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Autophagy is an intracellular self-degradation process that is essential for tissue development, cell differentiation, and survival. Nevertheless, the role of autophagy in tooth development has not been definitively identified. The goal of this study was to investigate how autophagy is involved in midkine (MK)–mediated odontoblast-like differentiation, mineralization, and tertiary dentin formation in a mouse tooth pulp exposure model. In vitro studies show that MK and LC3 have similar expression patterns during odontoblast-like cell differentiation. Odontoblast-like cell differentiation is promoted through MK-mediated autophagy, which leads to increased mineralized nodule formation. Subcutaneous transplantation of hydroxyapatite/tricalcium phosphate with rMK-treated human dental pulp cells led to dentin pulp–like tissue formation through MK-mediated autophagy. Furthermore, MK-mediated autophagy induces differentiation of dental pulp cells into odontoblast-like cells that form DSP-positive tertiary dentin in vivo. Our findings may provide 1) novel insight into the role of MK in regulating odontoblast-like differentiation and dentin formation in particular via autophagy and 2) potential application of MK in vital pulp therapy.

Dentin, which composes most of the tooth structure, is formed by odontoblasts, long-lived post-mitotic cells maintained throughout the entire life of the tooth. In mature odontoblasts, however, cellular activity is significantly weakened. Therefore, it is important to augment the cellular activity of mature odontoblasts to regenerate physiological dentin; however, no molecule regulating the cellular activity of mature odontoblasts has yet been identified. Here, we suggest that copine-7 (CPNE7) can reactivate the lost functions of mature odontoblasts by inducing autophagy. CPNE7 was observed to elevate the expression of microtubule-associated protein light chain 3-II (LC3-II), an autophagy marker, and autophagosome formation in the pre-odontoblast and mature odontoblast stages of human dental pulp cells. CPNE7-induced autophagy upregulated DSP and DMP-1, odontoblast differentiation and mineralization markers, and augmented dentin formation in mature odontoblasts. Furthermore, CPNE7 also upregulated NESTIN and TAU, which are expressed in the physiological odontoblast process, and stimulated the elongation of the odontoblast process by inducing autophagy. Moreover, lipofuscin, which progressively accumulates in long-lived post-mitotic cells and hinders their proper functions, was observed to be removed in recombinant CPNE7-treated mature odontoblasts. Thus, CPNE7-induced autophagy reactivated the function of mature odontoblasts and promoted the formation of physiological dentin in vivo. On the other hand, the well-known autophagy inducer, rapamycin, promoted odontoblast differentiation in pre-odontoblasts but did not properly reactivate the function of mature odontoblasts. These findings provide evidence that CPNE7 functionally reactivates mature odontoblasts and introduce its potential for dentinal loss-targeted clinical applications.

We aim to examine the effects of a newly developed peptide derived from CPNE7 (Cpne7-DP) in tertiary dentin formation and peritubular space occlusion, and comprehensively evaluate its potential as a bioactive therapeutic agent. Human dental pulp cells (HDPCs) and a mouse pre-odontoblast cell line, MDPC-23, were chosen for in vitro studies to characterize lineage-specific cell responses after Cpne7-DP treatment. Whether Cpne7-DP reproduces the dentin regenerative potential of CPNE7 was tested using a beagle dog model by generating dentinal defects of various degrees in vivo. Peritubular space occlusion was further examined by scanning electron microscopy and microleakage test, while overall mineralization capacity of Cpne7-DP was tested ex vivo. CPNE7 promotes tubular dentin formation under both shallow and deep dentinal defects, and the functional peptide Cpne7-DP induces odontoblast-like differentiation in vitro, mineralization ex vivo, and tubular dentin formation in in vivo beagle dog dentin exposure and pulp exposure models. Moreover, Cpne7-DP leads to peritubular space occlusion and maintains stability under different conditions. We show that CPNE7 and its derivative functional peptide Cpne7-DP promotes dentin regeneration in dentinal defects of various degrees and that the regenerated hard tissue demonstrates the characteristics of true dentin. Limitations of the current dental materials including post-operative hypersensitivity make biological repair of dentin a field of growing interest. Here, we suggest that the dual functions of Cpne7-DP in tubular dentin formation and peritubular space occlusion are promising for the treatment of dentinal loss and sensitivity.

