Analysis of the effect of aging on the response to hypoxia by cDNA microarray

doi:10.1016/S0047-6374(03)00166-0

Mechanisms of Ageing and Development

Volume 124, Issues 8–9, August–September 2003, Pages 941-949

https://doi.org/10.1016/S0047-6374(03)00166-0 Get rights and content

Abstract

Little is known about the detail of hypoxia-responsive gene expression patterns in advanced age, even though aging is thought to be partially associated with a decreased response to hypoxia. In the present study, we identified several hypoxia-inducible genes and investigated the effect of aging on hypoxic gene expression profiles using cDNA microarray analysis of young/old human diploid fibroblasts. Of 7458 genes in the microarray, we found that genes involved in angiogenesis, defense against oxidative stress, and transcription regulation are severely impaired in senescent cell, which is consistent with the fact that aged cells have attenuated responses to various stimuli.

Introduction

Aging is characterized by a deleterious and progressive decline in physiological efficiency. Since multiple factors are involved in the aging progress, it is still difficult to collectively understand the global network involved. Nonetheless, it is known that the aging process primarily occurs due to an altered gene expression by the loss or the altered activities of gene regulatory molecules (Roy et al., 2002). In particular, an aged organism is susceptible to various age-related diseases including vascular diseases and shows impaired angiogenesis under hypoxic conditions, which result from the reduced induction of vascular endothelial growth factor (VEGF) (Rivard et al., 1999), a potent hypoxia-targeted angiogenesis stimulator (Shweiki et al., 1992).

Hypoxia, a limited cellular oxygen tension, is important for the regulation of the expressions of essential genes under specific developmental or physiological conditions: energy metabolism, proliferation, survival/apoptosis, erythropoiesis and angiogenesis (Semenza, 2001). Hypoxia-inducible factor-1 (HIF-1) is a basic–helix–loop–helix protein and forms heterodimer composed of a constitutively expressed HIF-1β subunit and an O₂-regulated HIF-1α subunit (Wang et al., 1995). In the normoxic cells, HIF-1α protein, which is hydroxylated at proline residues in its oxygen-dependent degradation (ODD) domain by O₂-regulated prolyl hydroxylases, is subjected to ubiquitination and subsequent proteosomal degradation by von Hippel–Lindau (VHL) tumor suppressor protein. However, under hypoxic conditions, HIF-1α escapes from the degradation pathway, and forms a heterodimeric active complex that binds to hypoxia-responsive recognition element (HRE), which leads to the induction of hypoxia-responsive genes such as erythropoietin (EPO), VEGF, and the genes required for metabolic adaptation to an oxygen-limited environment (Semenza, 2001, Wenger, 2002).

Recent reports have suggested that the DNA binding activity of HIF-1α declines in old age, and that this is responsible for reduced VEGF expression and impaired angiogenesis (Frenkel-Denkberg et al., 1999). In addition, it is well known that the metabolic rate in advanced age is gradually reduced. Given that the unique characteristics of impaired angiogenesis and a slow rate of metabolic uptake in an aged organism appear to be deeply involved in the inefficiency of gene regulation under hypoxic conditions, it becomes apparent that aging exerts an influence on global gene expression patterns of hypoxia-targeted genes.

cDNA microarrays becomes a growing technology, which can be used to better understand the global networking of individual genes. Thus, in this study, we used a cDNA microarray to identify hypoxia-related genes expressed in an age-dependent fashion, and by analyzing gene expression profile, we attempt to explain the background of the impaired biological function of old organisms in hypoxia.

Here, we demonstrate that the inductions of genes, responsible for adaptation to hypoxia, are markedly declined in old human diploid fibroblasts (HDFs). Especially, those genes related to angiogenesis such as VEGF, PGAR, thromboplastin and PAI-1 and to those related to oxidative stress like heme oxygenase 1 (HO-1). These genes were found to be differentially expressed in ‘old’ and ‘young’ HDFs, and could be responsible for the impaired angiogenesis and susceptibility to ischemic injury observed in old age. In addition, we report, for the first time, that insulin induced gene 1 (INSIG-1), and bagpipe homeobox homologue 1 (BAPX1) are hypoxia-inducible genes.

