In our previous studies, we found that deletion of Pten
in murine bladder epithelium leads to an increase in p21 expression, and that the p21 mediates a significant decrease in cell proliferation [19
]. We therefore investigated whether p21 was similarly induced in human urothelial carcinoma cells by signaling through the PI3-kinase pathway. We selected two cell lines: UMUC-3 (PTEN negative) and UMUC-14 (PTEN positive)for our studies. These cell lines were originally derived from two independent urothelial carcinomas. The cells were serum starved for 24 hours in order to attenuate growth factor signaling. Then, we treated cells with two different growth factors that are known to activate PI3-kinase: epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) [33
]. When treated with EGF, p21 levels increased in both cell lines (Figure ). While UMUC-14 cells do express PTEN protein, the cell line is still clearly responsive to growth factor signaling. We also treated both cell lines with PDGF because if the p21 induction was dependent on PI3-kinase, then signaling through multiple growth factor receptors should induce p21. PDGF did induce p21 in both cell types, especially at the concentration of 100 ng/ml (Figure ).
Figure 1 Growth factors induce p21 and inhibit proliferation in bladder carcinoma cells. UMUC-3 and UMUC-14 human urothelial carcinoma cell lines were serum starved for 24 hours and then stimulated with growth factors. (A) Cells were lysed at intervals after addition (more ...)
It was important to see if the increased p21 in the cells affects cell proliferation, so we transfected the UMUC-3 cells with either control siRNA or p21 siRNA to knock down p21 expression. Two days after transfection, cells were washed in PBS and serum starved for 24 hours. Then EGF (10 ng/ml) was added to some of the wells for 24 hours, and an MTS assay was run to measure cell proliferation. As seen in Figure , EGF treatment actually significantly decreased the number of viable cells, and knock down of p21 prevented this effect of EGF (ANOVA; p = .002), showing that the induction of p21 in response to EGF results in reduced cell proliferation.
In order to verify whether this p21 induction was dependent on PI3-kinase and AKT signaling, cells were stimulated with EGF in the presence or absence of LY294002, a PI3-kinase inhibitor, or Akti-1/2, an AKT inhibitor (Figure ). Western blotting showed that p21 induction did not occur in the presence of AKT inhibitor, and there was very weak p21 induction in the presence of LY294002, indicating that p21 induction was in fact dependent on PI3-kinase and AKT activity. We confirmed that the inhibitors were effective at blocking signaling by testing for AKT phosphorylation at the critical activation site, serine 473 (Figure ).
Figure 2 EGF induces p21 in a PI3-kinase and AKT dependent manner. (A) UMUC-3 cells were pre-treated for one hour with PI3-kinase inhibitor LY294002 (30 μM) or AKT inhibitor AKTi-1/2 (10 μM) and then stimulated with EGF over a 180 minute time course. (more ...)
AKT is known to cause downstream mTOR activation [1
], and since mTOR increases translation of many proteins, we tested if mTOR was responsible for the increased p21 levels in the urothelial cell lines stimulated with growth factors. We pre-treated some of the UMUC-3 cells for one hour with 12.5 nM rapamycin, an mTOR inhibitor, and then stimulated the cells with EGF. Immunoblotting for phosphorylated ribosomal S6 protein, a critical downstream substrate that mediates mTOR's effects on protein synthesis, verified that the rapamycin suppressed mTOR activity (Figure ). We found that p21 was still induced by EGF in the presence of rapamycin in the UMUC-14 cells and possibly also in the UMUC-3 cells (Figure and ), although to a reduced extent. This indicated that the p21 induction caused by the PI3-kinase pathway is partially mediated by mTOR.
Figure 3 Induction of p21 is partially dependent on mTOR. (A) UMUC-3 cells were serum starved for 24 hours and then stimulated with 10 ng/ml EGF in the presence or absence of 1 hour pretreatment with 12.5 nM rapamycin. S6 phosphorylation at serines 235/236 was (more ...)
Since the PI3-kinase/AKT signaling pathway is known to inhibit GSK-3β activity [16
], and GSK-3β has been reported to cause the degradation of other proteins such as β catenin [17
] and SMAD1 [35
], we decided to investigate its involvement in p21 regulation in bladder cells. First, we examined if PI3-kinase/AKT signaling results in GSK-3 inhibition in UMUC-3 and UMUC-14 cells. While most studies have examined the role of GSK-3β in this pathway, the function of the GSK-3α isoform is less clear, so we investigated the status of both isoforms. AKT has been shown to directly phosphorylate GSK-3α and β at serines 9 and 21 [16
], respectively, resulting in an inhibition in GSK-3 activity. Treatment of both UMUC-3 and UMUC-14 cells with EGF resulted in increased phosphorylation of GSK-3α and β at serine 9 and 21(Figure and ). In both cell types, the phosphorylation of GSK-3α and β was PI3-kinase and AKT dependent. This result confirmed that GSK-3 is inhibited by PI3-kinase/AKT in human bladder cells and led to our subsequent investigation to determine if the GSK-3 inhibition affects p21 levels.
Figure 4 EGF stimulates inhibitory phosphorylation of GSK-3α and β in a PI3-kinase and AKT dependent fashion. Phosphorylation of GSK-3α and β at serines 9 and 21 respectively was tested by immunoblotting of (A) UMUC-3 and UMUC-14 (more ...)
In order to ascertain if GSK-3 inhibition causes an increase in p21 in bladder cells, UMUC-3 and UMUC-14 cells were serum starved for 24 hours to suppress PI3-kinase signaling and thereby promote GSK-3 activity. The cells were then treated with the GSK-3 inhibitor SB216763 over five hours. Levels of p21 were initially low after serum starvation, but increased in response to SB216763 treatment (Figure ), indicating that GSK-3 activity negatively regulates p21 expression. UMUC-3 cells contain a mutated p53, so the p21 induction in response to SB216763 is not p53-dependent in these cells.
