Article
Serum Deprivation-Induced Human GM3 Synthase (hST3Gal V) Gene Expression Is Mediated by Runx2 in Human Osteoblastic MG-63 Cells Hyun-Kyoung Yoon 1,† , Ji-Won Lee 1,† , Kyoung-Sook Kim 1 , Seo-Won Mun 1 , Dong-Hyun Kim 1 , Hyun-Jun Kim 2 , Cheorl-Ho Kim 3 and Young-Choon Lee 1, * Received: 31 October 2015; Accepted: 22 December 2015; Published: 29 December 2015 Academic Editor: Florian Lang 1
2 3
* †
Department of Medicinal Biotechnology, College of Health Sciences, Dong-A University, Busan 604-714, Korea;
[email protected] (H.-K.Y.);
[email protected] (J.-W.L.);
[email protected] (K.-S.M.);
[email protected] (S.-W.M.);
[email protected] (D.-H.K.) Department of Orthopaedic Surgery, College of Medicine, Dong-A University, Busan 604-714, Korea;
[email protected] Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, SungKyunKwan University, Kyunggi-Do 440-746, Korea;
[email protected] Correspondence:
[email protected]; Tel.: +82-51-200-7591; Fax: +82-51-200-6536 These authors contributed equally to this work.
Abstract: Serum deprivation (SD) is well known to induce G0/G1 cell cycle arrest and apoptosis in various cells. In the present study, we firstly found that SD could induce G1 arrest and the differentiation of human osteoblastic MG-63 cells, as evidenced by the increase of osteoblastic differentiation markers, such as bone morphogenetic protein-2 (BMP-2), osteocalcin and runt-related transcription factor 2 (Runx2). In parallel, gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 biosynthesis was upregulated by SD in MG-63 cells. The 51 -flanking region of the hST3Gal V gene was functionally characterized to elucidate transcriptional regulation of hST3Gal V in SD-induced MG-63 cells. Promoter analysis using 51 -deletion constructs of the hST3Gal V gene demonstrated that the ´432 to ´177 region functions as the SD-inducible promoter. Site-directed mutagenesis revealed that the Runx2 binding sites located side-by-side at positions ´232 and ´222 are essential for the SD-induced expression of hST3Gal V in MG-63 cells. In addition, the chromatin immunoprecipitation assay also showed that Runx2 specifically binds to the hST3Gal V promoter region containing Runx2 binding sites. These results suggest that SD triggers upregulation of hST3Gal V gene expression through Runx2 activation by BMP signaling in MG-63 cells. Keywords: serum deprivation; human GM3 synthase (hST3Gal V); MG-63 cells; runt-related transcription factor 2 (Runx2); transcriptional regulation
1. Introduction Serum deprivation (SD) has been widely used to produce a synchronized culture by cell cycle arrest in the G0/G1 phase [1–5]. Human glioblastoma T98G and ovarian cancer SK-OV-3 cells were reported to be arrested in G0 and G1, respectively, by SD [6,7]. In addition, it has been demonstrated in numerous studies that the cultured cells, including bone marrow-derived mesenchymal stem cells, undergo apoptosis by SD [8–15]. Moreover, SD induced not only cell differentiation in immortalized rat proximal tubule cells [16], but complete redifferentiation of human umbilical vascular smooth muscle cells [17]. Gangliosides are complex glycosphingolipids bearing sialic acid and, owing to the existence in the external leaflets of plasma membranes of animal cells, they play important roles in cell-cell interaction, Int. J. Mol. Sci. 2016, 17, 35; doi:10.3390/ijms17010035
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Gangliosides Int. J. Mol. Sci. 2016, 17,are 35
complex glycosphingolipids bearing sialic acid and, owing to the existence in 2 of 13 the external leaflets of plasma membranes of animal cells, they play important roles in cell-cell interaction, adhesion and cell signaling processes [18–21]. Ganglioside profiles, including its adhesion and cell signaling processes [18–21]. Ganglioside profiles, including its composition and composition and distribution, change dramatically during diverse biological processes, such as distribution, change dramatically during diverse biological processes, such as cell proliferation, cell proliferation, differentiation, development and apoptosis [18,20]. These changes are differentiation, development and apoptosis [18,20]. These changes are attributed to the strict attributed to the strict regulation of ganglioside biosynthesis by ganglioside synthases, a family of regulation of ganglioside biosynthesis by ganglioside synthases, a family of glycosyltransferases, glycosyltransferases, in the Golgi apparatus. It is well known that malignant tumor cells show a in the Golgi apparatus. It is well known that malignant tumor cells show a different expression different