| Fibroblast growth factors(FGFs) superfamily consists of 22 FGF genes in mice and humans. FGFs drive crucial biological functions such as early embryonic development and organogenesis via binding to FGF receptors(FGFRs). FGF2, also known as basic FGF, belongs to FGF superfamily and is recognized as an pro-angiogenic factor for new blood vessel formation and wound repair. Meanwhile, emerging evidence suggests that FGF2 functions as a potential oncogenic protein driving a variety of tumor malignancies. Human melanoma commonly expresses high levels of FGFR1 and FGF2.Antisense-mediated inhibition of FGF2 or FGFR1 led to growth regression of xenografts formed by human melanoma cells. Up-regulated FGF2-FGFR1 signaling is also implicated in the pathogenesis of prostate cancer, small cell lung cancer and glioblastoma multiforme(GBM).Previous study demonstrated that high FGF2 m RNA expression is observed in over 94% of human glioblastomas. It’s been shown that high FGF2 expression promotes human glioma’s malignancy. Studies suggested that FGF2 also exerts anti-apoptotic function through up-regulating anti-apoptotic genes BCL-2 and BCL-XL,making tumor cells resistant to chemotherapy.In human, FGF2 has five isoforms(18, 22, 22.5, 24 and 34 k Da), which are produced by alternative initiation of translation on the FGF2 m RNA. The translation of low molecular weight 18 k Da FGF2 is initiated from the classic Kozak AUG start codon. This 18K-FGF2 is made up of 155 amino acids,which is also the core domain for all FGF2 isoforms. The other four FGF2 isoforms are generally recognized as HMW FGF2 s and are translated from alternative upstream CUG start codons. Previous studies revealed that HMW-FGF2 is primarily localized in nucleus whereas 18K-FGF2 resides incytosol, and the nuclear FGF2 accumulation is associated with proliferation of human glioma cells. Mechanisms regulating such divergent cellular distribution of different FGF2 isoforms remain unclear.In this study, we investigated how HMW-FGF2 is translocated into the nucleus, as well as the impact of nuclear FGF2 on proliferation and survival of the T98 G GBM cell line. Transfected HMW-FGF2 is found to associate with Karyopherin-β2(Kapβ2), that belongs to the nuclear transport karyopherin protein family and also known as Transportin, functioning to transport macromolecule cargos between cytoplasm and nucleus. A small Ran GTPase,which regulates the Kapβ2-cargo interaction, is also involved in mediating the nuclear localization of HMW-FGF2. Compared to 18K-FGF2, HMW-FGF2 overexpression in T98 G cells resulted in a significantly higher proliferation rate. Together, our study demonstrated for the first time that Kapβ2 and Ran GTPase synergistically regulate the nuclear import of HMW-FGF2, which plays an important role in GBM cell proliferation.Part one The location analysis of 18K-FGF2 and HMW-FGF2 in GBMObjective: To study the localization of both 18 K and HMW FGF2 in glioblastoma cells, T98 G cell line derived from human glioblastoma was transfected with HA-tagged constructs expressing HMW-FGF2(HA-HMW)or 18K-FGF2(HA-18K)Methods:Plasmid construction:FGF2 c DNA sequence(NCBI Gene ID: 2247) was used as template of PCR. Primer pair used to amplify HMW-FGF2 ORF: 5’- CCC AGA TCT ATG TAC CCA TAT GAT GTT CCA GAT TAC GCT CTG GGG GAC CGC GGG CGC GGC CGC-3’(forward) and 5’- CCC TCT AGA TCA GCT CTT AGC AGA CAT TGG AAG-3’(reverse). Primer pair used to amplify 18K-FGF2ORF: 5’- CCC AGA TCT ATG TAC CCA TAT GAT GTT CCA GAT TAC GCT ATG GCA GCC GGG AGC ATC ACC ACG-3’, and same reverse primer was used. The above amplified ORF was cloned into p CMV2 vector using Bgl II and Xba I.Peptide translation of N-terminally HA-tagged HMW-FGF2 ORF:YPYDVPDYA-LGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTA APRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPK RLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVC ANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYV ALKRTGQYKLGSKTGPGQKAILFLPMSAKS.Peptide translation of N-terminally HA-tagged 18K-FGF2 ORF:YPYDVPDYA-MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGG FFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMK EDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQY KLGSKTGPGQKAILFLPMSAKS.Cell culture:The human glioblastoma cell line T98 G was obtained from ATCC. Cells were cultured in MEM-α medium(Invitrogen, USA) supplemented with 10%FBS and maintained at 37°C in a humidified atmosphere with 5% CO2. The medium used throughout this study is devoid of FGF2 and EGF.Immunofluorescence:T98G cells were grown on coverslips and transfected with 18 K or HMW FGF2 constructs. 