Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression.

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

Dexamethasone-induced activation of heat shock response ameliorates seizure susceptibility and neuroinflammation in mouse models of Lafora disease.

Heat shock response (HSR) is a conserved cytoprotective pathway controlled by the master transcriptional regulator, the heat shock factor 1 (HSF1), that activates the expression of heat shock proteins (HSPs). HSPs, as chaperones, play essential roles in minimizing stress-induced damages and restoring proteostasis. Therefore, compromised HSR is thought to contribute to neurodegenerative disorders. Lafora disease (LD) is a fatal form of neurodegenerative disorder characterized by the accumulation of abnormal glycogen as Lafora bodies in neurons and other tissues. The symptoms of LD include progressive myoclonus epilepsy, dementia, and cognitive deficits. LD is caused by the defects in the gene coding laforin phosphatase or the malin ubiquitin ligase. Laforin and malin are known to work upstream of HSF1 and are essential for the activation of HSR. Herein, we show that mice deficient for laforin or malin show reduced levels of HSF1 and their targets in their brain tissues, suggesting compromised HSR; this could contribute to the neuropathology in LD. Intriguingly, treatment of LD animals with dexamethasone, a synthetic glucocorticoid analogue, partially restored the levels of HSF1 and its targets. Dexamethasone treatment was also able to ameliorate the neuroinflammation and susceptibility to induced seizures in the LD animals. However, dexamethasone treatment did not show a significant effect on Lafora bodies or autophagy defects. Taken together, the present study establishes a role for HSR in seizure susceptibility and neuroinflammation and dexamethasone as a potential antiepileptic agent, suitable for further studies in LD.

Heat Shock Factor 1 Is a Direct Antagonist of AMP-Activated Protein Kinase.

Through transcriptional control of the evolutionarily conserved heat shock, or proteotoxic stress, response, heat shock factor 1 (HSF1) preserves proteomic stability. Here, we show that HSF1, a physiological substrate for AMP-activated protein kinase (AMPK), constitutively suppresses this central metabolic sensor. By physically evoking conformational switching of AMPK, HSF1 impairs AMP binding to the γ subunits and enhances the PP2A-mediated de-phosphorylation, but it impedes the LKB1-mediated phosphorylation of Thr172, and retards ATP binding to the catalytic α subunits. These immediate and manifold regulations empower HSF1 to both repress AMPK under basal conditions and restrain its activation by diverse stimuli, thereby promoting lipogenesis, cholesterol synthesis, and protein cholesteroylation. In vivo, HSF1 antagonizes AMPK to control body fat mass and drive the lipogenic phenotype and growth of melanomas independently of its intrinsic transcriptional action. Thus, the physical AMPK-HSF1 interaction epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine.

HSF1/HSP pathway in the hippocampus is involved in SIRT1-mediated caloric restriction-induced neuroprotection after surgery in aged mice.

Postoperative cognitive dysfunction is common in the elderly. Endoplasmic reticulum stress (ER-stress) increases neuronal apoptosis after surgery, and chaperone molecules, such as heat shock proteins (HSPs), help reduce unfolded protein reactions, thereby promoting protein homeostasis. Mammal sirtuin1 (SIRT1)-mediated deacetylation of heat shock factor 1 (HSF1) upregulates HSF1 binding to the HSP70 promoter. Caloric restriction (CR) improves cognition in many neurodegenerative models. In this study, we evaluated whether CR improves impaired learning and memory after surgery by attenuating ER-stress in an SIRT1-dependent manner. Male 18-month-old C57BL/6J mice receiving a 12-week CR or an ad libitum (AL) diet pre-intervention were challenged with tibial open fracture surgery and anesthesia or no treatment. We found a significant protective effect of CR on memory in contextual fear conditioning test after surgery compared with the AL group. CR alleviated ER-stress and neuronal apoptosis in the hippocampus induced by surgery. CR increased HSP70 expression through the HSF1/HSP pathway in a SIRT1-mediated manner, and inhibition of SIRT1 in the hippocampus by lentivirus injection partially reduced the benefits of CR (increased HSP70, deacetylated HSF1, reduced ER-stress, and improved memory). Taken together, our results showed that CR alleviates memory impairment postoperatively via attenuation of ER-stress in the hippocampus in an SIRT1-dependent manner, and the SIRT1/HSF1/HSP70 pathway is involved in this process.

