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J. Biol. Chem., Vol. 282, Issue 8, 5736-5748, February 23, 2007

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-Synuclein Stimulates Differentiation of Osteosarcoma Cells

RELEVANCE TO DOWN-REGULATION OF PROTEASOME ACTIVITY^*

Masayo Fujita, Shuei Sugama, Masaaki Nakai, Takato Takenouchi, Jianshe Wei, Tomohiko Urano^¶, Satoshi Inoue^¶, and Makoto Hashimoto¹

From the Laboratory for Chemistry and Metabolism, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan, Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan, and ^¶Department of Geriatrics and Gerontology, School of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Received for publication, June 28, 2006 , and in revised form, December 15, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Because a limited study previously showed that -synuclein (-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether -syn might be involved in the regulation of tumor differentiation. For this purpose, -syn and its non-amyloidogenic homologue -syn were stably transfected to human osteosarcoma MG63 cell line. Compared with -syn-overexpressing and vector-transfected cells, -syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in -syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In -syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in -syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of -syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of -syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of -syn regulation of tumor differentiation and neuropathological effects of -syn may considerably overlap with each other.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Synuclein (-syn)² is a presynaptic protein that belongs to the syn family of peptides with two other members, - and -syn (1, 2). These proteins are characterized by a native unfolded structure with a highly conserved N-terminal region and a divergent C-terminal acidic region (3). However, the most striking feature is that -syn possesses a highly hydrophobic domain in the middle region that was previously purified from Alzheimer disease brain amyloid (4). Due to this hydrophobic domain, -syn forms toxic protofibrils that might cause synaptic injury and dysfunction (5). The importance of -syn in the pathogenesis of neurodegenerative disorders was further augmented by identification of missense mutations of -syn in familial Parkinson disease (PD) (6-8). Subsequently, numerous histological studies have shown that -syn is a major constituent in Lewy bodies and dystrophic neuritis in sporadic PD and diffuse Lewy body disease (2). Immunoreactivity of -syn was also shown in glial cell inclusions in multiple system atrophy (2). By contrast, -syn, which lacks the majority of the hydrophobic domain in the middle region, was protective against protofibrils of -syn (9, 10). Thus, these results support the contention that - and -syn may play a central role in the pathogenesis of neurodegenerative diseases.

On the other hand, -syn, the less conserved third member of the syn family of peptides, was identified as a breast cancer-specific gene product (11) and has been extensively studied in a variety of cancers, such as breast cancer, ovarian cancer, liver cancer, pancreatic adenocarcinoma, and bladder cancer (11-15). Because expression levels of -syn were well correlated with the presence of metastatic lesions, it has been generally thought that -syn might regulate tumor invasiveness and metastasis (16, 17). Several studies indicated that -syn might be involved in the deregulation of cell cycle in cancer. For example, -syn interacted with a mitotic spindle checkpoint protein, BubR1, leading to decreased checkpoint function and tumor progression (18, 19). Moreover, -syn stimulated cell proliferation by augmenting estrogen receptor-mediated signaling in breast cancer cells (20). Thus, the role of -syn has been investigated mainly in the area of tumor biology.

A recent study, however, suggests that molecular pathways shared by neurodegenerative disease and cancer may be considerably overlapped than thought before. Supporting this notion, it has been recently shown that several familial PD-causing factors are involved in the pathogenesis of cancer (21). First, overexpression of the PARK2 parkin, which functions as an E3 ligase in the ubiquitin-proteasome system (UPS), resulted in growth suppression in hepatocellular carcinoma cells (22). Furthermore, ectopic expression of parkin reportedly reduced in vivo tumorigenesis in nude mice (23). Second, the PARK5 ubiquitin C-terminal hydrolase light chain-1, which acts as a de-ubiquitinating enzyme in UPS, was shown to suppress proliferation of a lung cancer cells (24). Other evidence suggests that ubiquitin C-terminal hydrolase light chain-1 might be involved in the degradation of p27, a cyclin-dependent kinase inhibitor (25). Third, the PARK7 DJ-1 was first identified as an oncogene product that stimulated transformation of NIH3T3 cells in coordination with Ras (26). DJ-1 was recently shown to up-regulate the phosphatidylinositol 3-kinase pathway through inhibition of the tumor suppressor phosphatase PTEN, leading to enhance survival of cancer (27).

In the same context one may wonder if - and -syn, the central players in the pathogenesis of PD, might play some important roles for the regulation of cancer. Indeed, -syn was widely expressed in a variety of brain tumors, such as medulloblastoma, neuroblastoma, pineoblastoma, and ganglioma (28, 29). Furthermore, both - and -syn were shown to be expressed in the peripheral cancers, including ovarian and breast cancers (30). Although the role of syn proteins in the pathogenesis of cancer is unclear, a limited number of studies suggest that -syn might be involved in the regulation of tumor differentiation. Supporting this possibility, -syn was preferentially expressed in brain tumors showing neuronal differentiation (28). In cell cultures expression of -syn was increased during the hematopoietic differentiation of K562 myelogenous leukemia cells (31). Similarly, it was shown that -syn was up-regulated during neural differentiation of pheochromocytoma PC12 cells (32). Thus, distinct from the possible role of -syn for tumor metastasis, -syn might be involved in tumor differentiation.

Accordingly, the main objective of the present study was to determine whether - and/or -syn might regulate growth and differentiation of cancer cells. For this purpose, - and -syn were stably overexpressed in MG63 human osteosarcoma cells. We found that compared with vector-transfected and -syn-overexpressing cells, -syn-overexpressing cells exhibited distinct phenotype of differentiated osteoblastic cells. Mechanistically, -syn may cause down-regulation of proteasome activity, leading to accelerate cellular differentiation. Further studies revealed that down-regulation of proteasome activity by -syn was regulated by alteration of various factors, including PKC signaling activities and the autophagy-lysosomal pathway in -syn-overexpressing cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagent—Chemical reagents include MG132 and lactacystin (purchased from EMD Biosciences, San Diego, CA), rotenone, 12-myristate 13-acetate (PMA), 4-phorbol 12,13-didecanoate (4PDD), leupeptin, chelerythrine chloride, and 3-methyladenine (3-MA) (obtained from Sigma), and caspase I inhibitor and III inhibitors (Ac-AAVALLPAVLLALLAP-YVAD-CHO, Ac-AAVALLPAVLLALLAP-DEVD-CHO) (Calbiochem) were applied to cell cultures at indicated concentrations.

