Heat Shock Proteins HSP70 And MRJ Cooperatively Regulate Cell Adhesion And Migration Through Soluble Urokinase Receptor

Overexpression of heat shock proteins （HSPs） have been found to be closely correlated with cancer progression and poor prognosis. HSPs assist to modify many important tumor-related proteins through abnormal gene expression. The HSP70 protein is a member of the DnaK/HSP70 class （HSP701A1,72KDa） （NM₀05345）. HSP40 are essential co-chapreones of HSP70, with the N-terminal J-domain necessary for its interaction with HSP70 and its weak ATPase activity. The MRJ （mammalian relative of DnaJ） protein is a member 6 of the DnaJ/HSP40 homolog subfamily B （DANJB6） （NM₀05494）. The uPAR protein belongs to the Ly-6/uPAR/α-neuotoxin protein domain family. It is a multifunctional receptor and regulate the extracellular matrix degradation, tumor growth, cell adhesion and migration.uPAR and HSPs HSP70 and MRJ （DNAJB6） have been implicated in cancer development and metastasis. We found that HSP70, MRJ and uPAR formed a triple complex by co-immunoprecipitation. Knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR mediated adhesion to vitronectin. This study further demonstrated that HSP70 colocalized with uPAR both in cytoplasm and on cell membrane using immunofluorence assays. In addition, knockdown of HSP70 and/or MRJ suppressed expression of cell surface uPAR. To understand the biological functions of the triple complex modulation, we determined whether regulation of HSP70 and MRJ has effects on uPAR mediated cell proliferation, cell cycle, cell migration and signal transduction. MTT and flow cytometry assays showed that knockdown HSP70 or MRJ had no significant effects on uPAR mediated cell proliferation or cell cycle. Knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR mediated cell migration, and suppressed mRNA level of invasion related genes MMP-2 and MMP-9. Overexpression of HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK, suggesting that MAPK pathway was involved in this regulation. The regulation of HSP70/MRJ on uPAR and uPAR associated celluler functions may provide a new insight into uPAR mediated cell migration and novel targets for cancer therapy.To investigate the effects of MRJ on cytoskeleton rearrangement and the expression of matrix metalloproteinase 9 （MMP-9）, we constructed a stable breast cancer cell line:MDA-MB-231/MRJsi. We transfected the MDA-MB-231 cells with the plasmid of MRJ scramble sequence （pRNAT-U6.1/neo） and MRJ siRNA （pRNAT-U6.1/neo-MRJsi） and used 600 μg/ml G418 to screen the stable knockdown cell line. The selected cell line was then subjected to real time PCR to quantitate the mRNA level. Then immunnofluorence was used to investigate the distribution of microfilaments using phalloidin dyes, respectively. Moreover, the enzyme activity of MMP-9 was measured using gelatin zymography and its gene expression was detected using quantitative real time PCR. The results showed that we successfully established MDA-MB-231/MRJsi stable cell line and destigated it as MDA-MB-231/MRJsi, while the MDA-MB-231/pRNAT-U6.1 was failed and was replaced with MDA-MB-231 cells. The MRJ mRNA in MDA-MB-231/MRJsi cells was reduced by 50% compared to the wild type MDA-MB-231 cells （P<0.05）. Furthermore, the cytoskeleton proteins in MDA-MB-231/MRJsi cells were redistributed when compared to wild type MDA-MB-231 cells. Furthermore, the enzyme activity of MMP-9 was enhanced after MRJ suppression while the activity of MMP-2 was not changed, suggesting the enzyme activity regulation was specific. In addition, MMP-9 mRNA was increased significantly （P<0.01） which was consistent with its enzyme activity. In conclusion, the establishment of stable MDA-MB-231/MRJsi cell line may be a fundamental cell model for further studies of MRJ function in oncogenesis and development of breast cancer.