24 hours after transfection, cells were washed with PBS,fixed in 4% paraformaldehyde for 45 min and then blocked and permeabilized for 90 min. Primary anti-HA antibody were incubated for 90 min followed by incubation of secondary antibody for 60 min. Stained cells were analyzed using confocal fluorescence microscopy.Nuclear and cytosolic fractionation:T98G cells were incubated in buffer containing 5m M sodium phosphate,p H 7.4, 50 m M Na Cl, 150 m M sucrose, 5m M KCl, 2m M dithiothreitol, 1m M Mg Cl2, 0.5m M Ca Cl2, and 0.1% digitonin. Lysates were extracted via scrapping. Nuclei fractionation was obtained by centrifuging through buffer(2.5m M Tris-HCl, p H 7.4, 10 m M Na Cl) containing 30% sucrose at 1000 g for10 min. The supernatant was used as cytoplasmic fraction.Immunoblotting:Cells were washed with PBS and lysed in RIPA lysis buffer(20m M Tris-HCl, p H 7.6, 150 m M Na Cl, 1% Triton X-100 and 2m M PMSF) with protease and phosphatase inhibitors, which were added upon use. Protein concentration was quantified by BCA assay system(Biorad, USA). 25μg of total protein extracts were loaded for electrophoresis. 25μg of total protein extracts were loaded for electrophoresis.Results:We conducted the 18-FGF2 and HMW-FGF2 vectors successfully and were verified by sequencing and immunoblotting.Immunostaining against HA showed that HA-HMW is primarily located in the nucleus while HA-18 K mainly resides in the cytosol. Even though there are three HMW FGF2 isoforms that display distinct molecular weights(22,24,34 k Da), the transfection of HMW-FGF2 construct led to the expression of a pure HMW-FGF2 population because of that HA tag addition at the N-terminal eliminates the alternative CTG start codon for HMW-FGF2 translation. This specific expression pattern of HMW-FGF2 excludes experimental variance and provides a favorable assay condition. Western blot analysis using nuclear and cytosolic fractionations extracted from HWM or18 K FGF2-transfected T98 G cells revealed that almost all of overexpressed18K-FGF2 is located in the cytosol. The majority of HMW-FGF2 is nucleus-localized, with a small amount of cytosolic HA-HMW FGF2.Conclusion: HMW-FGF2 is located in nucleus, however 18K-FGF2 is in cytoplasm.Part two The mechanism of Kapβ2 mediated nuclear localization of HAHMW FGF2Objective: The transport of proteins between nucleus and cytoplasm is mediated by nuclear transport factors such as Karyopherin-β(Kapβ) family proteins that recognize nuclear localization signals(NLS). To confirm whether Kapβ2 is involved in mediating the preferential nuclear localization of HA-HMW FGF2,which potentially contains a non-classical arginineglycine-rich NLS(see discussion for details), T98 G cells were transfected with HA-HMW, HA-18 K and empty plasmid before immunoprecipitation(IP) assay was performed to examine the association between Kapβ2 and transfected FGF2. T98 G cells were then co-transfected with si RNA1 and HA-HMW FGF2 followed by immunostaining using antibody against HA.Nuclear and cytosolic fractionations were isolated from si RNA1 and HA-HMW FGF2 co-transfected T98 G cells and subjected to western blotting analysis for HMW-FGF2. FGF2 signaling drives a wide range of oncogenic events such as proliferation, migration and survival in multiple cancer cell types.We next evaluated the role of FGF2 in the tumorigenic potential of T98 G cells.Methods:RNA interference:Si RNA duplex targeting Karyopherin-β2 is synthesized by Dharmacon,GE Healthcare. T98 G cells were cultured in serum-free medium and si RNA was transfected using Lipofectamine 2000(Invitrogen,USA). Subsequent analysis was conducted 48 hours after si RNA transfection.Quantitative real-time PCR:RNA was isolated using Trizol reagent(Invitrogen,USA). Reverse transcription was performed using RT kit from Promega. SYBR green system(Thermo Scientific,USA) was used to conduct the real-time PCR experiment.Gene expressions were normalized to GAPDH. 