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Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

ABSTRACT: Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (?47%) and plasma membrane (?31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

SUBMITTER: Bilen MA

PROVIDER: S-EPMC5860883 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

Bilen Mehmet Asim MA Pan Tianhong T Lee Yu-Chen YC Lin Song-Chang SC Yu Guoyu G Pan Jing J Hawke David D Pan Bih-Fang BF Vykoukal Jody J Gray Kavanya K Satcher Robert L RL Gallick Gary E GE Yu-Lee Li-Yuan LY Lin Sue-Hwa SH

Journal of proteome research 20170718 8

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of ...[more]

PMID: 28675788

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Project description:Patients with metastatic prostate cancer frequently develop bone metastases that elicit significant skeletal morbidity and increased mortality. The high tropism of prostate cancer cells for bone and their tendency to induce the osteoblastic-like phenotype are a result of a complex interplay between tumor cells and osteoblasts. Although the role of osteoblasts in supporting prostate cancer cell proliferation has been reported by previous studies, their precise contribution in tumor growth remains to be fully elucidated. Here, we tried to dissect the molecular signaling underlining the interactions between castration-resistant prostate cancer (CRPC) cells and osteoblasts using in vitro co-culture models. Transcriptomic analysis showed that osteoblast-conditioned media (OCM) induced the overexpression of genes related to cell cycle in the CRPC cell line C4-2B but, surprisingly, reduced androgen receptor (AR) transcript levels. In-depth analysis of AR expression in C4-2B cells after OCM treatment showed an AR reduction at the mRNA (p = 0.0047), protein (p = 0.0247), and functional level (p = 0.0029) and, concomitantly, an increase of C4-2B cells in S-G2-M cell cycle phases (p = 0.0185). An extensive proteomic analysis revealed in OCM the presence of some molecules that reduced AR activation, and among these, Matrix metalloproteinase-1 (MMP-1) was the only one able to block AR function (0.1 ng/ml p = 0.006; 1 ng/ml p = 0.002; 10 ng/ml p = 0.0001) and, at the same time, enhance CRPC proliferation (1 ng/ml p = 0.009; 10 ng/ml p = 0.033). Although the increase of C4-2B cell growth induced by MMP-1 did not reach the proliferation levels observed after OCM treatment, the addition of Vorapaxar, an MMP-1 receptor inhibitor (Protease-activated receptor-1, PAR-1), significantly reduced C4-2B cell cycle (0.1 μM p = 0.014; 1 μM p = 0.0087). Overall, our results provide a novel AR-independent mechanism of CRPC proliferation and suggest that MMP-1/PAR-1 could be one of the potential pathways involved in this process.

Project description:Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Dataset Information

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

Publications

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

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