Regulation of urokinase receptor expression by protein tyrosine phosphatases

Abstract

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a major role in several physiological processes such as cell migration, proliferation, morphogenesis, and regulation of gene expression. Many of the biological activities of uPA depend on its association with uPAR. uPAR expression and its induction by uPA are regulated at the posttranscriptional level. Inhibition of protein tyrosine phosphatase-mediated dephosphorylation by sodium orthovanadate induces uPAR expression and, with uPA, additively induces cell surface uPAR expression. Sodium orthovanadate induces uPAR by increasing uPAR mRNA in a time- and concentration-dependent manner. Both sodium orthovanadate and uPA induce uPAR mRNA stability, indicating that dephosphorylation could contribute to uPA-induced posttranscriptional regulation of uPAR expression. Induction of the tyrosine phosphatase SHP2 in Beas2B and H157 cells inhibits basal cell surface uPAR expression and uPA-induced uPAR expression. Sodium orthovanadate also increases uPAR expression by decreasing the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK) as well as by enhancing the interaction between a uPAR mRNA 3' untranslated sequence with heterogeneous nuclear ribonucleoprotein C (hnRNPC). On the contrary, overexpression of SHP2 in Beas2B cells increased interaction of PGK with the uPAR mRNA coding region and inhibited hnRNPC binding to the 3' untranslated sequence. These findings confirm a novel mechanism by which uPAR expression of lung airway epithelial cells is regulated at the level of mRNA stability by inhibition of protein tyrosine phosphatase-mediated dephosphorylation of uPAR mRNA binding proteins and demonstrate that the process involves SHP2.

Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury

“…Isolation of PGK and hnRNPC was performed as previously described (29). In brief, the cells were lysed in Western lysis buffer by three freeze-thaw cycles.…”

“…The PGK and hnRNPC proteins were isolated from the total lysates using specific antibody conjugated with agarose and then separated on 8% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane and subjected to Northwestern assay using 32 P-labeled coding region and 39 UTR transcripts, respectively, as described previously (29).…”

“…The PGK and hnRNPC proteins were isolated from Beas2B and mouse lung extracts using specific antibodies, as described previously (13,25,29). Briefly, isolated proteins were separated on SDS-PAGE and then blotted onto a nitrocellulose membrane.…”

“…The same membrane was later stripped and subjected to Western blotting using anti-phosphotyrosine antibody to determine the tyrosine phosphorylation status of PGK and hnRNPC. Lastly, membranes were stripped and developed with anti-PGK or anti-hnRNPC antibodies to normalize uPAR mRNA binding activities and tyrosine phosphorylation of PGK or hnRNPC proteins with their total expression, as previously described (13,24,25,29).…”

“…It has been previously reported that PMA, epidermal growth factor, LPS, and TNF-a increase the specific binding capacity of uPA and cell surface expression of uPAR on various cell lines (29,(32)(33)(34). To better understand the mechanism by which bacterial endotoxins regulates lung airway epithelial cell uPAR during ALI caused by sepsis, we exposed bronchial epithelial Beas2B cells to higher doses of LPS and analyzed the effect on the expression of uPAR protein and uPAR mRNA.…”