Section snippets

Cell culture and hypoxic exposure

HDFs were obtained from newborn foreskins, as described by Boyce and Ham (1983), and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 50 μU of penicillin/streptomycin. Cells with mean population doublings (MPDs) of less than 25 were used as young cells and the cells with MPDs of 65–70 MPD as old cells. Senescent cells were characterized by morphological changes, enhanced β-galactosidase activity, and a reduced rate of proliferation (Yeo et

General presentation of the results

To examine the aging effect on hypoxia-responsive gene expression, we performed cDNA microarray analysis on young and old HDFs (Fig. 1A). EST clones including 215 unassigned ESTs and the 20 ESTs of putative genes, we selected genes showing significant fold-differences between young and old HDFs. The expression fold of genes under hypoxia was plotted (Fig. 1B). The region A includes genes induced by hypoxia in both young and old HDFs and region B includes genes induced preferentially in young

Discussion

Under hypoxic conditions, organisms respond to maintain oxygen homeostasis by increasing oxygen delivery or by providing a metabolic adaptation, and by regulating cellular growth. Moreover, these responses are facilitated at the transcription level and are followed by posttranslational modification. In this study, we investigated the aging effect on the response to hypoxia by comparing the differential gene expressions of young and old HDFs upon exposure to hypoxia.

Of 7458 human ESTs examined,

Acknowledgements

This work was supported by Korean Research Foundation (KRF-2001-015-FP0042).

References (36)

A.J. Belanger et al.
Hypoxia up-regulates expression of peroxisome proliferator-activated receptor angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1
J. Mol. Cell. Cardiol.
(2002)
P.E. Di Cesare et al.
Matrix–matrix interaction of cartilage oligomeric matrix protein and fibronectin
Matrix Biol.
(2002)
T. Fink et al.
Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1
Blood
(2002)
G. Frenkel-Denkberg et al.
The function of hypoxia-inducible factor 1 (HIF-1) is impaired in senescent mice
FEBS Lett.
(1999)
Y. Lavrovsky et al.
Age-dependent increase of heme oxygenase-1 gene expression in the liver mediated by NF-kB
Mech. Ageing Dev.
(2000)
E. Minet et al.
c-Jun gene induction and AP-1 activity is regulated by a JNK-dependent pathway in hypoxic HepG2 cells
Exp. Cell Res.
(2001)
J.M. Muller et al.
Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway
J. Biol. Chem.
(1997)
A.K. Roy et al.
Impacts of transcriptional regulation on aging and senescence
Age Res. Rev.
(2002)
G.L. Semenza
HIF-1, O2 and the 3 PHDs: how animal cells signal hypoxia to nucleus
Cell
(2001)
T. Watanabe et al.
Angiogenesis induced by tissue factor in vitro and in vivo
Thrmob. Res.
(1999)

S.F. Yan et al.

Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6

J. Biol. Chem.

(1995)

S.F. Yan et al.

Hypoxia-associated induction of early growth response-1 gene expression

J. Biol. Chem.

(1999)

E.J. Yeo et al.

Reduction of UV-induced cell death in the human senescent fibroblasts

Mol. Cells

(2000)

W.G. An et al.

Stabilization of wild-type p53 by hypoxia-inducible factor 1α

Nature

(1998)

D. Bandyopadhyay et al.

Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes

Cancer Res.

(2002)

S.T. Boyce et al.

Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture

J. Invest. Dermatol.

(1983)

T.A. Chan et al.

14-3-3σ is required to prevent mitotic catastrophe after DNA damage

Nature

(1999)

G. Deng et al.

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release

J. Cell Biol.

(1996)

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Analysis of the effect of aging on the response to hypoxia by cDNA microarray

Abstract

Introduction

Section snippets

Cell culture and hypoxic exposure

General presentation of the results

Discussion

Acknowledgements

J. Mol. Cell. Cardiol.

Matrix Biol.

Blood

FEBS Lett.

Mech. Ageing Dev.

Exp. Cell Res.

J. Biol. Chem.

Age Res. Rev.

Cell

Thrmob. Res.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cells

Stabilization of wild-type p53 by hypoxia-inducible factor 1α

Nature

Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes

Cancer Res.

Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture

J. Invest. Dermatol.

14-3-3σ is required to prevent mitotic catastrophe after DNA damage

Nature

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release

J. Cell Biol.