Aberrant Regulation and Modification of Heat Shock Factor 1 in Senescent Human Diploid Fibroblasts Yoon Kwang Lee, 1,2 Diana J. Liu, 1 Jiebo Lu, 3 Kuang Yu Chen, 3 and Alice Y.-C. Liu 1 * 1 Department of Cell Biology and Neuroscience, Rutgers State University of New Jersey 604 Allison Road, Piscataway, New Jersey 08854 2 Department of Integrative Medical Sciences, Northeastern Ohio Universities Colleges of Medicine, 4209 State Route 44, Rootstown, Ohio 44272 3 Department of Chemistry and Chemical Biology, Rutgers State University of New Jersey 610 Taylor Road, Piscataway, New Jersey 08854 ABSTRACT Induction of the heat shock response (HSR), determined by hsp70-luciferase reporter and HSP70 protein expression, is attenuated as a function of age of the IMR-90 human diploid fibroblasts. To better understand the underlying mechanism, we evaluated changes in the regulation and function of the HSF1 transcription factor. We show that the activation of HSF1 both in vivo and in vitro was decreased as a function of age, and this was attributable to a change in the regulation of HSF1 as the abundance of HSF1 protein and mRNA was unaffected. HSF1 was primarily cytosolic in young cells maintained at 378C, and heat shock promoted its quantitative nuclear translocation and trimerization. In old cells, some HSF1 was nuclear sequestered at 378C, and heat shock failed to promote the quantitative trimerization of HSF1. These changes in HSF1 could be reproduced by treating young cells with H 2 O 2 to stunt them into premature senescence. Flow cytometry measurement of peroxide content showed higher levels in old cells and H 2 O 2 -induced premature senescent cells as compared to young cells. Experiments using isoelectric focusing and Western blot showed age-dependent changes in the mobility of HSF1 in a pattern consistent with its S-glutathiolation and S-nitrosylation; these changes could be mimicked by treating young cells with H 2 O 2 . Our results demonstrated dynamic age-dependent changes in the regulation but not the amount of HSF1. These changes are likely mediated by oxidative events that promote reversible and irreversible modification of HSF1 including S-glutathiolation and S-nitrosylation. J. Cell. Biochem. 106: 267–278, 2009. ß 2008 Wiley-Liss, Inc. KEY WORDS: AGING; HSF1; TRANSCRIPTION FACTOR REGULATION; POST-TRANSLATIONAL MODIFICATION; S-GLUTATHIOLATION; S-NITROSYLA- TION A ging is a near universal phenomenon. While the mechan- isms that underlie aging remain to be elucidated and defined, there is good evidence that genes and cell physiology that confer stress resistance prolong life-span [Lithgow and Kirkwood, 1996; Tatar et al., 1997; Kirkwood and Austad, 2000; Kirkwood et al., 2000]. This correlation between stress resistance and life-span suggests that a stress resistance gene may function as a longevity- assurance gene, and that resistance to stress can be used as a screen for isolating long-lived variants of the organism, as has been accomplished in Saccharomyces cerevisiae [Kennedy et al., 1995; Smeal and Guarente, 1997] and Caenorhabditis elegans [Murakami and Johnson, 1996]. One of the most studied responses to stress is induction of the heat shock transcriptional response [Morimoto, 2008]. We previously reported that induction of the heat shock response is attenuated in aging human diploid fibroblasts [Liu et al., 1989, 1996]. An attenuated response to stress is also observed in aging animal model systems and in cells/tissues derived from them [Heydari et al., 1993, 1996, 2000; Liu et al., 1996; Locke and Tanguay, 1996; Morimoto, 2008]. These considerations suggest that an attenuated heat shock response is a hallmark of aging in the dividing human diploid fibroblasts (replicative senescence), non-dividing post-mitotic cells (cellular senescence), and organisms. Given the well-known function of HSPs in protein homeostasis, including protein folding, Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 106:267–278 (2009) 267 Abbreviations used: EMSA, electrophoretic mobility shift assay; HDF, human diploid fibroblasts; HSR, heat shock response, HSF1, human heat shock factor 1; HSP, heat shock protein; DTT, dithiothreitol; FACS, fluorescent activated cell sorting; IAA, iodoacetate; IAM, iodoacetamide; IEF, isoelectric focusing; ROS, reactive oxygen species; SIPS, stress-induced premature senescence; DCFH-DA, 2 0 ,7 0 -dichlorofluorescin diacetate. Grant sponsor: NSF; Grant number: 02-40009. *Correspondence to: Dr. Alice Y.-C. Liu, Department of Cell Biology and Neuroscience, Rutgers State University of New Jersey, 604 Allison Road, Piscataway, NJ 08855-8082. E-mail: liu@biology.rutgers.edu Received 26 September 2008; Accepted 21 October 2008  DOI 10.1002/jcb.21997  2008 Wiley-Liss, Inc. Published online 18 December 2008 in Wiley InterScience (www.interscience.wiley.com).