Establishment of a New Cell Line from Lepidopteran Epidermis and Hormonal Regulation on the Genes

Abstract

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

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“…However, the receptor tyrosine kinase (RTK) inhibitor SU6668 25 affected neither intracellular calcium release nor extracellular calcium influx ( Figure 3D). These results suggest that 20E via ErGPCR-2 induces cellular Ca 21 increase, and various calcium channels are involved in this process.…”

“…20E mediates CDK10 phosphorylation to enhance formation of the EcRB1/USP1 transcription complex 36 . 20E via GPCR-, PLC-, Ca 21 -, and PKC-signaling mediates USP1 phosphorylation for gene transcription 12 . At the downstream of ligand activated-GPCR, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) 37 .…”

“…Such control is not exerted by directly or tightly binding to 20E. 20E via ErGPCR-2 regulates rapid intracellular Ca 21 increase and phosphorylation of USP1 and CDK10, which induce gene transcription in the 20E pathway, thereby regulating metamorphosis. After performing the task for 20E entrance, ErGPCR-2 is phosphorylated and internalized via GRK2 for degradation to desensitize 20E signaling (Figure 8).…”

“…In HaEpi cells 21 , ErGPCR-2 knockdown also blocked 20E-induced gene expression ( Figure 2F). These results suggest that ErGPCR-2 participates in 20E-regulated gene expression and metamorphosis.…”

“…The intracellular Ca 21 -ATPases inhibitor thapsigargin (TG), which depletes the stored intracellular calcium 24 , repressed the intracellular calcium release and extracellular calcium influx, but did not block extracellular calcium influx in 20E induction ( Figure 3C). The GPCR inhibitor suramin blocked both intracellular calcium release and extracellular Ca 21 influx. However, the receptor tyrosine kinase (RTK) inhibitor SU6668 25 affected neither intracellular calcium release nor extracellular calcium influx ( Figure 3D).…”