Journal of Cellular Biochemistry 104:2298–2309 (2008) Novel Surface Expression of Reticulocalbin 1 on Bone Endothelial Cells and Human Prostate Cancer Cells Is Regulated by TNF-a Carlton R. Cooper, 1 * Bianca Graves, 1 Freddie Pruitt, 1 Hassan Chaib, 7 Jill E. Lynch, 1 Andrew Koemeter Cox, 1 Linda Sequeria, 1 Kenneth L. van Golen, 1 Angelo Evans, 1 Kirk Czymmek, 1,2 Rebecca S. Bullard, 3 Carlton D. Donald, 3 Katia Sol-Church, 4 James D. Gendernalik, 5 Babette Weksler, 6 Mary C. Farach-Carson, 1 Jill A. Macoska, 7 Robert A. Sikes, 1 and Kenneth J. Pienta 5,7 1 Center for Translational Cancer Research and Department of Biological Sciences, University of Delaware, Newark, Delaware 2 Delaware Biotechnology Institute, Department of Biological Sciences, University of Delaware, Newark, Delaware 3 Department of Pathology and Lab Medicine, Medical University of Charleston, Charleston, South Carolina 4 Biomedical Research Department, Nemours Children Clinic, Wilmington, Delaware 5 Section of Hematology/Oncology, Departments of Internal Medicine and Urology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 6 Department of Medicine, Weill Medical College of Cornell University, New York, New York 7 Departments of Internal Medicine and/or Urology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan Abstract An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-a. J. Cell. Biochem. 104: 2298 – 2309, 2008. ß 2008 Wiley-Liss, Inc. Key words: endothelial cells; prostate; LNCaP; progression; neoplasm; bone metastasis; phage display; reticulocalbin 1 ß 2008 Wiley-Liss, Inc. Contract grant sponsor: NIHNCRRINBRE; Contract grant number: RR016472-04; Contract grant sponsor: NIH Extramural Research Facilities Program; Contract grant numbers: C06RR14516, RO1-HL55267. *Correspondence to: Dr. Carlton R. Cooper, PhD, Depart- ment of Biological Sciences, University of Delaware, 324 Wolf Hall, Newark, DE 19716. E-mail: crcooper@udel.edu Received 14 November 2007; Accepted 17 March 2008 DOI 10.1002/jcb.21785 Contract grant sponsor: Prostate Cancer SPORE; Contract grant number: P50 CA 69568; Contract grant sponsor: The University of Michigan Comprehensive Cancer Center; Contract grant number: CA 46592 (KP); Contract grant sponsor: NIH-K22 Career-Transition Award; Contract grant numbers: 5K22CA971117-3, CA096788; Contract grant sponsor: National Foundation for Cancer Research Center for Metastasis Research (CRC); Contract grant sponsor: University of Delaware; Contract grant sponsor: NIH/NCI; Contract grant numbers: PO1 CA098912, DAMD-17-00-1-0049, CA-105435, DK63919, CA-60948;