Abstract
TGF-β inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to inhibitory effects of TGF-β. The intracellular signaling mechanisms involved in differential effects of TGF-β during different stages are largely unknown. Using cell line models, we have shown that TGF-β inhibits proliferation in normal (RWPE-1) and prostate cancer (DU145) cells but does not have any effect on proliferation of prostate cancer (PC3) cells. We have investigated the role of Jun family proteins (c-Jun, JunB, and JunD) in TGF-β effects on cell proliferation. Jun family members were expressed at different levels and responded differentially to TGF-β treatment. TGF-β effects on JunD protein levels, but not mRNA levels, correlated with its effects on cell proliferation. TGF-β induced significant reduction in JunD protein in RWPE-1 and DU145 cells but not in PC3 cells. Selective knockdown of JunD expression using siRNA in DU145 and PC3 cells resulted in significant reduction in cell proliferation, and forced overexpression of JunD increased the proliferation rate. On the other hand, knockdown of c-Jun or JunB had little, if any, effect on cell proliferation; overexpression of c-Jun and JunB decreased the proliferation rate in DU145 cells. Further studies showed that down-regulation of JunD in response to TGF-β treatment is mediated via the proteasomal degradation pathway. In conclusion, we show that specific Jun family members exert differential effects on proliferation in prostate cancer cells in response to TGF-β, and inhibition of cell proliferation by TGF-β requires degradation of JunD protein.
Highlights
TGF- is a secreted cytokine that acts as a major anti-proliferative factor in the initial stages of prostate cancer, whereas in the advanced stages of prostate cancer, it acquires pro-oncogenic and pro-metastatic properties [1,2,3]
Effects of TGF-1 on Proliferation of Prostate Cell Lines—We have previously shown that TGF-1 exerts differential effects on proliferation of different prostate cancer cell lines [15, 64]
These results suggested that downregulation of JunD protein levels and/or up-regulation of c-Jun and JunB protein levels in DU145 cells may play a role in TGF-1 effects on inhibition of cell proliferation
Summary
Effects of TGF-1 on Proliferation of Prostate Cell Lines—We have previously shown that TGF-1 exerts differential effects on proliferation of different prostate cancer cell lines [15, 64]. As measured by [3H]thymidine incorporation, TGF-1 caused a significant dose-dependent inhibition of cell proliferation in RWPE1 and DU145 cells but not in PC3 and LNCaP cells. Using gene-specific primers to amplify mRNA encoding each member of this protein family, all members of the Jun family were detectable in all four prostate cell lines (Fig. 2A). At the protein level, TGF-1 exerted significant effects on the levels of JunD, c-Jun, and JunB in DU145 and/or PC3 cells in a time-dependent manner (Fig. 3, B and C). TGF-1 significantly up-regulated c-Jun in PC3 cells starting at 2 h (2.4 Ϯ 0.24-fold; p Ͻ 0.05), which stayed elevated for 24 h (1.8 Ϯ 0.46-fold, p Ͻ 0.05) but did not have any effect on c-Jun protein levels in DU145 cells. Cell cycle phase distributions of DU145 and PC3 cells after treatment with TGF-1 (5 ng/ml)
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