Insect Molecular Biology (2015) 24(5), 551–560

doi: 10.1111/imb.12182

Transcription factor CAAT/enhancer-binding protein is involved in regulation of expression of sterol carrier protein x in Spodoptera litura

L.-N. Liang, L.-L. Zhang, B.-J. Zeng, S.-C. Zheng and Q.-L. Feng Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China

These results suggest that the transcription factor C/ EBP may regulate the expression of SlSCPx by binding to the CRE in the promoter of this gene. Keywords: sterol carrier protein, transcription factor, promoter, cholesterol, Spodoptera litura.

Abstract

Introduction

The Spodoptera litura sterol carrier protein x (SlSCPx) gene is expressed in various tissues throughout the life cycle and plays important role in sterol absorption and transport. In this study, the effects of insect hormones (20-hydroexcdysone and juvenile hormone) and lipids (arachidonic acid, cholesterol) on the expression of SlSCPx was analysed by reverse-transcriptase PCR. The results showed that none of these substances significantly induced the expression of SlSCPx in Spodoptera litura-221 (Spli-221) cells. To identify the transcription factors responsible for regulation of SlSCPx expression, a 3311-bp promoter sequence of the gene was cloned. Transcriptional activity of the promoter was studied using an in vivo promoter/reporter system and a 29bp sequence between 21000 and 21029 nucleotides (nt) upstream of this gene was found to be responsible for the up-regulation of the gene. Over-expression of CAAT/enhancer-binding protein (C/EBP) in Spli221 cells increased the promoter activity 5.57-fold. An electrophoretic mobility shift assay showed that two nuclear proteins bound to this sequence. Recombinant C/EBP specifically bound with a putative cis-regulatory element (CRE). Mutation of the C/EBP CRE abolished the binding of the C/EBP with the CRE.

Sterols are one of the major components of cell membranes (Demel & De Kruyff, 1976) and the substrate for ecdysteroid synthesis in insects (Ritter & Nes, 1981; Svoboda, 1999; Gilbert et al., 2002). However, insects are not able to de novo synthesize cholesterol from simple organic compounds because of the lack of two key enzymes, squalene monooxygenase and lanosterol synthase, in the cholesterol biosynthesis pathway (Beydon & Lafont, 1987; Grieneisen, 1994; Zdobnov et al., 2002). Insects therefore rely on their host plants or symbiotic microbes to get sterols to meet the physiological demands for cholesterol (Ritter & Nes, 1981; Gilbert et al., 2002). Sterol carrier protein 2 (SCP2) is a key player in cellular cholesterol transport and distribution in both vertebrates and invertebrates (Gallegos et al., 2001) and therefore is critically important for their survival and growth. SCP2 is a family of sterol carrier proteins, which includes SCPx, SCP2, 17-hydroxysteroid dehydrogenase Type IV, SCP2-like, metallo-b-lactomase and stomatin (Gallegos et al., 2001). These proteins share a common SCP2 domain responsible for binding and transporting sterols and fatty acids (Seedorf et al., 2000). In insects, SCP2 protein is encoded by the SCP2 or SCPx gene, which encodes for both SCP2 (C-terminus of SCPx) and SCPt (N-terminus of SCPx) proteins (Krebs & Lan, 2003; Lan & Wessely, 2004; Takeuchi et al., 2004; Gong et al., 2006; Guo et al., 2009). SCPx and SCP2 genes have been identified and their expression profiles analysed in Aedes aegypti (Krebs & Lan, 2003; Lan & Wessely, 2004), Bombyx mori (Gong et al., 2006), Spodoptera littoralis (Takeuchi et al., 2004) and Spodoptera litura (Guo et al.,

First published online 15 July 2015. Correspondence: Qili Feng, Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. Tel./fax: 1 86 20 85215291; e-mail: [email protected] C 2015 The Royal Entomological Society V

551

552

L.-N. Liang et al.

2009). The midgut is the main tissue where uptake and absorption of cholesterol occur (Komnick & Giesa, 1994; Jouni et al., 2002) and SCPx and SCP2 genes are highly expressed there (Krebs & Lan, 2003; Lan & Wessely, 2004; Takeuchi et al., 2004; Gong et al., 2006; Guo et al., 2009). In S. litura, Slscpx was expressed at higher levels in the midgut than in other tissues during the feeding stage and the expression of Slscpx decreased during the larval moulting and larval–pupal transition stages (Guo et al., 2009). Over-expression of SlSCPx or SlSCPx-2 increased cholesterol uptake in Spli-221 cells, whereas Slscpx RNA interference (RNAi) decreased cholesterol content in the haemolymph and retarded the growth and development of the larvae (Guo et al., 2009). Expression of the SCPx gene appears to be regulated by hormones. In vertebrates, SCPx is expressed in different tissues (Baum et al., 1993; Pfeifer et al., 1993). Hormones such as estradiol (Pfeifer et al., 1993), adrenocorticotropic hormone (Wieslaw et al., 1987) and gonadotropins (Lopez et al., 2007) can induce expression of scpx through cyclic adenosine monophosphate. In Aedes aegypti, 20hydroxyecdysone (20E) at high concentrations decreased the mRNA level of AeSCPx in the gut tissue and in carcass cultures, whereas 20E at physiological concentrations had no effect (Lan & Wessely, 2004). 20E induced expression of AeSCP2 mRNA in a tissue- and time-specific manner. The up-regulation of AeSCP2 was mediated by the transcription factor hormone receptor-3, but not Beta Fushi-tarazu Factor-1 (bFTZ-F1) (Vyazunova & Lan, 2010). Two factors, thanatos-associated protein and activating transcription factor-2, antagonistically regulate AeSCP2 transcription activity in the midgut of feeding larvae of A. aegypti (Peng et al., 2012). In this study, we analysed the promoter sequence and transcription activity of SlSCPx in S. litura and found that the transcription factor C/EBP bound to a response element and up-regulated the expression of this gene. Results Cloning and analysis of regulatory region of SlSCPx To understand the mechanism of regulation of SlSCPx expression, a 3311-bp 50 -end upstream regulatory sequence of SlSCPx was cloned by Tail-PCR amplification (Fig. 1A). A transcription start site at 214 nt upstream of the open reading frame (ORF) was estimated by 50 -RACE PCR with guanosine (G) as the starting nucleotide. Several putative cis-regulatory elements (CREs), including Broad complex-Z1 (BRC-Z1), Broad complex-Z2 (BRC-Z2), Ets transcription factor 74 A (E74A), glial cells missing gene (GCM), E74-like factor-1 (Elf-1), deformed gene (Dfd) and complement factor 2-II (CF2-II), were predicted using both the MATINSPECTOR and TF SEARCH programs (Fig. 1A, B). Only those

