U2OS is a human osteosarcoma cell line that does not express ARF. When either ARF-GFP or ARF itself was expressed in U2OS cells, the level of NS significantly declined (A). Western blotting showed that ARF expression decreased the level of NS by ~50% in the cell population (C). Conversely, the level of NS became elevated when endogenous ARF was knocked down in HeLa cells (B). Thus, the level of nucleostemin responds to either an increase or decrease in ARF. Because knockdown of NS did not affect the level of ARF in HeLa cells (data not shown), it would seem that ARF is an upstream regulator of NS.
Figure 1. Down-regulation of nucleostemin by ARF expression. (A) U2OS cells were transfected with ARF-GFP or with ARF itself. After 24 h, NS was detected by immunostaining, and ARF was detected by immunostaining (right column) or by the fluorescence of ARF-GFP (more ...)
NS is thought to interact with p53 (Tsai and McKay, 2002
) whereas ARF is known to stabilize p53 by binding MDM2, a ubiquitin ligase that normally degrades p53 (Haupt et al., 1997
; Kubbutat et al., 1997
; Stott et al., 1998
). To examine the possible links among ARF, NS, and p53, we used the U2OS-derived cell line NARF6, in which ARF expression is inducible (Stott et al., 1998
). Expression of ARF in NARF6 cells resulted in a pronounced increase of p53 and a significant decline of NS (A). This result does not distinguish between a direct action of ARF that destabilizes NS versus an indirect effect in which ARF stabilizes p53 (vide supra
), which causes NS to be destabilized. To determine whether the level of NS is related to that of p53, U2OS cells were subjected to UV-induced DNA damage under conditions known to elevate p53. As can be seen in B, this resulted in a significant decrease of NS. This result is consistent with the interpretation that experimental elevation of ARF (A) may negatively impact NS via a stabilization of p53.
Figure 2. Down-regulation of NS coincides with up-regulation of p53. (A) NARF6 cells (a U2OS-derived ARF-inducible cell line) were immunostained for p53 and NS after 48-h treatment with 1 mM isopropyl-β-d-thiogalactopyranoside. (B) U2OS cells were subjected (more ...)
It was next logical to ask whether NS modulates p53. As shown in A, knockdown of NS led to an elevation of p53. Immunoblot analysis of these cells (C) revealed an ~60% knockdown of NS and an approximately threefold elevation of p53. To ask whether the increased p53 is functional, we examined the level of MDM2 after NS knockdown. Functional p53 acts as a transcriptional activator of MDM2 gene expression, so an elevation of MDM2 protein would imply functionality of the elevated p53. As can be seen in B, the level of MDM2 was indeed increased in cells in which NS was knocked down, in support of the hypothesis that the p53 induced by NS knockdown is functional. We also investigated whether knockdown of p53 affects the level of NS and found that it does not (Supplemental Figure 2), indicating that NS acts as an upstream regulator of p53.
Figure 3. Knockdown of NS leads to elevation of p53 and MDM2. Control or NS siRNAs were transfected into U2OS cells. (A) NS and p53 were detected by immunostaining after 48 h. (B) NS and MDM2 were detected after 48 h. (C) Western blot performed 48 h after transfection (more ...)
A role of NS in cell cycle progression is indicated by the fact that its depletion in U2OS cells reduces S phase entry, that blastocysts or fibroblasts from NS +/− embryos display haploinsufficiency with respect to growth rate, and that NS −/− blastocysts fail to enter S phase (Tsai and McKay, 2002
; Beekman et al., 2006
; Zhu et al., 2006
). Based on our finding that depletion of NS up-regulates p53, which plays a key role in the surveillance of cell cycle progression, we hypothesized that the p53 pathway might be involved in cell cycle arrest in NS-deficient cells. We therefore investigated cell cycle progression in U2OS (p53 positive, Rb positive, and ARF negative) and Saos-2 cells (p53 negative, Rb negative, and ARF negative) as a function of NS and p53 expression levels. Compared with cells transfected with a control siRNA, knockdown of NS caused a significant decrease in the percentage of cells entering S phase in U2OS cells but not in Saos-2 cells (, A and B). These results thus suggest that NS depletion-induced G1 cell cycle arrest may require p53. To address this issue, p53 was knocked down in U2OS cells (Supplemental Figure 2). As expected, this did not affect the percentage of cells traversing S as these cells are already cycling at a high rate (C, left two columns, and D). NS knockdown again reduced cell cycle progression (D). However, when p53 was knocked down in addition to NS, the percentage of cycling cells returned to the same high level as seen in control cells (D), providing direct evidence that cell cycle arrest induced by depletion of NS is mediated by the p53 pathway.