2-ΔΔCt method was used to analyze data.Immunoprecipitation and immunoblotting:Whole cell lysates were extracted from FGF2-transfected T98 G cells.Cells were washed with PBS and lysed in RIPA lysis buffer(20m M Tris-HCl,p H 7.6, 150 m M Na Cl, 1% Triton X-100 and 2m M PMSF) with protease and phosphatase inhibitors, which were added upon use. Protein concentration was quantified by BCA assay system(Biorad,USA). 25μg of total protein extracts were loaded for electrophoresis. Primary HA antibody was added to the whole cell protein lysates according to manufacturer’s instruction and incubated at4°C overnight. Protein beads A/G were added to pull down the HA antibody complex followed by centrifuge down the beads and boiling above 95°C.Supernatant lysates were loaded and subjected to immunoblotting analysis.Immunofluorescence:T98G cells were grown on coverslips and transfected with 18 K or HMW FGF2 constructs. 24 hours after transfection, cells were washed with PBS,fixed in 4% paraformaldehyde for 45 min and then blocked and permeabilized for 90 min. Primary anti-HA antibody were incubated for 90 min followed by incubation of secondary antibody for 60 min. Stained cells were analyzed using confocal fluorescence microscopy.Nuclear and cytosolic fractionation:T98G cells were incubated in buffer containing 5m M sodium phosphate,p H 7.4,50 m M Na Cl,150 m M sucrose,5m M KCl,2m M dithiothreitol,1m M Mg Cl2,0.5m M Ca Cl2,and 0.1% digitonin. Lysates were extracted via scrapping. Nuclei fractionation was obtained by centrifuging through buffer(2.5m M Tris-HCl, p H 7.4, 10 m M Na Cl) containing 30% sucrose at 1000 g for10 min. The supernatant was used as cytoplasmic fraction.MTT assay:For the measurement of cell proliferation,3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide(MTT) was used according to manufacturer’s instructions(Roche, Switzerland). T98 G cells were incubated with 0.5 mg/ml MTT in the incubator with 5% O2 at 37°C, and then incubated with lysis buffer overnight in the incubator. The optical density of solubilized formazan was measured at 570 nm on a plate reader Statistical analysis:Results were collected as the average of at least five independent experiments. All the data were presented by the mean ± SEM. The statistical significance of the difference between the values of control and treatment groups was determined by Student t test. Values of p<0.05 were considered statistically significant.Results:Staining analysis showed that Kapβ2 knockdown blocked the nuclear transport and led to a uniform subcellular distribution of HMW-FGF2.Quantification of fluorescent signal intensity showed that the percentage ration of nuclear to cytoplasmic HMW-FGF2 is 85% vs. 15% in Scramble group,whereas in si RNA group the difference of the ratio is not significant(approximately 51% vs. 49%). Similar to immunostaining data, the immunoblot results showed that the nuclear HMW-FGF2 level was significantly reduced upon Kapβ2 knockdown. The proliferation results showed that overexpression of both HMW-FGF2 and 18K-FGF2 led to increased cell proliferation while the effect of HMW-FGF2 is more profound.Kapβ2 knockdown in native T98 G cells led to reduced proliferation,indicating that Kapβ2 is required for maintenance of cell growth at the basal state. Next, we knocked down expression of Kapβ2 in HMW or 18 K FGF2-expressing T98 G cells using si RNA and found that Kapβ2 inhibition resulted in dramatically decreased proliferation in HMW FGF2-expressing cells. However, the proliferation of T98 G cells expressing 18K-FGF2 was not affected by Kapβ2 knockdown.Conclusion:1 Kapβ2 is required for efficient nuclear translocation of HMW FGF2.2 Kapβ2 mediated nuclear localization promoted the proliferation of T98 G.Part three Nuclear translocation of HMW-FGF2 requires Ran GTPase activitySubject:The Kapβ2 and cargo interaction, as well as nuclear-cytoplasmic transportation are regulated by Ran GTPase nucleotide cycle. To confirm the involvement of Ran GTPase in the