Figure 5 GSK-3 regulates p21 expression, and inhibition of GSK-3 reduces cell viabiliity in bladder carcinoma cell lines. (A) UMUC-3 cells were serum-starved, then treated with GSK-3 inhibitor SB216763 (2.5 μM) over a time course and lysates were immunoblotted (more ...)
In order to more directly test the effects of GSK-3α and β individually on p21, we transiently transfected GSK-3 expression vectors into UMUC-14 cells for 48 hours. The exogenous GSK-3β has a slightly higher molecular weight than does endogenous GSK-3β due to the presence of a C-terminal HA tag (Figure ). When p21 levels were tested in the presence of 10% fetal calf serum, no effects of GSK-3 expression on p21 were evident. However, when transfected cells were serum starved for 24 hours in order to minimize GSK-3 inhibition by AKT, exogenous expression of GSK-3β resulted in a massive decrease in p21 levels (Figure ). GSK-3α had a lesser effect but also appeared to diminish p21 levels in experimental replicates. This indicates that GSK-3β downregulates p21, but that activity is inhibited in the presence of serum and an active PI3-kinase pathway. Interestingly, β catenin levels were unaffected by exogenous GSK-3 expression (Figure ), which suggests that β catenin did not play a role in the induction of p21.
The ability of the GSK-3 inhibitor SB216763 to induce p21 led us to speculate that SB216763 should inhibit urothelial carcinoma cell proliferation. We further hypothesized that the combination of SB216763 with a PI3-kinase inhibitor such as LY294002 should more effectively inhibit cell proliferation and/or induce cytotoxicity than LY294002 alone. In order to test these hypotheses, we treated UMUC-3 and UMUC-14 cells with various concentrations of SB216763 in the presence or absence of LY294002. After 48 hours (for UMUC-14 cells) or 72 hours (for UMUC-3 cells), we performed an MTS assay to measure cell viability (Figure ). The UMUC-3 cells had a longer drug treatment because they showed relatively little cytotoxicity at 48 hours. After 72 hours, the UMUC-3 cells did not show significant decreases in viability due to treatment with LY294002 alone or SB216763 alone, even at the highest concentrations used in this assay, although there was a trend toward decreased viability for both drugs. A combination of the two drugs was most effective for cell cytotoxicity; 2 μM LY294002 plus SB216763 at any tested concentration caused significant decreases in UMUC-3 cell viability compared to 2 μM LY294002 alone (Oneway ANOVA; p < .0001). While treatment of the UMUC-14 cells with 2 μM LY294002 alone had no significant effect on cell viability compared to untreated cells, there was a dose-dependent cytotoxicity response to SB216763 alone (Oneway ANOVA; p < .0001), and there was a significant decrease in cell viability at 10 μM SB216763 (Tukey-Kramer HSD; p = .0004) and at 40 μM SB216763 (Tukey-Kramer HSD; p < .0001) compared to the untreated control cells. A combination of the two drugs was once again more effective for cytotoxicity; when 2 μM LY294002 was added to the UMUC-14 cells together with 10 μM SB216763, there was a significant further decrease in cell viability compared to LY294002 alone (Oneway ANOVA; p < .0001; Tukey-Kramer HSD; p = .0007). In addition, there was significantly more cytotoxicity at 40 μM SB216763 when comparing cells in the presence of LY294002 compared to the cells that received no LY294004 (Student's t-test; p = .02). This suggests that increased inhibition of cell viability can be attained with a combination of the LY294002 PI3-kinase inhibitor and the SB216763 GSK-3 inhibitor.
GSK-3β is known to directly phosphorylate substrates leading to ubiquitylation and subsequent proteasome dependent degradation of those proteins. It is unclear, however, if p21 is targeted for proteasomal destruction in these cells. If GSK-3 was regulating p21 in this manner in these cell lines, then we should expect to see evidence that the proteasome regulates p21 levels. We found that serum starvation to activate GSK-3, followed by proteasome inhibition using MG-132 leads to an increase in p21 in both UMUC-3 and UMUC-14 cells (Figure ), suggesting that p21 levels are regulated by proteasomal degradation.
Figure 6 Proteasome inhibition causes an increase in p21. (A) UMUC-3 cells were serum starved for 24 hours and then treated with 1 μM and 10 μM MG-132 over a 180 minute time course. Cells were lysed and immunoblotted for p21 and tubulin. (B) Quantitation (more ...)
Given the evidence that GSK-3β inhibition causes elevated p21 levels in PI3-kinase stimulated human urothelial cells, we examined if GSK-3β is also inhibited in mice that are conditionally deficient for Pten
)in bladder urothelium. These mice were previously shown to have elevated levels of nuclear p21 in urothelium [19
]. Immunohistochemical staining of Pten
deficient mouse bladders and their wild-type littermates showed not only higher levels of p21 positive cells in the bladder, but also greatly increased cytoplasmic staining of phospho-GSK3α and β at serines 9 and 21 (Figure ). This finding that GSK-3 exhibits large levels of inhibitory phosphorylation at sites that are known targets for AKT is consistent with the idea that GSK-3 inhibition contributes to elevated p21 levels in the Pten
-deficient mouse bladder.
Figure 7 Pten deletion causes an increase in cytoplasmic phosphorylated GSK-3 α/β ser9/21 in murine bladder epithelium. Sections of bladder from (A) three day old wild-type Ptenloxp/loxp mice and (B) Pten deficient Fabpl-Cre; Ptenloxp/loxp mice (more ...)