expression pattern of cell surface gangliosides as compared to normal cells, including pattern of cell surface gangliosides as compared to normal cells, including predominant expression of predominant expression of specific gangliosides [22], which led to the development of antibodies specific gangliosides [22], which led to the development of antibodies against specific gangliosides against specific gangliosides as immunotherapeutic agents [23]. Moreover, recent studies have as immunotherapeutic agents [23]. Moreover, recent studies have revealed that gangliosides are revealed that gangliosides are involved in neuronal and osteoblast differentiation of human involved in neuronal and osteoblast differentiation of human mesenchymal stem cells (hMSCs), mesenchymal stem cells (hMSCs), human adipose-derived stem cells (hADSCs) and human dental human adipose-derived stem cells (hADSCs) and human dental pulp-derived stem cells (hDPSCs), as pulp-derived stem cells (hDPSCs), as well as mouse embryonic stem cells (mESCs) [24–28]. well as mouse embryonic stem cells (mESCs) [24–28]. Gangliosides GM1 and GT1b expression was Gangliosides GM1 and GT1b expression was markedly elevated in the neural differentiation of markedly elevated in the neural differentiation of hMSCs and mESCs [24], whereas GD3 and GD1a hMSCs and mESCs [24], whereas GD3 and GD1a were highly expressed in the neural differentiation were highly expressed in the neural differentiation of hDPSCs [25]. During the differentiation of hMSCs of hDPSCs [25]. During the differentiation of hMSCs into osteoblasts, reduction of ganglioside GM3 into osteoblasts, reduction of ganglioside GM3 expression was observed, while GD1a expression was expression was observed, while GD1a expression was remarkably elevated [26–28]. remarkably elevated [26–28]. On the basis of these observations, we speculated that ganglioside synthesis might be associated On the basis of these observations, we speculated that ganglioside synthesis might be associated with osteoblast differentiation. To confirm this hypothesis, we investigated whether during with osteoblast differentiation. To confirm this hypothesis, we investigated whether during osteoblast osteoblast differentiation, gene expression of human ganglioside synthases is regulated in the human differentiation, gene expression of human ganglioside synthases is regulated in the human osteoblastic osteoblastic MG-63 cells. In this study, we have firstly found osteoblast differentiation by SD and MG-63 cells. In this study, we have firstly found osteoblast differentiation by SD and significant significant elevation of human GM3 synthase (hST3Gal V) gene expression in SD-induced elevation of human GM3 synthase (hST3Gal V) gene expression in SD-induced differentiation of differentiation of MG-63 cells, thereby enhancing ganglioside GM3 expression. To understand the MG-63 cells, thereby enhancing ganglioside GM3 expression. To understand the molecular basis of molecular basis of hST3Gal V gene expression induced by SD, furthermore, the promoter region to hST3Gal V gene expression induced by SD, furthermore, the promoter region to mediate transcriptional mediate transcriptional upregulation of hST3Gal V gene in serum-deprived cells was functionally upregulation of hST3Gal V gene in serum-deprived cells was functionally characterized, and we found characterized, and we found involvement of bone morphogenetic protein (BMP) BMP/Runx2 involvement of bone morphogenetic protein (BMP) BMP/Runx2 signaling in SD-induced hST3Gal V signaling in SD-induced hST3Gal V transcriptional activation. transcriptional activation. 2. Results 2. Results 2.1. 2.1. Effect Effect of of Serum Serum Deprivation Deprivation on on Cell Cell Proliferation Proliferation Prior Prior to to investigation investigation of of the the SD SD effect effect on on MG-63 MG-63 cell cell differentiation differentiation and and hST3Gal hST3Gal V V expression, expression, we first examined the cytotoxicity of SD in MG-63 cells by the tetrazolium salt reduction (MTT) we first examined the cytotoxicity of SD in MG-63 cells by the tetrazolium salt reduction (MTT)assay. assay. As shown shown Figure Figure1,1,cell cellviability viabilityfor fora 24-h a 24-h incubation was more than 80%, about viability As incubation was more than 80%, andand about 73%73% viability was was observed in incubation for 48 h, indicating that MG-63 cell proliferation was not significantly observed in incubation for 48 h, indicating that MG-63 cell proliferation was not significantly affected affected under serum-free conditions under serum-free conditions for 48 h. for 48 h.
