717. VCP Short Hairpin RNA Rescues ΔF508- CFTR and Suppresses IL8 Levels: Therapeutic Implications in Cystic Fibrosis

Abstract

Top of pageAbstract Background: Endoplasmic reticulum associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, |[Delta]|F508-CFTR, is destroyed by ERAD to cause cystic fibrosis (CF). Valosin containing protein (VCP) is an AAA ATPase that participates in retrograde translocation of misfolded proteins from the ER and degradation of polyubiquitinated protein by the proteasomes. We hypothesize that selective inhibition of VCP not only rescues |[Delta]|F508-CFTR but also suppresses IL8 cytokine levels, the major inflammatory cytokine in CF airways. Methods: The VCP shRNA was PCR amplified from oligo/template containing the VCP hairpin using universal primers which contain XhoI (5' prime) and EcoRI (3' prime) flank. The PCR fragment was then cloned into the hairpin cloning site of pSHAG-MAGIC2/pSM2 vector (Open Biosystems). IB3-1 (|[Delta]|F508/W1282X) bronchial epithelial cells were transiently transfected with VCP and gp78 shRNA/siRNA constructs for selective inhibition of ERAD. CFTR levels and chloride efflux were measured by metabolic labeling and MQAE fluorescence assay, respectively. Proteasome inhibition in IB3-1 cells was carried out using PS-341 (Bortezomib/Velcade) for 6hrs. The IL8 cytokine levels were measured in IL1-|[beta]| induced IB3-1 cells after ERAD or proteasome inhibition. Findings: We show here that p97/VCP and gp78 form complexes with CFTR during translocation from the ER for degradation by the cytosolic proteasome. Interference in the VCP/CFTR complex promotes accumulation of immature CFTR in the ER and partial rescue of functional chloride channels to the cell surface. Moreover, under these conditions, the cytokine whose expression is regulated by the proteasome, IL8, is reduced. Inhibition of the proteasome with bortezomib (PS-341/Velcade) also rescues CFTR and suppresses IL8 levels, but with less efficiency. Interpretation: We propose that selective inhibition of ERAD by VCP short hairpin RNA is superior to gp78 siRNA or proteasome inhibition in the correction of the |[Delta]|F508 CFTR trafficking defect.