KLF4：一个热休克反应和热休克蛋白表达的新调控介质

发布时间：2018-06-15 12:56

本文选题：KLF4 + 热休克反应　；参考：《中南大学》2006年博士论文

【摘要】： 热休克反应(heat shock response，HSR)是机体中普遍存在的一种应激反应。在热休克反应中，细胞内一组热休克蛋白(heat shock proteins，HSPs)诱导表达增加。HSPs作为重要的分子伴侣(molecular chaperone)，具有帮助蛋白质的折叠与移位，防止蛋白质聚集，帮助变性蛋白质的解聚及复性，促进严重受损蛋白质的降解等功能。热休克时HSPs的诱导表达主要在转录水平受热休克因子1(heat shock factor 1，HSF1)的调控。在热休克反应中，HSF1被活化并从胞浆移位到细胞核，从转录水平激活HSPs的表达。除HSPs外，HSF1还能直接调控多种其它基因的表达。此外，，采用HSF1基因敲除小鼠研究还发现HSF1在非活化状态下能够调控HSPs的组成型表达，但其机制目前尚不清楚。 Kruppel样因子4(Kruppel-like factor 4，KLF4)是Sp1／KLF锌指转录因子家族的成员之一。作为一个核转录因子，它能够与下游基因启动子区的GC盒，CACCC盒和基础转录元件(basic transcription elements)三种结合元件相结合，从而直接调控这些基因的表达。通过对下游基因表达的调控，KLF4在正常细胞的增殖和分化、胚胎发育以及肿瘤的发生发展过程中起着重要的作用。但是，KLF4与热休克反应、HSF1及HSPs之间有何关系，至今尚未见报道。为了探讨KLF4与热休克反应之间的关系，本研究第一部分采用RT-PCR和Western blot分别检测了KLF4 mRNA和蛋白质在热休克反应中的表达情况，结果发现热休克导致整体小鼠的多种组织以及多种离体培养细胞中KLF4的表达显著增加。根据上述结果，我们首次提出：KLF4可能是一个热休克反应相关基因。由于热休克反应中多个基因的表达都受到HSF1的调控，我们进一步采用HSF1基因敲除小鼠探讨了HSF1对KLF4 mRNA表达的可能影响，结果发现HSF1既可以促进KLF4的基础表达，又可以促进其在热休克时的诱导表达。此外，我们还在KLF4基因启动子区找到了HSF1结合元件(heat shock element，HSE)。由此可见，KLF4是HSF1调控的下游基因。 HSPs是热休克反应中受HSF1直接调控的重要功能蛋白。作为热休克反应中表达增加的转录因子，KLF4对HSPs基因表达的可能影响引起了我们的关注。以前的研究发现，Sp1／KLF家族的某些成员既可以调控HSPs的组成型表达，又可以调控其在热休克时的诱导表达。因此，本研究第二部分首先构建了KLF4过表达细胞株，并采用反义寡核苷酸技术建立了内源性KLF4表达抑制的模型；接着分别采用RT-PCR和Western blot在mRNA和蛋白质水平检测了KLF4过表达和内源性KLF4表达抑制对小分子HSPs家族的αB晶状体蛋白和HSP25，HSP70家族的HSP72和HSP73以及HSP90家族的HSP84和HSP86的组成型表达和诱导表达的影响。结果发现，KLF4过表达能够上调上述HSPs的组成型表达，而KLF4表达抑制则下调上述HSPs的组成型表达；但KLF4过表达或表达抑制均不影响上述HSPs的诱导表达。为了进一步阐明KLF4调控α晶状体蛋白、HSP25和HSP84组成型表达的机制，本研究采用EMSA和荧光素酶报告基因方法进行了深入探讨。结果发现，KLF4能够通过其DNA结合结构域与α晶状体蛋白、HSP25和HSP84基因启动子区的KLF4结合元件相结合而直接促进其转录，从而维持其组成型表达。为了更全面地了解KLF4能够调节哪些HSPs的组成型表达，我们进一步采用cDNA芯片检测了KLF4过表达对HSPs基因表达的影响，结果发现有9个HSPs的组成型表达可能受到KLF4的调控。此外，本研究还发现热休克反应中KLF4与HSPs基因启动子的结合显著减少，这可能是热休克时KLF4对HSPs失去调控作用的主要原因。综上所述，本研究首次发现KLF4是一个受HSF1调控的热休克反应相关基因。KLF4的组成型表达及热休克导致的诱导表达均受到HSF1的调控。KLF4通过其DNA结合结构域与αB晶状体蛋白、HSP25及HSP84等基因启动子区的KLF4结合元件相结合而直接调控上述HSPs的组成型表达，即KLF4能够介导HSF1对HSPs组成型表达的促进作用。而热休克时，由于KLF4与HSPs基因启动子的结合减少而导致其失去对HSPs的调控作用。上述研究揭示了KLF4在HSPs表达调控方面的新功能，完善了HSPs的表达调控机制。有关KLF4在生理状态下及热休克反应和其它应激反应中的功能和生物学意义值得进一步深入研究。
[Abstract]:Heat shock response (HSR) is a common stress response in the body. In the heat shock response, a group of heat shock proteins (heat shock proteins, HSPs) can induce the expression of.HSPs as an important molecular chaperone (molecular chaperone), which helps the folding and displacement of protein and prevents protein aggregation. Help the depolymerization and renaturation of denatured proteins and promote the degradation of severely damaged proteins. The induction of HSPs in heat shock is mainly regulated at the transcriptional level of heat shock factor 1 (heat shock factor 1, HSF1). In the heat shock response, HSF1 is activated and moved from the cytoplasm to the nucleus and activates the expression of HSPs from the transcriptional level. Except HS, the expression of HSPs is activated. HSF1 can also directly regulate the expression of a variety of other genes. In addition, the study of HSF1 knockout mice also found that HSF1 can regulate the expression of HSPs in the non activated state, but its mechanism is not yet clear.
Kruppel like factor 4 (Kruppel-like factor 4, KLF4) is one of the members of the Sp1 / KLF zinc finger transcription factor family. As a nuclear transcription factor, it can be combined with the three binding elements of the GC box, CACCC box and the basic transcriptional element (basic transcription elements) in the promoter region of the downstream gene, which directly regulates the expression of these genes. By regulating the expression of the downstream genes, KLF4 plays an important role in the proliferation and differentiation of normal cells, the development of embryo and the development of tumor. However, the relationship between KLF4 and heat shock response and the relationship between HSF1 and HSPs has not yet been reported.
