Basic—Alimentary TractProlyl Hydroxylase-3 Is Down-regulated in Colorectal Cancer Cells and Inhibits IKKβ Independent of Hydroxylase Activity
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Cell Culture and Reagents
Human colon cancer HCT116 and SW480 cells and human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% serum. PHD3 antibody was from Novus Biologicals (Littleton, CO). IKKβ and phosphorylated IKKβ antibodies were from Cell Signaling (Beverly, MA). HA, myc, and Hsp90 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). β-Actin antibody and cycloheximide were from Sigma (St. Louis, MO). Recombinant human TNF-α was from R&D Systems
Expression of PHD3 Is Decreased in Human Colorectal Cancer
Sixty pairs of human colorectal cancer samples, including primary colorectal carcinoma tissues and paired normal colorectal tissues, were tested. Decreased expression of PHD3 occurs in 37 of 60 (61.7%) of colorectal tumors compared with the paired normal colorectal tissues (Figure 1A). Univariate analysis indicates that messenger RNA (mRNA) levels of PHD3 are significantly different between paired normal colorectal tissues and tumors (P < .001). The decreased expression of PHD3 is associated
Discussion
Hydroxylase activity of PHD is modulated by oxygen availability. The hydroxylase activity of PHD is inhibited when under hypoxia or in the presence of DMOG. Our results show that both PHD3 and the hydroxylase-defective PHD3(H196A) inhibit hypoxia- and DMOG-induced activation of NF-κB (Figure 3C). Moreover, PHD3(H196A) also inhibits TNF-α-induced phosphorylation of IKKβ and activation of NF-κB (Figure 3). These results suggest that PHD3 inhibits IKKβ/NF-κB signaling independent of its
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Conflicts of interest The authors disclose no conflicts.
Funding Supported by National Natural Science Foundation of China (30870524), Chinese Academy of Sciences (KSCX2-YW-R-114), Chinese Ministry of Science and Technology (2007CB947100), and Shanghai Institute for Biological Sciences (2008KIP402).