Antibodies used are as follows. Monoclonal antibodies, anti--syn (syn-1), anti--syn, anti-PKC, and anti-retinoblastoma (Rb) were purchased from BD Biosciences. Monoclonal anti--actin (AC-15) was obtained from Sigma. Monoclonal antibodies anti-p21 and anti-cyclin B1, rabbit polyclonal antibodies anti-p15, anti-cyclin D1, anti-cyclin-dependent kinase 4, and anti-phosphorylated Rb, and goat polyclonal anti-osteocalcin antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-ubiquitin antibody was from Chemicon (Temecula, CA). Monoclonal anti-19 S proteasome S6a subunit antibody was from Biomol (Plymouth Meeting, PA). Rabbit polyclonal anti-cathepsin B was from Calbiochem. Rabbit polyclonal antibodies, anti-C-terminal -syn, and anti--syn were previously described (33, 34). Alexa fluor 488-conjugated anti-goat secondary antibody, Alexa fluor 488-conjugated anti-rabbit secondary antibody, and Alexa fluor 555-conjugated anti-mouse secondary antibody were from Invitrogen.

Cell Cultures and Transfection—MG63 and 293T cells were both cultured in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal calf serum (BioWest, Nuaille, France) and 1% v/v penicillin/streptomycin (Invitrogen) in a 5% CO₂, 95% air atmosphere. MG63 cells exhibited both - and -syn transcripts by RT-PCR analysis, but their protein expressions were hardly detectable by immunoblot analysis (data not shown). For stable transfection, MG63 cells were transfected with pCEP4 expression vector (Invitrogen) or pCEP4 containing either human -or -syn cDNA (34) using Lipofectamine 2000 (Invitrogen). After 23 weeks of incubation in the presence of 200 µg/ml hygromycin B (EMD Biosciences), the resistant colonies of cells (20) were isolated. These stable cell lines were routinely maintained in the presence of 50 µg/ml hygromycin B. 293T cells were transiently transfected with p-Target expression vector (Promega, Madison, WI) containing human p21 cDNA insert.³

Reverse Transcriptase (RT)-PCR—Total RNA was isolated from cultured cells using the RNA extraction buffer ISOGEN (Nippon Gene, Tokyo, Japan). cDNA was synthesized from 2 µg of total RNA using the Superscript III First-Strand Synthesis system (Invitrogen) according to the manufacturer's instruction.

PCR amplification was carried out using Taq PCR polymerase (ABgene, Tokyo, Japan), and the amplified products were resolved by agarose gel electrophoresis. The following primers were used for PCR. Human -syn (NM_000345 [GenBank] ): sense, 5'-ATGGATGTATTCATGAAAGGACTTTC-3' (47-72-oligonucleotide position), antisense, 5'-GGCTTCAGGTTCGTAGTCTTGATAC-3' (the 442-466 oligonucleotide position); human -syn (BT006627 [GenBank] ): sense, 5'-ATGGACGTGTTCATGAAGGGC-3' (1-21-oligonucleotide position), antisense, 5'-CTACGCCTCTGGCTCATACTC-3' (385-405-oligonucleotide position); human cyclophilin A (NM_021130 [GenBank] ): sense, 5'-TACTATTAGCCATGGTCAAC-3' (62-81-oligonucleotide position), anti-sense 5'-GTCTTGCCATTCCTGGACCC-3' (508-527-oligonucleotide position); human liver/bone/kidney-type alkaline phosphatase (ALP) (AB011406 [GenBank] ): sense, 5'-GGGGGTGGCCGGAAATACAT-3' (834-853-oligonucleotide position), anti-sense, 5'-GGGGGCCAGACCAAAGATAG-3' (1357-1376-oligonucleotide position); human osteocalcin (X51699 [GenBank] ): sense, 5'-ATGAGAGCCCTCACACTCCTC-3' (19-39-oligonucleotide position), antisense, 5'-GCCGTAGAAGCGCCGATAGGC-3' (292-312-oligonucleotide position).

Immunoblot and Co-immunoprecipitation Analyses—Immunoblot analysis was performed as previously described (34). Briefly, cells were harvested and dissolved in lysis buffer (1% Nonidet P-40, 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM sodium fluoride, and protease inhibitor mixture (Nakarai Tesque, Kyoto, Japan)). Protein concentrations of the cell lysates were determined Bio-Rad protein assay reagent. Cell extracts (10 µg) were then resolved by SDS-PAGE (10 or 16%) and electroblotted onto nitrocellulose membranes (GE Healthcare) with CAPS buffer (pH 11.0). The membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl) plus 0.2% Tween 20 followed by an incubation with primary antibodies in TBS containing 3% BSA. After washing, the membranes were further incubated with second antibody conjugated with horseradish peroxidase (GE Healthcare) in Tris-buffered saline (1:10,000). Finally, the target proteins were visualized with the ECL plus system (GE Healthcare). The intensity of the band was measured using BioMax 1D image analysis soft ware (Eastman Kodak Co.). In some experiments 293T cells were transfected with p-Target vector (Promega) with or without human p21 cDNA insert, and cell extracts were used for controls. Recombinant human - and -syn were also used for positive controls (9).

Immunoprecipitation was performed as previously described (35). Briefly, cell extracts (200 µg) were preabsorbed with protein G-Sepharose (GE Healthcare) for 1 h, and the precleared lysates were incubated with either syn-1 or mouse IgG control (each 1 µg) overnight at 4 °C followed by incubation with protein G-Sepharose. The immune complexes were then washed three times with the lysis buffer. The samples were then heated in the SDS sample buffer for 5 min and subjected to immunoblotting.

