Function of nuclear transport factor 2 and Ran in the 20E signal transduction pathway in the cotton bollworm, Helicoverpa armigera

Abstract

Background: Nuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known.

Results: A 703 bp encoding Ntf2 and a 1233 bp encoding Ran full-length cDNAs were cloned from Helicoverpa armigera, and named Ha-Ntf2 and Ha-Ran, respectively. Northern blot and immunoblotting revealed that Ha-Ntf2 had an obviously higher expression levels in the head-thorax and integument of the metamorphically committed larvae. In contrast, the expression of Ha-Ran did not show obvious variation at various developmental stages in four tissues by immunoblotting analysis, except in the midgut, which showed increased expression from 5th-36 h (molting) to 6th-48 h. Both expressions of Ha-Ntf2 and Ha-Ran could be upregulated by 20E in vitro. Immunohistochemistry revealed that Ha-Ntf2 and Ha-Ran were primarily localized in the nucleus of various tissues. Protein binding assay and co-immunoprecipitation indicated that Ha-Ntf2 and Ha-Ran can combine with each other in vitro and in vivo. Knock down of Ha-Ntf2 or Ha-Ran by RNAi resulted in the suppression of other 20E regulated genes including EcR-B1, USP1, E75B, BR-CZ2, HHR3 and Ha-eIF5c. In addition, the knockdown of Ha-Ntf2 resulted in Ha-Ran being prevented in the cytoplasm. The nuclear location of the ecdysone receptor b1 (EcR-B1) was also blocked after the knockdown of Ha-Ntf2 and Ha-Ran.

Conclusion: These evidences suggested that Ha-Ntf2 and Ha-Ran participated in the 20E signal transduction pathway by regulating the location of EcR-B1.

Figure 1

Expression profiles of Ha-Ntf2 and Ha-Ran in four tissues samples during developmental stages. A, Ha-Ntf2: a, Northern blot; b, 18s rRNA; c, immunoblotting. B, Ha-Ran: analyzed by immunoblotting. 5-12, 5-24, 5-36 (HCS), 6-0 (WH),6-24,6-48,6-72 (wandering d 1), 6-96 (wandering d 2) and 6-120 h (prepupae or quiescent d 1) indicated the different times of larvae; p0, pre-pupae; p1, 1st d pupae; p2 d, 2nd d pupae; Ht: head-thorax; It: integument; Mg: midgut; Fb: fat body; HCS, head capsule slippage; WH, white head capsule.

Figure 2

Immunohistochemical location of Ha-Ntf2 and Ha-Ran in the integument and fat body of the feeding 5th instar larva (5th-24 h). Panel A, negative controls with preserum replacing antiserum; panel B, same specimens stained by anti-Ha-Ntf2; panel C, stained by anti-Ha-Ran; panels a, b, c are the same as A, B and C, respectively, but stained with DAPI. Fb, fat body; Ep, epidermis; Cu, cuticle; Fb-Nu or Ep-Nu: the nucleus of the fatbody or epidermis, respectively.

Figure 3

Binding assay and co-immunoprecipitation assay to show the interaction between Ha-Ntf2 and Ha-Ran in vitro or in vivo. A, B: SDS-PAGE to show the direct binding assay between the rHa-Ntf2 and rHa-Ran in vitro. A, rHa-Ntf2 binds on His-rHa-Ran affiliated on Ni2⁺-NTA column. B, rHa-Ran binds on His-rHa-Ntf2 affiliated on Ni2⁺-NTA column. rHa-Ntf2 or rHa-Ran: removed His-tag by thrombin; dHis-rHa-ntf2: the degradation of His-rHa-ntf2 in the process of binding. C, D: immunoblotting to examine the interaction of Ha-Ntf2 and Ha-Ran produced by co-immunoprecipitation. C, Ha-Ntf2 detection from the co-immunoprecipitation (co-ippt) produced by anti-Ha-Ran antibody. Lane 1, protein extracts from the whole larvae of 6th-96 h; lane 2, sample from the last wash of the co-ippt; lane 3, eluted proteins from well-washed co-ippt produced by anti-Ha-Ran antibody. D, Ha-Ran detection from the co-ippt produced by anti-Ha-Ntf2 antibody. Lanes 1 and 2 are the same as in C; lane 3, eluted proteins from well-washed co-ippt produced by anti-Ha-Ntf2 antibody. M, protein marker.