sequences that were recognized by both programs were considered to be putative elements. Effects of hormones and lipids on expression of SlSCPx The SlSCPx gene transcribes three mRNA isoforms: 2.8, 2.0 and 0.9 kb (Guo et al., 2009). The 2.8 and 2.0 kb mRNA are thought to be pre-mature and mature mRNA products of SlSCPx, respectively, and the 0.9 kb mRNA is the transcript for SlSCPx-2. The expression of SlSCPx and SlSCPx-2 mRNAs in response to different compounds was analysed by reverse-transcriptase PCR (RTPCR) analysis of 2.8 and 0.9 kb mRNA. The effects of 20E, the juvenile hormone analogue methoprene, arachidonic acid and cholesterol on the expression of SlSCPx in Spli-221 cells were examined (Fig. 2). After treatments with 20E, methoprene or arachidonic acid for 8, 16 or 24 h, the mRNA levels of both SlSCPx and SlSCPx-2 did not significantly change (Fig. 2A–F). Treatment with cholesterol for 12 h also did not change the expression of the gene (Fig. 2G, H). The results indicated that neither the tested hormones nor lipids significantly induced the expression of SlSCPx in Spli-221 cells. Identification and localization of cis-regulatory elements in SlSCPx To determine whether or not there are CREs in the promoter region of SlSCPx, truncated fragments of the promoter were prepared by PCR and cloned into pGL3basic luciferase-expressing vector under the control of these fragments and luciferase expression activities of these constructs were assessed by transfecting Spli-221 cells. The results of the transient luciferase expression at 24 h post-transfection indicated that the expression activity was dramatically reduced when the region between 21186 and 21000 nt was deleted (Fig. 3A). Further analyses of the sequence truncation of this region showed that when the sequence between 21076 and 21000 nt was deleted, the luciferase expression activity was significantly decreased (Fig. 3B). To test whether or not there is a nuclear protein that binds with this region, three overlapping probes each of 35 bp were designed based on the sequence between 21076 and 21000 nt (Fig. 3C) and an electrophoretic mobility shift assay (EMSA) was carried out. The results showed that Probe 1 and Probe 3 bound with two similar nuclear proteins (P1 and P2), whereas Probe 2 bound with one extra abundant protein (P3) in addition to P1 and P2 (Fig. 3D). As all of the probes contained a common motif of TTNNAT/ATAT/AAT, it is possible that this motif could bind to P1 and P2. To further determine which region of the promoter is responsible for the activation, another truncated sequence was prepared. The result indicated that the active CRE is located between 21000 C 2015 The Royal Entomological Society, 24, 551–560 V

C/EBP is involved in regulation of SlSCPx

553

Figure 1. Cloning and analysis of the upstream regulatory sequence of the Spodoptera litura sterol carrier protein x (SlSCPx) gene. (A) The upstream regulatory sequence; the G in red represents the transcription start site, the first amino acid (AA) is underlined with an arrow, and the potential response elements predicted by MATINSPECTOR and TF SEARCH are underlined. (B) Schematic structure of the predicted response elements in the regulatory region of the SlSCPx gene. BRC-Z1, Broad complex-Z1; BRC-Z2, Broad complex-Z2; GCM, Glial cells missing gene; Elf-1, E74-like factor-1; Dfd, Deformed gene; CF2-II, complement factor 2-II; E74, Ets transcription factor 74.

and 21029 nt (Fig. 3E). Further EMSA with Probe 3 showed that the binding of the two nuclear proteins (P1 and P2) could be competitively inhibited by the cold probe (unlabelled Probe 3), but not by a nonspecific probe (Fig. 3F), indicating that the binding of these two proteins to the region (Probe 3) was specific. These results suggested that there was a putative CRE in this region that could specifically bind with two unknown nuclear proteins, regulating the expression of the gene. Analysis of the proteins binding with CRE To identify the two proteins (P1 and P2) that bound to Probe 3, the proteins in the two protein bands were isolated and sequenced using a shotgun strategy. More than 100 peptides were detected in each of the bands (data not shown). C 2015 The Royal Entomological Society, 24, 551–560 V

