The online version of this article (doi:10.1186/1476-4598-11-66) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
AG carried out major experiments including Western blotting with human normal and tumor tissue lysates, immunohistochemistry on TMA, analyses with conditioned medium (Western blotting and osteoclast differentiation), studies with inhibitors (αv and PKC) and SiRNA (Smad 5). AG also participated in the MS preparation, statistical analysis of the data, discussion and interpretation of results. WC generated CD44 knockdown stable PC3 cell lines. MAC conceived the study, confocal microscopy analysis of immunostained PC3 cells, RUNX2 knockdown experiments and manuscript preparation. All authors read and approved the final manuscript.
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.
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.
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.
Additional file 1: Figure S1. Analysis of the effects of SiRNA to RUNX2 on MMP9 and MMP2 RNA and protein levels (A-E) and revelation of major MMPs present in PC3 and LNCaP cells (F). A-D: We determined the effects of RUNX2 knockdown on the expression of MMP9 and MMP2 at mRNA (Figure S1-A) and protein levels (Figure S1D) in PC3 cells. Dose-dependent decrease in the levels of RUNX2 expression was observed in PC3 cells treated with SiRNA to RUNX2 at concentrations of 10, 20, and 50nM. The decrease was maximal (>90%) at 50nM RUNX2 SiRNA (A, lane 4). PC3 cells treated with scrambled RNAi (50nM) were used as control (A, lane 1). SiRNA to RUNX2 had very negligible effects on the changes in the levels of mRNA expression of MMP2 in PC3 cells (lane 6). GAPDH was used as internal control (Figure S1-B). A decrease in the expression of MMP9 at mRNA (Figure S1-A, lane 4) parallels with the MMP9 activity (~ 90kDa) in the conditioned medium isolated from cultures of PC3 cells treated with RUNX2 SiRNA (Figure S1-E, lane 3). MMP9 activity was determined by zymogram analysis. About 50μg membrane protein was used for the gelatin zymography to determine the activities of MMP9 (S1-E). As shown previously [Ref.28], only the active form of MMP-9 was observed in the conditioned medium (Figure S1-E, lanes 1-3). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 4 in S1-E). Furthermore, the decrease in the protein levels of RUNX2 (~55kDa) in SiRNA to RUNX2 treated cells (Figure S1-C, lane 3) corresponds with a decrease in the total cellular protein levels of MMP 9 (Figure S1-D, lane 3) but not MMP 2 (~72kDa). MMP 2 levels remain the same in control untreated as well as scrambled RNAi and SiRNA to RUNX2 treated cells (Figure S1- D). These results imply that the RUNX2 is not a direct binding factor to induce transcriptional activation of MMP 2.F: Zymogram analysis with normal prostatic epithelial cells (HPR1) was used as a control (lane 4) for prostate cancer cells derived from lymph node (LNCaP, lane 2) and bone (PC3, lane 3) metastases. The activity of a recombinant MMP2 and MMP9 protein containing pro and active bands (indicated by arrows) were used as an identification marker (lane 1). LNCaP cells demonstrated MMP2 as a major metalloproteases where as MMP9 was observed as major MMP although MMP2 was observed at mRNA (Figure 1A) and protein levels (Figure S1-D and F) in PC3 cells. About 75μg total cellular protein was used for zymogram analysis as shown previously [ref.[ 28]. Method: Gelatin zymography: Conditioned media collected from various PC3 cell lines were concentrated approximately 10-fold) with a centricon concentrator (Amicon, Beverly, MA). Ten micrograms of concentrated media protein in 10-20 μl were mixed with gel loading buffer with no reducing agent (βME or DTT) and incubated at RT for 10-15 min. SDS-PAGE containing 0.1% gelatin was used for electrophoresis. Samples were loaded without heating with sample buffer. After electrophoresis, gels were incubated overnight in a buffer containing 50 mM Tris-HCl, pH 7.6, 5 mM CaCl 2, 1 μM ZnCl 2, and 1% Triton X-100. Triton was used to remove SDS from the gel. Gels were then stained with Coomassie brilliant blue for 2-3 h and destained with 7% acetic acid or water. Gelatinolytic activity was detected as clear bands in the background of blue staining [ref.[ 28]. (DOC 122 KB)
Additional file 2: Figure S2.Immunoblotting analysis for Smad 2, 3, 5 and 6 proteins in PC3 cells. About 50μg total cellular lysate protein was used for immunoblotting with antibodies to phospho-Smad (p-Smad) -2 (60kDa; lane 1), -3 (52 kDa; lane 2), -5 (60kDa; lane 3) and -6 (62kDa; lane 4). Blots were reprobed with an antibody to GAPDH after stripping. Phosphorylation of 2, 3, and 5 was observed in PC3 cells. However, Smad- 5 phosphorylation is significantly more than Smad-2 and 3 (lanes 1 and 2). Phosphorylation of Smad-6 is really negligible or not observed. (DOC 70 KB)12943_2012_1043_MOESM2_ESM.doc
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- 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
Meenakshi A Chellaiah
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