Recombinant human insulin-like growth factor I (IGF-I; 291-G1; 10 ng/ml) and transforming growth factor-β2 (TGF-β2; 302-B2-010; 10 ng/ml) were obtained from R&D Systems. A selective inhibitor of ERK1/2 (U0126; 10 μM; Cell Signaling Technology, Inc.) and allosteric inhibitor of IGF-IR (AG1024; 1 µM; Sigma-Aldrich; Merck KGaA) were used in the present study for 1 h. Primary antibodies from Santa Cruz Biotechnology, Inc. were used, and these included anti-lumican (sc166871; mouse monoclonal; 1/100 dilution for western blot analysis or 1/50 for immunofluorescence), anti-β-catenin (sc7963; mouse monoclonal; 1/300 dilution), anti-ERK1/2 (sc514302; mouse monoclonal; 1/200 dilution), anti-IGF-IR (sc81464; mouse monoclonal; 1/100 dilution), anti-pERK1/2 (sc136521; mouse monoclonal; 1/100 dilution), anti-Smad2 (sc6200; goat polyclonal; 1/200 dilution) and anti-pSmad2 (sc101801; rabbit polyclonal; 1/200 dilution). In addition, anti-actin (MAB1501; mouse monoclonal; 1/5,000 dilution; EMD Millipore), anti-p-IGF-IR (PA5-37602; polyclonal rabbit; 1/500 dilution; Thermo Fisher Scientific, Inc.), keratan sulfate (KS; 270427; mouse monoclonal; 1/1,000 dilution; Seikagaku Corporation) and keratanase II (100812; 0,005 µ/ml; Seikagaku Corporation) were utilized. Secondary-HRP antibodies anti-rabbit (AP182PR) and anti-mouse (AP192PM) were used at a 1/5,000 dilution and obtained from Millipore.

In the present study, the HTB94 (ATCC^® HTB-94™) human chondrosarcoma cell line was utilized. Cells were grown in DMEM (Biosera AG; LM-D1111) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.; 10500-064; heat-inactivated), gentamycin (Invitrogen; Thermo Fisher Scientific, Inc.; 15710-049) and penicillin/streptomycin (100units/ml; Biosera LMA4118). Prior to the addition of treatments, cells were cultured in serum-free medium for 24 h at 37°C and 5% CO₂. Inhibitors, when used (ERK inhibitor or IGF-IR inhibitor), were added 1h prior to growth factor treatment.

Following the treatments, the cells were harvested, and 10,000 cells/well were seeded onto fibronectin (FN)-coated 96-well plates for 1 h. According to the manufacturer's instructions, the number of adherent cells was measured using the CyQUANT fluorometric assay (Molecular Probes; Thermo Fisher Scientific, Inc.). Fluorescence was measured on a Fluorometer (BioTek Instruments, Inc.) using the proposed excitation (485 nm) and emission filters (528 nm), as previously described (25). A separate standard curve was used to convert fluorescence units to cell numbers. All adhesion experiments were repeated at least 3 times and performed in triplicate.

HB94 cells were seeded in 24-well culture plates at a concentration of 10×10⁴ cells per well. The optimal concentration for plating was selected so that the cells would be confluent at almost 100% after 72 h of culture at 37°C and 5% CO₂. RNA interference was performed according to the protocol described in the section below entitled 'Transfection with siRNA' using siRNAs specific for lumican, or decorin, or biglycan. Serum-free medium was utilized for culture. The cell layer was scratched with a sterile 10 µl pipette tip. Detached cells were removed by washing the cell layer twice with serum-free medium. The wound closure was monitored at 6 and 12 h with a digital image processor connected to a microscope (Leica DMIL), at 5 different positions across the wound. Cell motility was quantified by ImageJ analysis (ImageJ 1.4.3.67 launcher; Symmetry Software).

Growing cells from confluent cultures were harvested and seeded in black 96-well plates (3603; Corning, Inc.) at a density of 5,000 cells per well in 200 µl of DMEM (10% FBS). The cell density number was selected from optimization experiments (data not shown). The cells were allowed to rest overnight. If necessary, transfection with short interfering RNAs (siRNAs) was performed in a serum-free medium without antibiotics for 24 h. This was then replaced with fresh medium (0% FBS) with antibiotics. All treatments (inhibitors and/or growth factors) were added for the next 24 h at 37°C and 5% CO₂ in 0% FBS. The cells were then lysed, and their number was calculated using the CyQUANT fluorometric assay (C7026; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Fluorescence was measured in a Fluorometer (BioTek Instruments, Inc.) using the proposed excitation (485 nm) and emission filters (528 nm). A separate standard curve was used to convert fluorescence units to cell numbers. All experiments were performed in triplicate.