For P2, the most abundant proteins were b-actin, 14-3-3 zeta and 14-3-3 epsilon. As b-actin is a structural protein, 14-3-3 zeta and 14-3-3 epsilon were selected for further research. 14-3-3 proteins are highly conserved and ubiquitously expressed in eukaryotes. They bind to phospho-serine- and phospho-threonine-containing ligands to regulate the functions of the ligands and are involved in many cellular processes such as cell cycle control, signal transduction and apoptosis (Kumagai & Dunphy, 1999; Fu et al., 2000; Van Hemert et al., 2001; Jin et al., 2004). To analyse whether or not 14-3-3 zeta and 14-3-3 epsilon are involved in the regulation of SlSCPx, cDNAs of 14-3-3 zeta and 14-3-3 epsilon were cloned from S. litura. 14-3-3 zetagreen fluorescent protein (14-3-3 zeta-GFP) and 14-3-3 epsilon-GFP expression vectors were constructed, and each of these GFP expressing vectors was used to cotransfect

554

L.-N. Liang et al.

Figure 2. Reverse-transcriptase PCR analyses of the induced expression of Spodoptera litura sterol carrier protein x (Slscpx) by different compounds in Spli-221 cells. (A–D) Induced expression of SlSCPx by 20-hydroxyecdysone (20E) (A, B) and the juvenile hormone (JH) analogue methoprene (C, D). The cells were exposed to the hormones at 0, 0.5, 2.0 or 8.0 lM and were harvested at 8, 16 or 24 h post-treatment. Both 20E and methoprene were dissolved in 0.1% dimethyl sulphoxide. (E, F) Induced expression of SlSCPx by arachidonic acid. The cells were incubated with arachidonic acid at 0, 1.0 or 10 lM and harvested at 8, 16 or 24 h post-treatment. (G) Induced expression of SlSCPx by cholesterol. The cells were exposed to cholesterol at 0, 1.0, 10 or 100 lM and harvested at 12 h posttreatment. Arachidonic acid and cholesterol were dissolved in 1% ethanol. Error bars show the SD from three biological replicates. No significant differences were detected amongst the treatments (Student’s t-test, P < 0.05).

C 2015 The Royal Entomological Society, 24, 551–560 V

C/EBP is involved in regulation of SlSCPx

555

Figure 3. Localization of active cis-regulatory elements (CREs) in the Spodoptera litura sterol carrier protein x (SlSCPx) regulatory sequence. (A, B) Localization of active CREs in the regulatory sequence between 21570 and 21000 nt. The regulatory sequences were firstly truncated and inserted into pGL3-basic vectors. The recombinant vectors were then transfected into Spli-221 cells. At 24 h post-transfection, the activities of the reporter luciferase were measured with an internal control. (C) Sequence of the probes and their positions. (D) Electrophoretic mobility shift assay (EMSA) of the probes binding with nuclear proteins isolated from Spli-221 cells. (E) Further localization of CREs between 21076 and 21000 nt. (F) EMSA analysis of binding of Probe 3 with nuclear proteins isolated from Spli-221 cells. Two shifted bands (P1 and P2) appeared on the gel. Binding of the P2 protein was totally and competitively inhibited by 503 specific cold probe; binding of the P1 protein could be incompletely competed with the specific cold probe. Neither of the bands were competitively inhibited by 1003 nonspecific probe.

expressing a luciferase reporter gene that is under the control of the promoter sequence between 21000 and 21076 nt. The data showed that although the proteins were efficiently expressed as indicated by enhanced GFP (EGFP) expression (data not shown), they did not change the expression of the reporter luciferase (Fig. 4A). In agreement with this, the recombinant proteins of 14-3-3 zeta and 14-3-3 epsilon were expressed in an Escherichia coli protein expression system and EMSA was conducted with the purified proteins and Probe 3. The results showed that neither of the purified proteins (14-3-3 zeta and 14-3-3 epsilon) bound to the probe (Fig. 4B). Thus, taken together these data suggest that 14-3-3 zeta and 14-3-3 epsilon do not bind to the region between 21000 and 21029 nt in SlSCPX. Identification of C/EBP as an activator of SlSCPx expression Owing to the failure to identify the nuclear proteins that bind to the region between 21000 and 21029 nt by the protein C 2015 The Royal Entomological Society, 24, 551–560 V

sequencing approach, an alternative strategy was employed. Prediction of putative CREs revealed several CREs, including Elf-1, C/EBP and others. To determine whether or not Elf-1 and C/EBP proteins can bind with the DNA sequence, the cDNA fragments that encoded BmElf-1 and BmC/EBP were cloned. In the cotransfection experiment, the expression of BmC/EBP significantly increased the expression of the luciferase reporter, suggesting that it could bind to and regulate the transcriptional activity of the region that contained a C/EBP CRE (Fig. 5A), whereas the expression of Elf-1 did not activate the expression of luciferase (Fig. 5B). To test the specificity of DNA binding with BmC/EBP, three mutated versions of CRE in Probe 3, with changes to the first, middle and last three nucleotides of the putative consensus binding motif of (Ryden & Beemon, 1989; Sourmeli et al., 2003), mainly pyrimidine to purine and purine to pyrimidine, were prepared and tested. The mutants of the C/EBP CRE from TATTGAAAT to GGCTGAAAT (Mutant 1), to TATGAGAAT (Mutant 2) or to

556

L.-N. Liang et al.

