Hsa_circ_0099630 knockdown induces the proliferation and osteogenic differentiation and attenuates the apoptosis of porphyromonas gingivalis lipopolysaccharide-induced human periodontal ligament fibroblasts.

Background: Periodontitis is an inflammatory destructive bone disease and is the most critical cause of tooth loss in adults. Recent studies have reported that circular RNAs (circRNAs) are essential in periodontitis. However, the influence and mechanism of hsa_circ_0099630 on periodontitis are not clear. Methods: Normal periodontal tissues and inflammatory periodontal tissues were obtained from healthy patients and patients with periodontitis, respectively. Hsa_circ_0099630 was 1st identified by polymerase chain reaction (PCR) and sanger sequencing, and hsa_circ_0099630 expression was determined by real-time (RT)-quantitative PCR in periodontitis. Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) was used to construct an inflammation model in vitro. Next, cell proliferation, apoptosis, and osteogenic differentiation were monitored using Cell Counting Kit-8, flow cytometry, and western blot in the Pg-LPS-induced human periodontal ligament fibroblasts (HPLFs). The microRNA (miRNA)/messenger RNA (mRNA) axis of hsa_circ_0099630 was predicted and screened, and the function of the target genes was analyzed by a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Results: The identified hsa_circ_0099630 was upregulated in the gingival tissue of patients with periodontitis. Next, an inflammation model was constructed using Pg-LPS in the HPLFs. We discovered that Pg-LPS or hsa_circ_0099630 overexpression suppressed cell proliferation and osteogenic differentiation, and induced apoptosis in HPLFs. Additionally, hsa_circ_0099630 knockdown induced proliferation and osteogenic differentiation and prevented apoptosis in the Pg-LPS-induced HPLFs. We also screened the vast miRNA/mRNA axis associated with hsa_circ_0099630. Conclusions: The current study uncovered the crucial role of hsa_circ_0099630 in periodontitis.

Pluripotency of periodontal ligament and dental pulp cells is induced by intercellular communication via the CDX2/Oct-4/Sox2 pathway.

Background: Intercellular communication in the environments of mature or aged cells can restore and regenerate their function and promote the expression of pluripotency markers. The regeneration of dental tissue is stimulated by periodontal ligament cells (PDLCs) and dental pulp cells (DPCs). However, the communication networks between the cells and their microenvironments are poorly understood. Methods: In this study, gene expression was analyzed by polymerase chain reaction, and chromatin immunoprecipitation assays, dual-luciferase assays, and electrophoretic mobility shift assays were used to analyze the signaling pathways associated with pluripotency after the knockdown or overexpression of caudal-type homeobox transcription factor 2 (CDX2). Results: Elevated levels of SRY-box transcription factor 2 (Sox2) and octamer-binding transcription factor 4 (Oct-4) were observed in the co-culture system, while the levels of CDX2 were significantly reduced. The overexpression of CDX2 promoted cell apoptosis and reduced the synthesis stage of the cell cycle. CDX2 was shown to bind directly to the promoter regions of Sox2 and Oct-4. The silencing of CDX2 promoted calcium deposition, adipogenic differentiation, and elevated alkaline phosphatase (ALP) activity in the DPCs. Conclusions: These findings demonstrate the enhancement of DPC and PDLC pluripotency by intercellular communication. CDX2 plays a significant part in the regulation of DPC and PDLC pluripotency via its regulation of Oct-4 and Sox2 expression.

Pluripotency of Dental Pulp Cells and Periodontal Ligament Cells Was Enhanced through Cell-Cell Communication via STAT3/Oct-4/Sox2 Signaling.

