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Function Analysis of Rab4B, Rab32and Protein Kinase C Delta (PKCδ) During Metamorphosis in Helicoverpa Armigera

Author: HouLi
Tutor: ZhaoXiaoFan
School: Shandong University
Course: Biochemistry and Molecular Biology
Keywords: Ecdysone juvenile hormone insulin metamorphosis Rab4b Rab32 PKCδ
CLC: Q966
Type: PhD thesis
Year: 2012
Downloads: 136
Quote: 0
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BackgroundInsects are the largest biological species with a maximum population on earth, which have a close relationship with humans. Insect development is a complicated physiological process regulated by hormones, including molting and metamorphosis. In holometabolous insects, insect molting contains molt from larvae-larvae, larvae-pupa and pupa-adult. The larva to pupae transition is called metamorphosis. This process is controlled by two kinds of hormones:20E and JH. At larval stage,20E initiate molting, and JH maintains larval growth. At the final larval stage,20E provokes metamorphosis when JH level decreases. Previous studies have uncovered the mechanism of20E and JH regulated metamorphic transition. Whereas, recent finding indicates that insulin play a crucial role in larvae-pupae transform. Insulin could cooperate with20E signal and regulating insect development. However, the molecular mechanism that insulin interacts with20E during insect metamorphosis is not fully understood.During insect metamorphosis, tissues undergo remodeling under the control of20E and JH. In this process, the larval tissues, such as midgut and fat body undergo degradation by20E induced programmed cell death (PCD), and then the new tissues form by imaginal cell proliferation, whereas, JH antagonizes20E effects. The foregoing investigations have revealed the hormone regulations on tissue remodeling. Nevertheless, molecules involved in this process need to be further identified. In mammalian, the Small GTPases are widely found to take part in multiple physiological processes, such as visecle trafficking, cytoskeleton activity and signal transduction. Howerve, the function of Rab proteins are not well studied in insect. The protein kinase C is also reported to play important roles in cell growth, proliferation and apoptosis. Recently studies indicate the protein particiapates in20E signaling. Pending ProblemsWhat are the functions of Rab4b and Rab32in hormones regulated metamorphosis? And how is the difference between Rab4b and Rab32? Whether PKCδ in invovoled in20E pathway and tissue remodeling during larval-pupal transition.Significance of the ResearchThe rapid development of molecular technologies brings us a convenient platform for further study the insect development. Insect undergoes molting and metamorphosis during its life. The larval-pupal transition is very crucial process for insect mature and reproduction, which is mainly regulated by20E and JH. However, more and more researches find that insulin signaling play vital role in metamorphosis by interacting with20E pathway. Thus, questions remained. The mechanism of the crosstalk between20E and insulin pathway is not well understood. Therefore, it is very meaningful to have a better conmand of the interaction of the two pathways. Moreover, tissue remodeling is an important physiological process during metamorphosis. Charactiaziton of the effetors involved in the process is with great importance and providing new thought for pest-cpntrolMethods and Acquired ResultsIn the current study, we characterized a small GTPase Rab4b, participates in metamorphosis by regulating20E and insulin signals. It provided a target molecule for further understand the crosstalk between20E and insulin pathway. Moreover, we found another Rab protein, Rab32, is involved in imaginal midgut formation. Besides, our study demonstrated PKC delta (PKCδ) takes part in20E induced tissue degradation through cell death. Our investigations provide a theoretical basis for further reveal the molecular mechanism of metamorphosis and tissue reconstruction.1. Rab4b participates in metamorphosis by regulating glycogen level and gene transcription insulin and20E signaling.In the present study, we found that a small GTPase Rab4b from a lepidopteran insect participates in signal transduction in the two pathways. RT-PCR results showed that Rab4b was present consistently in various