Effects of α-Syn on the activation of tyrosinase (TYR) induced by UVB light.

<p>After cells were exposed to UVB light (120 mJ/cm²) and post-cultured for 24 h, the TYR activity was measured spectrophotometrically at 475 nm using iMark Microplate Reader. Cells without UVB light exposure (non-UVB) were used as a control. A: A375 melanoma cells; B: SK-MEL-28 melanoma cells; C: SH-SY5Y dopaminergic neuronal cells; D: PC12 dopaminergic neuronal cells. *: p<0.05; **: p<0.01 as compared to its non-UVB control (a) or non-α-Syn control (b). Dox  =  doxycycline.</p

Flow chat of the linkage between PD and melanoma mediated by α-Syn.

<p>In skin melanocytes, UVR causes DNA damage, leading to the initiation of melanoma. UVR also induces melanin synthesis which is catalyzed by TYR. Increased melanin prevents UVR-induced DNA damage, reducing the vulnerability to development of melanoma. α-Syn expression in skin melanocytes may interact with TYR, inhibiting UVR-induced TYR activation, leading to the reduction of melanin synthesis, which may enhance the susceptibility of skin melanocytes to develop melanoma. In brain dopaminergic neurons, impaired degradation of α-Syn or α-Syn multiplications causes accumulation/aggregation of α-Syn, leading to the loss of α-Syn function, thereafter, the decrease of DA release and increase of cellular DA content, which may be converted into melanin in lysosome. The increased melanin content in dopaminergic neurons (neuromelanin) enhances the susceptibility to oxidative stress-induced neuronal injury relevant to PD.</p

Roles of α-Syn in melanin synthesis in melanoma and dopaminergic neuronal cells.

<p>A375 melanoma cells (A) and dopaminergic neuronal SH-SY5Y and PC12 cells (C, D) with or without wt-α-Syn over-expression, SK-MEL-28 melanoma cells (B) with or without suppression of endogenous α-Syn, were exposed to UVB light (120 mJ/cm²) or non-UVB light and post-cultured for 24 h. The protein levels of α-Syn were determined by western blot assay (A, B, C, D, upper panel). Melanin content was determined spectrophotometrically at 415 nm using iMark Microplate Reader (A, B, C, D, lower panel). *: p<0.05; **: p<0.01; *** p<0.001 as compared to its non-UVB control (a), non-α-Syn control (b), or vector/<i>#3</i> siRNA control (c). Dox  =  doxycycline.</p

UVB light-induced changes in gene expression and protein level of TYR.

<p>A375 (A) and SH-SY5Y cells (B) with or without α-Syn expression were exposed to UVB light (120 mJ/cm²) or non-UVB light and post-cultured for 24 h. Gene expression of TYR was determined by real-time quantitative PCR assay (A, B). *: p<0.05; **: p<0.01 as compared to its non-UVB control (a) or non-α-Syn control (b). The protein levels of TYR were determined by western blot assay (C, D).</p

UVB light-induced loss of cell viability.

<p>A375 and SK-MEL-28 melanoma cells were exposed to UVB light at the doses of 30 mJ/cm² (A), 120 mJ/cm² (B) and 240 mJ/cm²(C) and post-cultured for 4 h, 24 h and 48 h (A, B, C). Cell viability was determined by MTT assay. The results were expressed as percentage of non-UVB control. *: p<0.05; **: p<0.01 as compared to its non-UVB control (0 h).</p

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

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

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

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

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

<div><p>Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an <i>in vivo</i> metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.</p></div

Expression of Cad11 in 786-O cell lines derived from metastases to various organs.

<p>(A) Quantitative PCR for the message levels of <i>Cad11</i> in the four 786-O cell lines. (B) Western blotting for the protein levels of Cad11 in four 786-O cell lines. Upper panel: A representative image of Western blot. Lower panel: Quantification of band density using Image J software. Data were expressed as folds of parental 786-O cells and the values were the Mean ± S.E. n = 5. *: <i>p</i><0.05; **: <i>p</i><0.01 as compared to parental 786-O cells. (C) FACS for surface expression of Cad11 in the four 786-O cell lines. Data were expressed as percentage of gated cells. (D) Immunofluorescence staining of cells with anti-Cad11 antibody. P, Parental 786-O; Liv, Liv-786-O; LN, LN-786-O, and Bo, Bo-786-O RCC cells.</p

Immunohistochemical staining of human RCC specimens with anti-Cad11 antibody.

<p>(A) Representative image of primary RCC; (B) Representative image of bone metastatic RCC.</p