Alternation in culture environment due to cell-cell communications can rejuvenate the biological activity of aged/differentiated cells and stimulate the expression of pluripotency markers. Dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) are promising candidates in dental tissue regeneration. However, the molecular network that underlies cell-cell communications between dental-derived cells and the microenvironment remains to be identified. To elucidate the signaling network that regulates the pluripotency of DPCs and PDLCs, proliferation, apoptosis, cell cycle, and the expression of Oct-4/Sox2/c-Myc in DPCs and PDLCs with indirect/direct coculture were examined. PCR arrays were constructed to identify genes that were differentially expressed, and the results were confirmed by a rat model with injury. Further research on the mechanism of the related signaling pathways was investigated by overexpression/silence of STAT3, ChIP, the dual-luciferase reporter assay, and EMSA. We found that the proliferation and apoptosis of DPCs and PDLCs were inhibited, and their cell cycles were arrested at the G0/G1 phase after coculture. Oct-4, Sox2, and STAT3 expression significantly increased and PAX5 expression decreased in the coculture systems. Oct-4/Sox2/STAT3/PAX5 was actively expressed in the rat defect model. Moreover, STAT3 was directly bound to the Oct-4 and Sox2 gene promoter regions and activated the expression of those genes. Our data showed that the pluripotency of DPCs and PDLCs was enhanced through cell-cell communication. STAT3 plays essential roles in regulating the pluripotency of DPCs and PDLCs by targeting Oct-4 and Sox2 both in vitro and in vivo.

Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells.

Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.

Luteolin and apigenin activate the Oct-4/Sox2 signal via NFATc1 in human periodontal ligament cells.

Identifying small molecules to activate the Oct-4/Sox2-derived pluripotency network represents a hopeful and safe method to pluripotency without genetic manipulation. Luteolin and apigenin, two major bioactive flavonoids, enhance reprogramming efficiency and increase expression of Oct-4/Sox2/c-Myc, albeit the detailed mechanism regulating pluripotency in dental-derived cells remains unknown. In the present study, to elucidate the effect of luteolin/apigenin on pluripotency of periodontal ligament cells (PDLCs) through interaction with downstream signals, we examined cell cycle, proliferation, apoptosis, expression of Oct-4/Sox2/c-Myc, and multilineage differentiation of PDLCs with luteolin/apigenin treatment. Moreover, we profiled the differentially expressed pluripotency genes by PCR arrays. Our results demonstrated that luteolin/apigenin restrained cell proliferation, increased apoptosis, and arrested PDLCs in G2/M and S phase. Luteolin and apigenin activated expression of Oct-4, Sox2, and c-Myc in a time- and dose-dependent pattern, and repressed lineage-specific differentiation. PCR arrays profiled multiple signals in PDLCs with luteolin/apigenin treatment, among which NFATc1 was the major upregulated gene. Notably, blocking of the NFATc1 signal with INCA-6 significantly decreased mRNA and protein expression of Oct-4, Sox2, and c-Myc in PDLCs with luteolin/apigenin treatment, indicating that NFATc1 may act as an upstream modulator of Oct-4/Sox2 signal. Taken together, this study showed that luteolin and apigenin effectively maintain pluripotency of PDLCs through activation of Oct-4/Sox2 signal via NFATc1.

XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc.

Introduction. Xeroderma pigmentosum group C (XPC), essential component of multisubunit stem cell coactivator complex (SCC), functions as the critical factor modulating pluripotency and genome integrity through interaction with Oct-4/Sox2. However, its specific role in regulating pluripotency and multilineage differentiation of human dental pulp cells (DPCs) remains unknown. Methods. To elucidate the functional role XPC played in pluripotency and multilineage differentiation of DPCs, expressions of XPC in DPCs with long-term culture were examined by real-time PCR and western blot. DPCs were transfected with lentiviral-mediated human XPC gene; then transfection rate was investigated by real-time PCR and western blot. Cell cycle, apoptosis, proliferation, senescence, multilineage differentiation, and expression of Oct-4/Sox2/c-Myc in transfected DPCs were examined. Results. XPC, Oct-4, Sox2, and c-Myc were downregulated at P7 compared with P3 in DPCs with long-term culture. XPC genes were upregulated in DPCs at P2 after transfection and maintained high expression level at P3 and P7. Cell proliferation, PI value, and telomerase activity were enhanced, whereas apoptosis was suppressed in transfected DPCs. Oct-4/Sox2/c-Myc were significantly upregulated, and multilineage differentiation in DPCs with XPC overexpression was enhanced after transfection. Conclusions. XPC plays an essential role in the modulation of pluripotency and multilineage differentiation of DPCs through regulation of Oct-4/Sox2/c-Myc.