Figure 4. Effects of 14-3-3 zeta and 14-3-3 epsilon on the expression of the reporter luciferase driven by regulatory sequences of Spodoptera litura sterol carrier protein x (SlSCPx) and electrophoretic mobility shift assay (EMSA) of the purified 14-3-3 zeta and 14-3-3 epsilon proteins binding to the putative response element. (A) Effects of 14-3-3 zeta and 14-3-3 epsilon on the expression of the reporter luciferase in Spli-221 cells. Expression vectors of 14-3-3 zeta-enhanced green fluorescent protein (14-3-3 zeta-EGFP) or 14-3-3 epsilon-EGFP were cotransfected with the promoter-pGL3 vector into Spli-221 cells and the expression of the reporter luciferase was detected. pEGFP-N1 was a control vector that was used for construction of the 14-3-3 protein expressing vector. Data are mean 6 SD of three independent assays. (B) EMSA analysis of binding of nuclear proteins extracted from Spli-221 cells, the purified recombinant 14-3-3 zeta and 14-3-3 epsilon proteins to probe 3. Error bars show SD from three biological replicates. No significant differences were detected amongst the treatments (Student’s t-test, P < 0.05).

TATTGATCC (Mutant 3) did not bind to P2, but still bound to P1 (Fig. 5C), which suggested that C/EBP CRE is specific to P2. Recombinant BmC/EBP was produced and EMSA was conducted. The results showed that the recombinant protein bound to Probe 3 exclusively and the resultant complex shifted higher than P2 in the C/EBP EMSA gel (Fig. 5D). This occurred because the recombinant C/EBP (31 kDa) was fused with a 16 kDa peptide tag at the N-terminus and a 3 kDa peptide tag at the C-terminus, with a total size of 50 kDa. Taken together, these data suggest that C/EBP bound to a CRE between 21000 and 21029 nt in the promoter of SlSCPx. The common motif TTNNAT/ ATAT/AAT may be responsible for the binding with BmC/EBP. Discussion The functions of SCPx and SCP2 in lipid metabolism have been intensively studied in humans. In insects, the proteins of the SCP2 family have been reported to be involved in the absorption and transportation of sterols and lipids. SCPx and SCP2 genes are regulated by 20E (Vyazunova & Lan, 2010), juvenile hormone III (JH III; Lan & Wessely, 2004) and the substrate cholesterol (Kraemer et al., 1995; Guo et al., 2009). However, neither cholesterol starvation nor feeding cholesterollowering agents, such as cholestyramine and mevinolin, affected the levels of either SCPx-2 or SCPx protein in

rat livers (Paulussen et al., 1989; Baum et al., 1993). Enriching cholesterol in vascular smooth muscle cells increased the mRNA level of SCPx-2 but not SCPx (Kraemer et al., 1995). These results suggest that the response of SCPx to cholesterol is complicated, and is perhaps tissue- and/or species-specific. In S. litura, SlSCPx-2 had high affinity to cholesterol and fatty acids (Zhang et al., 2014). Over-expression of SlSCPx and SlSCPx-2 increased cholesterol uptake into Spli-221 cells from a sterol-containing medium (Guo et al., 2009). In the present study, the expression of SlSCPx was not induced by 20E, the JH analogue methoprene, arachidonic acid or cholesterol in Spli-211 cells (Fig. 2). These results suggest that there are differences in the response to hormone and substrate treatments between larval tissues and in vitro cells. The results of the present study indicate that there are at least two nuclear proteins in Spli-211 cells that bind with a regulatory region of the promoter of the SlSCPx gene (Fig. 3D, F). 14-3-3 zeta, 14-3-3 epsilon and Elf-1 appeared not to be the proteins that bind with the promoter (Fig. 4), although they are considered to be the putative regulators of gene expression based on analysis of protein sequencing and response element prediction. Instead, the transcription factor C/EBP was found to bind with the regulatory region of the promoter of SlSCPx and enhanced the expression of the marker gene luciferase (Fig. 5A). The C/EBP gene family, which contains at least six members, named from C/EBPa to C/EBPf, is characterized by a C 2015 The Royal Entomological Society, 24, 551–560 V

C/EBP is involved in regulation of SlSCPx

557

Figure 5. Effects of the recombinant E74-like factor-1 (Elf-1) and CAAT/enhancer-binding protein (C/EBP) on the expression of the reporter luciferase driven by regulatory sequences of Spodoptera litura sterol carrier protein x (Slscpx) and electrophoretic mobility shift assay (EMSA) of binding of C/EBP protein binding to Probe 3. (A, B) The expression of the reporter luciferase in the Slscpx promotor-pGL3 expression vector after cotransfection with Elf-1enhanced green fluorescent protein (Elf-1-EGFP) (A) or C/EBP-EGFP (B) expression vector. The cells were harvested at 24 h post-cotransfection for luciferase assays. pRL-SV40 was used as an internal control. Data are mean 6 SD of three independent assays. (C) Mutation analysis of Probe 3 binding to nuclear proteins of Spli-221 cells. Wild-type C/EBP cis-regulatory element Probe 3 TATTGAAAT (lane 1) was changed to GCCTGAAAT in Mutant 1 (M1) (lane 2), to TATGAGAAT in Mutant 2 (M2) (lane 3) and to TATTGATCC in Mutant 3 (M3) (lane 4). (D) EMSA analysis of binding of Probe 3 to nuclear proteins extracted from Spli-221 cells (lane 1) and to the purified recombinant C/EBP protein (lanes 2–6). Control cell proteins were the proteins isolated from the bacterial cells transformed with the control vector pET32a (lane 7). The cold probe 3 was a non-labelled probe 3 (lanes 3–6).

