HSF1 and HSF3 cooperatively regulate the heat shock response in lizards

Ryosuke Takii, Mitsuaki Fujimoto, Yuki Matsuura, Fangxu Wu, Namiko Oshibe, Eiichi Takaki, Arpit Katiyar, Hiroshi Akashi, Takashi Makino, Masakado Kawata, Akira Nakai, Suzannah Rutherford
2017 PLoS ONE  
Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family
more » ... s possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs. conserved transcription factor heat shock factor (HSF), which binds to the heat shock response element (HSE) in eukaryotes [6] [7] [8] . In contrast to the single HSF gene in yeast, worm, and fruit fly, four HSF genes (HSF1, HSF2, HSF3, and HSF4) have been isolated in vertebrates, including chicken, mouse, and human [9, 10]. Among vertebrate HSFs, HSF1 is a master regulator of HSP expression during proteotoxic stresses, including heat shock, in mammals. HSF1 mostly remains as an inert monomer in unstressed conditions. In response to heat shock, it is converted to an active trimer that binds to HSEs in HSP promoters, and robustly induces the transcription of these genes [11, 12] . The induction of HSPs is associated with increased proteostasis capacity and resistance to cell death. Mammalian HSF2, HSF3, and HSF4 are also involved in the maintenence of proteostasis capacity by regulating the expression of non-HSP genes such as αB-and γ-crystallins and PDZK3 [13] [14] [15] [16] . However, they are dispensable for the induction of HSP expression during proteotoxic stresses [14, 17, 18] , although they modify it to some extent [19, 20] . Of particular note, chicken HSF1 is dispensable for stress-induced HSP expression, indicating that it is functionally different from mammalian HSF1. Instead, chicken HSF3 (cHSF3) is required for the induction of HSP expression [21-23]. cHSF3 remains as an inert dimer in unstressed conditions, and forms an active trimer in response to heat shock [24] . Thus, cHSF3 has unique properties and plays a pivotal role in the regulation of the chicken HSR. However, the potential to induce HSP expression had been lost in mouse HSF3, and the human HSF3 gene is a pseudogene [14] . Thus, the indispensable role of HSF3 has so far been considered to be specific in birds. Recently, whole genomic sequences of a variety of animal species have been determined and released to the public. It was revealed that all four HSF genes exist in vertebrate species, including in reptiles and amphibians [10] . To uncover roles of HSFs in the HSR in vertebrates, we isolated complementary DNAs of lizard and frog HSF family members and examined their potential to induce HSP expression during heat shock. Unexpectedly, we found that both HSF1 and HSF3 have the potential to induce HSP expression in lizards and frogs. Materials and methods Cell cultures and treatments Immortalized wild-type (stock #10) or HSF1-/-(stock #4) mouse embryonic fibroblasts (MEFs) [25], human HEK293 cells, and chicken DF-1 fibroblast (ATCC CRL-12203) [26] cell lines were maintained at 37˚C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; Gibco/Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Sigma Life Science). Lizard gekko lung-1 (GL-1) epithelial cells isolated from Gekko gecko (ATCC CCL-111) [27] were maintained at 30˚C in 5% CO 2 in Ham's F-12K medium (Gibco/Life Technologies) supplemented with 15% FBS. Western clawed frog epithelial-like Speedy cells derived from Xenopus tropicalis (or Silurana tropicalis) tadpole hindlimbs [28] were provided by Dr. Nicolas Pollet (Institute of Systems and Synthetic Biology, Genopole, CNRS, France), and maintained at 28˚C in Leibovitz's L-15 Medium (Sigma Life Science)/double distilled water (2:1) supplemented with 10% FBS. To induce heat shock in the cultured cells, the culture dishes were sealed with parafilm and submerged in a water bath at indicated heat shock temperature. Care and treatment of lizards and frogs Adult female brown anole lizards, Anolis sagrei, were collected in Havana city, Cuba during 2009 to 2011 (for RNA isolation), and transported alive to Tohoku University, Japan as described previously [29] . Briefly, we collected A. sagrei in Havana City, which was not a Characterization of heat shock factors in lizards PLOS ONE | https://doi.
doi:10.1371/journal.pone.0180776 pmid:28686674 pmcid:PMC5501597 fatcat:u65ln27ncfeprbau5j2zijfisu