Immunofluorescence Study—Cells were seeded onto poly-L-lysine-coated glass coverslips, grown to 70% confluence, fixed in 4% paraformaldehyde for 30 min and pretreated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min. Fixed cells were blocked with PBS containing 3% goat serum and 5% bovine serum albumin at room temperature. For staining, cells were incubated overnight at 4 °C with primary antibody (or antibodies for double staining). After washing, cells were incubated with Alexa fluor-conjugated secondary antibody (or antibodies for double staining) (Invitrogen) for 1 h at room temperature. Control experiments included immuno-staining in the absence of primary antibody. Coverslips were air-dried, mounted on slides with Gel/Mount (Biomeda Corp., Foster City, CA), and imaged with the laser-scanning confocal microscope (LSCM) (Olympus, FV1000, Tokyo, Japan).

Cell Cycle Analysis Using Flow Cytometry—Cells seeded at 1 x 10⁵ cells in 6-well cell culture plates were incubated under low serum (0.1%) conditions for 24 h, treated with 10% serum for 6, 12, 18, 24, and 96 h, and harvested using trypsin-EDTA. After washing with PBS, cells were fixed by ice-cold ethanol to final concentration of 70%. The cells were then resuspended in PBS containing 0.5 mg/ml RNaseA (Nakarai Tesque), incubated for 30 min at 37 °C, and followed by staining with propidium iodide (10 µg/ml) for 10 min at room temperature. Before flow cytometry analysis, the stained cells were filtrated with nylon mesh. The fluorescence signals of 2 x 10⁴ cells were recorded by EPICS ALTRA (Beckman Coulter, Fullerton, CA). The distribution of cell cycle phase was analyzed by Multicycle software (Phoenix Flow Systems, San Diego, CA).

Measurement of ALP Activity—Cellular activities of ALP were determined as described previously with some modifications (36). Briefly, cells were grown until confluency under the 10% serum conditions in 6-well cell culture plates. Cells were then washed twice, harvested into PBS, and sonicated by ultra-sonic disruptor (TOMY, Tokyo, Japan) for 20 s. After centrifugation of the cell preparations at 15,000 rpm for 10 min, the supernatants were recovered and assessed for protein concentration. The supernatants (10 µg) were then incubated in the ALP assay buffer containing 10 mM p-nitrophenyl phosphate (Sigma), 100 mM glycine, 1 mM ZnCl₂, and 1 mM MgCl₂ (pH 10.4). After 90 min of incubation at 37 °C, ALP activity was determined by monitoring the amount of released p-nitrophenol at 415 nm. Dissolved p-nitrophenol in assay buffer was used to establish the standard for quantification. The released p-nitrophenol was adjusted by the amount of protein and described as nmol/min/mg of protein.

Increased levels of the cellular ALP activities were further confirmed by direct stain of the cells. The cells reached confluency were fixed with 70% ethanol, washed twice with Tris-buffered saline, and incubated with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt ALP substrate solution (Roche Applied Science) for 2 h. The visualized image was then photographed.

Mineralization Assay—Mineralization of MG63 cells was performed as previously described (36) with some modifications. Briefly, cells were grown until confluency under the 10% serum conditions in 24-well cell culture plates. The cells were further incubated in -minimal essential medium (Invitrogen) plus 10% serum in the presence of 10 mM -glycerophosphate (Sigma) and 50 µg/ml ascorbic acid with or without the addition of various chemical reagents. The media were regularly changed twice a week. After 4 weeks, cells were evaluated for the extent of matrix mineralization by either alizarin red staining or von Kossa staining. For alizarin red staining, the cells were fixed with 70% ethanol for 30 min at room temperature. Then the cells were incubated with 40 mM alizarin red-S (Sigma) for 10 min and washed 4 times with distilled water. The visualized image was photographed. For von Kossa staining, the cells were fixed with 10% neutralized formaldehyde for 30 min at room temperature. The fixed cells were incubated with 5% silver nitrate for 5 min under the exposure to the sunlight. The reaction was stopped by the addition of 5% sodium thiosulfate. The stained cells were washed four times with distilled water and imaged with microscope.

Evaluation of Proteasome and Cathepsin B Activity—Measurement of proteasome and cathepsin B activity was done as previously described with modifications (37). Briefly, cells reaching confluency in 6-well cell culture plates were incubated under the low serum (0.1%) conditions for 24 h and treated with 10% serum with or without treatment of various reagents. After the indicated times (0, 6, or 12 h), the cells were harvested in buffer containing 50 mM HEPES (pH 7.4), 10 mM EDTA, and 10 mM NaCl, subjected to freezing and thawing to rapture cell membranous structures, and then centrifuged at 15,000 rpm for 10 min. For the measurement of proteasome activity, 10 µg of the supernatants were incubated in assay buffer containing 50 mM HEPES (pH 7.4), 10 mM EDTA, 10 mM NaCl, and 40 µM benzyloxycarbonyl-Leu-Leu-Glu-amidomethylcoumarin fluorogenic proteasome substrate (Chemicon). For cathepsin B activity, 10 µg of the supernatants were incubated in buffer containing 50 mM HEPES (pH 6.0), 10 mM EDTA, 10 mM NaCl, and 40 µM benzyloxycarbonyl-Arg-Arg-amidomethylcoumarin fluorogenic cathepsin B substrate (Chemicon). These enzyme activities were assayed by continuous recording of the fluorescence activity released from fluorogenic substrate using Berthold Mithras LB940 microplate reader (Berthold, Bad Wildbad, Germany) for 1 h at 37 °C (excitation, 380 nm; emission, 460 nm), and the reaction rate was analyzed. These enzyme activities were described as arbitrary unit/min/mg of protein.

Evaluation of PKC Activity—PKC activity was determined using PepTag nonradioactive PKC assay kit (Promega) according to the manufacture's instruction. Briefly, cells reaching confluency in 6-well cell culture plates were treated with or without PMA for 20 min and harvested in extraction buffer (25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol, 100 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Nakarai Tesque)). Cell extracts were then sonicated by ultrasonic disruptor (TOMY) for 20 s and centrifuged at 15,000 rpm for 15 min. The supernatants were used for the assay. The reaction was performed at 30 °C for 30 min in assay solution including, PepTag C1 peptide (0.4 µg/µl) and 5 µg of protein sample. The samples were separated on the 0.8% agarose gel and visualized on a transilluminator. The intensity of fluorescence of the phosphorylated peptides were quantified using BioMax 1D image analysis soft ware (Kodak).

