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| Effect of Calmodulin (CaM) on Expression of Inducible Heat Shock Protein (HSP70) in Mice(Mus musculus) Preimplantation Embryo |
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Abstract Heat shock protein(HSP)70 is synthesized in the preimplantation embryos of cow(Bos tarurs), ewe(Ovis aries) and mice(Mus musculus) under the conditions of heat shock in vitro, which enhances their thermotolerance to protect them from further heat damage. And Calmodulin(CaM)is the one of most important multifunctional receptor of Ca2+ and plays a key role in regulating the important gene expression in cell activities. To clarity whether the CaM participating in the expression of HSP70 in the mice embryos with heat shock and verify its mechanism, mice embryos were cultured in vitro to the blastocyst stage and then were randomly divided into control group(37℃), group treated with W7 without heat shock(37℃+W7), heat shock group(39℃)and heat shock group treated with W7. The method of RT-PCR was used to detect the gene expression of CaM, HSP70 and heat shock factor1(HSF1), and the method of Western blot was used to detect the expression of CaM, HSP70 and HSF1. The combinations of both HSP70-CaM and HSP70-HSF1 were detected by using co-immunoprecipitation(Co-IP). The results showed that the CaM and HSP70 mRNA expression significantly increased in the mice embryos treated with 39℃ for 1 h (P<0.05). However, the CaM and HSP70 mRNA expression significantly decreased in both groups of embryo cultured at 37℃ (P<0.05) and 39℃ (P<0.01) treated with W7; W7 had no effect on the HSF1 mRNA expression in any groups of embryo. The synthesis of CaM, HSP70 and HSF1 significantly increased in the embryos treated at 39℃ for 1 h (P<0.05), and the CaM synthesis significantly decreased in the embryos cultured both at 37℃ and 39℃ and treated with W7 (P<0.05). W7 had no effect on neither HSP70 nor HSF1 synthesis in the embryos cultured at 37℃ (P>0.05) while it inhibited both HSP70 and HSF1 synthesis in the embryos cultured at 39℃(P<0.05). The study also found that CaM combined with HSP70 and formed CaM-HSP70 complex in the mice embryos. The identification of interactions between CaM and HSP70,HSF1 and HSP70 in the embryos cultured both at 37℃ and 39℃ by using Co-IP showed that the quantity of HSP70-CaM complex had the tendency of increase while the HSP70-HSF1 complex decreased in the heat shocked embryos. The above results indicate that CaM regulates HSP70 expression by the way of competitive binding HSP70 and releasing HSF1, which finally activates HSP70 expression in heat shocked mice embryos. The research results reveal one of the ways that CaM participates in HSP70 expression in heat shock response of mice embryos which will provide a theoretical basis for the study of thermotolerance and heat shock signal transduction of the mice embryos.
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Received: 25 February 2013
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