The JAK3 inhibitor CP-690,550 blocks the phosphorylation of STAT5 by IL-2/JAK3 but not by IL-3/JAK2
In murine IL-3-dependent myeloid progenitor 32D cells stably expressing the human IL-2 receptor β chain (32D/IL-2Rβ), JAK2 and JAK3 undergo transient phosphorylation in response to IL-3 and IL-2, respectively (). Subsequently STAT5 becomes activated by JAK2 or JAK3, suggesting that measuring STAT5 activity induced by IL-2 or IL-3 in the cells can faithfully monitor the activity of these kinases. To assess whether this cell line can be used as a cellular model for the identification of low-molecular-weight inhibitors specific for JAK3 over JAK2, we examined IL-2- or IL-3-induced STAT5 activity in the cells treated with the selective JAK3 inhibitor CP-690,550, or the pan-JAK inhibitor Curcumin. The cells were incubated with various concentrations of CP-690,550 for 16 hours in the absence of WEHI-3 cell-conditioned medium and then stimulated by recombinant IL-2 or IL-3. While phospho-STAT5 was barely detectable in the cells without cytokine stimulation, as assessed by its reactivity to an antibody specific for Tyr694, its levels were increased in response to IL-2 or IL-3 treatment (). CP-690,550 efficiently blocked the phosphorylation of STAT5 by IL-2, which is JAK3-dependent, in a dose-dependent manner (). In fact, phospho-STAT5 levels were decreased by more than 50% at 25 nmol/L of CP-690,550, as compared with those of control, and were undetectable at 50 nmol/L. As expected, this reagent potently inhibited IL-2-induced JAK3 phosphorylation (). This demonstrates that CP-690,550 inhibited IL-2-induced activation of JAK3, which subsequently blocked the activation of STAT5. Importantly, CP-690,550 failed to affect the levels of phospho-STAT5 following treatment of cells with IL-3, which activates JAK2, at the concentrations up to 100 nmol/L (). Consistent with this result, treatment of 32D/IL-2Rβ cells with CP-690,550 did not show any inhibitory effect on the levels of phospho-JAK2 following IL-3 treatment at the concentrations up to 100 nmol/L (). We next examined the effect of Curcumin, which inhibits the activation of all JAK kinases by cytokines, on IL-2-induced phospho-JAK3/STAT5 and IL-3-induced phospho-JAK2/STAT5 activity. As expected, treatment of 32D/IL-2Rβ cells with this pan-JAK inhibitor resulted in a decrease in both IL-2-induced JAK3/STAT5 and IL-3-induced JAK2/STAT5 phosphorylation ().
Fig. 1 CP-690,550 inhibits the phosphorylation of STAT5 by IL-2/JAK3 but not by IL-3/JAK2. Effect of the selective JAK3 inhibitor CP-690,550 on JAK3/STAT5 phosphorylation induced by IL-2 (A) and on JAK2/STAT5 phosphorylation induced by IL-3 (B) in 32D/IL-2Rβ (more ...)
To confirm the selective inhibitory effect of CP-690,550 on JAK3, we next examined the cellular distribution of phospho-STAT5 in the 32D/IL-2Rβ cells treated with CP-690,550 and either IL-2 or IL-3. In resting cells, STAT5 resides mainly in the cytoplasm. However, in response to cytokine or growth factor, it becomes phosphorylated on Tyr694 and rapidly translocates to the nucleus, where activated STAT5 dimers alter the expression of numerous target genes. Since CP-690,550 selectively blocked STAT5 phosphorylation induced by IL-2 but not by IL-3 (), we hypothesized that treatment of 32D/IL-2Rβ cells with CP-690,550 would interfere with the nuclear distribution of phospho-STAT5 only in cells treated with IL-2. In untreated 32D/IL-2Rβ cells, only basal levels of phosho-STAT5 were observed and signal was confined to the cytoplasm (). By contrast, significant accumulation of phosphorylated STAT5 was observed in the nuclei of the cells treated with either IL-2 or IL-3 (). Importantly, the nuclear accumulation of phospho-STAT5 in cells treated with IL-2 was almost completely abolished by co-administration of CP-690,550 (100 nmol/L) (). By contrast, this compound failed to affect the distribution of nuclear phospho-STAT5 in cells treated with IL-3 (). These data are consistent with the findings that CP-690,550 selectively decreases STAT5 activation by JAK3 but not by JAK2 (). Taken together, these results suggest that 32D/IL-2Rβ cells can serve as a useful tool for the evaluation and discovery of small molecule inhibitors preferentially inhibit JAK3 and not JAK2.