In order to investigate the relationship between KLF4 and heat shock response, the first part of this study used RT-PCR and Western blot to detect the expression of KLF4 mRNA and protein in the heat shock response. The results showed that the heat shock resulted in a significant increase in the expression of KLF4 in various tissues of the whole mice and in a variety of isolated cultured cells. For the first time, we suggest that KLF4 may be a related gene for heat shock response. Because the expression of multiple genes in the heat shock response is regulated by HSF1, we further use HSF1 knockout mice to explore the possible influence of HSF1 on the expression of KLF4 mRNA. The results show that HSF1 can promote both the basic expression of KLF4 and the promotion of the expression of the KLF4. In addition, we also found the HSF1 binding element (heat shock element, HSE) in the promoter region of the KLF4 gene. Thus, KLF4 is a downstream gene for HSF1 regulation.
HSPs is an important functional protein regulated by HSF1 in the heat shock response. As a transcriptional factor expressed in the heat shock response, the possible influence of KLF4 on the expression of HSPs gene has aroused our attention. Previous studies have found that some members of the Sp1 / KLF family can regulate the composition of HSPs and regulate their thermal rest in the heat shock. Therefore, the second part of this study first constructed the KLF4 overexpressed cell lines, and established a model of endogenous KLF4 expression inhibition by antisense oligonucleotides, and then used RT-PCR and Western blot to detect KLF4 overexpression and endogenous KLF4 expression inhibition to small molecule HSPs at mRNA and protein levels, respectively. The family's alpha B crystallin and HSP25, the HSP72 and HSP73 of the HSP70 family, and the expression and induction of HSP84 and HSP86 in the HSP90 family. The results showed that the overexpression of KLF4 overexpressed the expression of the above-mentioned HSPs, while the inhibition of KLF4 expression downregulated the expression of the above-mentioned HSPs. In order to further elucidate the mechanism of KLF4 regulation of the expression of alpha crystallin, HSP25 and HSP84, this study uses EMSA and luciferase reporter gene method to further explore the mechanism of the expression of alpha crystallin, HSP25 and HSP84. The results show that KLF4 can be used in the promoter region of the alpha crystallin, HSP25 and HSP84 genes through its DNA binding domain. In order to understand the composition expression of which HSPs can be regulated by KLF4, we further use cDNA chip to detect the effect of KLF4 overexpression on the expression of HSPs gene, and we found that the expression of 9 HSPs components may be regulated by KLF4 in order to understand more fully the expression of which HSPs expression can be regulated in a more comprehensive way. In addition, this study also found that the combination of KLF4 and HSPs gene promoter in the heat shock response decreased significantly, which may be the main cause of the loss of KLF4 on HSPs during heat shock.
To sum up, this study for the first time found that KLF4 is a HSF1 regulated heat shock response related gene.KLF4 and the induced expression of heat shock induced by HSF1 is regulated by.KLF4 through its DNA binding domain and the binding of KLF4 binding elements in the promoter region of the alpha B lens protein, HSP25 and HSP84. The component expression of the above HSPs, that is, KLF4 can mediate the promotion of HSF1 on the expression of HSPs, and the decrease in the binding of KLF4 and HSPs gene promoters to the regulation of HSPs. These studies reveal the new functions of KLF4 in the regulation of HSPs expression, and improve the mechanism of the expression and regulation of HSPs. The functional and biological significance of LF4 in physiological state, heat shock response and other stress responses deserves further study.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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