Kapβ2-HMW-FGF2 nuclear transport, we employed treatments of GTPγS and GDPβS. GTPγS is a GTP analogue that is non-hydrolyzable and keeps Ran in GTP-bound state, whereas GDPβS is a GDP analogue that cannot be phosphorylated and locks Ran in inactive GDP-bound state. Both treatments block GTPase activity and the GTP-GDP cycle. To verify the important roles of Ran GTPase in Kapβ2-HMW-FGF2 nuclear transport promoted proliferation, we tested the proliferation of T98 G cells with GTPγS or GDPβS treatment.Treatments of GDPβS and GTPγS:T98G cells were transfected with HA-HMW-FGF2. Following transfection, culture medium was replaced by fresh medium containing 0.1n M GTPγS or GDPβs or sterile water as control. Cells were incubated for 30 mins under 37°C, followed by whole cell lysate extraction or nuclearcytoplasmic fractionation for western blot analysis or MTT assay.Immunofluorescence:T98G cells were grown on coverslips and transfected with 18 K or HMW FGF2 constructs. 24 hours after transfection, cells were washed with PBS,fixed in 4% paraformaldehyde for 45 min and then blocked and permeabilized for 90 min. Primary anti-HA antibody were incubated for 90 min followed by incubation of secondary antibody for 60 min. Stained cells were analyzed using confocal fluorescence microscopy.Nuclear and cytosolic fractionation:T98G cells were incubated in buffer containing 5m M sodium phosphate,p H 7.4, 50 m M Na Cl, 150 m M sucrose, 5m M KCl, 2m M dithiothreitol, 1m M Mg Cl2, 0.5m M Ca Cl2, and 0.1% digitonin. Lysates were extracted via scrapping. Nuclei fractionation was obtained by centrifuging through buffer(2.5m M Tris-HCl, p H 7.4, 10 m M Na Cl) containing 30% sucrose at 1000 g for10 min. The supernatant was used as cytoplasmic fraction.Immunoprecipitation and immunoblotting:Whole cell lysates were extracted from FGF2-transfected T98 G cells.Cells were washed with PBS and lysed in RIPA lysis buffer(20m M Tris-HCl,p H 7.6, 150 m M Na Cl, 1% Triton X-100 and 2m M PMSF) with protease and phosphatase inhibitors, which were added upon use. Protein concentration was quantified by BCA assay system(Biorad, USA). 25 μg of total protein extracts were loaded for electrophoresis. Primary HA antibody was added to the whole cell protein lysates according to manufacturer’s instruction and incubated at4°C overnight. Protein beads A/G were added to pull down the HA antibody complex followed by centrifuge down the beads and boiling above 95°C.Supernatant lysates were loaded and subjected to immunoblotting analysis.Methods:MTT assay:For the measurement of cell proliferation,3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide(MTT) was used according to manufacturer’s instructions(Roche, Switzerland). T98 G cells were incubated with 0.5 mg/ml MTT in the incubator with 5% O2 at 37°C, and then incubated with lysis buffer overnight in the incubator. The optical density of solubilized formazan was measured at 570 nm on a plate reader.Statistical analysis:Results were collected as the average of at least five independent experiments. All the data were presented by the mean ± SEM. The statistical significance of the difference between the values of control and treatment groups was determined by Student t test. Values of p<0.05 were considered statistically significant.Results : Immunostaining against HA showed that both GTPγS and GDPβS treatments reduced HMW-FGF2 nuclear accumulation in T98 G cells overexpressing HMW-FGF2(Fig. 3A). Western blot analysis using nuclear and cytoplasmic fractions from HWM-FGF2-expressing T98 G cells further confirmed that inhibiting Ran GTPase activity blocks HMW-FGF2 nuclear transport(Fig. 3B). Immunoprecipitation results showed that Ran interacts with HMW-FGF2 upon treatment of GDPβS, but not GTPγS, Similar with si RNA-mediated Kapβ2 knockdown, inhibiting Ran GTPase activity and FGF2 nuclear transport by GTPγS or GDPβS treatments significantly reduced proliferation rate in HMW-FGF2-overexpressing T98 G cells.Conclusion:1 Nuclear translocation of HMW-FGF2 requires Ran GTPase activity.2 The binding of Ran GDP and FGF2 mediates the cytoplasm- nucleus transportion.Part four Nuclear HMW FGF2 activates Akt signaling pathway.Subject : To