Growing cells from confluent cultures were harvested and seeded in 96-well plates at a density of 12,000 cells per well in 200 µl of DMEM (10% FBS). The cells were allowed to rest overnight. Transfection with siRNAs was performed in a serum-free medium without antibiotics for 24 h (siLum) or 12 h (siDec, siBig). This medium was then replaced with fresh medium (0% FBS) and cells were incubated at 37°C and 5% CO₂ in 0% FBS for the following 24 h. The Vybrant MTT Cell Proliferation Assay (cat. no. 11465007001; Roche Diagnostics) was performed according to the manufacturer's instructions. In brief, the medium was replaced with 100 μl of fresh medium (0% FBS) and 10 µl of MTT stock solution (12 Mm) were added to each well. Following 4-h incubation at 37°C, 50 µl of DMSO was added to cells for the next 10 min. The absorbance was measured at 540 nm using a Synergy HTX multimode microplate reader (BioTek). A separate standard curve was used to convert absorbance to cell number. All experiments were performed in triplicate.

For transfection experiments, the cells were plated in serum- and antibiotic-free medium in either 96-well plates (5,000 cells/well) or T25 flasks (1:8 dilution of a 90% confluent T75 flask). siRNA specific for lumican (siLum; stealth siRNAs HSS106200; Invitrogen; Thermo Fisher Scientific, Inc.), decorin (siDec; (stealth siRNAs HSS102673; Invitrogen; Thermo Fisher Scientific, Inc.) and biglycan (siBig; stealth siRNAs HSS184531; Invitrogen; Thermo Fisher Scientific, Inc.) and RNAi negative control (siScr; medium GC content negative control; Invitrogen; Thermo Fisher Scientific, Inc.). For transfection, siRNA, and Lipofectamine 2000 (11668-027; Invitrogen; Thermo Fisher Scientific, Inc.) were diluted in Opti-MEM I Reduced Serum Medium (31985-070; Invitrogen; Thermo Fisher Scientific, Inc.). Following 5 min of incubation at room temperature, diluted Lipofectamine 2000 was mixed with diluted siRNA (100 nm) for 20 min at room temperature to allow siRNA-liposome complexes to form and added to cell layers. Transfection was allowed to take place during 24 h for siLum or 12 h for siDec and siBig, when the medium was replaced with fresh (0% FBS) containing antibiotics and the incubation period continued for 24 or 36 h, respectively. Cells were then harvested, and mRNA or protein was extracted. When necessary, treatments were performed at the 24 h point after the initial transfection period. All transfection experiments were repeated at least 3 times and performed in triplicate.

According to the manufacturer's instructions, total ribonucleic acid isolation was performed using TRIzol (15596026; Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was added for cDNA synthesis using the Takara (RR037A) RT cDNA synthesis kit. For semi-quantification of the genes of interest, qPCR reactions were performed on a Mx300P cycler using the Universal qPCR kit (KK4602; KAPA Biosystems) in a total volume of 20 µl. The thermocycling conditions were as follows: 94°C for 15 min, 40 cycles at 94°C for 20 sec, 55°C for 30 sec, 72°C for 30 sec, 72°C for 10 min. The PCR primer sequences were as follows: GAPDH forward, 5′-GGAAGGTGAAGGTCGGAGTCA-3′ and reverse, 5′-GTCATTGATGGCAACAATATCCACT-3′; lumican forward, 5′-CTTCAATCAGATAGCCAGACTGC-3′ and reverse, 5′-AGCCAGTTCGTTGTGAGATAAAC-3′; decorin forward, 5′-TCAATGGACTGAACCAGATGA-3′ and reverse, 5′-CCTTGA GGAATGCTGGTGAT-3′; biglycan forward, 5′-TCTGAA GTCTGTGCCCAA-3′ and reverse, 5′-TCTGAGATGCGCAG GTA-3′; p53 forward, 5′-CGTCGTGGCTTCTTGCAT TC-3′ and reverse, 5′-AAGACCTGCCCTGTGCAGC-3′; p21WAF1/ CIP1 forward, TGGAGACTCTCAAGGTCGAAA-3′ and reverse, 5′-AAGATCAGCCGGCGTTTG-3′. Standard curves were run in each optimized assay, which produced a linear plot of the threshold cycle Ct (dRn) against initial quantity (copies). The amount of each target was semi-quantified based on the concentration of the standard curve and was presented as arbitrary units. GAPDH was utilized as a housekeeping gene.