Figure 1. Effect of serum serumdeprivation deprivation(SD) (SD)on onthe theviability viabilityofof MG-63 cells. The cytotoxic effects of SD Figure 1. Effect of MG-63 cells. The cytotoxic effects of SD on on MG-63 assessed the assay. MTT assay. Cellsgrown were in grown in serum-free for the MG-63 cellscells werewere assessed by thebyMTT Cells were serum-free medium medium for the indicated indicated timeand periods, and their at absorbance at measured 540 nm was on animmunosorbent enzyme-linked time periods, their absorbance 540 nm was on anmeasured enzyme-linked immunosorbent assay The (ELISA) reader. The results expressedofascell percentages of cell proliferation assay (ELISA) reader. results are expressed as are percentages proliferation in the control (0 in h) the control (0 h) and represent the means ± SEM of three independent experiments. * p < 0.05 and represent the means ˘ SEM of three independent experiments. * p < 0.05 (compared to the control); (compared to the ** p < 0.01; *** p < control); 0.001. ** p < 0.01; *** p < 0.001.
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2.2. Serum Deprivation (SD) Induces G1 Arrest of the Cell Cycle in MG-63 Cells 2.2. Serum (SD) Induces G1 Arrest of the is Cell Cycle in MG-63 Cells of the cell cycle [29], we Since Deprivation the differentiation of mammalian cells preceded by G1 arrest examined SD inducesofG1 arrest of the cellis cycle in MG-63 After MG-63 Since whether the differentiation mammalian cells preceded by G1cells. arrest of the cell cells cyclewere [29], incubated under serum-free conditions for various times, cells were collected, and the cell cycle we examined whether SD induces G1 arrest of the cell cycle in MG-63 cells. After MG-63 cells were profile was analyzed by flow cytometry. As shown in Figure 2A, the percentage of cells in the G1 incubated under serum-free conditions for various times, cells were collected, and the cell cycle profile phase was increased time dependently by SD, whereas the percentages of S and G2 phases were was analyzed by flow cytometry. As shown in Figure 2A, the percentage of cells in the G1 phase was decreased. Cells in the sub-G1 phase were not observed, indicating that SD did not cause cell death. increased time dependently by SD, whereas the percentages of S and G2 phases were decreased. Cells In addition, the morphology of MG-63 cells became branched and elongated time dependently in the sub-G1 phase were not observed, indicating that SD did not cause cell death. In addition, the (Figure 2B). morphology of MG-63 cells became branched and elongated time dependently (Figure 2B).
Figure 2. Typical histograms of the DNA content and cell morphology of MG-63 cells cultured under Figure 2. Typical histograms of the DNA content and cell morphology of MG-63 cells cultured under serum-free conditions. Cells were grown in serum-free medium for the indicated time periods. (A) DNA serum-free conditions. Cells were grown in serum-free medium for the indicated time periods. content was analyzed by flow cytometry; (B) Cell morphological images were taken by phase-contrast (A) DNA content was analyzed by flow cytometry; (B) Cell morphological images were taken by microscope (400ˆ) at each time point. phase-contrast microscope (400×) at each time point.