Effect of luteolin and apigenin on the expression of Oct-4, Sox2, and c-Myc in dental pulp cells with in vitro culture.

INTRODUCTION: Dental pulp cells (DPCs) are promising cell source for dental tissue regeneration. Recently, small molecules which optimize microenvironment or activate the reprogramming network provide a new way to enhance the pluripotency. Two promising bioflavonoids luteolin and apigenin were reported to enhance reprogramming efficiency in mouse embryonic fibroblast (MEF). However, their effect and underlying mechanism in cell fate determination of human DPCs remain unclear. METHODS: To elucidate the effect of luteolin and apigenin on the cell fate determination of DPCs, we explored the cell proliferation, cell cycle, senescence, apoptosis, expression of pluripotency markers Oct-4, Sox2, and c-Myc, and multilineage differentiation capability of DPCs with luteolin or apigenin treatment. RESULTS: We demonstrated that luteolin and apigenin inhibited cell proliferation, arrested DPCs in G2/M and S phase, and upregulated PI value and apoptosis. Moreover, luteolin and apigenin increased telomerase activity, maintained DPCs in a presenescent state, and activated the expression of Oct-4, Sox2, and c-Myc at a dose- and time-dependent pattern in DPCs even at late passages, albeit repressed lineage-specific differentiation. CONCLUSIONS: Addition of luteolin and apigenin in the culture medium might provide an effective way to maintain DPCs in an undifferentiated stage and inhibit lineage-specific differentiation.

Expression of Oct-4, SOX-2, and MYC in dental papilla cells and dental follicle cells during in-vivo tooth development and in-vitro co-culture.

During tooth development, the special structure of dental follicle and dental papilla enables dental papilla cells (DPCs) and dental follicle cells (DFCs) to make contact with each other. Octamer-binding transcription factor 4 (Oct-4), sex determining region Y box-2 (SOX-2), and cellular homologue of avian myelocytomatosis virus oncogene (MYC) (OSM) are associated with reprogramming and pluripotency. However, whether the expression of OSM could be activated through cell-cell communication is not known. In this study, the distribution of OSM in rat tooth germ was investigated by immunohistochemical staining. An in-vitro co-culture system of DPCs and DFCs was established. Cell proliferation, cell apoptosis, cell cycle stages, and expression of OSM were investigated by Cell Counting Kit 8 (CCK8) analysis, flow cytometry, real-time PCR, and immunohistochemical staining. We found that Oct-4 and SOX-2 were strongly expressed in tooth germ on days 7 and 9 after birth, whereas MYC was expressed only on day 9. Cell proliferation and apoptosis were inhibited, the cell cycle was arrested in the G0/G1 phase, and the propidium iodide (PI) value was downregulated. Expression of Oct-4 and SOX-2 was significantly elevated in both cell types after 3 d of co-culture, whereas expression of MYC was not significantly elevated until day 5. These results indicate that the optimized microenvironment with cell-cell communication enhanced the expression of reprogramming markers associated with reprogramming capacity in DPCs and DFCs, both in vivo and in vitro.

[The expression and modulatory role of reprogramming markers on the in vivo regeneration of dental pulp and periodontal ligament tissue after injury].

PURPOSE: The purpose of this study was to investigate the expression of reprogramming markers including Oct-4, Sox2 and c-Myc in rat dental pulp and periodontal ligament tissues undergoing tissue regeneration after injury. METHODS: Lewis rat model with dental pulp and periodontal ligament injury was created. HE staining was applied to investigate the structure of newly formed dentin-pulp complex and periodontium. The distribution of Oct-4, Sox2 and c-Myc was determined by immunofluorescent staining. RESULTS: HE staining showed that newly formed dentin-pulp complex and periodontium was constructed 4 weeks after injury. Immunofluorescent staining revealed Oct-4, Sox2 and c-Myc signal was activated in the newly formed dental pulp and periodontal ligament tissue after injury compared with the normal ones. Oct-4 and Sox2 showed similar expression pattern. CONCLUSIONS: Oct-4, Sox2 and c-Myc signaling may play an important modulatory role in the dental tissue regeneration after injury. Oct-4 and Sox2 may have operative interaction whereas c-Myc may play independent role during this process.