Human aging alters the first phase of the molecular response to stress in T-cells

Abstract

This study examines how age affects the first phase of the heat shock response in human T-cells. To understand how age alters transcriptional regulation of the heat shock genes, a cross-sectional study was conducted utilizing human T-cells enriched from peripheral blood lymphocytes of healthy young (20–40 years old) and old (>70 years old) donors. Nuclear run-on analysis revealed a 66% reduction in hsp70 transcription rates in old compared to young nuclei harvested from T-cells exposed to a brief 42 °C heat shock. To determine if one or more protein transactivators of the proximal and distal promoter regions of the hsp70 gene were affected by age, gel shift analysis was performed. Both HSF1 and SP1 DNA-binding were reduced with age but no reduction was noted in CCAAT–DNA binding. Western blot analysis indicated that HSF1 but not HSF2 protein levels were reduced in aged donor samples. These data suggest that human T-cell senescence involves a multi-factorial mechanism that diminishes an important transcriptional response to thermal stress. The results are discussed relative to recent studies that support a multi-factorial mechanism for age-dependent attenuation of the heat shock transcription factor.

Introduction

Human aging exhibits diminished responses to physiological stress. Epidemiological studies consistently reveal a disproportionate number of deaths in the elderly population during environmental stress (Anonymous, 1995, Fish et al., 1985, Marmor, 1975, Schuman, 1972). These data suggest that age alters homeostasis. Thus, an important question is how age alters physiological stress at the cellular and molecular level.

Numerous conditions induce cellular stress. Unfavorable changes in temperature, oxygenation, and metabolism can damage intracellular proteins and signal new transcriptional and translational activities (Engelberg et al., 1994, Morimoto et al., 1990). Well-characterized cellular defenses include glucose-regulated, ultraviolet light, and heat shock responses (Halliwell et al., 1988, Morimoto et al., 1992, Roy and Lee, 1995). Each stress response leads to distinct molecular switches that increase expression of cell survival genes.

Thermal stress, a component of inflammation and infection, causes heat shock protein (HSP) accumulation. These proteins serve vital functions and confer cellular protection. They disaggregate damaged proteins, protect newly synthesized polypeptides and act as chaperones (Hightower, 1991, Martin et al., 1992, Pelham, 1990). Maximal HSP induction during stress requires activation of the heat shock transcription factor (HSF1) (Rabindran et al., 1994). This transactivator of heat shock gene expression unfolds from its latent form, translocates into the nucleus, self-associates as homotrimers, and binds to heat shock elements located in the promoter region of heat shock and other stress-inducible genes (Conway et al., 1994, Schuetz et al., 1991, Westwood et al., 1991). Additional modifications of HSF1 such as phosphorylation play a role in the positive and negative regulation of transcription (Chu et al., 1996, Hoj and Jakobsen, 1994, Sorger and Pelham, 1988). In addition to HSF1 regulation of the heat shock promoter region, other cooperative factors play a role in HSP mRNA expression. CCAAT–box-binding transcription factor (CTF) and the transcription factor Sp1, two factors known to regulate a large number of genes, have binding sites in human hsp70 promoter and thus are candidates for age-dependent changes in heat shock gene expression (Chu and Ferro, 2005, Morgan et al., 1987, Taira et al., 1997). In sum, a fairly well characterized sequence of events occurs during thermal stress that can potentially go awry during senesce.

To further understand how age affects stress responses in the immune system, this report focuses on thermal control of heat shock gene expression. Quiescent T-cells enriched from peripheral blood lymphocytes were isolated from healthy human donors and exposed to a non-lethal and transient heat shock in vitro. Young donors include subjects 20–40 years of age, whereas, old donors are 70 years old and older. Health of the subjects was ascertained by a health survey based on the SIENEUR protocol. A side-by-side analysis was conducted of young and old donor T-cells for baseline (37 °C) and inducible (42 °C) factors that comprise the first phase of the heat shock response. These analyses include measurements of the transcriptional rate of heat shock genes, DNA-binding levels of the Heat Shock Transcription Factor 1 (HSF1), and protein levels of DNA-binding proteins that intersect with the proximal and distal promoter of the hsp70 gene.

Results from this study reveal for the first time how senescence selectively impacts both the proximal and distal promoter region of the hsp70 gene. The mechanism for an age-dependent decline in the transcription rates of inducible heat shock genes appears to be multi-factorial. Changes in both protein–DNA binding properties as well as changes in transcription factor protein levels during thermal stress are observed in heat shocked T-cells from elderly donors. These data are discussed in reference to age-dependent declines in the immune system and its response to physiological stress and inflammation.