2.3. Effect Effect of of SD SD on on Osteoblast-Related Osteoblast-Related Marker Marker Gene 2.3. Gene Expression Expression in in MG-63 MG-63 Cells Cells To investigate To investigate whether whether SD SD induces induces the the expression expression of of marker marker genes genes related related to to osteoblast osteoblast differentiation, MG-63 cells were incubated under serum-free conditions for various times. AsAs shown in differentiation, MG-63 cells were incubated under serum-free conditions for various times. shown Figure 3A,3A, SD increased BMP-2 and osteocalcin mRNAmRNA levels in a time-dependent manner. manner. In addition, in Figure SD increased BMP-2 and osteocalcin levels in a time-dependent In qPCR results showed that mRNA levels of BMP-2, osteocalcin and Runx2 were also enhanced in a addition, qPCR results showed that mRNA levels of BMP-2, osteocalcin and Runx2 were also time-dependent manner, and peak levelsand of osteocalcin Runx2 wereand reached after SDreached for 24 hafter and enhanced in a time-dependent manner, peak levelsand of osteocalcin Runx2 were decreased (Figure 4). Moreover, levels Runx2levels wereof increased in proportion SD for 24 hthereafter and decreased thereafter (Figurethe 4). protein Moreover, theof protein Runx2 were increased to the increment of its mRNA level by SD (Figure 3C). Taken together, these results indicate SD in proportion to the increment of its mRNA level by SD (Figure 3C). Taken together, thesethat results induces G1 arrest not cell death,but in not MG-63 indicate thatcell SDcycle induces G1and cell differentiation, cycle arrest andbut differentiation, cell cells. death, in MG-63 cells. 2.4. Effect of SD on hST3Gal V Expression in MG-63 Cells 2.4. Effect of SD on hST3Gal V Expression in MG-63 Cells To check whether ganglioside synthesis is associated with osteoblast differentiation, we evaluated To check whether ganglioside synthesis is associated with osteoblast differentiation, we the effect of SD on the gene expression of human ganglioside synthases in MG-63 cells. As shown in evaluated the effect of SD on the gene expression of human ganglioside synthases in MG-63 cells. As Figure 3A,B, the mRNA levels of hST3Gal V catalyzing ganglioside GM3 synthesis were markedly shown in Figure 3A,B, the mRNA levels of hST3Gal V catalyzing ganglioside GM3 synthesis were increased by SD, and their enhancements were in a time-dependent manner. In addition, these markedly increased by SD, and their enhancements were in a time-dependent manner. In addition, increments were confirmed by qPCR in which the highest level of mRNA expression was observed these increments were confirmed by qPCR in which the highest level of mRNA expression was after SD for 24 h and decreased thereafter (Figure 4). These results clearly revealed that hST3Gal V observed after SD for 24 h and decreased thereafter (Figure 4). These results clearly revealed that gene expression was induced by SD. hST3Gal V gene expression was induced by SD.
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Figure 3. 3. Effect ofofSD SD onon the expression levels of osteoblastic osteoblastic markers and hST3Gal hST3Gal V. Total Total RNARNA from Figure on the expression levels of markers and V. RNA from Figure 3. Effect Effectof SD the expression levels of osteoblastic markers and hST3Gal V. Total MG-63 cells was isolated after incubation in serum-free medium for the indicated time periods (A) or MG-63 cells was after incubation in serum-free medium for thefor indicated time periods (A) or from MG-63 cellsisolated was isolated after incubation in serum-free medium the indicated time periods afterorculture culture for 24 24 hhfor in medium medium containing various concentration concentration of FBS FBS (B), (B),ofand and mRNA transcripts after for in containing various of mRNA transcripts (A) after culture 24 h in medium containing various concentration FBS (B), and mRNA of osteoblastic markers and hST3Gal V were detected by reverse transcription-polymerase chain of osteoblastic markers and hST3Gal V were detected by reverse transcription-polymerase chain transcripts of osteoblastic markers and hST3Gal V were detected by reverse transcription-polymerase reaction (RT-PCR). As an internal control, parallel reactions were performed to measure the levels of reaction (RT-PCR). As an internal control,control, parallelparallel reactions were performed to measure the levelsthe of chain reaction (RT-PCR). As an internal reactions were performed to measure the housekeeping gene β-actin; (C) Equal amounts of cell lysates (20 μg) were separated on sodium the housekeeping gene β-actin; Equal(C) amounts of cell lysates μg) were separated on sodium levels of the housekeeping gene(C) β-actin; Equal amounts of cell(20 lysates (20 µg) were separated on dodecyldodecyl sulfate sulfate (SDS)-polyacrylamide gels and and transferred to aatopolyvinylidene polyvinylidene fluoride (PVDF) dodecyl sulfate (SDS)-polyacrylamide gels transferred to sodium (SDS)-polyacrylamide gels and transferred a polyvinylidenefluoride fluoride(PVDF) (PVDF) membrane. The Themembrane membrane was probed with specific antibodies against Runx2 and hST3Gal hST3Gal V. membrane. The membrane was probed specific antibodies against and V. was probed withwith specific antibodies against Runx2Runx2 and hST3Gal V. GAPDH GAPDH was used as an internal control. GAPDH used as ancontrol. internal control. was usedwas as an internal