further explore molecular mechanisms responsible for enhanced proliferation by FGF2, we focused on the PI3K/AKT signaling cascade, which is the major mitogenic pathway in response to growth factor signaling. To demonstrate that nucleus-localized HMW-FGF2, but not cytoplasmic 18K-FGF2, is capable of activating the mitogenic AKT signaling and facilitating cell proliferation in T98 G cells. We genetically engineered the construct expressing 18K-FGF2 that is tagged by the classic lysine-rich NLS,PKKKRKV, and transfected it into T98 G cells to verify its function.Methods:Immunofluorescence:T98G cells were grown on coverslips and transfected with 18 K or HMW FGF2 constructs. 24 hours after transfection, cells were washed with PBS,fixed in 4% paraformaldehyde for 45 min and then blocked and permeabilized for 90 min. Primary anti-HA antibody were incubated for 90 min followed by incubation of secondary antibody for 60 min. Stained cells were analyzed using confocal fluorescence microscopy.Nuclear and cytosolic fractionation T98 G cells were incubated in buffer containing 5m M sodium phosphate,p H 7.4, 50 m M Na Cl, 150 m M sucrose, 5m M KCl, 2m M dithiothreitol, 1m M Mg Cl2, 0.5m M Ca Cl2, and 0.1% digitonin. Lysates were extracted via scrapping. Nuclei fractionation was obtained by centrifuging through buffer(2.5m M Tris-HCl, p H 7.4, 10 m M Na Cl) containing 30% sucrose at 1000 g for10 min. The supernatant was used as cytoplasmic fraction.Immunoprecipitation and immunoblotting:Whole cell lysates were extracted from FGF2-transfected T98 G cells.Cells were washed with PBS and lysed in RIPA lysis buffer(20m M Tris-HCl,p H 7.6, 150 m M Na Cl, 1% Triton X-100 and 2m M PMSF) with protease and phosphatase inhibitors, which were added upon use. Protein concentration was quantified by BCA assay system(Biorad, USA). 25 μg of total protein extracts were loaded for electrophoresis. Primary HA antibody was added to the whole cell protein lysates according to manufacturer’s instruction and incubated at4°C overnight. Protein beads A/G were added to pull down the HA antibody complex followed by centrifuge down the beads and boiling above 95°C.Supernatant lysates were loaded and subjected to immunoblotting analysis.MTT assay:For the measurement of cell proliferation,3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide(MTT) was used according to manufacturer’s instructions(Roche, Switzerland). T98 G cells were incubated with 0.5 mg/ml MTT in the incubator with 5% O2 at 37°C, and then incubated with lysis buffer overnight in the incubator. The optical density of solubilized formazan was measured at 570 nm on a plate reader Statistical analysis:Results were collected as the average of at least five independent experiments. All the data were presented by the mean ± SEM. The statistical significance of the difference between the values of control and treatment groups was determined by Student t test. Values of p<0.05 were considered statistically significant.Results:Our results showed that overexpression of 18K-FGF2 resulted in reduced PTEN expression and HMW-FGF2 overexpression inhibit PTEN level to a further extent. As a result, p-Akt level was markedly increased with FGF2 overexpression, indicating that PTEN/p-Akt cascade might mediate the enhanced cell proliferation, downstream of FGF2 signaling. Kapβ2knockdown in HMW-FGF2-expressing T98 G cells gave rise to elevated PTEN and declined p-Akt levels. We discovered that additional NLS sequence forced18K-NLS-FGF2 translocate into the nucleus with undetectable cytosolic level,whereas 18K-FGF2 is dominantly cytoplasm-localized. Western blot analysis showed 18K-NLS-FGF2 and HMW-FGF2 transfection led to significantly decreased PTEN expression, accompanied by increased p-Akt level.Subsequent growth curve analysis revealed that the proliferation rate of nuclear 18K-NLS-FGF2-transfected T98 G cells is significantly higher than that of other FGF2 forms-overexpressing cells, specifically HMWFGF2-transfected T98 G cells.Conclusion:1 Nuclear HMW-FGF2 activates Akt signaling pathway.2 NLS plays a crucial role in facilitating the nuclear entry of HMW-FGF2. |
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