The total protein secreted into the serum-free culture medium was concentrated using Amicon Ultra 15ml (UFC901024; 10 kDa cut-off) centrifugal concentrator tubes. The initial volume of 3ml serum-free medium collected from culture, to isolate secreted proteins, was concentrated to a final volume of 500 µl whereas, harvested cells were lysed with RIPA solution (50 mM Tris-HCl, 1% NP-40, 0.25% Na-Deoxycholate, 150 mM NaCl, 1 mM EDTA with protease and phosphatase inhibitors). Equal amounts of protein, either cell extracts or secreted, were subjected to SDS-PAGE using 10% polyacrylamide gels under reducing conditions. Separated protein bands were transferred to nitrocellulose membranes in 10 mM CAPS (pH 11), containing 10% methanol. Membranes were blocked overnight at 4°C with PBS containing 0.1% Tween-20 (PBS-Tween) and 5% (w/v) low-fat milk powder. The membranes were incubated for 1 h at room temperature with the primary antibodies in PBS containing 0.1% Tween-20 (PBS-Tween) and 1% (w/v) low-fat milk powder. The immune complexes were detected following incubation with the appropriate peroxidase-conjugated secondary antibody diluted (1:5,000) in PBS-Tween, 2% low-fat milk for 1 h at room temperature, using the LumiSensor Chemiluminescent HRP substrate kit (Genscript; L00221V500), according to the manufacturer's instructions. The protein expression of actin was used to correct for the amount of each sample analyzed using ImageJ Analysis Software.

HTB94 cells were seeded on round coverslips placed in 24-well plates, at a concentration of 70,000 cells/well, and incubated in complete medium for 24 h. Subsequently, the cells were incubated for 48 h at 37°C and 5% CO₂ in 0% FBS medium. The cells were fixed with 5% formaldehyde and 2% sucrose in PBS for 10 min at room temperature. Following 3 washes with PBS supplemented with 1% FBS, the permeabilizing agent Triton X-100 was added for 10 min and then washed prior to the addition of the primary antibody for 1h at room temperature. Coverslips not incubated with the primary antibody were utilized as negative controls. The coverslips were rewashed and incubated for 1 h, in the dark at room temperature, with anti-mouse Alexa Fluor 488 (A21206; 1/200 dilution; Molecular Probes). TO-PRO-3 iodide (Molecular Probes; T3605) diluted 1:300 in de-ionized H₂O was applied for 40 min to stain the nuclei. The coverslips were then placed onto slides using glycerol as a mountant and visualized using confocal microscopy.

Statistical significance was evaluated using a Student's t-test, or one-way ANOVA analysis of variance with Tukey's post-test, using GraphPad Prism (version 4.0) software.

The expression of decorin, biglycan and lumican in HTB94 chondrosarcoma cells was estimated at the mRNA and protein level. The results of RT-qPCRdemonstrated that the HTB94 chondrosarcoma cells expressed lumican, decorin and biglycan at the mRNA level (Fig. 1A). The lumican transcripts were several fold higher than the low expression levels of decorin and biglycan (Fig. 1A). GAPDH was utilized as a housekeeping gene. To determine SLRP expression in HTB94 cells at the protein level, total protein was extracted from the cell culture medium, as well as from harvested cells. Western blot analysis of the proteins secreted by the HTB94 cells using specific antibodies revealed that the 3 SLRPs were secreted at discrete levels (Fig. 1B). The most abundant SLRP secreted by the HTB94 cells was lumican, with decorin and biglycan exhibiting low levels of expression, as demonstrated by densitometric analysis (Fig. 1C) and by transcript data. In all cases, in addition to the approximately 40 kDa band representative of the protein core, bands of higher molecular weight were present, indicating that the 3 SLRPs are also secreted as variously glycosylated protein products (Fig. 1B). In continuation, the present study focused on lumican, the main member of the SLRPs, secreted by the HTB94 chondrosarcoma cells. As shown in Fig. 1C, the bands representing glycosylated lumican were mostly localized in the 55 to 80 kDa molecular weight range. The monoclonal antibody (5D4) specific for KS chains was utilized to characterize the type of lumican substitution. To further evaluate the production of lumican by the HTB94 cells, respective cell extracts were probed with an anti-lumican antibody. Western blot analysis (Fig. 1D) revealed lumican specific bands of various molecular weights indicative of different stages of protein glycosylation. The utilization of fluorescence likewise demonstrated the deposition, of under synthesis, lumican to the cytoplasm of HTB94 cells (Fig. 1E). As shown in Fig. 2, the use of the 5D4 antibody gives the specific 55-80 kDa band pattern, identical to that obtained upon probing with the anti-lumican antibody. In continuation, secreted proteins were subjected to enzymic treatments with keratanase II, which cleaves within sulfated lactosamine residues, and subjected to SDS-PAGE and western blot analysis with anti-KS antibody to confirm the nature of the carbohydrate component. The specific KS-reaction was markedly attenuated in the samples treated with keratanase II, demonstrating that lumican secreted by HTB94 was partially substituted with KS (Fig. 2).