Figure 4. 4. Quantitative Quantitative real-time real-time PCR analysis of of the the expression expression levels levels of of osteoblastic osteoblastic markers markers and and Figure Quantitative real-time PCR PCR analysis analysis of hST3Gal V in SD-induced MG-63 cells. Total RNA from MG-63 cells was isolated after incubation in hST3Gal V in SD-induced MG-63 cells. Total RNA from MG-63 cells was isolated after incubation in hST3Gal V in SD-induced MG-63 cells. Total RNA from MG-63 cells was isolated after incubation serum-free medium for the indicated time periods, and mRNA transcripts of hST3Gal V (A) and serum-free medium forfor thethe indicated time in serum-free medium indicated timeperiods, periods,and andmRNA mRNAtranscripts transcriptsofofhST3Gal hST3GalVV (A) (A) and osteoblastic markers markers (B–D) (B–D) were were was was analyzed analyzed by by quantitative quantitative real-time real-time PCR. PCR. The The transcript transcript copy copy osteoblastic osteoblastic markers (B–D) real-time numbers of of osteoblastic osteoblastic markers markers and and hST3Gal hST3Gal V V were were normalized normalized to to the the β-actin β-actin transcript transcript copy copy numbers numbers osteoblastic markers and hST3Gal number for foreach eachsample. sample. Experiments were repeated three times to check check the reproducibility reproducibility of number for each sample. Experiments were repeated three to the of Experiments were repeated three timestimes to check the reproducibility of results. results. ** p < 0.01 (compared to the control); *** p < 0.001. results. ** p(compared < 0.01 (compared to the control); *** p < 0.001. ** p < 0.01 to the control); *** p < 0.001.
As the the next next step, step, the the correlation correlation between between mRNA mRNA and and protein protein expression expression levels levels of of hST3Gal hST3Gal V V by by As SD was was evaluated evaluated by by Western Western blot blot analysis analysis using using an an hST3Gal hST3Gal V-specific V-specific monoclonal monoclonal antibody. antibody. As As SD shown in in Figure Figure 3C, 3C, protein protein levels levels of of hST3Gal hST3Gal V V were were also also increased increased in in aa time-dependent time-dependent manner. manner. shown
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As the next step, the correlation between mRNA and protein expression levels of hST3Gal V by SD was by17, Western blot analysis using an hST3Gal V-specific monoclonal antibody. As shown Int. J.evaluated Mol. Sci. 2016, 35 5 ofin 13 Figure 3C, protein levels of hST3Gal V were also increased in a time-dependent manner. These results These results indicate thatofthe induction of hST3Gal V expression regulated at bothand the indicate that the induction hST3Gal V expression by SD is regulatedby at SD bothisthe transcriptional transcriptional and translational levels. translational levels. 2.5. 2.5. Effect Effect of of SD SD on on Ganglioside Ganglioside GM3 GM3 Expression Expression in in MG-63 MG-63 Cells Cells To ofof hST3Gal VV byby SDSD leads to to thethe enhancement of Tocheck checkwhether whetherorornot notthe theincreased increasedexpression expression hST3Gal leads enhancement ganglioside GM3GM3 levellevel synthesized by hST3Gal V in MG-63 we analyzed cellularthe expression of ganglioside synthesized by hST3Gal V in cells, MG-63 cells, we the analyzed cellular level of ganglioside GM3 by immunofluorescence confocal microscopy using the mouse anti-GM3 expression level of ganglioside GM3 by immunofluorescence confocal microscopy using the mouse monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse IgM as the anti-GM3 monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse secondary toantibody visualize to GM3 expression by SD in MG-63 cells. As shown Figure 5, IgM as theantibody secondary visualize GM3induced expression induced by SD in MG-63 cells.inAs shown the expression of ganglioside was increased in MG-63 cells in incubated serum-freein medium in Figure 5, thelevel expression level of GM3 ganglioside GM3 was increased inincubated MG-63 cells serumfor h, but not in 10% FBS-containing medium. free24medium forin 24MG-63 h, but cells not inincubated MG-63 cells incubated in 10% FBS-containing medium.
