Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells.

BACKGROUND/PURPOSE: Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. MATERIALS AND METHODS: Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. RESULTS: HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. CONCLUSION: These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

Autophagy upregulates inflammatory cytokines in gingival tissue of patients with periodontitis and lipopolysaccharide-stimulated human gingival fibroblasts.

BACKGROUND: Periodontitis is an inflammatory disease caused by multiple disease-associated bacterial species in periodontal tissues. Autophagy is known to modulate various inflammation-driven diseases and inflammatory responses, but the role of autophagy related to the pathogenesis of periodontitis is not fully established. We investigated whether autophagic flux regulated the expression of inflammatory cytokines in the gingiva of periodontitis patients and lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs) and the underlying mechanism. METHODS: The mRNA and protein expression of proinflammatory cytokines was assessed in human gingival tissues collected from patients with periodontitis and HGFs treated with LPS. The expression of signaling molecules related to autophagy was evaluated by immunofluorescence and Western blot analyses. RESULTS: The expression of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1) was increased in the gingival tissues of patients with periodontitis. LC3B-positive cells, a typical autophagic marker, were increased in the gingival tissues of periodontitis patients and LPS-treated HGFs. The conversion ratio of LC3-I to LC3-II was higher in the gingival tissues associated with periodontitis and LPS-treated HGFs compared to the controls. The autophagy inhibitor 3-methyladenine (3MA) significantly abrogated the LPS-sustained inflammatory effect by reducing the expression of IL-6, TNF-α, COX-2, and ICAM-1 in HGFs. The phosphorylation of protein kinase B (AKT) and protein S6K1 (S6), signals involved in the mTOR-dependent mechanism, was decreased in gingiva derived from periodontitis patients and LPS-treated HGFs. CONCLUSIONS: Autophagy augmented the production of inflammatory cytokines by mTOR inactivation via the AKT signaling pathway in the gingival tissues of patients with periodontitis and LPS-stimulated HGFs. These findings would provide a better understanding of the mechanism by which autophagy regulates the inflammatory response associated with periodontal pathogenesis.

Mineral trioxide aggregate-induced AMPK activation stimulates odontoblastic differentiation of human dental pulp cells.

AIM: To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). METHODOLOGY: In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. RESULTS: Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P < 0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P < 0.05) and DMP1 (P < 0.05) as well as formation of mineralized nodules (P < 0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P < 0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P < 0.01) and DMP1 (P < 0.05), but increased the phosphorylation of mTOR (P < 0.05), p70S6 (P < 0.01) and Unc-51-like kinases 1 (ULK1) (ser757) (P < 0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. CONCLUSION: MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.

Autophagy-Related Protein MAP1LC3C Plays a Crucial Role in Odontogenic Differentiation of Human Dental Pulp Cells.

BACKGROUND: Autophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs. METHODS: After tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining. RESULTS: After tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs. CONCLUSION: Our results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.

A Coil-to-Helix Transition Serves as a Binding Motif for hSNF5 and BAF155 Interaction.

Human SNF5 and BAF155 constitute the core subunit of multi-protein SWI/SNF chromatin-remodeling complexes that are required for ATP-dependent nucleosome mobility and transcriptional control. Human SNF5 (hSNF5) utilizes its repeat 1 (RPT1) domain to associate with the SWIRM domain of BAF155. Here, we employed X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and various biophysical methods in order to investigate the detailed binding mechanism between hSNF5 and BAF155. Multi-angle light scattering data clearly indicate that hSNF5171-258 and BAF155SWIRM are both monomeric in solution and they form a heterodimer. NMR data and crystal structure of the hSNF5171-258/BAF155SWIRM complex further reveal a unique binding interface, which involves a coil-to-helix transition upon protein binding. The newly formed αN helix of hSNF5171-258 interacts with the ß2-α1 loop of hSNF5 via hydrogen bonds and it also displays a hydrophobic interaction with BAF155SWIRM. Therefore, the N-terminal region of hSNF5171-258 plays an important role in tumorigenesis and our data will provide a structural clue for the pathogenesis of Rhabdoid tumors and malignant melanomas that originate from mutations in the N-terminal loop region of hSNF5.

Autophagy induction plays a protective role against hypoxic stress in human dental pulp cells.

Human dental pulp exposed to hypoxic conditions induces cell death accompanied by autophagy. However, the role of hypoxia-induced autophagy in human dental pulp cells (HDPCs) is unclear. The present study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of HDPCs. Cobalt chloride (CoCl2 ) treated HDPCs, to mimic hypoxic conditions, decreased cell viability. Also, apoptosis-related signal molecules, cleaved caspase-3 and PARP levels, were enhanced in CoCl2 -treated HDPCs. HDPCs exposed to CoCl2 also promoted autophagy, showing upregulated p62 and microtubule-associated protein 1 light chain 3 (LC3)-II levels, typical autophagic markers, and increased acidic autophagolysosomal vacuoles. Autophagy inhibition by 3 methyladenine (3MA) or RNA interference of LC3B resulted in increased levels of cleaved PARP and caspase-3, and the release of cytochrome c from mitochondria into cytosol in the CoCl2 -treated HDPCs. However, autophagy activation by rapamycin enhanced the p62 and LC3-II levels, whereas it reduced PARP and caspase-3 cleavage induced by CoCl2. These results revealed that CoCl2 -activated autophagy showed survival effects against CoCl2 -induced apoptosis in the HDPCs. CoCl2 upregulated HIF-1α and decreased the phosphorylation of mTOR/p70S6K. HIF-1α inhibitor, YC-1 decreased p62 and LC3-II levels, whereas it augmented PARP and caspase-3 cleavage in response to CoCl2 . Also, YC-1 enhanced the phosphorylation of mTOR and p70S6K suppressed by CoCl2 , demonstrating that CoCl2 -induced autophagy via mTOR/p70S6K is mediated by HIF-1α. Taken together, these finding suggest that CoCl2 -induced autophagy mediated by the mTOR/p70S6K pathway plays a protective role against hypoxic stress in HDPCs.

Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation.

Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells.

Epigallocatechin gallate protects against nitric oxide-induced apoptosis via scavenging ROS and modulating the Bcl-2 family in human dental pulp cells.

Nitric oxide (NO) is produced by three different isoforms of the enzyme NO synthase (NOS). NOS isoforms are expressed in many cell types, including human dental pulp cells (HDPC). NO acts as an intracellular messenger at physiological levels although it can be cytotoxic at higher concentrations. Epigallocatechin gallate (EGCG), a major green tea polyphenol, has diverse pharmacological activities in cell growth and death. This study is aimed to investigate the apoptotic mechanism by NO and effects of EGCG on NO-induced apoptosis in HDPC. Sodium nitroprusside (SNP), an NO donor, decreased the cell viability of HDPC in a dose- and time-dependent manner. EGCG was administered for 1 hr before the SNP treatment, resulting in increased cell viability and reactive oxygen species (ROS) production inhibition. Expression of Bax, a pro-apoptotic Bcl-2 family, was upregulated, whereas expression of Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated in SNP-treated HDPC. SNP augmented the release of cytochrome c from mitochondria into cytosol and enhanced caspase-9, and -3 activities, a marker of the apoptotic executing stage. EGCG ameliorated caspase-9 and -3 activities and cytochrome c release increased by SNP. These results suggest that EGCG has a protective effect against NO-induced apoptosis in HDPC by scavenging ROS and modulating the Bcl-2 family.