Serum heat shock protein 70 level as a biomarker of exceptional longevity

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Abstract

Heat shock proteins are highly conserved proteins that, when produced intracellularly, protect stress exposed cells. In contrast, extracellular heat shock protein 70 (Hsp70) has been shown to have both protective and deleterious effects. In this study, we assessed heat shock protein 70 for its potential role in human longevity. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that: (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences. In this cross-sectional study, we assessed serum Hsp70 levels from participants enrolled in either the New England Centenarian Study (NECS) or the Longevity Genes Project (LGP): 87 centenarians (from LGP), 93 centenarian offspring (from NECS), and 126 controls (43 from NECS, 83 from LGP). We also examined genotypic and allelic frequencies of polymorphisms in HSP70-A1A and HSP70-A1B in 347 centenarians (266 from the NECS, 81 from the LGP), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls; LGP: 79 spousal controls). The adjusted mean serum Hsp70 levels (ng/mL) for the NECS centenarian offspring, LGP centenarians, LGP spousal controls, and NECS controls were 1.05, 1.13, 3.07, 6.93, respectively, suggesting that a low serum Hsp70 level is associated with longevity; however, no genetic associations were found with two SNPs within two hsp70 genes.

Introduction

Heat shock proteins (HSPs) are highly conserved proteins that function by chaperoning, transporting, and folding proteins when cells are exposed to a variety of stresses. Normally, HSP is expressed at low levels within all known cells; however, in response to various stressful conditions, a self-protective reaction occurs resulting in the synthesis of intracellular HSPs (Lindquist and Craig, 1988, Villar et al., 1993). One particular HSP, the inducible 70-kd heat shock protein (Hsp70), preserves cell viability by binding to polypeptide chains and preventing protein denaturation and incorrect assembly intracellularly (Georgopoulos and Welch, 1993, Hartl, 1996). Intracellular Hsp70 is also thought to exert an anti-apoptotic function by binding to the tumor suppressor protein p53 and the cellular transcription factor, c-myc, thereby inhibiting the activation of the caspase cascade (Pinhasi-Kimhi et al., 1986, Koskinen et al., 1991).

Although the numerous functions of intracellular Hsp70 have been elucidated, only recently has the role of extracellular Hsp70 (also referred to as serum Hsp70) begun to be addressed. Accumulating evidence indicates that extracellular Hsp70 activates innate immune cells leading to an inflammatory cascade. Prior research has shown that extracellular Hsp70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca2+ flux, activate the transcription factor NF-κB nuclear translocation, augment the expression and release of pro-inflammatory cytokines (Basu et al., 1998, Asea et al., 2000a, Asea et al., 2000b, Binder et al., 2000), induce nitric oxide release (Panjwani et al., 2002), upregulate co-stimulatory molecule expression (Asea et al., 2002), and induce dendritic cell maturation (Kuppner et al., 2001, Asea et al., 2002, Noessner et al., 2002). These functions can be protective if activated in the context of a cellular insult; however, they can also be detrimental if they lead to excessive inflammation.

Various studies have examined the relationship of extracellular Hsp70 with aging. In their cross-sectional study, Rea et al. (2001) examined serum Hsp70 in 60 individuals with ages ranging from 20 to 96 years. They demonstrated a progressive decline in serum Hsp70 levels in older age groups. Similarly, Jin et al. (2004), in their study of 327 healthy male donors aged between 15 and 50 years, demonstrated a decline in serum Hsp70 at older ages (between 30 and 50) although at younger ages, they noted a positive correlation with age. These findings suggest that serum Hsp70 levels decline at older ages although they do not indicate whether levels decline over time within an individual. They also do not indicate whether there is a decline in Hsp70 function over time although in vitro studies suggest that cell lines from centenarians, when exposed to heat stress, have similar Hsp70 synthesis (Marini et al., 2004), have a similar transcriptional response to the hsp70 gene (Ambra et al., 2004), and are less prone to heat-induced apoptosis when compared to cell lines from younger individuals.

In this study we analyzed serum Hsp70 levels in a sample of centenarian subjects, their offspring, and unrelated controls. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences.

Few studies have examined the role of genetics in the relationship between Hsp70 and survival to very old age. Altomare et al. (2003) found that a polymorphism within the hsp70 gene promoter was associated with longevity only in females. Subsequently, Marini et al. (2004) reported that in centenarians, the A/A genotype of the (A/C)-110 genetic polymorphism in the promoter region was associated with a lower expression of Hsp70 protein after heat stress, in comparison to the C/C genotype. In addition to analyzing serum Hsp70, we examined two single nucleotide polymorphisms (SNPs) of hsp70 genes for their potential role in exceptional longevity.

Section snippets

Study population

The criteria for eligibility, methods of recruitment, and study outcomes of the New England Centenarian Study (NECS) have been published elsewhere (Perls et al., 1999, Terry et al., 2003). Briefly, the NECS is an United States nationwide study of centenarians, their offspring, and controls. The controls include (a) spouses of the centenarian offspring and (b) offspring of individuals whose parents were born in the same years as the centenarians but who died at the average life expectancy for

Demographics and HSP analyses

As expected, the centenarians are older, predominantly female, and generally sicker than the centenarian offspring and the controls (Table 1). The youngest “centenarian” is 95-years-old, while the oldest is a female, age 108. The oldest male is 106-years-old. The age range for the NECS centenarian offspring is 54–88 years, for the NECS controls is 57–89, and for the LGP controls is 39–74. Despite being of similar age (p = 0.497), a higher proportion of NECS centenarian offspring are disease-free

Serum Hsp70 findings

This study demonstrates that centenarians and centenarian offspring have significantly lower mean serum Hsp70 levels than unrelated controls. Centenarians and centenarian offspring have similar unadjusted levels of Hsp70. Unfortunately, since the offspring of centenarians are unrelated to the centenarians in our study, we can only speculate that the similarity in their Hsp70 levels is consistent with Hsp70 being a heritable trait.

After examining age, gender, race, income, alcohol,

Conflicts of Interest

None.

Acknowledgements

The authors thank Susana Fiorentino for helpful discussions and Edwina Asea and Diana T. Page for expert technical assistance. This work was supported in part by the National Institute on Aging grant K08AG22785 (to DFT); Paul Beeson Physician Faculty Scholar in Aging Awards (to DFT, TP, NB), the Ellison Medical Foundation Senior Scholar Award and RO1 AG-18728-01A1 (to N.B); the National Institutes of Health grant RO1CA91889, institutional support from Scott & White Memorial Hospital and Clinic,

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