title: Integrin αvβ3 and CD44 pathways in metastatic prostate cancer cells support osteoclastogenesis via a Runx2/Smad 5/receptor activator of NF-κB ligand signaling axis.
abstract: BACKGROUND
  Bone loss and pathological fractures are common skeletal complications associated with androgen deprivation therapy and bone metastases in prostate cancer patients. We have previously demonstrated that prostate cancer cells secrete receptor activator of NF-kB ligand (RANKL), a protein essential for osteoclast differentiation and activation. However, the mechanism(s) by which RANKL is produced remains to be determined. The objective of this study is to gain insight into the molecular mechanisms controlling RANKL expression in metastatic prostate cancer cells.
  
  
  RESULTS
  We show here that phosphorylation of Smad 5 by integrin αvβ3 and RUNX2 by CD44 signaling, respectively, regulates RANKL expression in human-derived PC3 prostate cancer cells isolated from bone metastasis. We found that RUNX2 intranuclear targeting is mediated by phosphorylation of Smad 5. Indeed, Smad5 knock-down via RNA interference and inhibition of Smad 5 phosphorylation by an αv inhibitor reduced RUNX2 nuclear localization and RANKL expression. Similarly, knockdown of CD44 or RUNX2 attenuated the expression of RANKL. As a result, conditioned media from these cells failed to support osteoclast differentiation in vitro. Immunohistochemistry analysis of tissue microarray sections containing primary prostatic tumor (grade2-4) detected predominant localization of RUNX2 and phosphorylated Smad 5 in the nuclei. Immunoblotting analyses of nuclear lysates from prostate tumor tissue corroborate these observations.
  
  
  CONCLUSIONS
  Collectively, we show that CD44 signaling regulates phosphorylation of RUNX2. Localization of RUNX2 in the nucleus requires phosphorylation of Smad-5 by integrin αvβ3 signaling. Our results suggest possible integration of two different pathways in the expression of RANKL. These observations imply a novel mechanistic insight into the role of these proteins in bone loss associated with bone metastases in patients with prostate cancer.
authors.affiliation: Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA.
authors.forename: Aditi
authors.initials: A
authors.lastname: Gupta
authors.forename: Wei
authors.initials: W
authors.lastname: Cao
authors.forename: Meenakshi A
authors.initials: MA
authors.lastname: Chellaiah
mesh_terms: D001665:Binding Sites; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008297:Male; D009362:Neoplasm Metastasis; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000556:secondary*; D011485:Protein Binding; D021381:Protein Transport; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism*; D015398:Signal Transduction; D051902:Smad5 Protein / Q000378:metabolism*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D018960:Hyaluronan Receptors; D039302:Integrin alphaVbeta3; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D051902:Smad5 Protein