Figure5.5.Confocal Confocal analysis of ganglioside GM3 expression in SD-induced MG-63 After Figure analysis of ganglioside GM3 expression in SD-induced MG-63 cells. Aftercells. incubation incubation for 24 h in standard medium containing 10% FBS or 0% FBS, cells were immunostained for 24 h in standard medium containing 10% FBS or 0% FBS, cells were immunostained with anti-GM3 with anti-GM3 (FITC; green). Nuclei with DAPI (blue) and analyzed by antibodies (FITC;antibodies green). Nuclei were stained with were DAPI stained (blue) and analyzed by confocal microscopy. confocal Scale bar: 20 μm. Scale bar:microscopy. 20 µm.
2.6. Analysis Analysis of of Transcriptional TranscriptionalActivity Activityofofthe thehST3Gal hST3GalVVPromoter Promoterby bySD SDin inMG-63 MG-63Cells Cells 2.6. Since the the mRNA mRNA transcript transcript levels levels of of hST3Gal hST3Gal V V were were significantly significantly increased increased by by SD SD in in MG-63 MG-63 Since cells (Figures 3 and 4), using the luciferase reporter gene assay system, the transcriptional activity of cells (Figures 3 and 4), using the luciferase reporter gene assay system, the transcriptional activity thethe hST3Gal V promoter was evaluated to clarify whether the the transcriptional activity of hST3Gal V is of hST3Gal V promoter was evaluated to clarify whether transcriptional activity of hST3Gal controlled in SD-induced MG-63 cells. As shown in Figure 6A, a markedly enhanced luciferase V is controlled in SD-induced MG-63 cells. As shown in Figure 6A, a markedly enhanced luciferase activity was was observed observed in in cells cells harboring harboring the the pGL3-432 pGL3-432 construct construct under under serum-free serum-free conditions conditions for for activity 24 h, h, about in in 10% FBS-containing medium. However, SD in cells 24 about 3.0-fold 3.0-foldhigher higherthan thancells cellsincubated incubated 10% FBS-containing medium. However, SD in carrying other promoter constructs and the pGL3-basic (negative control) did not trigger the cells carrying other promoter constructs and the pGL3-basic (negative control) did not trigger the remarkableincrease increaseof oftheir theirluciferase luciferaseactivities. activities.These Thesedata datasuggest suggestthat thatthe theregion regionbetween between´432 −432 and and remarkable −178 is indispensable for the SD-responsive promoter function of hST3Gal V in MG-63 cells. ´178 is indispensable for the SD-responsive promoter function of hST3Gal V in MG-63 cells. 2.7. Identification Identification of of the the SD-Responsive SD-Responsive Element Element in in the the Functional Functional´432/´178 −432/−178 Region 2.7. Regionofof the hST3Gal VthePromoter hST3Gal V Promoter To Toidentify identifythe theSD-responsive SD-responsiveelement elementin inthe thenucleotide nucleotide´432 −432 to to ´178 −178 region of the hST3Gal hST3Gal V V gene, gene, based based on on the the above above results, results, we we firstly firstly focused focused on on transcription transcription factor factor Runx2, Runx2, which which is is essential essential for differentiation and and isisexpressed expressedininallallosteoblasts osteoblasts[30]. [30]. DNA sequence analysis, for osteoblast differentiation ByBy DNA sequence analysis, we we found two potential Runx2 binding sites in the promoter region between ´432 and ´177 of the found two potential Runx2 binding sites in the promoter region −432 −177 the hST3Gal hST3Gal V V gene. gene. Two Two are are located locatedside-by-side side-by-side at atpositions positions´232 −232 and and ´222 −222 in the hST3Gal hST3Gal V V gene gene (Figure (Figure 6C). 6C). These These DNA DNA sequences, sequences, ACCGCACCGCA, ACCGCACCGCA, closely closely match match the the consensus consensus Runx Runx binding binding motif, 5′-PuACCPuCA-3′ [31]. To clarify whether these binding sites are closely associated with SD-induced expression of hST3Gal V in MG-63 cells, one mutant plasmid (pGL3-432muRunx2) was constructed (Figure 6B) and then transfected into MG-63 cells, and luciferase assays were performed. As shown in Figure 6C, pGL3-432muRunx2 remarkably decreased transcriptional activity to more than two-fold of pGL3-432. This result indicates that these Runx2-binding sites are indispensable for