tissues during larval development. Knocked down of Rab4b caused53%larvae to die before pupation,24%failed to pass the metamorphic transition and became malformed pupae, and part of thelarvae transformed to smaller pupae with lower body weight. These results indicate that Rab4b is critical to larval survival and metamorphosis. Glycogen levels declined in the larval body after Rab4b silencing. Knockdown of Rab4b suppressed the gene transcription in the20E (Br and HR3) and insulin pathways (GS), whereas, FOXO increased after Rab4b interference, RNAi in HaEpi cells confirmed the results in vivo. Immunocytochemistry and western bolt showed that insulin kept FOXO located in the cytoplasm after1h treatment, whereas FOXO could not be arrested in the cytoplasm after insulin induction after knockdown of Rab4b. Overexpression experiment revealed that insulin could not alter the cytoplasm location of Rab4b. However, the20E induced Rab4b moving toward the cell membrane. Hormone regulation results indicate that20E did not effect the transcription of Rab4b, but insulin enhanced the transcription of Rab4b in3to12h after injection. Besides, the Rab4b transcription depended on the glucose level.2. Rab32and the remodeling of the imaginal midgut in Helicoverpa armigeraWe got the cDNA sequence of Rab32, RT-PCR results show that Rab32is up-regulated in epidermis and midgut during metamorphosis. Its expression could be up-regulated by20E, but not by JHA. RNAi in HaEpi cells demonstrated Rab32could be regulated by20E signal transduction pathway through EcRB1as a receptor of20E. Immunocytochemistry showed that Rab32mostly existed in the cytoplasm in normal condition,20E induced Rab32accumulation in the cytosolic granular astructures. Western blot showed20E enhanced Rab32in cytoplasm. Immunohistochemistry results revealed that the Rab32signal was detected in the cytoplasm of midgut cells in both the5th feeding larvae and metamorphic committed larvae. In the5th feeding larval midgut, the intensive signal was detected around the basement of the midgut and the apical region of the epithelium. In the metamorphic committed midgut at the6th120h larval stage, the most intensive signal was detected at the apical region of the epithelium cells of the imaginal midgut, suggesting Rab32may relate to imaginal midgut formation by nutrient absorption or signal transduction. Knockdown of Rab32 expression disrupts the imaginal midgut morphogenesis during metamorphosis. These data suggest that Rab32takes part in imaginal midgut formation.3. Protein Kinase C delta participates in tissue degradation during metamorphosis in Helicoverpa armigeraHere we find a novel type Protein Kinase C δ (PKCδ) takes part in the apoptosis in midgut and the fat body in a lepidopteran insect Helicoverpa armigera. PKCδ was highly expressed in several tissues during metamorphic stage, and could be stimulated by20E and Juvenile hormone (JHⅢ). Knockdown of EcR-B1in HaEpi cells blocked20E induced PKCδ transcription, but interference of JH candidate receptor, Met, did not effect the JHIII induced PKCδ transcription, suggesting20E regulates PKCδ through EcR-B1, whereas JH effects PKCδ via other way except Met. Immunohistochemistry and HE staining indicated that the fat body and the midgut did not experience degradation in the PKCδ knockdown larvae. RT-PCR analysis demonstrated the apoptosis related gene Caspase8transcription was blocked after PKCδ knockdown, whereas, the inhibitor of apoptosis, Survivin, was enhanced in the RNAi larvae. The data suggest PKCδ participate in the hydrolyzing of the midgut and the fat body during metamorphosis. Overexpress functional domain of PKCδ in HaEpi cells induced apoptosis, and the indicator of apoptosis Caspase8was upregulated, meanwhile the IAP, Survivin was inhibited, which further evidenced the active role of PKCδ in HaEpi cells apoptosis. In PKCδ overexpression cells, FOXO displayed much stronger signal in the nuclei comparing with that in the normal cells. RFP was also used as control to confirm the effect of PKCδ on subcellular location of FOXO. The data suggest the overexpressed PKCδ functional domain enhance the nuclear location of FOXO. RNAi of PKCδ in HaEpi cells surpressed the20E and JHIII induced expression of USP1and Calponin, Which suggest that PKCδ involved in the20E and JH signal transduction.

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