Electron Microscopy—Electron microscopy analysis was performed as previously described with minor modifications (38). Briefly, cells were harvested using trypsin-EDTA and fixed by 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C for 2 h. After centrifugation, cells were washed with 0.1 M sodium cacodylate buffer 3 times. Cell pellets were obtained by centrifuge, post-fixed in 1% osmium tetroxide and 1% potassium ferrocyanide at room temperature for 2 h, and processed for embedding in Quetol 812 (Nisshin EM, Tokyo, Japan). Ultrathin sections were stained with uranyl acetate and lead nitrate and observed by a Hitachi H-7500 electron microscope.

Statistical Analysis—All values in the figures are expressed as means ± S.D. To determine statistical significance, the values were compared by two group t tests. The differences were considered as significant if p values were less than 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression of Syn in MG63 Osteosarcoma Cells—To investigate the effect of syn on growth and differentiation of tumor cells, MG63 osteosarcoma cells were stably transfected with -or -syn cDNA. Three high expressors of -syn (clones 1, 5, and 15) and -syn (clones 5, 8, and 13) were selected on the basis of immunoblot analysis together with empty vector-transfected cells (clones v2, v5, and v10) (Fig. 1A). For the majority of experiments, clone 1, the high expresser of -syn, and clones 5 and v2 were used. Immunoblot analysis also showed that the immunoreactivities of both - and -syn were almost exclusively those of their monomers (Fig. 1A). To further analyze expression and distribution of syn proteins, double immunolabeling/LSCM was performed using at least two antibodies for each syn (Fig. 1B). The immunoreactivity of -syn in -syn-overexpressing cells (1) was diffuse in the cell bodies as detected by both monoclonal syn-1 and polyclonal C-terminal antibodies (a and b). Immunoreactivity of -syn in -syn-overexpressing cells (5) was also localized diffusely in the cell bodies by monoclonal anti--syn antibody (e) but was detected with a shift to perinuclear regions by polyclonal anti--syn antibody (f). In none of these cases, inclusion bodies were detected. Notably, expression levels of -syn were heterogeneous (c), and similar patterns were consistently observed in all the three clones of -syn-overexpressing cells (data not shown). By contrast, immunoreactivities of -syn were homogeneous in -syn-overexpressing cells (g). No immunoreactivities of syn proteins were detected in vector-transfected cells (v2) (d and h).

To determine whether cell proliferation was affected by overexpression of syn, the stable clones were evaluated for their cell growth rates. The doubling times of cells in log phase of growth under 10% serum conditions were 20-24 h for -syn-overexpressing cells and 14-17.5 h for both -syn-overexpressing and vector-transfected cells (data not shown). Cells were then incubated under the low serum (0.1%) for 24 h to synchronize at G₁ in cell cycle and then added with 10% serum followed by counting of cell numbers at the indicated times. The result showed that that the average cell numbers of -syn-overexpressing cells at day 4 were significantly decreased compared with both -syn-overexpressing and vector-transfected cells (Fig. 1C).

To determine whether decreased cell proliferation of -syn-overexpressing cells is due to alteration of cell cycle profile, cell cycle analysis was performed using flow cytometry at varying time periods (0, 6, 12, 18, 24, and 96 h) after the addition of 10% serum to serum-deprived cells. The result showed that compared with both -syn-overexpressing and vector-transfected cells, -syn cells in G₀/G₁ phases were significantly increased (p < 0.05), whereas those in both S and G₂/M phase were decreased at 24 and 96 h (p < 0.05) (Fig. 1D). Essentially similar results were observed at 96 h. However, compared with the profile at 24 h, there were more cells in G₀/G₁ phase, fewer cells in S phase, and more cells in G₂/M at 96 h. One possible reason for the difference between 24 and 96 h could be that at 24 h many cells might have not yet reached G₂/M phase. Furthermore, a relatively high ratio of cells in G₀/G₁ phase at 96 h could be due to the decreased nutrients in the culture medium after long time culture. Taken together, these results suggested that overexpression of -syn, but not of -syn, suppressed cell proliferation in MG63 osteosarcoma cells.