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motif, 51 -PuACCPuCA-31 [31]. To clarify whether these binding sites are closely associated with SD-induced expression of hST3Gal V in MG-63 cells, one mutant plasmid (pGL3-432muRunx2) was constructed (Figure 6B) and then transfected into MG-63 cells, and luciferase assays were performed. As in2016, Figure 6C, pGL3-432muRunx2 remarkably decreased transcriptional activity to more6than Int. shown J. Mol. Sci. 17, 35 of 13 two-fold of pGL3-432. This result indicates that these Runx2-binding sites are indispensable for the the SD-induced expression of hST3Gal V. To confirm further confirm Runx2 could bind to these SD-induced expression of hST3Gal V. To further whether whether Runx2 could bind to these sequences sequences in theVhST3Gal V we promoter, wechromatin used the chromatin immunoprecipitation (ChIP) assaythe to in the hST3Gal promoter, used the immunoprecipitation (ChIP) assay to exam exam the in vivo association of Runx2 with promoter region between −432 and −177 of the in vivo association of Runx2 with promoter region between ´432 and ´177 of the hST3Gal V gene hST3Gal V gene in SD-induced MG-63 cells.6D, As as shown FigurePCR 6D, products as the specific PCR products in SD-induced MG-63 cells. As shown Figure the specific obtained with input obtained with input amplification DNA, Runx2-specific amplification PCRcells wasincubated observedunder in MG-63 cells DNA, Runx2-specific by PCR was observed in by MG-63 serum-free incubated under serum-free medium for 24 h to induce the expression of the hST3Gal V gene. medium for 24 h to induce the expression of the hST3Gal V gene. However, distinct binding in a However, distinct binding control assaywas with FBS or These IgG antibody was notthat observed. These control assay with 10% FBS in or aIgG antibody not10% observed. results indicate the hST3Gal results indicate that the hST3GalMG-63 V gene expression in SD-induced MG-63of cells is upregulated by V gene expression in SD-induced cells is upregulated by direct binding Runx2 to the hST3Gal direct binding of Runx2 to the hST3Gal V promoter region containing Runx2 binding sites. V promoter region containing Runx2 binding sites.
Figure Figure 6. 6. Analysis Analysis of of the the hST3Gal hST3Gal V V promoter promoter activity activity in inSD-induced SD-induced MG-63 MG-63cells. cells. The The schematic schematic diagrams diagrams represent represent DNA DNA constructs constructs (A) (A) containing containing various various lengths lengths of of the the wild-type wild-type hST3Gal hST3Gal V V promoter (B,C) with with the the mutant mutantRunx2 Runx2sequence sequenceininthe the5′-flanking 51 -flanking region, upstream promoter or or constructs constructs (B,C) region, upstream of of a luciferase reportergene; gene;the thetranscription transcriptionstart startsite site is is designated designated +1. The pGL3-basic a luciferase reporter +1. The pGL3-basic construct, construct, which did not contain a promoter or an enhancer, was used as a negative control. construct which did not contain a promoter or an enhancer, was used as a negative control. EachEach construct was was transfected into MG-63 cells, with pRL-TK co-transfected as an internal control. The transfected transfected into MG-63 cells, with pRL-TK co-transfected as an internal control. The transfected cells cells incubated the presence bar) or absence ofFBS 10%for FBS 24 h. Relative werewere incubated in theinpresence (open(open bar) or absence (solid(solid bar) ofbar) 10% 24for h. Relative firefly firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System, and all luciferase activity was measured using the Dual-Luciferase Reporter Assay System, and all firefly firefly activity was normalized to the Renilla luciferase activity derived from pRL-TK. The values activity was normalized to the Renilla luciferase activity derived from pRL-TK. The values represent represent the±means SD of independent three independent experiments triplicate measurements. the means SD of˘three experiments withwith triplicate measurements. ** **pp
