Ionizing radiation (IR) induces DNA double strain breaks (DSB) in mammalian cells, with subsequent cell cycle arrest, apoptosis and/or senescence, depending on the cell type and stage of development. We demonstrated that features of the response of hematopoietic stem and progenitor cells and hematopoietic niche osteoblast cells to γ radiation are different. IR caused death of primary human hematopoietic CD34+ cells through apoptosis 
, whereas it induced senescence in human fetal osteoblast cell line cells. The stress-induced senescent cell markers, including proliferative (clonogenicity)-inhibition, SA-β-gal activity and senescence-associated secretory phenotype (SASP) were markedly displayed in hFOB cells after 4 and 8 Gy γ irradiation. In contrast, indications of radiation-induced apoptosis in hFOB cells (S1) were relatively lower than in CD34+ cells 
. Furthermore, we found that a novel cell stress response gene REDD1 
is highly induced in mouse bone marrow osteoblastic cells (unpublished data) and hFOB cells in response to γ radiation. The molecular mechanisms of REDD1 were further studied in hFOB cells. Knockdown of REDD1 by siRNA resulted in hFOB cell number decreases. In contrast, over-expression of REDD1 inhibited mTOR and p21 expression, suppressed inflammatory factor secretion and senescent cell marker SA-β-Gal activity in hFOB cells, and protected these cells from γ radiation-induced senescence (). It has been suggested that the tumor suppressor protein p53 and its downstream p21 and p16 signal transduction cascades in human cells mediate the activation of the senescence program, and therefore have been used as biomarkers to identify senescent cells 
. In general, activation of p53 and its downstream signaling molecule p21 in cells undergoing senescence occurred prior to the expression of p16. Overexpression of REDD1 inhibited p21 expression in irradiated hFOB cells, which confirmed its anti-senescence and host defense effects in these cells. At present, how REDD1 inhibited p21 expression is not clear. However, p53 has bind site in the promoter region of p21 
and REDD1 
gene and regulates their expression. According to Hill et al. 
, the nature of DNA damage enables p53 to selectively discriminate between promoters in the induction of target genes, thereby regulating their expression and subsequent cellular outcome. Whether overexpressed REDD1 inhibits transcriptional activity of p53 on p21 gene or enhances p21 protein degradation are under investigation.
Furthermore, our study showed that overexpression of REDD1 in hFOB cells suppressed mTOR and phosphorylation of its downstream target 4EBP-1 (mTOR signal inhibitor); this suggests inhibition of radiation-induced mTOR signal pathway activation. mTOR is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis. Braunstein et al 
, suggested that early, transient mTOR-induced cap-dependent mRNA translation after IR contributed to DNA repair and cell survival. Our recent study confirmed the protective effect of mTOR on γ radiation-induced apoptosis in human hematopoietic CD34+ cells and mouse hematopoietic cells 
. However, recent studies from Demidenko et al. 
demonstrated the mTOR pathway is involved in cellular senescence. Their hypothesis is that when the cell cycle is inhibited by stress (such as radiation or DNA damage), induction of p53 and its downstream target p21 inhibit cell proliferation. However, if mTOR is still active as a result of stress-induced growth factor secretion, it will cause cell hypertrophy and senescence 
. Eventually, lysosomal enzymes, such as β-D-galactosidase activity, will result in the senescent cell’s lysosomal membrane breakdown and the release of lysosomal proteases into the cytosolic compartment. Blocking of the mTOR activity and hypophosphorylation of 4EBP1 before radiation-induced cellular senescence commences is necessary for saving energy and for assembly of the DNA damage response machinery 
. Hence REDD1, as an essential negative regulator of mTOR 
, may play an important role in suppressing mTOR-induced protein synthesis 
and cell senescence.
We further investigated REDD1 regulation in irradiated hFOB cells. Immunoprecipitation assays demonstrated that the stress response proteins p53, RPA2 and NFkB were associated with REDD1 in hFOB cells. Knockdown of NFkB
gene by siRNA dramatically suppressed endogenous REDD1 protein expression in irradiated hFOB cells, indicating that REDD1 was regulated by both factors. Moreover, overexpression of REDD1 did not change expression and phosphorylation of p53 or NFkB after irradiation, suggesting their activation is REDD1- independent. The stress-activated p53 and NFkB signaling pathways are key players in the regulation of cellular senescence and organismal aging 
. Accumulated evidence has indicated that p53 signaling is functionally antagonistic to the NFkB system. However, the tumor suppressor p53 is an important trigger of cellular senescence and NFkB signaling is involved in the induction of the SASP. Interestingly, we report for the first time that REDD1 expression is regulated by both p53 and NFkB simultaneously. Whether REDD1 inhibition of SASP is NFkB-dependent, or whether there is a feedback loop which results in REDD1 inhibition of NFkB activation, needs further study. Furthermore, the interaction of REDD1 and RPA2 in irradiated hFOB cells supports the survival-promoting role of REDD1 in these cells. RPA, the primary single-stranded DNA (ssDNA) binding protein, is indispensible for DNA repair (including SSBs and DSBs) and replication after DNA damage in eukaryotes. RPA is a heterotrimer composed of 70 kDa (RPA1), 32 kDa (RPA2), and 14 kDa (RPA3) subunits 
. RPA2 is hyperphosphorylated after exposure to radiation 
through ATM and DNA-PK regulation, and is preferentially recruited to DSB repair in a checkpoint-dependent manner. p53 and RPA complexes after DNA damage are linked with DNA repair and p53-dependent checkpoint control 
. Our data are consistent with this model and suggest that REDD1 may be involved in p53 and RPA survival signaling in response to IR.
Our data also show that endogenous REDD1 was expressed at 4 to 48 h after IR, with peak expression at 4 h in osteoblast cells. This p53- and NFkB-induced expression of REDD1 at a relatively early stage of the response to IR could inhibit p21 and mTOR activation and protect cells from senescence. Previous reports suggested that p53 can suppress senescence through inhibition of mTOR 
. Our data further suggest that the effect of p53 on inhibition of mTOR may be through upregulation of REDD1 in irradiated hFOB cells. This hypothesis is under investigation.
In conclusion: REDD1 is regulated by p53 and NFkB signaling in response to radiation and plays an important role in suppressing p21- induced cell proliferation arrest and mTOR-induced protein synthesis, hence protects osteoblast cells from radiation-induced premature senescence.