Figure 4

Regulation of 20E in the expression of Ha-Ntf2 or Ha-Ran. A: RT-PCR analysis the regulation of 20E in the expression of Ha-Ntf2. B: Regulation of 20E in the expression of Ha-Ran. Other signs are: C stands for the normal 6th-0 h larvae; D1, D3, D6, D12 and D24 are 1, 3, 6, 12 and 24 h after injected by the DMSO, respectively; E1, E3, E6, E12 and E24 stand for 1, 3, 6, 12 and 24 h after injected by the 20E, respectively. β-actin is used as a quantitative control. Error bars represent the standard deviation in three independent experiments. An asterisk indicates significant differences (Student's t-test, *: p < 0.05).

Figure 5

Effects on the genes in the HaEpi cell after the knockdown of Ha-Ntf2 or Ha-Ran. A. RT-PCR analysis the expressions of other genes in the normal cell, the cells plus 20E, the cells with dsGFP added, dsNtf2 and dsRan, respectively. B: Statistical analysis of results in after the knockdown of Ha-Ntf2 comparing to the control adding with the dsGFP. C: Statistical analysis of results in after the knockdown of Ha-Ran comparing to the control adding with the dsGFP. β-actin is used as a quantitative control. Error bars represent the standard deviation in three independent RNAi experiments. Asterisks stand for statistically significant differences. (*: p < 0.05 by T-test, n = 3), respectively.

Figure 6

The location of Ha-Ran in the HaEpi cells after knock down of Ha-Ntf2. The cells were immunostained with polyclonal antibody against Ha-Ran (green color). Nuclei were counterstained with DAPI (blue color). Arrow indicates the magnified cell in B, C and D, respectively. Nu: stand for the nucleus; Cy:cytoplasm. Size bars = 50 μm.

Figure 7

Variation of the subcellular localization of EcR-B1 in the HaEpi cell after the knockdown of Ha-Ntf2 or Ha-Ran. Panel A': The EcR-B1 from the HaEpi cell extract could be detected by the monoclonal antibody of EcR-B1 from the M. sexta using immunoblotting. Panels A-F: The red color indicates EcR-B1 stained with anti-EcR-B1 monoclonal antibody; the blue color indicates nuclei counterstained with DAPI. Neg, negative control without first antibody; nor, normal cells cultured in Grace's medium; nor+20E, the cells cultured in Grace's medium plus 20E; dsGFP+20E, the cells cultured in Grace's medium plus dsGFP and 20E; dsRan+20E, the cells cultured in Grace's medium plus dsRan and 20E; dsNtf2+20E, the cells cultured in Grace's medium plus dsNtf2 and 20E. Nu: nucleus; Cy: cytoplasm. Size bars = 50 μm.

Figure 8

The subcellular localization of USP1 in the HaEpi cell after the knockdown of Ha-Ntf2 or Ha-Ran. Panel A': The USP1 from the HaEpi cell extract could be detected by the monoclonal antibody of USP1 from the D. melanogaster using the immunoblotting. The red color indicates USP1 stained with anti-USP1 monoclonal antibody; the blue color indicates nuclei counterstained with DAPI. Nu: nucleus; Cy: cytoplasm. Size bars = 50 μm.

Figure 9

Model of how the nuclear transport factor 2 and Ran may function in the import of the EcR-B1 in the 20E signal transduction pathway. 1, the EcR-B1 transport through the NPC by binding with the importinα1 and importinβ and goes into the nucleoplasm; 2, in the nucleoplasm, 20E binds with EcR-B1 and formed trimeric complex with USP to regulate the transcriptions of genes. In the cytoplasm, NTF2 binds with RanGDP and transports RanGDP into the nucleoplasm, where RanGDP is converted into RanGTP by RCC1; RanGTP then is transported back into the cytoplasm by importinβ and hydrolyzed to RanGDP by RanGAP [44]; this process results in the energy gradient for the transport of EcR-B1 and the accumulation of EcR-B1 in the nucleus. NPC: nuclear pore complex; RCC1: Ran nucleotide exchange factor; RanGAP: Ran GTPase-activating protein. EcRE: ecdysone response elements.