Subcytotoxic H2O2 Stress Triggers a Release of Transforming Growth Factor-β1, Which Induces Biomarkers of Cellular Senescence of Human Diploid Fibroblasts

Abstract

Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H 2 O 2 , with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-␤1 (TGF-␤1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated ␤-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-␤1 or its receptor (TGF-␤ RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated ␤-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-␤1 in stressful conditions, leading to SIPS and overexpression of these four genes.Normal human diploid fibroblasts (HDFs) 1 exposed to various types of noncytotoxic oxidative stress display a senescentlike phenotype coined "stress-induced premature senescence" or SIPS (1, 2). Such stressful conditions include exposure to hydrogen peroxide (3, 4), tert-butylhydroperoxide (t-BHP) (5), hyperoxia (6), UV light (7), and radioactivity (8). Many biomarkers of replicative senescence appear in SIPS: typical cell morphology (5), irreversible growth arrest, lack of response to mitogenic stimuli (4), sharp decrease of the DNA synthesis, and an increase in cells positive for the senescent-associated ␤-galactosidase activity (SA ␤-gal) (9). A long term overexpression of the cyclin-dependent kinase inhibitor p21waf-1 was observed in SIPS induced by H 2 O 2 (10) or t-BHP (9). p21waf-1 inhibits the cyclin D/cyclin-dependent kinase 4 and 6 complexes, leading to hypophosphorylation of the retinoblastoma protein (Rb). A long term hypophosphorylation of Rb over several weeks was indeed observed in SIPS induced by H 2 O 2 or t-BHP, explaining the block of the cell cycle, through Rb-mediated inhibition of the E 2 F transcription factor (9, 10). Last, several genes overexpressed in senescent HDFs, such as fibronectin, osteonectin, SM22, and apolipoprotein J (clusterin), displayed a similar increase in mRNA level in SIPS induced by t-BHP or H 2 O 2 (9).In different experimental models, an overexpression of either SM22 (11), apolipoprotein J (12), osteonectin (13), or fibronectin (14) is induced by extracellular addition of transforming growth factor-␤1 (TGF-␤1). Moreover, incubation of HDFs with TGF-␤1 triggers the appearance of a senescent-like morphology (15, 16) and growth arrest (17).Two main arguments favor the hypothesis that oxidative stress-induced premature senescence could be triggered by a pRb-mediated TGF-␤1 overexpression. First, it has been shown that AT...

UVB-induced premature senescence of human diploid skin fibroblasts

“…At 750 mJ/cm 2 , this proportion increased by more than three-fold. Similar results were obtained when foetal lung HDFs were exposed to subcytotoxic concentrations of H 2 O 2 [15] or t-BHP [10]. According to this criteria, skin HDFs exposed to UVB and foetal lung HDFs exposed to H 2 O 2 or to t-BHP behave like presenescent cells.…”

Oxidative Stress Induces Premature Senescence by Stimulating Caveolin-1 Gene Transcription through p38 Mitogen-Activated Protein Kinase/Sp1–Mediated Activation of Two GC-Rich Promoter Elements

“…We have previously shown that treatment with 150 Amol/L H 2 O 2 for 2 hours is sufficient to promote premature senescence in NIH 3T3 cells 7 to 10 days after oxidative stress (33). It is also well known that senescent cells display a typical large and flat morphology (41)(42)(43). Thus, we evaluated the cell morphology of NIH 3T3 cells treated with subcytotoxic levels of hydrogen peroxide in the presence or absence of p38 MAPK inhibitor 7 days after oxidative stress.…”

“…Acid h-galactosidase activity (at pH 6) is a well-established biochemical marker that is associated with the senescent cell phenotype (41)(42)(43). A representative field is shown in Fig.…”

Inonotus obliquus Protects against Oxidative Stress-Induced Apoptosis and Premature Senescence

“…Oxidative stress has been shown to induce stress-induced premature senescence in fibroblasts (Dasari et al, 2006;Frippiat et al, 2001). Senescent cells show different features from proliferative cells.…”

“…Oxidative stress has been shown to induce stress-induced premature senescence in fibroblasts (Dasari et al, 2006;Frippiat et al, 2001). Stress-induced premature senescence was recently invoked as an explanation of irreversible growth arrest characterized by senescence-specific cell morphology and gene expression, similar to the phenomenon of replicative senescence (Touissant et al, 2000).…”