Fig. 2 CP-690,550 disrupts the nuclear translocation of phospho-STAT5 induced by IL-2 but not by IL-3 in 32D/IL-2Rβ cells. The cells were deprived of WEHI-3 cell-conditioned medium for 6 hours. The cells were then seeded into 48-well plates and incubated (more ...)
Development of a cell line 32D/IL-2Rβ/6×STAT5 stably expressing a STAT5 reporter gene
The identification of specific JAK3 antagonists is essential both for basic research and for developing therapeutic strategies for the treatment of various human disorders, including immune-related diseases and hematopoietic neoplasms. Notably, many of the newly developed JAK3 inhibitors that have significant off-target effects on JAK2 were identified by structure-based inhibitor designs or in vitro biochemical JAK3 enzymatic assays. Small molecules identified from these cell-free assays may not elicit the same effects in cellular assays, due to issues of membrane permeability, off-target effects, and cytotoxicity. By contrast, results obtained from small molecule screening in cell-based assays reflect the physiological effect of a compound in the context of a normal cellular milieu. In particular, luciferase reporter gene assays provide simple, quantitative, sensitive, and cost-effective means for monitoring the activity of target molecules in a cellular context. To develop a tool that affords quantitative measurement of STAT5 activity in 32D/IL-2Rβ cells, we established a 32D/IL-2Rβ cell line stably expressing a STAT5 reporter. We placed six tandem repeats of the STAT5 response element of β-casein gene promoter upstream of a TATA-box minimal promoter and a firefly luciferase gene. 32D/IL-2Rβ cells stably expressing this reporter are hereafter referred to as 32D/IL-2Rβ/6×STAT5 cells (see also Materials and Methods). We first assessed if this reporter gene is responsive to JAK/STAT signaling by treating these cells with IL-2 or IL-3. Specifically, 32D/IL-2Rβ/6×STAT5 cells were deprived of WEHI-3 cell-conditioned medium for 6 hours and then treated with IL-2 or IL-3 at various concentrations for an additional 16 hours. We observed that both IL-2 and IL-3 treatment led to an increase in STAT5 reporter activity in a dose-dependent manner and that activity of the reporter was saturated at higher cytokine concentrations (). In fact, treatment of 32D/IL-2Rβ/6×STAT5 cells with 1 μg/mL of IL-2 or 5 ng/mL of IL-3 resulted in almost 17- or 50-fold increase in the reporter activity, respectively, compared with those of controls. We next determined the time course of activation of the reporter after treatment of 32D/IL-2Rβ/6×STAT5 cells with IL-2 or IL-3. Both cytokines increased the reporter activity in a time-dependent manner, with approximately 40- and 100-fold increase in maximum at 24 hours for IL-2 and IL-3, respectively (). These observations indicate that the STAT5 reporter activity in 32D/IL-2Rβ/6×STAT5 cells is robust and sensitive to cytokine stimulation.
Fig. 3 Establishment of a cell line 32D/IL-2Rβ/6×STAT5 that stably expresses a STAT5 reporter gene. (A-D) Dose and time effects of IL-2 or IL-3 on STAT5 reporter activity in 32D/IL-2Rβ/6×STAT5 cells. (A, B) The cells were deprived (more ...)