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression.

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1-/- cells than in the wild-type cells at both mRNA and protein levels. In Nrf2-/- cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40-42 °C in Nrf2-/- cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2-/- cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.

Downregulation of heat shock factor 4 transcription activity via MAPKinase phosphorylation at Serine 299.

Dysfunction of HSF4 is associated with congenital cataracts. HSF4 transcription activity is turned on and regulated by phosphorylation during early postnatal lens development. Our previous data suggested that mutation HSF4b/S299A can upregulate HSF4 transcription activity in vitro, but the biological significance of posttranslational modification on HSF4/S299 during lens development remains unclear. Here, we found that the mutation HSF4/S299A can upregulate the expression of HSP25 and alpha B-crystallin at both protein and mRNA levels in mouse the lens epithelial cell line, but HSF4/S299D does not. Using the rabbit polyclonal antibody against phospho-S299 of HSF4, we found that EGF and ectopic expression of MEK1 can increase the phosphorylation of HSF4/S299 and induce HSF4 sumoylation, and these effects are inhibited by U0126. ERK1/2 can phosphorylate the S299 in HSF4/wt but not in HSF4/S299A in the in vitro kinase assay. Functionally, ectopic MEK1 can inhibit HSF4-controled alpha B-crystallin expression but has less effect on HSF4/S299A. EGF can upregulate phospho-HSF4/S299 and downregulate alpha B-crystallin expression in P3 mouse lens, and this downregulation is suppressed by U0126. During mouse lens development, phosphorylation of HSF4/S299 is downregulated in P3 lens and upregulated in P7 and P14 lens. However, in 2 months old lens, both phosphorylation of HSF4/S299 and total HSF4 protein are decreased. Interestingly, ERK1/2 activity is lower in P3 lens than in P7 and P14 lens, which is in line with the phosphorylation of HSF4/S299. Taken together, our data demonstrate that HSF4/299 is a phosphorylation target of MEK1-ERK1/2, and phosphorylation of S299 is responsible for tuning down HSF4 transcription activity during postnatal lens development.

Acute HSF1 depletion induces cellular senescence through the MDM2-p53-p21 pathway in human diploid fibroblasts.

Heat shock transcription factor 1 (HSF1) regulates the expression of a wide array of genes, controls the expression of heat shock proteins (HSPs) as well as cell growth. Although acute depletion of HSF1 induces cellular senescence, the underlying mechanisms are poorly understood. Here, we report that HSF1 depletion-induced senescence (HDIS) of human diploid fibroblasts (HDFs) was independent of HSP-mediated proteostasis but dependent on activation of the p53-p21 pathway, partly because of the increased expression of dehydrogenase/reductase 2 (DHRS2), a putative MDM2 inhibitor. We observed that HDIS occurred without decreased levels of major HSPs or increased proteotoxic stress in HDFs. Additionally, VER155008, an inhibitor of HSP70 family proteins, increased proteotoxicity and suppressed cell growth but failed to induce senescence. Importantly, we found that activation of the p53-p21 pathway resulting from reduced MDM2-dependent p53 degradation was required for HDIS. Furthermore, we provide evidence that increased DHRS2 expression contributes to p53 stabilization and HDIS. Collectively, our observations uncovered a molecular pathway in which HSF1 depletion-induced DHRS2 expression leads to activation of the MDM2-p53-p21 pathway required for HDIS.

HSF1 deficiency accelerates the transition from pressure overload-induced cardiac hypertrophy to heart failure through endothelial miR-195a-3p-mediated impairment of cardiac angiogenesis.

