy that, in these studies, that expression of Myr-Akt via adenoviral transfer was found to enhance TRB3 mRNA expression in T-antigen immortalized hepatocytes. The reason for the discrepancy between our and those studies was not clear. However, it could be due to either cell type differences (Fao cell in our study vs antigen immortalized hepatocytes) or alternatively the different ways to express Myr-Akt (retrovirus vs adenovirus). In spite of this discrepancy, our findings that MEFs that are either Akt1-null or Akt2-null have a higher level of TRB3 expression argue that Akt negatively regulates TRB3 expression. Because insulin-induced TRB3 expression depends on PI3K kinase and Akt plays a negative role in TRB3 expression, we have sought to investigate the positive signal activated by PI3K for insulin mediated TRB3 expression. Our data suggest that atypical PKCζ could be the kinase that mediates TRB3 expression as atypical PKC inhibitor R032-0432 specifically abolished insulin-induced TRB3 expression This was further demonstrated that overexpression of PKCζ enhanced whereas that of dominant negative PKCz and PKC shRNA suppressed TRB3 promoter activity and TRB3 protein expression. At present, it is not clear how PKCζ enhances TRB3 expression. One of interesting recent finding is that PCKζ can regulate SP1 transcription activity [37]. There are several putative SP1 binding sites in TRB3 promoter. It will be greatly interesting in the future studies to determine whether PKCζ mediates TRB3 expression via phosphorylation of SP1. In spite of this, the potential role of PCKζ in TRB3 expression may provide the insight into cell type specific TRB3 expression. PKCζ is mainly activated via PI3K-IRS-2 pathway [38,39]. IRS-2 is the major isoform of IRS protein family in hepatocytes and adipocytes. Thus, it is likely that PKCζ is specifically activated in hepatocytes and adipocytes, thereby, activating TRB3 expression. PKCζ blunted insulin signaling via interacting with Akt [40] or phosphorylating IRS-1 [41]. Interestingly, inhibition of Akt by PKCζ depends on PI3K [41,42]. As TRB3 negatively regulates Akt activation, mediation of TRB3 expression induced by insulin by PCKζ may provide an additional means for PKCζ to negatively regulate insulin signaling in hepatic cells. In any signal transduction pathway, protein kinase activation needs to be balanced as improper kinase activity is often associated with different pathological conditions. Here we have demonstrated that insulin transmits both negative and positive signals to regulate TRB3 expression. We suggest that
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the level of TRB3 expression induced by insulin depends on relative level Akt activation and PKCζ activation. Thus, TRB3 expression may represent a balanced action of insulin. [14]
Acknowledgments We thank Dr. PN Tsichlis for providing mouse embryonic fibroblasts, Dr. RV Farese for providing PKCζ expression vectors and Dr. R. Cunard for TRB3 promoter constructs. KD is the recipient of Thomas R Lee career development award from The American Diabetes Association.
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