The HTB94 cells were transfected with lumican-, decorin- and biglycan-specific siRNAs to estimate their putative biological roles. The downregulation of SLRP mRNA expression was verified by RT-qPCR. Transfection of the HTB94 cells with siLum, siDec, and siBig for 24h resulted in the potent inhibition of mRNA expression (P≤0.001), respectively (Fig. 3A). The downregulation of lumican, decorin and biglycan transcripts was followed by a significant decrease in lumican protein secretion, as demonstrated by western blot analysis (Fig. 3B and C).

The downregulation of lumican secretion following transfection of the HTB94 cells with lumican siRNA resulted in the potent inhibition of growth as compared to the cells transfected with scramble siRNA (P≤0.001; Fig. 4). However, the downregulation of the low endogenous expression of decorin and biglycan did not affect the growth of the HTB94 cells, as shown by the CyQUANT fluorometric and MTT assays (P=NS) (Fig. 4).

SLRPs have previously been shown to affect cell motility and adhesion by modulating the cell-matrix interaction of cells (26). The present study thus examined the effect of endogenous decorin, biglycan and lumican production on the motility of HTB94 cells using a 'wound healing' assay and transfection with siRNA, as previously demonstrated (26). In the present study, the HTB94 cells, however, migrated with equal efficiency in the presence or absence of decorin, biglycan and lumican siRNA, indicating that these SLRP members do not affect their migratory capabilities (Fig. 5A and B). In continuation, the adhesion ability of these cells was examined utilizing a 96-well plate adhesion assay, as previously described (27). The results demonstrated that transfection with decorin, biglycan and lumican siRNA did not affect the ability of the cells to attach to the fibronectin substrate (Fig. 5C).

TGF-β2 has previously been shown to negatively mediate the growth of physiological chondrocytes (28,29) and human chondrosarcoma cells (30). In the present study, treatment of the HTB94 cells with TGF-β2 resulted in a potent decrease in growth (P≤0.001; Fig. 6A). As SLRP members may affect cell proliferation by modulating TGF-β2 signaling, both lumican and scramble siRNA-transfected cells were treated with TGF-β2, and cell proliferation was measured. The lumican siRNA-transfected HTB94 cells treated with TGF-β2 exhibited lower growth rates than the siScr-transfected cells treated with TGF-β2, suggesting an additive effect of lumican on the TGF-β2-dependent decrease in cell growth. Therefore, the difference in growth between the lumican- and scramble siRNA-transfected cells treated with TGF-β was attributed to the effect of lumican. These results suggest that the regulation of HTB94 cell proliferation by lumican is TGF-β2-independent.

To further examine the putative interaction between lumican and TGF-β2 in HTB94 cells growth, the TGF-β2 activation of Smad2, an established downstream mediator of TGF-β2 action (31), was assessed. To characterize Smad2 expression and phosphorylation levels, specific anti-Smad2 and anti-phospho-Smad2antibodies were used. As was expected, treatment ofthe HTB94 cells with TGF-β2 enhanced the Smad2 phosphorylation levels (Fig. 6B and C). In continuation, we examined the possible effects of lumican downregulation on Smad2 activation. Western blot analysis showed that neither Smad2 protein expression (specific 65 kDa band) nor the expression of phospho-Smad2 (specific 60 kDa band) was affected by lumican downregulation (Fig. 6B and C). Exogenously added TGF-β2 increased Smad 2 phosphorylation to a similar extent in both scramble and lumican-deficient cells (Fig. 6B and C). Densitometric analysis of the respective bands is presented in Fig. 6C. Protein amounts were normalized against actin. These results revealed that lumican affected neither the TGF-β2 receptor-restricted, Smad2 signaling, or consecutively the TGF-β2-dependent growth of HTB94 cells.