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Analysis of syn expression in transfected MG63 osteosarcoma cells. A, immunoblot analysis of syn proteins. Cell extracts were analyzed by immuno-blotting using syn-1 (upper panel), anti--syn monoclonal antibody (middle panel), and anti-actin antibody (lower panel). Three vector-transfected clones (v2, v5, v10, lanes 1-3), three high expresser clones for -syn (1, 5, 15, lanes 4-6), and -syn (5, 8, 13, lanes 7-9) are shown. Recombinant - and -syn proteins were used as positive controls (lanes 10 and 11). B, immunofluorescence/LSCM analysis of syn expression. -Syn-overexpressing cells (1) (a-c), -syn-overexpressing cells (5) (e-g), and vector-transfected cells (v2) (d and h) were immunostained with antibodies against -syn (green, a-d), -syn (green, e-h), and actin (red, c, d, g, and h) followed by observation by LSCM. -Syn was diffusely distributed in the cell bodies with a slight shift to cell membrane as detected by both syn-1 (a) and polyclonal c-terminal antibody (b). -Syn was similarly detected in the cell bodies by monoclonal anti--syn antibody (e), but strong stains of perinuclear regions were observed by polyclonal anti--syn antibody (f). Immunoreactivities of -syn were heterogeneous (c), whereas those of -syn were almost even in all cell populations (g). No immunoreactivities of syn proteins were detected in vector-transfected cells (d and h). Bars represent either 20 µm for high magnification (a, b, e, and f) or 50 µm for low magnification (c, d, g, and h). C, evaluation of cell proliferation. Cells (v2, v5, v10, 1, 5, 15, 5, 8, and 13) were incubated at 1 x 105 cells/well in the 6-well plates under the low serum (0.1%) conditions for 24 h to synchronize. Cells were then treated with 10% serum, and cell numbers were counted at 96 h. Open circles represent the group mean values. Data shown are the mean ± S.D. (n = 3). **, p < 0.01. D, flow cytometry for cell cycle analysis. Cells were prepared as described in C, and DNA contents were analyzed at the indicated times (0, 6, 12, 18, 24, and 96 h) after serum stimulation. The upper figures are a typical cell cycle profile at 24 h for v2, 1, and 5. The x axis represents the fluorescent intensities proportional to the amount of DNA, where as the y axis indicates the cell number. The lower column figures show that G0/G1 phases at 24 and 96 h in -syn cells were significantly increased, whereas those in both S and G2/M phase were decreased compared with both -syn-overexpressing and vector-transfected cells. Data presented are the mean ± S.D. of triple determinations. *, p < 0.05.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Up-regulation of the expression of osteoblastic differentiation markers in -syn-overexpressing MG63 cells. A, measurement of ALP activity. Cells (v2, v5, v10, 1, 5, 15, 5, 8, and 13) under the confluent conditions were harvested, and cell extracts were incubated with p-nitrophenyl phosphate at 37 °C for 90 min. The amount of released nitrophenol was monitored by absorbance at 415 nm. -Syn-overexpressing cells (1) showed significantly higher ALP activity compared with other cell types (v2 and 5). Data shown are mean ± S.D. (n = 5). *, p < 0.05; **, p < 0.01. B, evaluation of ALP activity by staining. Cells (v2, 1, and 5) were stained with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solution and photographed. C, immunostaining of osteocalsin. Cells (v2, 1, and 5) were immunostained with anti-osteocalsin antibody followed by observation with LSCM. The bar represents 50 µm. D, RT-PCR analysis of ALP and osteocalcin mRNAs. Cyclophilin mRNA was used as an internal control. E, in vitro mineralization assay. Cells (v2, v5, v10, 1, 5, 15, 5, 8, and 13) were seeded onto 24-well cell culture plates (1 x 105 cells/ml) and maintained in -minimal essential medium containing 10 mM -glycerophosphate and 50 µg/ml ascorbic acid for 4 weeks. Matrix mineralization was evaluated by either alizarin red staining (middle panel) or von Kossa staining (right panel). Each clone number is indicated in the squire matrix (left panel). Please note that the strongest staining was observed in clone 1, the highest -syn expresser.

To test this possibility, we evaluated ALP since this molecule had been widely used as a marker for osteoblastic differentiation. As we suspected, all three clones of -syn-overexpressing cells displayed significantly higher activity of ALP compared with both -syn-overexpressing and vector-transfected cells (Fig. 2A). Activation of ALP was also assessed by direct stain of cells using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as substrate. The result showed that the intensity of the staining in -syn-overexpressing cells was much stronger than those of -syn-overexpressing and vector-transfected cells (Fig. 2B). Next, we analyzed expression of osteocalcin, another marker for osteoblastic differentiation. The immunofluorescence/LSCM study showed that immunoreactivity of osteocalcin was strongly observed in -syn-over-expressing cells but not in other cell types (Fig. 2C). Furthermore, RT-PCR analysis revealed that up-regulation of ALP and osteocalcin in -syn-overexpressed cells might be attributed to increased transcription (Fig. 2D). To further corroborate that cellular differentiation is accelerated in -syn-overexpressing cells, we evaluated formation of mineral deposition in the matrix that may mimic the calcification by osteoblasts in vivo. For this purpose, cells were incubated in the presence of -glycerophosphate and ascorbic acid for 4 weeks followed by staining with alizarin red to assess calcium incorporation. The result showed that mineralization was clearly detectable in -syn-overexpressing cells but not in -syn-overexpressing nor vector-transfected cells (Fig. 2E). Similar results were obtained by von Kossa staining for the assessment of bone nodule formation (Fig. 2E). Taken together, -syn-overexpressing cells specifically exhibited a differentiated phenotype compared with both -syn-overexpressing and vector-transfected cells.

Alteration of G₁ Cell Cycle Regulators and Decreased Proteasome Activity in -Syn-overexpressing MG63 Cells—Because prolonged G₁ period in cell cycle is prerequisite for cell entry into G₀ and further differentiation, it was predicted that expressions and activities of various G₁ cell cycle regulators might be altered in -syn-overexpressing cells.

To test this possibility, cells were incubated under the low serum (0.1%) conditions for 24 h to synchronize at G₁ followed by 10% serum treatment. Cells were then harvested, and expression of various cell cycle regulators were analyzed at the indicated times (Fig. 3A). Notably, expression of the cyclin-dependent kinase inhibitor p21, one of the key molecules which negatively regulate cell cycle progression from G₁ to S phase, was significantly up-regulated in -syn-overexpressing cells. In these cells p21 was constitutively expressed without serum stimulation and was further increased in response to serum, reaching the maximum around 6-12 h followed by gradual decrease. By contrast, in vector-transfected cells expression of p21 was transiently up-regulated at 6 h and immediately decreased. Consistent with this, phosphorylation of Rb protein was compromised in -syn-expressing cells compared with vector-transfected cells. Furthermore, cyclin B1, a marker for G₂/M phase, was up-regulated earlier in vector-transfected cells than in -syn-overexpressing cells. As for other cyclin-dependent kinase inhibitors, p15 level was slightly elevated at 24- and 48-h time points, whereas expression of p27 was not up-regulated in -syn-overexpressing cells compared with vector-transfected cells (Fig. 3A and data not shown). Expression of other cell cycle regulators such as cyclin D1 and cyclin-dependent kinase 4 were not much different between both cell types. Similar expression patterns of p21 and phosphorylated Rb were observed between -syn-overexpressing cells and vector-transfected cells (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Altered expression of cell cycle regulators and decrease of proteasome activity in -syn-overexpressing MG63 cells. A, immunoblot analysis of cell cycle regulators. Vector-transfected (v2) and -syn-overexpressing cells (1) were incubated under the low serum (0.1%) conditions for 24 h to synchronize and then treated with 10% serum for the indicated times (6, 12, 24, and 48 h). Cells were then harvested, and cell lysates were analyzed by immunoblotting with anti-p21, Rb, phospho Rb, cyclin (cyc) B, cyclin D1, cyclin-dependent kinase (Cdk4), p15, and ubiquitin. B, evaluation of the stability of p21 protein by cycloheximide experiments. The synchronized cells (v2 and 1) were treated with 10% serum with or without cycloheximide (CHX) for the indicated times (1, 2, and 4 h), and cell lysates were analyzed by immunoblotting (upper panel). Each band was quantified using BioMax 1D image analysis soft ware (lower panel). Data shown are the mean ± S.D. Similar results were obtained by three independent experiments. C, measurement of proteasome activity. Cells (v2, 1, and 5) were incubated under the low serum (0.1%) conditions for 24 h and then treated with 10% serum for the indicated times (6 and 12 h). To evaluate proteasome activity, cell extracts (10 µg) were incubated with fluorogenic substrate (benzyloxycarbonyl-Leu-Leu-Glu-amidomethylcoumarin) at 37 °C. Released fluorescence (excitation 380 nm, emission 460 nm) was monitored by 5 min intervals up to 60 min. The fluorogenic value at each time point was plotted, and the slope was calculated. Data shown are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. D, immunoblot analysis of ubiquitylated proteins. Vector-transfected (v2) and -syn-overexpressing cells (1) were prepared as described in A and analyzed by immunoblotting using anti-ubiquitin antibody. Both types of cells treated with lactacystin for 6 h were included as positive controls.

