Cytoplasmic-nuclear shuttling of the urokinase mRNA binding protein regulates message stability.
Treatment of small airway epithelial (SAEC) cells or lung epithelial (Beas2B) cells with TNF-alpha or PMA induces urokinase-type plasminogen activator (uPA) expression. Treatment of these cells with TNF-alpha, PMA or cycloheximide but not TGF-beta increased steady-state expression of uPAmRNA. TNF-alpha, PMA or cycloheximide caused 8-10 fold extensions of the uPAmRNA half-life in SAEC or Beas2B cells treated with DRB, a transcriptional inhibitor. These findings suggest that uPA gene expression involves a post-transcriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 30 kDa uPA mRNA binding protein (uPA mRNABp) that selectively binds to a 66 nt protein binding fragment of uPA mRNA containing regulatory information for message stabilization. Binding of cytoplasmic uPA mRNABp to uPA mRNA was abolished after treatment with TNF-alpha but not TGF-beta. In addition, we found the accumulation of 30 kDa uPAmRNABp in the nuclear extracts of TNF-alpha but not TGF-beta treated cells. The uPA mRNABp starts moving to the nucleus from the cytoplasmic compartment as early as three hours after TNF-alpha treatment. Complete translocation is achieved between 12-24 h, which coincides with the maximal expression of uPA protein effected by cytokine stimulation. Treatment of Beas2B cells with NaF inhibited TNF-alpha-mediated translocation of uPA mRNABp from the cytoplasm to the nucleus and concomitant inhibition of uPA expression. TNF-alpha stabilizes uPA mRNA by translocating the uPA mRNABp from the cytoplasm to the nucleus. Our results demonstrate a novel mechanism governing uPA mRNA stability through shuttling of uPA mRNABp between the nucleus and cytoplasm. This newly identified pathway may have evolved to regulate uPA-mediated functions of the lung epithelium in inflamation or neoplasia.
Shetty
Sreerama
S
Department of Specialty Care Services, The University of Texas Health Center at Tyler, 75708, USA. Shetty@uthct.edu
D021581:Active Transport, Cell Nucleus; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014466:Ultraviolet Rays; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D016207:Cytokines; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016211:Transforming Growth Factor alpha; D016212:Transforming Growth Factor beta; D012313:RNA; D003513:Cycloheximide; D014568:Urokinase-Type Plasminogen Activator