JAK3 inhibitors selectively block the activation of the STAT5 reporter by IL-2/JAK3 in 32D/IL-2Rβ/6×STAT5 cells in a high-throughput format
We next examined whether JAK inhibitors can block the activation of the STAT5 reporter by IL-2 or IL-3 in 32D/IL-2Rβ/6×STAT5 cells. The cells were incubated in the absence of WEHI-3 cell-conditioned medium for 6 hours. The cells were then re-suspended without WEHI-3 cell-conditioned medium (4×105
cells/mL), mixed with IL-2 (20 ng/mL) or IL-3 (1 ng/mL) to activate JAK3 and JAK2, respectively, seeded into 96-well plates (~2.2×104
cells/well) containing varying concentrations of JAK inhibitors, and incubated for 16 hours. We showed that IL-2 or IL-3 enhances the reporter activity in a time-dependent manner (), but we optimized the assay condition by incubating the reporter cells for 16 hours in the presence of a compound to obtain relatively robust reporter activity and eliminate non-specific effects of the compound. Consistent with our earlier results, CP-690,550 selectively decreased in a dose-dependent manner activation of the STAT5 reporter in cells treated with IL-2 but not in those treated with IL-3 (). In fact, 25 nmol/L of CP-690,550 reduced the reporter activity by almost 70%. By contrast, the same compound failed to inhibit IL-3-induced reporter activity, which is JAK2-dependent, at the concentrations up to 100 nmol/L. We next generated the full dose response curve of CP-690.550 in IL-2- and IL-3-induced reporter activity. The IC50
value of CP-690.550 in IL-2- and IL-3-induced reporter activity was 18.4 nmol/L and 562.2 nmol/L, respectively, indicating the selectivity of CP-690.550 for JAK3 over JAK2 (). Interestingly, these results are consistent with the report that CP-690,550 shows 20-fold greater selectivity for JAK3 over JAK2 in ex vivo
JAK3 kinase assay,11
suggesting that our cell-based reporter assay is as efficacious as in vitro
enzymatic assays. To further demonstrate that JAK inhibitors can inhibit the activation of the STAT5 reporter by IL-2 or IL-3 in 32D/IL-2Rβ/6×STAT5 cells, we tested the effect of JAK3 inhibitor VI, which can block JAK3 kinase activity at nanomolar levels and exhibits much higher selectivity for JAK3 than JAK2,18
on IL-2-induced JAK3 and IL-3-induced JAK2 activity. As expected, while 1 μmol/L JAK3 inhibitor VI reduced the IL-2-induced reporter activity by almost 50%, the same compound showed no inhibitory effect on IL-3-induced JAK2 at the concentrations up to 10 μmol/L (). We next generated the full dose response curve of JAK3 inhibitor VI in IL-2- and IL-3-induced reporter activity. The IC50
value of JAK3 inhibitor VI in IL-2- and IL-3-induced reporter activity was 0.9 μmol/L and 40 μmol/L, respectively (data not shown). This result is comparable to the reported observation that JAK3 inhibitor VI exhibits ~16-fold greater selectivity for JAK3 over JAK2. On the other hand, the pan-JAK inhibitor Curcumin at the concentrations between 15 and 20 μmol/L reduced both IL-2- and IL-3-induced STAT5 reporter activity by 50% (). We found that other pan-JAK inhibitors, such as AG490 and WP1066, also non-selectively inhibited both IL-2- and IL-3-induced STAT5 reporter activity (data not shown). These data suggest that luciferase activity in the reporter cells can reflect JAK2 or JAK3 activity induced by IL-3 or IL-2, and that JAK3 inhibitors show high specificity for IL-2-induced JAK3 over IL-3-induced JAK2 in our reporter assay.
Fig. 4 CP-690,550 selectively inhibits STAT5 reporter activity induced by IL-2 but not IL-3 in 32D/IL-2Rβ/6×STAT5 cells. (A, B) Effect of CP-690,550 on STAT5 reporter activity induced by IL-2 or IL-3. CP-690,550 decreased IL-2-induced STAT5 reporter (more ...)
We next determined Z′ values to assess whether our assay is suitable for high-throughput screening. The Z′ value for the assay performed in 96-well plates was 0.738±0.03 (Supplementary Fig. 1A
). In addition, we found no significant day-to-day variation in Z′ values over a one week period (Z′=0.715±0.031) (Supplementary Fig. 1B
). Furthermore, we could not detect any significant edge effects in our assay as the entire incubation time after the treatment of compound is a mere 16 hours (data not shown). Taken together, these results demonstrate that our assay using 32D/IL-2Rβ/6×STAT5 cell line is very likely to be suitable and reliable for use in a full-scale, high-throughput screen of chemical libraries to identify new JAK3 inhibitors.
It is worth noting that 32D/IL-2Rβ/6×STAT5 cells were designed to test the selectivity of a compound for JAK3 over JAK2. Therefore, compounds identified through this reporter assay will be needed to examine their effects on JAK1 and TYK2, by assessing, for example, if a lead compound can inhibit the phosphorylation levels of STAT1 in U266 myeloma cells following treatment of interferon-α (IFN-α), which activates JAK1 and TYK2. In sum, our findings suggest that the 32D/IL-2Rβ/6×STAT5 cell line represents a powerful cellular tool for large-scale primary chemical screens to identify specific low-molecular-weight inhibitors of JAK3.