3.1. Molecular Changes in Osteoblasts Co-Cultured with Prostate Cancer Cells
To understand how factors secreted by prostate cancer cells regulate gene expression in osteoblasts we co-cultured rat osteosarcoma derived osteoblastic cells (UMR-106) with an osteolytic prostate cancer cell line (PC3) on transwell plates without physical contact (Figure 1A). UMR cells were also cultured alone as the control. By comparing gene expression in osteoblast co-cultures to monocultures we identified 113 up- and 63 down-regulated genes (Figure 1B, Table S2). Differentially expressed genes included several known regulators of bone metabolism, such as Gpnmb [20], Fhl2 [21], Mgp [22], Enpp1 [23] and Phex [24].
Figure 1  Co-culture of UMR-106 osteoblastic cells with prostate cancer cells (PC3) promotes changes in gene expression. (A) UMR-106 cells were cultured alone or with PC3 cells in transwells, and gene expression changes were quantified using microarrays; (B) 113 and 63 genes were found differentially expressed between UMR cells co-cultured with PC3 cells compared to UMR cells alone; (C) a subset of these differentially transcribed genes were confirmed using qPCR; and (D) immunocytochemistry showed a reduction in Sost protein expression in UMR cells co-cultured with PC3 cells (UMR + PC3) compared to UMR cells cultured alone (UMR alone).   Factors secreted by osteoblasts have been shown to play a major role in regulating cancer metastasis to bone [25,26]. This prompted us to investigate the changes in the regulation of the osteoblast secretome in response to osteoblast-prostate cancer interactions. Our analysis identified 41 genes encoding secreted proteins (Table 1) that are more than two-fold up- or down-regulated in osteoblast co-cultures compared to monocultures. We also found Wnt pathway inhibitors Sost [27] and its paralog Sostdc1 [28] with altered expression in co-cultured osteoblasts. Sost expression was significantly down-regulated, while its paralog Sostdc1 was significantly up-regulated in co-cultured osteoblasts (Table 1). Using qPCR we confirmed the differential expression of a subset of genes, including Mmp13, Ctgf, Il-6, Adamts1, Sost and Sostdc1 (Figure 1C), in osteoblast co-cultures.
microarrays-04-00503-t001_Table 1 Table 1  Genes encoding secreted proteins more than two-fold up- or down-regulated in UMR-106 osteoblastic cells co-cultured with PC3 cells compared to UMR cells cultured alone.

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