For the dentin regeneration, dental epithelial cells are indispensible and must possess odontoblastic induction capability. Epithelial cell-like stem cells were recently identified in human deciduous dental pulp (DPESCs). However, their cellular characteristics remain poorly defined. The purpose of this study was to characterize DPESCs compared to HAT-7 ameloblastic cells. Expression levels of ameloblast-specific markers [odontogenic ameloblast-associated protein (Odam), matrix metalloproteinase (Mmp)-20, amelogenin, and ameloblastin] were detected in DPESCs. Co-culturing odontoblastic MDPC-23 cells with DPESCs increased expression of odontoblast differentiation markers (Dmp1 and Dspp) from days 4 to 10, while the expression of bone sialoprotein rapidly decreased. MDPC-23 cells cultured in DPESC-conditioned medium (CM) showed increased Dspp promoter activity compared with control MDPC-23 cultures. Mineralization was first observed in the CM groups from day 4 and proceeded rapidly until day 14, whereas mineralized nodules were found from day 7 in control media-cultured cells. In conclusion, DPESCs in human deciduous pulp possess ameloblast-like characteristics and differentiation properties, and substances derived from DPESCs promote odontoblastic differentiation. Thus, our results indicate that DPESCs can be a realistic epithelial source for use in odontoblastic induction and dentin formation of dental mesenchymal cells.

The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.

The aim of the present study was to examine the regulation of interleukin (IL)-11 expression, as well as the role of IL-11, during ovulation in gonadotropin-primed immature rats. Injection of equine chorionic gonadotropin (eCG), followed by human CG (hCG) to induce superovulation stimulated expression of the Il11 gene in theca cells within 6 h, as revealed by northern blot and in situ hybridisation analyses. Real-time reverse transcription–polymerase chain reaction analysis showed that the IL-11 receptor, α subunit gene was expressed in granulosa and theca cells and that injection of hCG had no effect on its expression. IL-11 protein expression was stimulated in theca cells by hCG. LH-stimulated increases in Il11 mRNA levels in cultured preovulatory follicles were inhibited by protein kinase A and mitogen-activated protein kinase kinase inhibitors. Toll-like receptor (TLR) 2 and TLR4 were detected in preovulatory follicles, and the TLR4 ligand lipopolysaccharide, but not the TLR2 ligand Pam3Cys, increased Il11 mRNA levels in theca cells, but not in granulosa cells. Treatment of preovulatory follicles with IL-11 stimulated progesterone production and steroidogenic acute regulatory protein (Star) gene expression. Together, these results indicate that IL-11 in theca cells is stimulated by mitogen-activated protein kinase signalling and TLR4 activation, and increases progesterone production during ovulation.

Aims The aim of the present study was to characterize genes regulated by protein kinase C (PKC)ζ inhibitor in the preovulatory granulosa cells following LH stimulation in the rat ovary. Main methods Annealing control primer (ACP)-based polymerase chain reaction (PCR) method was used to identify differentially expressed genes in granulosa cells of preovulatory follicles cultured in the presence of luteinizing hormone (LH) and myristoylated PKCζ pseudosubstrate peptide or a similarly sized control peptide. Key findings Among the 16 genes identified, five (testin, glypican-4, retrovirus SC1, aminolevulinic acid synthase 1 and serum-inducible kinase) experienced rapid and transient stimulation of gene expression upon exposure to human chorionic gonadotropin (hCG) in the ovary of immature rats primed with pregnant mare's serum gonadotropin (PMSG). In situ hybridization analysis revealed that hCG administration induced expression of these five genes in granulosa cells of preovulatory follicles. The Western analysis showed that the protein levels of testin and serum-inducible kinase were also increased by hCG. Expression of the eleven remaining genes in the ovary remained high at 24–72 h following hCG treatment. Significance The present data demonstrate the gonadotropin stimulation of genes differentially expressed by PKCζ inhibitor, implicating that PKCζ pathway possibly plays a role in controlling the ovulation process.

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.

We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 microM), but not by low doses RO 31-8220 (0.1-1.0 microM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCzeta activity. Interestingly, like LH, forskolin was also able to activate PKCzeta. Treatment with the cell-permeable PKCzeta-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCzeta. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCzeta pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCzeta in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCzeta, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.

NGFI-B is an immediate-early gene that encodes an orphan nuclear receptor. In the rat ovary, the preovulatory surge of LH induces NGFI-B expression in granulosa cells of preovulatory follicles, reaching a peak within 1 h and declining to control levels at 6 h. The LH-stimulated NGFI-B expression is abolished by α-amanitin, but superinduced by cycloheximide. Similarly, treatment of human luteinized granulosa cells with LH causes a rapid and transient stimulation of NGFI-B expression. Interestingly, the induction of NGFI-B expression in response to LH stimulation in preovulatory granulosa cells requires signaling through protein kinase Cζ. Furthermore, two other NGFI-B family members, Nurr1 and Nor1, are also rapidly stimulated by LH in granulosa cells of preovulatory follicles through the activation of protein kinase Cζ. The cell-type specific expression and LH induction of NGFI-B suggests a potential role of NGFI-B in the ovulatory process.

Objective Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). Methods We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). Results Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. Conclusion We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.