Caveolin‐1 facilitates cyclooxygenase‐2 protein degradation

Abstract

Cyclooxygenase-2 (COX-2) plays major roles in diverse physiological and pathological processes such as inflammation and tumorigenesis. Transcriptional control of COX-2 has been extensively investigated and characterized, but its post-translational control is less clear. Here, we report a novel mechanism by which COX-2 is degraded. Protein levels of caveolin-1 (Cav-1) and COX-2 showed an inverse relation in colon cancer cell lines. COX-2 proteins in lung and colon tissues were higher in Cav-1 null mice than in wild-type mice. RNAi knockdown of Cav-1 increased COX-2 protein level and decreased ubiquitinated COX-2 accumulation. In addition, deletion of the carboxy (C)-terminus of COX-2, which contains a unique 19-amino acid segment compared with COX-1, resulted in reduced Cav-1 binding and attenuated COX-2 degradation. COX-1 and green fluorescence protein containing the C-terminus of COX-2 resulted in enhanced degradation. Our findings suggest that Cav-1 binds COX-2 in endoplasmic reticulum (ER) and carries it for degradation via ER associated degradation. The C-terminal region of COX-2 is required for Cav-1 binding and degradation. These results indicate a novel function of Cav-1 in controlling COX-2 expression, which may regulate physiological functions and have tumor suppression effects.

Caveolin-1 Interacts with Derlin-1 and Promotes Ubiquitination and Degradation of Cyclooxygenase-2 via Collaboration with p97 Complex

“…Construction of Plasmids-Plasmid vectors of pCMV14-3-FLAG and pXJN-HA and expression vectors encoding COX-1, COX-1-myc, COX-1 ϩ32aa -myc, COX-2, COX-2-FLAG, COX-2 ⌬32aa -myc, and Cav-1-HA were constructed as described (25). COX-2 N594A mutation was generated by PCR amplification with primers (GCTCA TTAAA ACAGT CACCA TCGCT GCAAG TTCTT CC and GGAAG AACTT GCAGC GATGG TGACT GTTTT AATGA GC).…”

“…In Vivo Ubiquitination Assay-HEK293 or H1299 cells were transfected to overexpress COX-2 and His-tagged ubiquitin plus Derlin-1 or p97 siRNA for 24 h and then treated with MG-132 (20 M) for another 8 h. The ubiquitinated proteins were bound by nickel-agarose beads for protein ubiquitination assay as described (25).…”

“…unique sequence containing an N-glycosylation site at Asn-594 reported to be crucial for COX-2 degradation via ERAD (25,32,37). To determine whether this region is involved in Derlin-1-mediated COX-2 degradation, we co-expressed Derlin-1 with FLAG-tagged COX-2 (COX-2-FLAG) or FLAG-tagged COX-2 with C-terminal 32 aa deletion (COX-2 ⌬32aa -FLAG), with Asn-594 and the adjacent 18 aa deleted, in H1299 cells.…”

“…facilitate cyclooxygenase 2 (COX-2) degradation in vivo and in vitro (25). These studies suggest that Cav-1 plays important roles in various cellular and physiological functions.…”

“…Moreover, expression of COX-2 is controlled by both transcription and post-translational regulation (31). Although proteasome-mediated COX-2 degradation through ERAD has been suggested for some time (25,32), the cellular machinery remains mostly unknown.…”