title: Heat shock proteins HSP70 and MRJ cooperatively regulate cell adhesion and migration through urokinase receptor.
abstract: BACKGROUND
  The urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein.
  
  
  METHODS
  We performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay.
  
  
  RESULTS
  HSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR.
  
  
  CONCLUSIONS
  These findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.
authors.forename: Yuli
authors.initials: Y
authors.lastname: Lin
authors.forename: Nana
authors.initials: N
authors.lastname: Peng
authors.forename: Hongqin
authors.initials: H
authors.lastname: Zhuang
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, Jiangsu, P,R, China. yao_wang_au@yahoo.com.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D057809:HEK293 Cells; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D019096:Vitronectin / Q000378:metabolism
chemical_list: C485565:DNAJB6 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D055293:Receptors, Urokinase Plasminogen Activator; D019096:Vitronectin; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9