To further determine whether increased stability of p21 was due to compromised degradation by UPS in -syn-overexpressing cells, syn-transfected cells were analyzed for proteasome activities. The result showed that the proteasome activities of -syn-overexpressing cells were decreased to 50% of those of vector-transfected cells, whereas those of -syn-overexpressing cells were little affected (Fig. 3C). Under the same experimental conditions, immunoreactivities of polyubiquitylated proteins were significantly stronger in -syn-overexpressing cells compared with those in vector-transfected and -syn-overexpressing cells (Fig. 3D and data not shown). Taken together, expression of cell cycle regulators such as p21 and phosphorylated Rb were altered in -syn-overexpressing cells, which might be attributed to the decreased proteasome activity.

Proteasome Inhibitors Stimulate Differentiation of Wild-type MG63 Cells—If suppression of proteasome activity by -syn plays a causative role for stimulation of differentiation in MG63 osteosarcoma cells, then it is possible that down-regulation of proteasome activity by other experimental procedures might similarly stimulate differentiation in these cells.

To test this possibility, wild-type MG63 cells were treated with proteasome inhibitors, including MG132 and lactacystin followed by evaluation of cellular differentiation. Immunoblot analysis revealed that administration of these reagents at 10 µM clearly up-regulated p21 expression at 12 h (Fig. 4A). Under the same experimental conditions, ALP activity was significantly up-regulated, and immunoreactivity of osteocalcin became detectable (Fig. 4B and data not shown). In addition, the expression of ALP and osteocalcin mRNA was up-regulated in lactacystin-treated cells (Fig. 4C). Furthermore, cells were induced to form matrix mineralization with lower concentrations of proteasome inhibitors (0.01-0.1 µM for MG132 and 0.1-1.0 µM for lactacystin) in long-term cultures. High concentration (10 µM) treatment of these reagents caused prominent cell death within 48 h (data not shown). By contrast, sublethal concentrations (0.01-0.1 µM) of rotenone, an inhibitor of mitochondria complex I, caspase I, and III inhibitors (0.1-1.0 µM), and leupeptin (1-10 µM) had little effect on cellular differentiation (Fig. 4D). Taken together, treatment of wild-type MG63 cells with proteasome inhibitors resulted in stimulation of cellular differentiation.

Down-regulation of PKC Activity in -Syn-overexpressing MG63 Cells—Our hypothesis was that alteration of signal transduction might be involved in the suppression of proteasome activity in -syn-overexpressing cells. In this regard we especially focused on the potential role of PKC since it was recently shown that treatment of skeletal muscle by phorbol ester resulted in stimulation of proteasome activity (39) and it was previously shown that -syn bound with PKC and down-regulated the activity of PKC in -syn overexpressing neuroblastoma cells (40).

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Proteasome inhibitors induce cellular differentiation in wild-type MG63 cells. Wild-type MG63 were incubated under the low serum (0.1%) conditions for 24 h and then treated with 10% serum in the presence of either MG132 or lactacystin. A, immunoblot analysis of p21. Cells were treated with various concentrations of either MG132 (0, 0.1, 1.0, 10 µM, lanes 3-6) or lactacystin (0, 1.0, 10, 20 µM, lanes 7-10) and harvested at 12 h. Cell lysates were analyzed by immunoblotting using anti-p21 antibody. Extracts of 293T cells transfected with or without p21 expression vector are shown as controls (lanes 1 and 2). B, measurement of ALP activity. Cells were treated with various concentrations (0, 10, 20 µM) of lactacystin for 12 h. To evaluate ALP activity, cell extracts were incubated with p-nitrophenyl phosphate at 37 °C for 90 min. The amount of released nitrophenol was monitored by the absorbance at 415 nm. Data shown in the left panel are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. C, RT-PCR analysis of ALP and osteocalcin mRNAs. Cells were treated with lactacystin (10 µM) for 24 h. Cyclophilin mRNA was used as an internal control. D, in vitro mineralization assay. Cells were treated with various reagents, including MG132 (0, 0.01, 0.1 µM), lactacystin (0, 0.1, 1.0 µM), rotenone (0, 0.1, 0.1 µM), caspase I inhibitor (0, 0.1, 1.0 µM), caspase III inhibitor (0, 0.1, 1.0 µM), and leupeptin (0, 1, 10 µM) for 4 weeks. Matrix mineralization was evaluated by alizarin red staining. Please note that positive staining was observed for cells treated with proteasome inhibitors. Acridine orange/ethidium bromide staining revealed that cells were alive during mineralization (not shown).

