Heat shock response, which is characterized by the induction of a set of heat shock proteins, is essential for induced thermotolerance and is regulated by heat shock transcription factors (HSFs). Curiously, HSF1 is essential for heat shock response in mammals, whereas in avian HSF3, an avian-specific factor is required for the burst activation of heat shock genes. Amino acid sequences of chicken HSF1 are highly conserved with human HSF1, but those of HSF3 diverge significantly. Here, we demonstrated that chicken HSF1 lost the ability to activate heat shock genes through the amino-terminal domain containing an alanine-rich sequence and a DNA-binding domain. Surprisingly, chicken and human HSF1 but not HSF3 possess a novel function that protects against a single exposure to mild heat shock, which is not mediated through the activation of heat shock genes. Overexpression of HSF1 mutants that could not bind to DNA did not restore the susceptibility to cell death in HSF1-null cells, suggesting that the new protective role of HSF1 is mediated through regulation of unknown target genes other than heat shock genes. These results uncover a novel role of vertebrate HSF1, which has been masked underthe roles of heat shock proteins.All living organisms respond to elevated temperatures by inducing a set of highly conserved proteins, heat shock proteins (Hsps). This response is called the heat shock response and is believed to be a universal and fundamental mechanism for cell protection against stresses such as heat shock. The heat shock response is regulated mainly at the level of transcription by heat shock transcription factors (HSFs) in eukaryotes, which bind to heat shock elements on upstream sequences of heat shock genes (45). It is well known that cells can survive an exposure to lethal temperatures when cells are preincubated at sublethal high temperatures. This phenomenon is now called induced thermotolerance. Numerous studies suggest that Hsp induction is crucial to the acquisition of the induced thermotolerance (19). Finally, heat shock response regulated by HSF is shown to be necessary for acquisition of the induced thermotolerance in the fruit fly (15), mouse embryo fibroblast cells (21), and chicken B lymphocyte DT40 cells (42).HSFs do more than activate heat shock genes in response to elevated temperatures. It was shown that in Drosophila HSF is required under normal growth conditions for oogenesis and early development (15). Mice deficient in HSF1 show abnormal placental development, growth retardation, and female infertility (7, 46). Furthermore, mice deficient in HSF2 exhibit abnormalities in brain development and defects in spermatogenesis and oogenesis (16). In all of these cases, developmental functions of HSFs are not mediated through the induction of Hsps, suggesting that HSFs regulate unknown genes related to development. Recently, it was found that HSFs can regulate only a specific heat shock gene under normal growth conditions. In chicken DT40 cells, HSF1 and HSF3 regulate only Hsp90␣ expression in a...

DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C‐terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N‐terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient‐derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR 1204–1207) at the DAD C‐terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID‐DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG‐tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID‐DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.

Heat shock transcription factors (HSFs) play roles not only in heat shock response but also in development of the reproductive organs, brain, and lens. Here, we analyzed sensory organs and found abnormalities of the olfactory epithelium in adult HSF1-null mice, which is developmentally related to the lens. The olfactory epithelium was normal until postnatal 3 weeks but was not maintained later than 4 weeks in HSF1-null mice. The olfactory epithelium was atrophied with increased cell death of olfactory sensory neurons. Analysis of the epithelium revealed that induction of HSP expression and reduction of LIF expression are lacking in adult HSF1-null mice. We found that DNA binding activity of HSF1 is induced in the olfactory epithelium later than 4 weeks and that HSF1 binds directly to Lif gene and inhibits its expression. HSF4 has opposing effects on LIF expression and olfactory neurogenesis. These data indicate that HSF1 is required for the precise expression of Hsp and cytokine genes that is obligatory for maintenance of olfactory neurogenesis in adult mice and suggest that stress-related processes are involved in its maintenance.Heat shock response is characterized by induction of a set of heat shock proteins (HSPs) 2 and is an evolutionary conserved response inherent in all organisms from bacteria to human. In eukaryotes, this response is regulated mainly by heat shock transcription factor (HSF), which can bind to a heat shock element (HSE) composed of at least three inverted repeats of consensus sequence nGAAn (1). In vertebrates, three mammalian Hsf genes (Hsf1, Hsf2, and Hsf4) and three avian HSF genes (HSF1, HSF2, and HSF3) have been identified (2, 3), and their expression profiles are temporally and spatially different during development (3, 4). In unstressed cells HSF1 is present mostly as a monomer and is converted to a trimer that binds to HSE in response to heat shock, whereas HSF4 is constitutively a trimer. A single mutation of Hsf1, Hsf2, or Hsf4 genes or combined mutations of Hsf genes in mice showed that HSFs are involved in development of the reproductive organs (5-10), brain (6, 10, 11), and lens (12-14) and in immune response (15,16). In addition to regulating HSP expression, HSFs are involved in development partly by regulating expression of growth factor genes such as Fgf and Il-6 genes directly (13,15). Although HSF1 binds to many genes in vivo in unstressed cells (17), we have little information about when and how HSF activity is regulated during development and maintenance of tissues.We previously identified that HSF4 is required for development of lens in the eye partly by regulating Hsp and Fgf genes (13). HSF1 is also involved in lens development and competes with HSF4 for expression of the same Fgf genes. These observations highlighted sensory organs that receive stimuli from the external environment and are susceptible to stress. Here, we examined development of other sensory organs in HSF1-and HSF4-null mice. Surprisingly, we found abnormalities in the nasal cavity in adult HSF1-nu...

Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.