Abstract
Accelerated senescence is a primary response to cellular stresses including DNA damaging agents (e.g., ionizing radiation) and is widely believed to be caused by continuous proliferative signaling in the presence of cell cycle arrest. Studies of signal transduction pathways leading to accelerated senescence have revealed that inhibition of mammalian target of rapamycin (mTOR) by rapamycin rescues cells from senescence. However, the molecular mechanisms upstream of mTOR following ionizing radiation (IR) are not well defined. We investigated signal transduction leading to IR-induced accelerated senescence in human pulmonary artery endothelial cells (HPAEC). Exposure of HPAEC to X-rays (10 Gy, 2.4 Gy/min) upregulated senescence markers including p53, p21/waf1, and senescence-associated beta galactosidase (SA-β-gal). Ly294002 (a phosphatidylinositol-3-kinase [PI3K] inhibitor) or rapamycin (an mTOR inhibitor) blocked the induction of cellular senescence markers suggesting roles for PI3K and mTOR. Pathway-directed microarrays revealed increased transcription of insulin-like growth factor I (IGF-1), a modulator of cell growth and proliferation upstream of mTOR. qRT-PCR confirmed that both IGF-1 and IGF-2 mRNA were increased in response to X-rays, and ELISA showed increased secretion of IGF-1 protein into the medium of irradiated HPAEC. Consistent with upregulation of these ligands, we found that X-ray exposure led to hyperphosphorylation of IGF-1R, the receptor for IGF-1 and -2. Treatment with AG1024, an IGF-1R inhibitor, suppressed IR-induced upregulation of p53, p21/waf1, and SA-β-gal. Together these findings suggest that IGF-1R is a key regulator of IR-induced accelerated senescence in a pathway that requires intact mTOR activity upstream of both p53 and p21/waf1.
Highlights
Accelerated senescence is a well-recognized cellular response to environmental stresses that damage biological molecules especially DNA
Our data indicated that human pulmonary artery endothelial cells (HPAEC) underwent cellular senescence upon exposure to 10 Gy X-rays as determined by increased SA-β-gal activity, detected cytochemically as blue perinuclear staining at 4 days post-irradiation. (Figure 1A,B)
To determine whether insulin like growth factor-1 receptor (IGF-1R) signaling is upstream of mammalian target of rapamycin (mTOR) activation following radiation exposure, we examined the effect of AG1024 on phosphorylation of S6 ribosomal protein, a known target of S6 kinase which is downstream of activated mTOR [48]
Summary
Accelerated senescence is a well-recognized cellular response to environmental stresses that damage biological molecules especially DNA. An mTOR inhibitor, prevents accelerated senescence in cells exposed to DNA-damaging agents [5,9] Paradoxically, both mitogen activated protein kinase (MAPK) p42/p44 and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways which play roles in cell survival and proliferation have been shown to positively regulate the development of senescence [10,11,12]. MAPK p42/p44 mediates thrombopoietin-induced senescence during megakaryocytic maturation [13] Both signaling pathways appear to require increased expression of the cell cycle checkpoint protein p21/ waf for the induction of cellular senescence [13,14]
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