highly conserved basic-leucine zipper (bZIP) domain at the C-terminus. This domain is involved in dimerization and DNA binding. This family of proteins plays important roles in cell proliferation, differentiation, nutrition metabolism, signal transduction, stress reaction and immune response (Darlington et al., 1998; Hu et al., 1998; Lekstorm-Himes & Xanthopoulos, 1998; Arizmendi et al., 1999; Cloutier et al., 2009). In insects, C/EBP was first identified in Drosophila melanogaster as Slbo, because the hypomorphic alleles of the gene caused the delayed onset of the border cells migration (Montell et al., 1992). Three C/EBP-like factors have been discovered in B. mori follicular cells (BmC/EBP, BmCBZ and BmC/EBPg). BmC/ EBP was found to bind to the C/EBP response elements in the promoters of chorion genes and to be involved in the C 2015 The Royal Entomological Society, 24, 551–560 V

regulation of the expression of early and late genes throughout choriogenesis (Sourmeli et al., 2005a; Papantonis et al., 2008). BmC/EBP binds to the homologous binding sites of promoters of chorion genes in other moths and flies, such as Dms18 from fruit fly, Ccs15 from medfly, and Ape18/401 and Apo292/10 from two Antheraea species (Sourmeli et al., 2005a). This factor is also required for differentiation during embryogenesis (Rørth & Montell, 1992) and plays an important role in defending against bacterial infection. Many antimicrobial peptide genes, including Cecropins, Lebocin and Moricin, and nitric oxide synthase contain C/EBP binding sites (Meredith et al., 2006; Seiichi et al., 2012). BmC/EBP, whose expression is confined to the ovaries in B. mori, can bind to the C/EBP element as a homodimer with low affinity, but can bind to

558

L.-N. Liang et al.

other motifs efficiently. BmCBZ, recognized as an insectspecific bZIP protein, cannot bind to the C/EBP element, but greatly increases BmC/EBPg binding to the C/EBP element by forming a BmCBZ/BmC/EBP complex (Sourmeli et al., 2005b). The present study found for the first time that this protein bound to the promoter of SlSCPx in S. litura, although the binding and activation mechanisms still need to be elucidated. It has previously been reported that BmC/EBP can bind to a consensus motif of TKNNGY/AAAK/C (K 5 T or G, Y 5 T or C) (Ryden & Beemon, 1989; Sourmeli et al., 2003). Sequence analyses in the present study indicated that the CRE of the promoter of SlSCPx that BmC/EBP bound with contained a similar C/EBP motif (TATTGAAAT). In addition, the shifted binding band in this study was larger than the expected one based on the molecular mass of C/EBP as a monomer (50 kDa), suggesting that it may form a dimer to bind to the promoter. This is consistent with a report that C/EBP binds to a CRE as a dimer in Drosophila (Rørth, 1994). Finally, although SlSCPx was shown to be regulated by BmC/EBP in the present study, how C/EBP manipulates the tissueand time-specific expression of SlSCPx and whether or not other isoforms of C/EBP can bind to the promoter of SlSCPx need further research.

wells at final concentrations of 0.5, 2.0 and 8.0 lM. The control was cells treated with 0.1% dimethyl sulphoxide. The cells were harvested after incubation of 8, 16 or 24 h post-treatment. For the treatments with arachidonic acid and cholesterol, cells were seeded at 105 cells/ml in 12-well plates and cultured for 12 h. The cells then were treated with arachidonic acid at final concentrations of 1.0 and 10 lM were also harvested at 8, 16 or 24 h post-treatment. For cholesterol treatment, the final concentrations were set at 1, 10, 50 and 100 lM and the cells were collected after 12 h. For arachidonic acid and cholesterol treatments, ethanol was used as a control. RT-PCR Total RNA was isolated from the harvested cells using Trizol reagent according to the manufacturer’s protocol (TaKaRa, Dalian, China). For RT-PCR, 2 lg RNA was treated with 2 units DNase I to remove trace amounts of genomic DNA. Reverse transcription was performed using a Reverse Transcriptase M-MLV Kit according to the manufacturer’s protocol (TaKaRa). The primers used for amplifying SlSCPx and SlSCPx-2 cDNA are listed in Table 1. b-actin cDNA was used as an internal control. PCR products were separated on 1.2% agarose gels and stained with ethidium bromide. The quantitative measurement of the corresponding PCR products from three independent experiments was performed using a Gel-Pro analyzer (xx). Construction of recombinant DNA plasmids expressing luciferase or GFP

Experimental procedures Cloning and analysis of regulatory sequences of SlSCPx An upstream 3.3 kb sequence of the SlSCPx gene was cloned by Tail-PCR and analysed using the MatINSPECTOR program (http://www.genomatrix.de) and TF SEARCH (http://www.cbrc.jp/ research/db/TFSEARCH.html) in order to identify putative cisresponse elements. Cell culture and treatment The Spli-221 cell line was obtained from The Entomology Institute of SUN YAT-SEN University, Guangzhou, China. The Spli-221 cells were cultured at 288C in Grace’s insect medium (Invitrogen, Carlsbad, CA, USA) containing 10% foetal bovine serum (Hyclone, Logan, UT, USA). Cells were passaged every two days using a 1:2 dilution. For the treatments with different compounds, cells were seeded at 105 cells/ml in 12-well plates and cultured for 12 h. 20E or methoprene was added into the