Heat shock transcription factor 1 (HSF1) deficiency aggravates cardiac remodeling under pressure overload. However, the mechanism is still unknown. Here we employed microRNA array analysis of the heart tissue of HSF1-knockout (KO) mice to investigate the potential roles of microRNAs in pressure overload-induced cardiac remodeling under HSF-1 deficiency, and the profiles of 478 microRNAs expressed in the heart tissues of adult HSF1-KO mice were determined. We found that the expression of 5 microRNAs was over 2-fold higher expressed in heart tissues of HSF1-KO mice than in those of wild-type (WT) control mice. Of the overexpressed microRNAs, miR-195a-3p had the highest expression level in HSF1-null endothelial cells (ECs). Induction with miR-195a-3p in ECs significantly suppressed CD31 and VEGF, promoted AngII-induced EC apoptosis, and impaired capillary-like tube formation. In vivo, the upregulation of miR-195a-3p accentuated cardiac hypertrophy, increased the expression of ß-MHC and ANP, and compromised systolic function in mice under pressure overload induced by transverse aortic constriction (TAC). By contrast, antagonism of miR-195a-3p had the opposite effect on HSF1-KO mice. Further experiments confirmed that AMPKα2 was the direct target of miR-195a-3p. AMPKα2 overexpression rescued the reduction of eNOS and VEGF, and the impairment of angiogenesis that was induced by miR-195a-3p. In addition, upregulation of AMPKα2 in the myocardium of HSF1-null mice by adenovirus-mediated gene delivery enhanced CD31, eNOS and VEGF, reduced ß-MHC and ANP, alleviated pressure overload-mediated cardiac hypertrophy and restored cardiac function. Our findings revealed that the upregulation of miR-195a-3p due to HSF1 deficiency impaired cardiac angiogenesis by regulating AMPKα2/VEGF signaling, which disrupted the coordination between the myocardial blood supply and the adaptive hypertrophic response and accelerated the transition from cardiac hypertrophy to heart failure in response to pressure overload.

Use of conditioned media is critical for studies of regulation in response to rapid heat shock.

Heat shock response (HSR) maintains and restores protein homeostasis when cells are exposed to proteotoxic heat stress. Heat shock (HS) triggers a rapid and robust change in genome-wide transcription, protein synthesis, and chaperone activity; and therefore, the HSR has been widely used as a model system in these studies. The conventional method of performing instantaneous HS in the laboratory uses heated fresh media to induce HSR when added to cells. However, addition of fresh media to cells may evoke additional cellular responses and signaling pathways. Here, we compared the change in global transcription profile when HS is performed with either heated fresh media or heated conditioned media. We found that the use of heated fresh media induces transcription of hundreds of genes that HS alone does not induce, and masks or partially masks HS-mediated downregulation of thousands of genes. The fresh-media-dependent upregulated genes encode ribosomal subunit proteins involved in translation and RNA processing factors. More importantly, fresh media also induce transcription of several heat shock protein genes (Hsps) in a heat shock factor 1 (HSF1)-independent manner. Thus, we conclude that a conventional method of HS with heated fresh media causes changes in transcription regulation that confound the actual change caused solely by elevated temperature of cells.

HSF1-deficiency affects gait coordination and cerebellar calbindin levels.

Heat shock proteins (HSPs) play an important role in cell homeostasis and protect against cell damage. They were previously identified as key players in different ataxia models. HSF1 is the main transcription factor for HSP activation. HSF1-deficient mice (HSF1-/-) are known to have deficiencies in motor control test. However, little is known about effects of HSF1-deficiency on locomotor, especially gait, coordination. Therefore, we compared HSF-deficient (HSF1-/-) mice and wildtype littermates using an automated gait analysis system for objective assessment of gait coordination. We found significant changes in gait parameters of HSF1-/- mice reminiscent of cerebellar ataxia. Immunohistochemical analyses of a cerebellum revealed co-localization of HSF1 and calbindin in Purkinje cells. Therefore, we tested the hypothesis of a potential interconnection between HSF1 and calbindin in Purkinje cells. Calbindin levels were analyzed qualitatively and quantitatively by immunohistochemistry and immunoblotting, respectively. While quantitative PCR revealed no differences in calbindin mRNA levels between HSF1+/+ and HSF1-/- mice, calbindin protein levels, however, were significantly decreased in a cerebellum of HSF1-/- mice. A pathway analysis supports the hypothesis of an interconnection between HSF1 and calbindin. In summary, the targeted deletion of HSF1 results in changes of locomotor function associated with changes in cerebellar calbindin protein levels. These findings suggest a role of HSF1 in regular Purkinje cell calcium homeostasis.