Next, to further determine whether -syn colocalize with PKC, cells were double-immunolabeled with anti--syn rabbit polyclonal antibody and anti-PKC antibody followed by observation with LSCM. As shown in Fig. 5B, immunoreactivities of -syn and PKC considerably overlapped in the cytoplasm of -syn-overexpressing cells. We observed that PKC and PKC were highly expressed in addition to moderate expression of other PKC family of peptides in MG63 cells (data not shown). Similar results were obtained by both co-immunoprecipitation and double immunolabeling/LSCM studies for PKC (data not shown).

Finally, to determine whether PKC activity was compromised in -syn-overexpressing cells, the syn-transfected cells were treated with or without PMA and analyzed for their PKC activities (Fig. 5C). The result showed that proteasome activities of -syn-overexpressing cells were significantly lower than those of -syn-overexpressing and vector-transfected cells. Importantly, PMA treatment of -syn-overexpressing cells restored the PKC activity to the basal levels of those in other cell types. Taken together, these results suggested that -syn might directly suppress the PKC activity in -syn-overexpressing cells.

Because previous studies suggested an alternative possibility that -syn bound with S6 subunit of 19 S proteasome, leading to interference with proteasome functions (41-43), we investigated the association of -syn with S6a. The results of co-immunoprecipitation experiment confirmed the association of these molecules (Fig. 5A). Double immunolabeling/LSCM showed that overlapping of the immunoreactivities of these molecules was partially detected in the cell bodies, since S6a was considerably localized in the nucleus (Fig. 5B).

Phorbol Ester Treatment of -Syn-overexpressing MG63 Cells Restores Proteasome Activity and Suppresses Cellular Differentiation—If suppression of PKC activity by -syn is causative for down-regulation of proteasome activity and enhanced differentiation in -syn-overexpressing cells, then stimulation of PKC might restore the proteasome activity and abrogate cellular differentiation in these cells.

Accordingly, -syn-overexpressing cells were treated with phorbol ester followed by evaluation of proteasome activity. The results showed that the compromised activity of proteasome in -syn-overexpressing cells was significantly improved by treatment with PMA both at 10 and 50 nM but not with an inactive analogue 4PDD (Fig. 6A). On the other hand, although proteasome activity of vector-transfected cells was increased by treatment with 50 nM PMA, there were little effects observed at 10 nM PMA (Fig. 6A). Thus, -syn-overexpressing cells responded more sensitively to PMA treatment, suggesting the effect of PMA on proteasome was more specific for -syn-overexpressing cells, which showed decreased proteasome activity. The stimulatory effects of PMA on the proteasome activity in -syn-overexpressing cells reached the maximum at 50 nM (data not shown) and were completely abrogated in the presence of PKC inhibitor, chelerythrine chloride (Fig. 6A), implying that PMA stimulated proteasome activity through phosphorylation but not through depletion of PKC.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Association of -syn with PKC and down-regulation of PKC activity in -syn-overexpressing MG63 cells. A, co-immunoprecipitation (IP) of -syn with either PKC (upper) or S6a (middle). Vector-transfected (v2) and -syn-overexpressing cells (1) were harvested, and cell extracts (300 µg) were immunoprecipitated with anti--syn antibody or mouse IgG control followed by immunoblotting (IB) with either anti-PKC (upper), anti-S6a (middle), or syn-1 (low). Cell extracts (5% of input) were used as positive controls. B, immunofluorescence/LSCM of -syn association with either PKC or S6a in -syn-overexpressing cells. Cells (1) were doubly stained with anti--syn antibody (green) and either anti-PKC or S6a (red) and observed by LSCM. Please note that -syn (red) and PKC (green) were well co-localized in the cell bodies (c). Bars represent 50 µm. C, measurement of PKC activity. Exponentially growing cells (v2, 1, and 5) were treated with or without PMA for 20 min, and cell extracts (10 µg) were analyzed for PKC activity using fluorogenic peptides as substrates as described under "Experimental Procedures." Data shown are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. DMSO, dimethyl sulfoxide.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Phorbol ester treatment restores proteasome activity and abrogates differentiation in -syn-overexpressing MG63 cells. A, measurement of proteasome activity. Vector-transfected (v2) and -syn-overexpressing cells (1) were incubated under the low serum (0.1%) conditions for 24 h and then treated with 10% serum in the presence of any of vehicle (0.1% Me2SO (DMSO)), PMA (10 and 50 nM), 4PDD (50 nM), chelerythrine chloride (PKC inhibitor, 50 nM), or PMA (50 nM) plus chelerythrine chloride (50 nM). Cell extracts (5 µg) were harvested at 12 h after treatment and incubated with fluorogenic proteasome substrate at 37 °C. Released fluorescence (excitation, 380 nm; emission, 460 nm) was monitored each 5 min up to 60 min. Fluorogenic intensity of each time point was plotted, and the slope was calculated. Data shown are the mean ± S.D. (n = 3). **, p < 0.01. B, measurement of ALP activity. Vector-transfected (v2) and -syn-overexpressing cells (1) were treated as described in A. Cell extracts at 12 h treatment were incubated with p-nitrophenyl phosphate at 37 °C for 90 min. The amount of released nitrophenol was monitored by the absorbance at 415 nm. Data shown are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. C, in vitro mineralization assay. -Syn-overexpressing cells (1) were treated with either PMA or 4PDD at the indicated concentrations (0, 10, 20, 50 nM). Matrix mineralization was then evaluated by alizarin red staining. Please note that the staining was mitigated by PMA but not by 4PDD. Similar results were obtained by three independent experiments.

Up-regulation of Lysosomal Activity in -Syn-overexpressing MG63 Cells—Because a recent study has shown that autophagy-lysosomal pathway may play an important role for aggregate-prone proteins, including -syn and Huntingtin (44), it is reasonable to speculate that down-regulation of proteasome activity by -syn might be further modulated by this pathway.

