| Background:Human dental pulp stem cells(hDPSCs)are considered to be ideal seed cells for dental pulp regeneration due to their characteristics of good tooth formation potential,easy access,and wide sources.Evidence indicates mutations of the Mg2+transporters TRPM7 and CNNM4 result in deficient dentin mineralization,confirming Mg2+involvement in tooth development.Animals fed low Mg2+diets show deficient dentin and enamel mineralization,demonstrating that Mg2+plays an important role in odontogenic differentiation of tooth.Studies have found high extracellular Mg2+ and its transporter regulate odontogenic differentiation in hDPSCs,with involvement in dentin mineralization.However,the regulatory mechanisms of Mg2+effects on odontogenic differentiation in hDPSCs remain undefined.The study was to test magnesium(Mg2+)-enriched microenvironment’ effect on odontogenic differentiation of hDPSCs’ migration and proliferation ability and the cDNA sequencing and KEGG pathway analysis during odontogenic differentiation of hDPSCs.The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway.Methods:hDPSCs were isolated and cultured used modified tissue explant collagenase method.Cell Counting Kit-8 was applied as directed by the manufacturer.Scratch test was tested Migration ability of hDPSCs.Alizarin red S staining was carried out as proposed by the manufacturer.After 14 days,mineralization nodules were assessed by Alizarin red S staining.Destaining was performed with 10%cetylpyridinium chloride monohydrate buffer for 30 min,and optical density was obtained at 575 nm.After 7 days,the protein amounts of RUNX2,DSP,and DMP-1 were determined by automated immunoblot.Intracellular magnesium amounts in hDPSCs were assessed with Mag-Fluo-4-AM as directed by the manufacturer.Briefly,Mag-Fluo-4-AM was added at 2 μM and incubated for 30 min.This was followed by three cell washes.Fluorescence intensities were obtained by flow cytometry in triplicate assays.hDPSCs were cultured in odontogenic medium with 5mM Mg2+.After 7 days,RNA-seq was carried out according to our previous work.Suitable fragments were amplified,and an Illumina HiSeqTM 2000 was employed for the cDNA sequencing.To assess MAPK and BMP2/Smads signaling involvement,hDPSCs underwent culture in 12-well plates in odontogenic medium with the addition of 0,1,5,or 10mM Mg2+ for 7 days,respectively.Then,total protein isolated from hDPSCs was to measure the protein amounts of ERK,p-ERK,JNK,p-JNK,p38,p-p38,BMP2,BMPR1 and p-Smad1/5/9 by automated immunoblot.To assess 2-APB involvement,hDPSCs underwent culture in 12-well plates in odontogenic medium with the addition of 0,5mM Mg2+or 100 μM 2-APB for 7 days,respectively.Total protein from hDPSCs was obtained with RIPA.Automated immunoblot was carried out with Simple Wes as directed by the manufacturer.Primary antibodies targeting ERK,p-ERK,BMP2,BMPR1 and p-Smad 1/5/9 were probed.The compass software(ProteinSimple)was utilized for analysis.To assess U0126 involvement,during odontogenic differentiation of hDPSCs administered 5mM Mg2+ for 7 days,ERK/MAPK and BMP2/Smads signaling was suppressed with 50 μM U0126 for 2h every 2 days.Total protein from hDPSCs was isolated to measure the protein amounts of ERK,p-ERK,JNK,p-JNK,p38,p-p38 and BMP2,BMPR1 and p-Smad1/5/9 by automated immunoblot.Results:The Primary and subcultured hDPSCs were observe under microscope.The cells were spindle shape.The cells were capable of subculture until cell confluence was approximately 80%.The scratch test results show that Mg2+concentration affects the migration ability of hDPSCs.Compared with the control group,the migration ability of hDPSCs was significantly enhanced when 1,5,and 10 mM Mg2+was added to the culture medium(P<0.05).5 and 10 mM Mg2+had no significant difference on the effect of hDPSCs migration capacity(P>0.05).Cell Counting Kit-8 analysis showed that high extracellular Mg2+ did not affect hDPSCs proliferation(P>0.05).To analyze the impact of high extracellular Mg2+on their differentiation,hDPSCs were