An upstream regulatory sequence (from 23097 to 1190 nt) of SlSCPx was amplified by PCR with a forward primer SlSCPx-F3097 and a reverse primer SlSCPx-R1190. The PCR product was cloned into pMD-18T vector (TaKaRa). A series of truncated fragments of the SlSCPx regulatory sequence starting at position 1190 nt and extending to 21000, 21186, 21339, 21443 and 21570 nt was generated by PCR amplification using the SlSCPxps-(23097/1190 nt) plasmid as template and a series of forward primers (SlSCPx-F-1000, SlSCPx-F-1186, SlSCPx-F-1339, SlSCPx-F-1443 and SlSCPx-F-1570) and the same reverse primer. The amplified DNA fragments were ligated to pMD-18T vectors (TaKaRa) for sequencing to confirm the fragments. The fragments were then inserted into the luciferase-expressing pGL3-basic vector (Promega, Madison, WI, USA) to controlling the reporter expression. cDNAs of BmSl14-3-3 zeta, BmSl14-3-3 epsilon, BmElf and BmC/EBP were cloned from B. mori and inserted into the transfection vector pEGFP-N1 individually. The

Table 1. Primers used in this study Name Slscpx-F Slscpx-R Slscpx-2-F b-actin-F b-actin-R Slscpx-F-3097 Slscpx-R1190 14-3-3 zeta F

Sequence(50 -30 )

Name

Sequence(50 –30 )

ATGGGGGCAGAGTAGTAGTG GGTTGTCGGTGTCATTTGC GCTGTCGTCTTTGTCAAGAGG CTCCCTCGAGAAGTCCTA CGAACT0 GGATGCCGCACGATTCCATAC ACGCGTGACGATTTCCCATTAGT CTCGAGTTGACAAAGACGACAGC CGATG TCCGTCGACAAGGA

14-3-3 zeta R 14-3-3 epsilon F 14-3-3 epsilon R Elf-1 F Elf-1 R C/EBP F C/EBP R

TTAGTTGTCGCCGCCCTC CAATGTCGGAAAGGGAAGATAAT CCATTACGAGACGTCCTGGTC ATGCTGATGTTC AGAGAAGAGAAAT ATGCTCTAAGGGCAGTTCCAC ATGGAGTCTCCCCAGATGTAC0 AAGCGTGTGGTCCTGGG

C/EBP, CAAT/enhancer-binding protein; Elf-1, E74-like factor-1; Slscpx, Spodoptera litura sterol carrier protein x.

C 2015 The Royal Entomological Society, 24, 551–560 V

C/EBP is involved in regulation of SlSCPx EGFP at the C-terminus of the recombinant protein acted as an indicator of gene expression. Transfection For transfection, S. litura Spli-221cells were seeded at 105 cells/ml in 12-well tissue culture plates and cultured for 12 h. A mixture of 4.5 lg of the reporter plasmid DNA, 0.5 lg internal control plasmid (pRL-SV40 vector) (Promega) and 5 ll Lipofectamine 2000 (Invitrogen) in 100 ll Opti-MEM medium (Invitrogen) was used to transfect the cells. For cotransfection, a mixture of 3 lg reporter plasmid DNA, 3 lg EGFP-protein plasmid, 0.6 lg internal control plasmid and 5 ll Lipofectamine 2000 in 100 ll TC-100 medium (Invitrogen) without FBS was used to infect the cells. After culturing for 8 h, the transfection mixture was replaced with 1 ml fresh TC-100 medium with 10% fetal bovine serum (FBS). The cells were cultured for an additional 24 h and harvested for luciferase activity determination. Transfection and cotransfection were repeated three times (n 5 3) and the average expression level of the target genes was expressed as mean 6 SD. The statistical significance of regulatory activities between the treatment and control was analysed using a Student’s t-test. Measurement of luciferase activity For measurement of luciferase activity, the treated cells were washed twice using 13 phosphate-buffered saline (PBS). The cells were then treated with 200 ll lysis buffer (Promega). A dualluciferase reporter assay was conducted according to the manufacturer’s protocol (Promega) in a microplate luminometer (IBA7300, Veritas, Turner Biosystems, Sunnyvale, CA, USA). Luciferase activity of the treatments and control was normalized to Renilla luciferase activity and represented as means 6 SD. Extraction of nuclear proteins Preparation of nuclear protein extracts from Spli-221 cells was conducted using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocol (Thermo Scientific, Waltham, MA, USA). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to examine the efficiency of isolation. Protein concentration was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). EMSA To test the binding of the proteins to the regulatory sequence, EMSA was performed using a LightShift chemiluminescent EMSA kit according to the manufacturer’s instructions (Thermo Scientific). For analysis of the binding of the nuclear proteins to the putative response elements, three complementary doublestranded DNA probes of 35 nt were synthesized based on the sequence between 21000 and 21076 nt and labelled with 50 biotin. The sequence of cold probes was the same but not labelled. The nonspecific cold probe was a random oligonucleotide with the same length as the labelled probes. Expression and purification of recombinant proteins The ORFs of the transcription factors 14-3-3 zeta and 14-3-3 epsilon were cloned with the cDNA isolated from Spli-221 cells. C 2015 The Royal Entomological Society, 24, 551–560 V