IGF-I is a well-established stimulator of chondroblast growth (32) and a moderate stimulator of chondrosarcoma proliferation (33). In initial experiments, it was verified, utilizing western blot analysis, that the HTB94 cells expressed a functional IGF-IR receptor with the ability to respond to IGF-I stimulation (Fig. 7A and B). Subsequently, the HTB94 cells were treated with IGF-I and IGF-IR inhibitor or their combination. IGF-I treatment significantly enhanced cell growth (P≤0.01), whereas the blockage of IGF-IR induced a marked attenuation of HTB94 cell basal and IGF-I-dependent growth (P≤0.01; Fig. 7C). When the lumican siRNA-transfected HTB94 cells were exposed to an IGF-IR inhibitor, no further attenuation of cell growth was detected (Fig. 7D). As SLRP members have earlier been shown to affect IGF-IR-dependent growth processes (12), both lumican and scramble siRNA-transfected cells were treated with IGF-I, and cell proliferation was measured. The lumican siRNA-transfected HTB94 cells treated with IGF-I exhibited complete abolishment of IGF-I-dependent growth stimulation (Fig. 8A), suggesting that the effects of lumican are partially mediated through IGF-IR-dependent signaling. The lumican-deficient cells were probed with IGF-IR and pIGF-IR antibodies to verify the contribution of lumican to IGF-IR signaling in the regulation of HTB94 cell growth. This approach revealed that IGF-IR activation was significantly downregulated in the lumican-deficient cells (P≤0.01; Fig. 8B and C).

Subsequently, the present study examined critical IGF-I downstream mediators focusing on ERK1/2, a well-established IGF-I downstream conduit in the regulation of tumor cell growth, migration, and adhesion (27,34). The utilization of a specific ERK1/2 inhibitor led to the significant suppression of the HTB94 basal level of cell proliferation (P<0.001), and the complete abolishment of IGF-I-dependent growth (P<0.01; Fig. 9A). To determine the effects of lumican on IGF-I-dependent ERK1/2 activation in HTB94 cells, the ERK1/2 phosphorylation levels in both the control and IGFI-treated lumican-deficient cellswere examined. As shown in Fig. 9B and C, lumican participation was found to be crucial for ERK1/2 phosphorylation, as the lumican-deficient cells exhibited an attenuated ERK1/2 activation (P<0.05). Furthermore, when the lumican-deficient cells were treated with the ERK1/2 inhibitor, no further downregulation of cell growth was evident, demonstrating that ERK1/2 participation is necessary for the lumican effect (Fig. 9D). Upon activation of the upstream effectors the degradation of β-catenin was inhibited, resulting in its increased expression in the cytoplasm and enhanced translocation to the nucleus. No changes in the total β-catenin expression in the lumican-deficient cells were detected by western blot analysis and immunofluorescence (data not shown). On the other hand, β-catenin signaling did not participate in the lumican/IGF-IR-mediated growth effects (Fig. S1).

The expression of 3 cell cycle-related genes was then analyzed to characterize the intracellular molecular mechanisms involving the Erk1/2 pathway on lumican-induced chondrosarcoma cell growth. The mRNA levels of p21WAF1/CIP1 and p53 were estimated in lumican-deficient cells to investigate the possible effects of lumican. RT-qPCR demonstrated that the levels of p53 tumor suppressor were significantly upregulated in the siLum-treated cells as compared to the siScr cells (P≤0.05). On the other hand, no change was demonstrated in the expression of p21WAF1/CIP1. These results collectively suggest that lumican is involved in transcriptional control of cell cycle-related genes (Fig. 10).

In separate experiments, the HTB94 cells were treated with TGF-β2 (10 ng/ml) and IGF-I (10 ng/ml), and the expression of lumican in HTB94 cells was estimated. The data obtained by western blot analysis revealed that TGF-β2 decreased (P≤0.001) and IGF-I enhanced (P≤0.01) lumican protein expression (Fig. 11).