Cloning and identification of grass carp transcription factor HSF1 and its characterization involving the production of fish HSP70

摘要

In mammals, heat shock transcription factor 1 (HSF1) is well documented as the critical transcript factor to regulate heat shock protein 70 (HSP70) expression under different stresses, such as heat shock or bacterial infection. In fish, Hsf1 responses to physiological and environmental stresses and regulates Hsp70 expression under thermal exposure. However, the functional role of Hsf1 in Hsp70 production is still elusive under bacterial infection. In the present study, a coding sequence of grass carphsf1(gchsf1) gene was cloned and identified. UsingCtenopharyngodon idelluskidney (CIK) cells as the model, we found that lipopolysaccharide (LPS) exerted stimulatory effects on the expression of grass carphsp70(gchsp70) andhsf1, implying possible relationship of Hsp70 and Hsf1 under immune stimulation in fish. To validate the hypothesis, overexpression of gcHsf1 was performed in CIK cells, and the effects of overexpressing gcHsf1 on the expression of gcHsp70 in the absence or presence of LPS were examined. Results showed that LPS significantly upregulated the transcription and protein synthesis of gcHsp70, and these stimulatory effects were further amplified when overexpression of gcHsf1 was performed. Furthermore, luciferase reporter assays in CIK cells revealed that both overexpression of Hsf1 and LPS upregulatedgchsp70transcription, and their combined treatment further enhanced thegchsp70promoter activity. Moreover, the regions responsive to these treatments were mapped to the promoter ofgchsp70. Besides transcriptional level and cellular protein contents, gcHsp70 secretion was measured by competitive ELISA, uncovering that gcHsf1 enhanced the release of gcHsp70 induced by LPS in the same cells. These data not only demonstrated the enhancement of Hsf1 in Hsp70 production but also initially revealed the involvement of Hsf1-Hsp70 axis in mediating inflammatory response in fish.