559

The ORFs of Elf-1 and C/EBP were cloned using mixed cDNA from fifth instar larvae of B. mori. The primers for 14-3-3 zeta (GenBank accession no.: EF210316.1) and 14-3-3 epsilon (GenBank accession no.: EF112400.1), Elf-1 (GenBank accession no.: XM_004930259.1) and C/EBP (GenBank accession no.: DQ149981.1) were designed based on the sequences. The ORFs of 14-3-3 zeta, 14-3-3 epsilon and Elf-1 were inserted into pPROEX HTa expression vector (Life Technologies, Burlington, Canada) in fusion with a 6xHis tag at the N-terminus, generating the recombinant expression vectors, pPROEXa-14-3-3 zeta, pPROEXa-14-3-3 epsilon and pPROEXa-Elf-1. The ORF of C/EBP was inserted into pET-32a with a 16 kDa peptide fused to the N-terminus and a 3 kDa peptide fused to the C-terminus. Escherichia coli cells (DH5a and Bl-21) were transformed with the recombinant plasmids and protein expression was induced by adding isopropyl-b-D-thiogalactopyranoside at a final concentration of 1 mM. Recombinant proteins were analysed by SDS-PAGE and protein purification was conducted using a His-Bind Kit according to the manufacturer’s instructions (Novagen, Darmstadt, Germany).

Acknowledgements This research was supported by grants from the National Basic Research Program of China (no.: 2012CB114602) and National Natural Science Foundation of China (31071688). References Arizmendi, C., Liu, S., Croniger, C., Poli, V. and Friedman, J.E. (1999) The transcription factor CCAAT/enhancer- binding protein b regulates gluconeogenesis and phosphoenolpyruvate carboxy-kinase (GTP) gene transcription during diabetes. J Biol Chem 274: 13033–13040. Baum, C.L., Kansal, S. and Davidson, N.O. (1993) Regulation of sterol carrier protein-2 gene expression in rat liver and small intestine. J Lipid Res 34: 729–739. Beydon, P. and Lafont, R. (1987) Long-term cholesterol labeling as a convenient means for measuring ecdysteroid production and catabolism in vivo: application to the last larval instar of Pieris brassicae. Arch Insect Biochem Physiol 5: 139–154. Cloutier, A., Guindi, C., Larivee, P., Dubois, C.M., Amrani, A. and McDonald, P.P. (2009) Inflammatory cytokine production by human neutrophils involves C/EBP transcription factors. J Immunol 182: 563–571. Darlington, G.J. Ross, S.E. and MacDougald, O.A. (1998) The role of C/EBP genes in adipocyte differentiation. J Biol Chem 273: 30057–30060. Demel, R.A. and De Kruyff, B. (1976) The function of sterols in membranes. Biochim Biophys Acta 457: 109–132. Fu, H., Subramanian, R. and Masters, S. (2000) 143-3 proteins: structure, function, and regulation. Ann Rev Pharmacol Toxicol 40: 617–647. Gallegos, A.M., Atshaves, B.P., Storey, S.M., Starodub, O., Petrescu, A.D., Huang, H. et al. (2001) Gene structure, intracellular localization, and functional roles of sterol carrier protein-2. Prog Lipid Res 40: 498–563. Gilbert, L.I., Rybczynski, R. and Warren, J.T. (2002) Control and biochemical nature of the ecdystroid-ogenic pathway. Ann Rev Entomol 47: 883–916.

560

L.-N. Liang et al.

Gong, J., Hou, Y., Zha, X.F., Lu, C., Zhu, Y. and Xia, Q.Y. (2006) Molecular cloning and characterization of Bombyx mori sterol carrier protein x/sterol carrier protein 2 (SCPx/SCP2) gene. DNA Seq 17: 326–333. Grieneisen, M.L. (1994) Recent advances in our knowledge of ecdysteroid biosynthesis in insects and crustaceans. Insect Biochem Mol Biol 24: 115–132. Guo, X.R., Zheng, S.C., Liu, L. and Feng, Q.L. (2009) The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses. BMC Mol Biol 10: 102–120. van Hemert, M.J., Steensma, H.Y. and van Heusden, G.P. (2001) 143-3 proteins: key regulators of cell division, signaling and apoptosis. Bioessays 23: 936–946. Hu, H.M., Baer, M., Williams, S.C., Johnson, P.F. and Schwartz, R.C. (1998) Redundancy of C/EBP-a, -b, and -d in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J Immunol 160: 2334–2342. Jin, J., Smith, F.D., Stark, C., Wells, C.D., Fawcett, J.P., Kulkarni, S. et al. (2004) Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr Biol 14: 1436–1450. Jouni, Z.E., Zamora, J. and Wells, M.A. (2002) Absorption and tissue distribution of cholesterol in Manduca sexta. Arch Insect Biochem Physiol 49: 167–175. Komnick, H. and Giesa, U. (1994) Intestinal absorption of cholesterol, transport in the haemolymph, and incorporation into the fat body and Malpighian tubules of the larval dragonfly Aeshna cyanea. Comp Physiol 107A: 553–557. Kraemer, R., Pomerantz, K.B., Kesav, S., Scallen, T.J. and Hajjar, D.P. (1995) Cholesterol enrichment enhances expression of sterol-carrier protein-2: implications for its function in intracellular cholesterol trafficking. J Lipid Res 36: 2630–2638. Krebs, K.C. and Lan, Q. (2003) Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti. Insect Mol Biol 12: 51–60. Kumagai, A. and Dunphy, W.G. (1999) Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev 13: 1067–1072. Lan, Q. and Wessely, V. (2004) Expression of a sterol carrier protein-x gene in the Yellow fever mosquito, Aedes aegypti. Insect Mol Biol 13: 519–529. Lekstorm-Himes, J. and Xanthopoulos, K.G. (1998) Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273: 28545–28548. Lopez, D., Niesen, M., Bedi, M., Hale, D. and McLean, M.P. (2007) Activation of the SCPx promotor in mouse adrenocortical Y1 cells. Biochem Biophys Res Comm 357: 549–553. Meredith, J.M., Munks, R.J.L. Grail, W., Hurd, H., Eggleston, P. and Lehane, M.J. (2006) A novel association between clustered NFjB and C/EBP binding sites is required for immune regulation of mosquito defensin genes. Insect Mol Biol 15: 393–401. Montell, D.J., Rorth, P. and Spradling, A.C. (1992) Slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell 71: 51–62. Papantonis, A., Sourmeli, S. and Lecanidou, R. (2008) Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements. Biochem Biophys Res Comm 369: 905–909.