cultured in Mg2+-enriched odontogenic medium at different concentrations(0mM,1 mM,5mM and 10 mM).After 7 days,mineralization nodules were also markedly increased.However,there was no difference between the 5 and 10mM Mg2+ groups.These results showed that the Mg2+-enriched microenvironment promoted the mineralization nodules of hDPSCs.RUNX2,DMP-1,and DSP protein amounts were markedly elevated in hDPSCs treated with 1mM,5 mM and 10mM Mg2+,respectively,compared with 0mM Mg2+.Intracellular magnesium levels in hDPSCs treated with Mg2+-enriched odontogenic media at different concentrations(0 mM,1 mM,5 mM,and 10 mM)were assessed flow cytometrically using Mag-Fluo-4-AM.Intracellular Mg2+was starkly increased in hDPSCs treated with 1mM,5mM,and 10mM Mg2+,compared with the 0mM Mg2+group.However,there was no difference in intracellular magnesium of hDPSCs treated with 5mM and 10mM Mg2+,which is consistent with their comparable effects on odontogenic differentiation.Mg2+ entry was significantly blunted by 2-APB(100μM,inhibiting TRPM7).The positive effect of high extracellular Mg2+(5mM Mg2+)on odontogenic differentiation of hDPSCs was also blocked by 2-APB.These results indicated that high extracellular Mg2+ promoted odontogenic differentiation in hDPSCs via intracellular Mg2+increase.Compared with control cells(0mM Mg2+),exposure to high extracellular Mg2+(5mM Mg2+)for 7 days resulted in 734 DEGs in hDPSCs,including 250 upregulated and 484 downregulated.KEGG pathway analysis indicated that the DEGs retrieved participated in many pathways,e.g.,MAPK and TGF-β pathways.As shown above,KEGG pathway analysis revealed MAPK signaling activation by high extracellular Mg2+ during odontogenic differentiation of hDPSCs.To verify this finding,hDPSCs were incubated in odontogenic medium with the addition of 0,1,5 and 10mM Mg2+for 7 days,respectively.The results showed that ERK phosphorylation was markedly enhanced in hDPSCs treated with 1,5 and 10mM Mg2+ compared with the 0mM Mg2+group(P<0.05),but p38 and JNK phosphorylation showed no change(P>0.05).The results showed that ERK phosphorylation was markedly decreased in hDPSCs treated with 100μM 2-APB compared with the 5mM Mg2+ group.In accordance,the positive effect of high extracellular Mg2+ on ERK phosphorylation was blocked by 2-APB.To verify the above KEGG pathway data,we performed Western blot to determine whether BMP2/Smads signaling is activated by high extracellular Mg2+.The results showed that BMP2,BMPR1,and phosphorylated Smad 1/5/9 were significantly increased in hDPSCs cultured in odontogenic medium containing high extracellular Mg2+.The positive effects of high extracellular Mg2+on BMP2,BMPR1,and phosphorylated Smad1/5/9 were blocked by 2-APB.We inhibited ERK signaling with U0126,and mineralization nodules were markedly decreased in hDPSCs treated with U0126 than Mg2+-enriched odontogenic medium(5mM Mg2+)for 7 days group.Accordingly,RUNX2,DSP,and DMP-1 protein amounts in hDPSCs treated with Mg2+-enriched odontogenic medium(5mM Mg2+)for 7 days.Interestingly,BMP2,BMPR1 and phosphorylated Smad 1/5/9 amounts were also significantly decreased by U0126,indicating that BMP2/Smads acted as downstream of ERK.Taken together,these findings suggested that Mg2+-enriched microenvironment promoted odontogenic differentiation in hDPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+increase.Conclusions:The Mg2+-enriched microenvironment could promote migration of hDPSCs.Cell Counting Kit-8 analysis showed that high extracellular Mg2+did not affect hDPSC proliferation.High extracellular Mg2+alters the transcriptome of hDPSCs,there were 734 DEGs in hDPSCs,including 250 upregulated and 484 downregulated;KEGG pathway analysis indicated that the DEGs retrieved participated in many pathways,e.g.,MAPK and TGF-β pathways.Taken together,these findings suggested that Mg2+-enriched microenvironment promoted odontogenic differentiation in hDPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. |
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