KLF4: A Novel Modulator Of Heat Shock Response And The Expression Of Heat Shock Proteins

The heat shock response (HSR) was discovered in 1962 byF.Ritossa, who detected a new puffing pattern upon heat shock in thepolytene chromosomes of the fruit fly Drosophila buschii. Today it iswell known that all organisms share a common molecular stress responsethat includes a dramatic change in the pattern of gene expression and theelevated synthesis of a family of stress-induced proteins called heat shockproteins(HSPs). Expression of HSPs usually results in repair of damagedproteins and survival of the cell, mainly through the chaperone functionof HSPs. Considering the important role of HSPs in cells, it is of utmostimportance to elucidate the regulatory mechanisms responsible for HSPsexpression. The inducible HSPs expression is regulated by the heat shocktranscription factor 1(HSF1). In vertebrates, HSF1 has been identified asthe HSF that mediates stress-induced heat shock gene expression on thebasis of the ability of HSF1 to display inducible DNA binding activity tothe heat shock element (HSE), oligomerization, and nuclear localizationin response to various inducers such as elevated temperatures, oxidants,heavy metals, and bacterial and viral infections. Besides HSPs, HSF1 canalso modulate many other stress response genes. In addition, theconstitutive expression ofαB-crystallin, HSP25 and HSP70 could bemodulated by HSF1 as indicated by the study in HSF1 knockout mice,but the mechanism is not clear.The Kruppel-like factor 4 (KLF4) is a member of a zinc-fingerfamily of transcription factors that contains three C₂H₂ zinc fingers. As atranscription factor, KLF4 is usually found to bind to the CACCCelement, GC-box or the basic transcription element (BTE) of thepromoters of its downstream genes and modulate the expression of thegenes. By regulating its downstream genes, KLF4 plays a critical role in regulating critical aspects of cell growth, proliferation, differentiation,and embryogenesis. However, the relationship between KLF4 and HSR,HSF1 or HSPs remains unknown.To illustrate the response of KLF4 to HSR, in the first part of thisstudy, Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR) were performed to determine the changes in KLF4 expressionafter HSR[in intact mice(42℃, 15min)and four cultured cell lines (43℃,1h) respectly]. The results showed that HSR up-regulated KLF4messenger RNA and protein levels in a time-dependent manner indifferent tissues of the intact animals and the four cell lines. This processoccurred rapidly, indicating that KLF4 is a heat shock response gene.Moreover, a study with HSF1 gene knockout mice indicated that theinduction of KLF4 in response to HSR was mediated by HSF1. And then,a series of HSEs were found in the promoter region of KLF4 gene. Theseresults indicated that HSF1 could directly regulat the expression of KLF4gene during HSR. In addition, HSF1 deficiency resulted in decrease ofconstitutive expression of KLF4, suggesting HSF1 is also a regulator ofKLF4 expression under the normal conditions. These results indicatedthat KLF4 was a heat shock response gene regulated by HSF1.As HSPs are the most important proteins regulated by HSF1 inresponse to HSR, it is interesting to study whether KLF4 regulates theexpression of HSPs. Previous studies have shown that several membersof the Sp1/KLF family can regulate the constitutive expression as well asthe inducible expression of HSPs. So in the second part of this study,KLF4 overexpressing cell lines were established by stable transfectedwith the pcDNA3.1-KLF4 plasmids and the expression of KLF4 wasinhibited by antisence oligodeoxynucleotides. And then RT-PCR andWestern blot were performed to detect the effect of KLF4 overexpressionor KLF4 deficiency on the expression ofαB-crystallin, HSP25(members of small HSP family); HSP72, HSP73(members of HSP70 family) andHSP84, HSP86(members of HSP90 family) in the mRNA level as well asthe protein level. The results showed that KLF4 overexpressionup-regulated the constitutive expression of the above HSPs, and KLF4deficiency down-regulated the constitutive expression of the HSPs.However, neither KLF4 overexpression nor KLF4 deficiency affected theinducible expression of the HSPs. To understand howαB-crystallin,HSP25 and HSP84 expression was regulated by KLF4, we cloned andcharacterized the promoter regions of these three mouse genes. Analysisof the promoters was conducted in the C₂C₁₂ myocytes or the RAW264.7macrophages by transient transfection of luciferase reporter genes withserially deleted and site-directed mutated promoter constructs andelectrophoretic mobility shift assays (EMSA). The results shown inEMSA indicated that KLF4 was able to bind to the KLF4 bindingelements in theαB-crystallin, HSP25 and HSP84 promoter. In addition,transient cotransfection experiments showed that overexpression of KLF4specifically increased the promoter activities ofαB-crystallin, HSP25 andHSP84 as showed by luciferase reporter gene assay. These resultsindicate that KLF4 can act to increase the constitutive expression ofαB-crystallin, HSP25 and HSP84 genes by its DNA binding-dependentmechanisms. To further understand the effect of KLF4 on the regulationof the expression of HSP genes, cDNA microarray containing 14,000genes was used to detect the changes of HSPs expression profile in KLF4overexpressing cells, and the expression of nine HSP genes was foundup-regulated in the KLF4-overexpressing cells. Interestingly, in currentstudy we also found that during HSR the ability of KLF4 to bind to thepromoters of HSPs was decreased, which might be the main reasonresponsible for the inability of KLF4 in regulating the inducibleexpression of HSPs during HSR. Taken together, for the first time we found KLF4 was a heat shockresponse gene regulated by HSF1. The constitutive expression as well asthe inducible expression of KLF4 was regulated by HSF1. And KLF4regulated the basal expression ofαB-crystallin, HSP25, HSP73, HSP84,and HSP86, but had no effect on their inducible expression. In addition,the constitutive expression ofαB-crystallin, HSP25 and HSP84 wasdirectly increased by KLF4 in a DNA binding-dependent manner. DuringHSR, KLF4 lost its ability to regulate the inducible expression of the HSPgenes because of its inability of binding to the promoters of HSPs.However, more investigations were needed to further understand the rolesof KLF4 under the normal conditions and in response to HSR or otherstressors.