Paulussen, R.J., Geelen, M.J., Beynen, A.C. and Veerkamp, J.H. (1989) Immunochemical quantitation of fatty-acid-binding proteins. Tissue and intracellular distribution, postnatal development and influence of physiological conditions on rat heart and liver FABP. Biochim Biophys Acta 1001: 201–209. Peng, R., Fu, Q., Hong, H., Schwaegler, T. and Lan, Q. (2012) THAP and ATF-2 regulated sterol carrier protein-2 promotor activities in the larval midgut of the yellow fever mosquito, Aedes aegypti. PLoS ONE 7: e46948. Pfeifer, S.M., Furth, E.E., Ohba, T., Chang, Y.J., Rennert, H., Sakuragi, N. et al. (1993) Sterol carrier protein 2: a role in steroid hormone synthesis? J Steroid Biochem Mol Biol 47: 167–172. Ritter, K.S. and Nes, W.R. (1981) The effects of cholesterol on the development of Heliothis zea. J Insect Physiol 27: 175–181. Ryden, T.A. and Beemon, K. (1989) Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein. Mol Cell Biol 9: 1155–1164. Rørth, P. (1994) Specification of C/EBP function during Drosophila development by the bZIP basic region. Science 266: 1878–1881. Rørth, P. and Montell, D.J. (1992) Drosophila C/EBP: a tissuespecific DNA-binding protein required for embryonic development. Genes Dev 6: 2299–2311. Seedorf, U., Ellinghaus, P. and Roch, N.J. (2000) Sterol carrier protein-2. Biochim Biophys Acta 1486: 45–54. Seiichi, F., Hiromitsu, T., Aki, S., Jun, I. and Minoru, Y. (2012) Both jB and C/EBP binding sites are indispensable for full expression of a nitric oxide synthase gene in the silkworm Bombyx mori. J Insect Biotechnol Sericol 81: 13–20. Sourmeli, S., Kravariti, R. and Lecanidou, R. (2003) In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors. Insect Biochem Mol Biol 33: 525–540. Sourmeli, S., Papantonis, A. and Lecanidou, R. (2005a) A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis. Biochem Biophys Res Commun 337: 713–719. Sourmeli, S., Papantonis, A. and Lecanidou, R. (2005b) BmCbZ, an insect-specific factor featuring a composite DNA-binding domain, interacts with BmC/EBPc. Biochem Biophys Res Commun 338: 1957–1965. Svoboda, J.A. (1999) Variability of metabolism and function of sterols in insects. BioChem 34: 49–57. Takeuchi, H., Chen, J.H., Jenkins, J.R., Bun-Ya, M., Turner, P.C. and Rees, H.H. (2004) Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans. Biochem J 382: 93–100. Trzeciak, W.H., Simpson, E.R., Scallen, T.J., Vahouny, G.V. and Waterman M.R. (1987) Studies on the synthesis of sterol carrier protein-2 in rat adrenocortical cells in monolayer culture. Regulation by ACTH and dibutyryl cyclic 3’,5’-AMP. J Biol Chem 262: 3713–3717. Vyazunova, I. and Lan, Q. (2010) Yellow fever mosquito sterol carrier protein-2 gene structure and transcriptional regulation. Insect Mol Biol 19: 205–215. Zdobnov, E.M., von Mering, C., Letunic, I., Torrents, D., Suyama, M. and Copley, R.R. (2002) Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298: 149–159. Zhang, L., Li, D., Xu, R., Zheng, S., He, H., Wan, J. et al. (2014) Structural and functional analyses of a sterol carrier protein in Spodoptera litura. PLoS ONE 9: e81542.

C 2015 The Royal Entomological Society, 24, 551–560 V

enhancer-binding protein is involved in regulation of expression of sterol carrier protein x in Spodoptera litura.

The Spodoptera litura sterol carrier protein x (SlSCPx) gene is expressed in various tissues throughout the life cycle and plays important role in ste...
642KB Sizes 0 Downloads 8 Views