Figure 4. Loss of p53 restores cell cycle progression in NS knocked down cells. (A) U2OS cells (p53 positive) and Saos-2 (p53 null) were transfected with control or NS siRNAs, respectively, and 10 μM BrdU was added 72 h later. After 24 h, the cells were (more ...)
We next asked whether NS might also control cell cycle through the Rb pathway. In contrast to our finding with p53, we found that NS depletion-induced G1 arrest was not rescued by Rb depletion (, A and B, and Supplemental Figure 3), thus indicating that NS does not operate via the Rb pathway, at least in U2OS cells.
Figure 5. Depletion of Rb does not restore cell cycle progression in NS knocked down cells. (A) Rb siRNA, NS siRNA, or NS + Rb siRNAs were transfected into U2OS cells. Seventy-two hours later, BrdU was added, and after another 24 h, NS and BrdU were detected. (B) (more ...)
p53 is a pivotal regulatory protein that evokes cell cycle arrest in response to numerous stress signals including hypoxia, nutrient depletion, heat shock, and DNA damage (Vogelstein et al., 2000
; Bensaad and Vousden, 2005
; Vousden, 2006
). Because these stimuli often trigger nucleolar disorganization and p53 activation, it has been suggested that the nucleolus plays a role in modulating the cell cycle's response to stress (Rubbi and Milner, 2003
; Horn and Vousden, 2004
; Raska et al., 2006
). This idea has received additional support from the finding that under such conditions the level of p53 can be coordinated by a variety of nucleolar proteins, including the ribosomal proteins S6, L5, L11, L23, and L26 as well as TIF-IA, nucleolin, Bop1, B23 (nucleophosmin), ARF, PML, and WRN (Blander et al., 1999
; Pestov et al., 2001
; Colombo et al., 2002
; Daniely et al., 2002
; Lohrum et al., 2003
; Bernardi et al., 2004
; Dai and Lu, 2004
; Jin et al., 2004
; Sulic et al., 2005
; Takagi et al., 2005
; Yuan et al., 2005
; Raska et al., 2006
). It is also of interest to recall that the nucleolus contains mitogenic growth factors such as fibroblast growth factor and angiogenin (Pederson, 1999
), an observation that now might be productively revisited given the connections that have been made among p53, Rb, and myc in the nucleolus (Raska et al., 2006
). In our experiments, NS depletion did not lead to nucleolar disruption as judged either by phase-contrast microscopy (see figures) or immunostaining for the nucleolar marker proteins UBF, fibrillarin, and B23 (data not shown). This suggests that NS depletion per se does not constitute a nucleolar stress signal, notwithstanding the fact that p53 is elevated in response to NS depletion as it is in other situations in which nucleolar effects are observed. This consideration supports the initial idea that NS and p53 operate primarily through a nucleoplasmic interaction (Tsai and McKay, 2002
). Nonetheless, it would now be all the more relevant to track the dynamics and molecular interactions of NS, p53 and related proteins within the nucleus of living cells.
NS −/− mice abort before blastula and NS +/− fibroblasts display reduced NS levels and slower growth, but normal levels of p53 (Zhu et al., 2006
). In NS +/− fibroblasts, it is possible that the haploinsufficiency of NS as regards optimal growth rate nevertheless does not result in a depletion of NS sufficient to evoke the stabilization of p53. Alternatively, NS may be operating in a p53-independent manner. The possibility that NS can function via a p53-independent pathway during embryogenesis is also indicated by finding that p53 loss in NS −/− blastocysts does not rescue embryonic lethality (Beekman et al., 2006
). Based on previous studies and the current investigation, it seems plausible at present that there are p53-dependent and -independent roles of NS in cell proliferation.