Prolyl Hydroxylase-3 Is Down-regulated in Colorectal Cancer Cells and Inhibits IKKβ Independent of Hydroxylase Activity  Jing Xue, Xuebing Li, Shi Jiao, Ye Wei, Guohao Wu, Jing Fang  Gastroenterology  Volume 138, Issue 2, Pages 606-615 (February 2010) DOI: 10.1053/j.gastro.2009.09.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Expression of PHD3 is decreased in human colorectal cancer. (A) Determination of PHD3 mRNA by real-time PCR. ΔCt(N): Ct value of GAPDH was subtracted from Ct value of PHD3 of normal tissue. ΔCt(T): Ct value of GAPDH was subtracted from that of PHD3 of colorectal tumor. Bar value (ΔCt(N) − ΔCt(T)) represents the difference of PHD3 mRNA between normal tissue and paired tumor. Bar value = −1 indicates that PHD3 mRNA of tumor is 2−1−fold of that of paired normal tissue. Bar value = 1 indicates that PHD3 mRNA of tumor is 21−fold of that of paired normal tissue. Bar value ≤ −1 indicates that the expression of PHD3 is decreased in tumors. Bar value ≥ 1 indicates that the expression of PHD3 is increased in tumors. (B) Determination of PHD3 protein. The tissues frozen in liquid nitrogen were crushed and transferred to a tube. Cold lysis buffer was added, and the tissues were incubated on ice for 0.5 hours. Specimens were centrifuged, and the supernatants were collected. (C) Immunohistochemical staining of tissues with PHD3 antibody. Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 PHD3 inhibits TNF-α-induced NF-κB activity through IKKβ. (A) Overexpression of PHD3 attenuated TNF-α-induced NF-κB activity. HCT116, SW480, and 293T cells were transfected with NF-κB-Luc, β-galactosidase, and myc-PHD3 plasmids. In 24 hours, the cells were treated with TNF-α (10 ng/mL) for 6 hours. Luciferase activity was determined, and relative NF-κB activity was calculated. *P < .05. (B) Knockdown of PHD3 activated NF-κB. HCT116 cells were transfected with NF-κB-Luc, β-galactosidase, and PHD3 siRNA oligoes. In 48 hours, the cells were treated with TNF-α (10 ng/mL) for 6 hours. Next, the cells were harvested for luciferase assay. *P < .05; #P < .05 vs control siRNA (open bar). (C) PHD3 inhibits NF-κB through IKKβ. 293T cells were transfected with NF-κB-Luc, β-galactosidase, myc-PHD3, or IKKβ (or p65) plasmids as indicated. In 24 hours, the cells were harvested for luciferase assay. *P < .05. (D) Overexpression of PHD3 inhibited TNF-α-induced phosphorylation of IKKβ. 293T cells were transfected with myc-PHD3 plasmid. In 24 hours, the cells were stimulated with TNF-α (10 ng/mL) for 10 minutes. (E) Knockdown of PHD3 enhanced phosphorylation of IKKβ. 293T cells were transfected with PHD3 siRNA oligoes. In 48 hours, the cells were stimulated with TNF-α (10 ng/mL) for 10 minutes. (F) PHD3 did not influence expression of IKKβ. 293T cells were transfected with PHD3 plasmid or PHD3 siRNA oligoes. In 48 hours, the cells were harvested for immunoblotting. Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 PHD3 inhibits phosphorylation of IKKβ independent of hydroxylase activity. (A) PHD3(H196A) inhibited TNF-α-induced NF-κB activity. HCT116, SW480, and 293T cells were transfected with NF-κB-luc, β-galactosidase, and PHD3(H196A) plasmids. In 24 hours, the cells were treated with TNF-α (10 ng/mL) for 8 hours. *P < .05 vs TNF-α alone. (B) PHD3(H196A) inhibited phosphorylation of IKKβ. 293T cells were transfected with PHD3(H196A). In 24 hours, the cells were stimulated with TNF-α (10 ng/mL) for 10 minutes. (C) PHD3 and PHD3(H196A) inhibited both hypoxia- and DMOG-induced NF-κB activities. SW480 and 293T cells were transfected with NF-κB-luc, β-galactosidase, and PHD3 [or PHD3(H196A)] plasmids. The transfected cells were incubated under hypoxia (1% O2) or 1 mmol/L of DMOG for 24 hours. *P < .05 vs Con. mutPHD3, PHD3(H196A). Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 PHD3 impairs the interaction between IKKβ and Hsp90. (A) PHD3 impairs the interaction between Hsp90 and IKKβ. 293T cells were transfected with myc-PHD3. In 24 hours, the cells were harvested. (B) PHD3(H196A) inhibits the IKKβ-Hsp90 interaction. 293T cells were transfected with myc-PHD3(H196A). In 24 hours, the cells were harvested. (C) Knockdown of PHD3 increased the Hsp90-IKKβ interaction. 293T cells were transfected with PHD3 siRNA oligoes. In 48 hours, the cells were harvested for immunoprecipitation. (D) PHD3 does not influence the interaction between IKKβ and IKKγ. 293T cells were transfected with HA-IKKγ and myc-PHD3 plasmids. In 24 hours, the cells were harvested for immunoprecipitation. Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 PHD3 associates with IKKβ and competes against Hsp90 for IKKβ binding. (A) The endogenous IKKβ and PHD3 coimmunoprecipitated. 293T cell lysates were used for immunoprecipitation. (B) The exogenous IKKβ and PHD3 coimmunoprecipitated. 293T cells were transfected with HA-IKKβ and myc-PHD3 plasmids. In 24 hours, the cells were harvested for immunoprecipitation. (C) PHD3 binds to IKKβ(1–307) and IKKβ(308–580). 293T cells were transfected with plasmids as indicated. In 24 hours, the cells were harvested. (D) A direct interaction between PHD3 and IKKβ. Equal amounts of bacterial lysates (containing His-PHD3) were incubated with the glutathione-Sepharose beads that had already captured GST, GST-IKKβ(1–307), GST-IKKβ(308–580), or GST-IKKβ(581–756). The beads were washed, and His-PHD3 retained on beads was determined by immunoblotting. (E) Hsp90 binds to IKKβ(1–307) and IKKβ(308–580). GST-IKKβ was captured on glutathione-Sepharose beads. The beads were incubated with 293T cell lysates, allowing the interaction between GST-IKKβ and Hsp90 from 293T cells. The beads were then washed and incubated in SDS-PAGE loading buffer. The resolved proteins were detected by immunoblotting. (F) PHD3 and Hsp90 compete for binding to IKKβ. 293T cells grown in 6-well plates were transfected with 0, 1, and 3 μg of myc-PHD3 plasmid. In 24 hours posttransfection, the cells were harvested, and lysates were prepared. Equal amounts of the lysates were incubated with glutathione-Sepharose beads containing GST-IKKβ(308–580) at 4°C for 3 hours. The beads were washed and incubated in SDS-PAGE loading buffer. The resolved proteins were analyzed by immunoblotting. Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Knockdown of PHD3 enhances cytokine resistance and tumorigenesis. (A) Knockdown of PHD3 leads to TNF-α resistance. HCT116 cells were transfected as indicated. The next day, the transfected cells were plated on 96-well plates. Cell viability was determined as described under the Materials and Methods section. *P < .05 vs control. (B) Knockdown of PHD3 enhanced tumor growth. HCT116 cells were infected with FG12-PHD3-siRNA or control virus. The infected cells were transplanted to nude mice as described under the Materials and Methods section. Measurement of tumor volume was started from day 10. Data are presented as mean ± SEM. #P < .01 vs control; *P < .05 vs control. Gastroenterology 2010 138, 606-615DOI: (10.1053/j.gastro.2009.09.049) Copyright © 2010 AGA Institute Terms and Conditions