View larger version (91K): [in this window] [in a new window]   FIGURE 7. Up-regulation of lysosomal activity in -syn-overexpressing MG63 cells. A, measurement of cathepsin B activity. Cell extracts (10 µg) were prepared as described under "Experimental Procedures" were analyzed for cathepsin B activity using fluorogenic substrates. Released fluorescence (excitation, 380 nm; emission, 460 nm) was monitored each 5 min up to 60 min. Fluorogenic intensity of each time point was plotted, and slope was calculated. Data shown are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. B, immunoblot analysis of cathepsin B. Cell extracts (v2, 1, and 5) were analyzed by immunoblotting using anti-cathepsin B antibody (upper panel) and anti-actin antibody (lower panel). C, electron microscopic analysis. Typically, -syn-overexpressing cells (1: a, c, e, and f) exhibited numerous enlarged electron-dense lysosomes (white arrowheads), vacuoles that might have already discharged their contents (white arrows), autolysosome-like body (black arrow), and myelinosome-like structures (black arrowheads). The enclosed area in panel e is magnified in panel f. Fewer lysosomal structures were found in vector-transfected cells (v2, b) and -syn-overexpressing cells (5, d). Bars represent 8 µm (a and b, x1500), 2 µm (c and d, x5000), 1 µm (e, x13000), or 0.5 µm (f, x26000).

Autophagy-lysosomal Inhibitor Treatment Results in Down-regulation of Proteasome Activity, Leading to Acceleration of Cellular Differentiation in -Syn-overexpressing MG63 Cells—If up-regulation of lysosomal activity in -syn-overexpressing cells may reflect the compensatory mechanism against the increased level of -syn, then suppression of autophagy-lysosomal pathways may result in compromised clearance of -syn, leading to decrease proteasome activity and accelerate cellular differentiation in those cells.

We first evaluated expression levels of - and -syn proteins in the presence of autophagy-lysosomal inhibitors. The results of immunoblot analysis showed that - and -syn proteins were up-regulated by macroautophagy inhibitor 3-MA as well as by ammonium chloride, which inhibits lysosomal activity independently of the form of autophagy. By contrast, proteasome inhibitor lactacystin had little effect on the expression of both - and -syn proteins (Fig. 8A). Thus, these results suggested that - and -syn were preferentially degraded by autophagy-lysosomal pathway.

Then, to determine whether down-regulation of autophagy-lysosomal activity affects proteasome function, proteasome activity was evaluated by treatment with autophagy-lysosomal inhibitors. The result showed that treatments with autophagy-lysosomal inhibitors significantly decreased proteasome activities in -syn-overexpressing cells but not in other cell types (Fig. 8B). By contrast, inhibition of proteasome by lactacystin had little effect on cathepsin B activity (data not shown). Similarly, treatment of PMA, which was shown to stimulate proteasome activity (Fig. 6), had little effects on cathepsin B activity (data not shown).

Finally, it was shown that treatment with both 3-MA and ammonium chloride significantly stimulated ALP activity, osteocalcin expression, and matrix mineralization in -syn-overexpressing cells compared with other cell types (Fig. 8, C and D, data not shown). Taken together, inhibition of autophagy-lysosomal activity in -syn-overexpressing cells resulted in down-regulation of proteasome activity, leading to stimulate cellular differentiation. Thus, these results suggest that autophagy-lysosomal pathway may mitigate the down-regulation of proteasome activity by -syn, therefore leading to abrogate cellular differentiation by -syn.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Although -syn was previously shown to be expressed in the brain tumors that preferentially displayed differentiation rather than proliferation, little has been documented regarding the role of -syn for tumor differentiation. The present study showed that -syn-overexpressing MG63 osteosarcoma cells specifically exhibited phenotype of osteoblastic differentiation. Compared with both -syn-overexpressing and vector-transfected cells, -syn-overexpressing cells were characterized by decreased growth rates, associated with increased expression of p21 and reduced phosphorylated Rb (Figs. 1 and 3), indicating that the progression from G₁ to S phase was compromised in these cells. The prolonged G₁ phase would be prerequisite for transition from G₁ to G₀ and subsequent cellular differentiation. Consistent with this view, osteoblastic differentiation markers, including ALP and osteocalcin, were significantly up-regulated in -syn-overexpressing cells compared with other cell types (Fig. 2). Furthermore, -syn-overexpressing cells, but not other cell types, were induced to form matrix mineralization during long-term cultures (Fig. 2). Taken together, our results using osteosarcoma cells support the contention that -syn might stimulate tumor differentiation.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Autophagy-lysosome inhibitor treatment decreases proteasome activity and stimulates cellular differentiation in -syn-overexpressing MG63 cells. A, immunoblot analysis of syn proteins. Cells (1, and 5) were incubated under the low serum (0.1%) conditions for 24 h and then treated with 10% serum in the presence of lactacystin (10 µM), ammonium chloride (NH4Cl) (20 mM), and 3-MA (10 mM) for 24 h. Cell lysates were analyzed by immunoblotting using syn-1 (upper panel) and anti--syn monoclonal antibody (low panel). Blots were reprobed with anti-actin antibody. Similar results were obtained by three independent experiments. B, measurement of proteasome activity. Cells (v2, 1, and 5) were prepared as described in A. Cell extracts (5 µg) were harvested and incubated with fluorogenic proteasome substrate at 37 °C. Released fluorescence (excitation, 380 nm; emission, 460 nm) was monitored each 5 min up to 60 min. Fluorogenic intensity of each time point was plotted, and slope was calculated. Data shown are the mean ± S.D. (n = 3). Please note that proteasome activity was significantly decreased in -syn-overexpressing cells by ammonium chloride treatment (*, p < 0.05). C, measurement of ALP activity. Vector-transfected (v2) and -syn-overexpressing cells (1) were prepared as described in A. Cell extracts at 12 h of treatment were incubated with p-nitrophenyl phosphate at 37 °C for 90 min. The amount of released nitrophenol was monitored by the absorbance at 415 nm. Data shown are the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. D, in vitro mineralization assay. Vector-transfected (v2) and -syn-overexpressing cells (1) were treated with ammonium chloride (10 mM), 3-MA, (10 mM), or lactacystin (1 µM) for 2-4 weeks (2W-4W). Matrix mineralization was then evaluated by alizarin red staining. Please note that the staining was stimulated by ammonium chloride and to a lesser extent by 3-MA in -syn-overexpressing cells but not in vector-transfected cells. Similar results were obtained by three independent experiments.