Advertisement

A WWP2–PTEN–KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice

  • Jing Fu
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China

    Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
    Search for articles by this author
  • Xiaobo Zhang
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China

    Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
    Search for articles by this author
  • Huiwen Zheng
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China

    Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
    Search for articles by this author
  • Guobin Yang
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
    Search for articles by this author
  • Zhi Chen
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
    Search for articles by this author
  • Guohua Yuan
    Correspondence
    For correspondence: Guohua Yuan
    Affiliations
    The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China

    Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
    Search for articles by this author
Open AccessPublished:June 30, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102220
      WW domain–containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2–PTEN–KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

      Keywords

      Abbreviations:

      2W (2 weeks), ALP (alkaline phosphatase), ARS (alizarin red S), co-IP (coimmunoprecipitation), IF (immunofluorescence), IHC (immunohistochemistry), KLF5 (Kruppel-like factor 5), mDPC (mouse dental papilla cell), PLA (proximity ligation assay), PN (postnatal), PTEN (phosphatase and tensin homolog), WWP2 (WW domain–containing E3 Ubiquitin-protein ligase 2)
      Odontoblasts are tooth-specific, terminally differentiated mesenchymal cells responsible for synthesis and secretion of collagens and noncollagenous proteins (
      • Mayor R.
      • Theveneau E.
      The neural crest.
      ,
      • Bleicher F.
      Odontoblast physiology.
      ). DSPP and DMP1 are two noncollagenous proteins and well recognized as important markers of odontoblast differentiation. They both contribute to the mineralization of predentin into dentin (
      • Butler W.T.
      Dentin matrix proteins and dentinogenesis.
      ). And mineralized dentin plays essential roles in providing support for enamel and cementum and protecting dental pulp from outside stimuli (
      • Goldberg M.
      • Kulkarni A.B.
      • Young M.
      • Boskey A.
      Dentin: structure, composition and mineralization.
      ). Therefore, unveiling the mechanisms of odontoblast differentiation and dentinogenesis is a hotspot in dental research field.
      As a crucial form of posttranslational modifications, ubiquitination has been found to mediate various cellular activities such as cell differentiation and cell fate determination (
      • Herhaus L.
      • Dikic I.
      Expanding the ubiquitin code through post-translational modification.
      ,
      • Rape M.
      Ubiquitylation at the crossroads of development and disease.
      ). When being ubiquitinated, the lysines of the substrate are modified with one or more ubiquitin(s). Then the target protein can be sent for proteolysis, or its function or cellular localization will be altered. Ubiquitin ligases (also called E3 ligases; E3s) play a significant role in the ubiquitination process (
      • Lorenz S.
      Structural mechanisms of HECT-type ubiquitin ligases.
      ). WW domain–containing E3 Ubiquitin-protein ligase 2 (WWP2) is a multifunctional E3 ubiquitin ligase and participates in various developmental processes including osteogenesis and palatogenesis (
      • Xu H.M.
      • Liao B.
      • Zhang Q.J.
      • Wang B.B.
      • Li H.
      • Zhong X.M.
      • et al.
      Wwp2, an E3 ubiquitin ligase that targets transcription factor Oct-4 for ubiquitination.
      ,
      • Zhu W.
      • He X.
      • Hua Y.
      • Li Q.
      • Wang J.
      • Gan X.
      The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.
      ,
      • Fang L.
      • Zhang L.
      • Wei W.
      • Jin X.
      • Wang P.
      • Tong Y.
      • et al.
      A methylation-phosphorylation switch determines Sox2 stability and function in ESC maintenance or differentiation.
      ,
      • Ambrozkiewicz M.C.
      • Schwark M.
      • Kishimoto-Suga M.
      • Borisova E.
      • Hori K.
      • Salazar-Lazaro A.
      • et al.
      Polarity acquisition in cortical neurons is driven by synergistic action of Sox9-regulated Wwp1 and Wwp2 E3 ubiquitin ligases and intronic miR-140.
      ,
      • Nakamura Y.
      • Yamamoto K.
      • He X.
      • Otsuki B.
      • Kim Y.
      • Murao H.
      • et al.
      Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25.
      ). Kruppel like factor 5 (KLF5) is an important odontoblast transcription factor. It is expressed in the odontoblast layer of mouse molars and incisors and can directly upregulate the expression of odontoblast marker genes, Dspp and Dmp1 (
      • Chen Z.
      • Zhang Q.
      • Wang H.
      • Li W.
      • Wang F.
      • Wan C.
      • et al.
      Klf5 mediates odontoblastic differentiation through regulating dentin-specific extracellular matrix gene expression during mouse tooth development.
      ,
      • Chen Z.
      • Couble M.L.
      • Mouterfi N.
      • Magloire H.
      • Chen Z.
      • Bleicher F.
      Spatial and temporal expression of KLF4 and KLF5 during murine tooth development.
      ,
      • Han N.
      • Chen Z.
      • Zhang Q.
      Expression of KLF5 in odontoblastic differentiation of dental pulp cells during in vitro odontoblastic induction and in vivo dental repair.
      ). WWP2 has been reported to facilitate odontoblastic differentiation of mouse dental papilla cells (mDPCs) through augmenting KLF5 transcriptional activity on Dspp and Dmp1 in a nonproteolytic monoubiquitination manner (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). However, the in vivo role of WWP2 during odontoblast differentiation and dentinogenesis is still unknown.
      Phosphatase and tensin homolog (PTEN) is a protein and lipid phosphatase that functions as a tumor suppressor. On the one hand, PTEN suppresses phosphatidylinositol 3 kinase/AKT signaling by hydrolyzing phosphatidylinositol (3,4,5)-trisphosphate to phosphatidylinositol (4,5)-bisphosphate, thus regulating various cellular behaviors including cell proliferation, differentiation, and migration. On the other hand, PTEN also mediates cell cycle, genomic stability, and gene expression in a phosphatidylinositol (3,4,5)-trisphosphate–independent manner (
      • Di Cristofano A.
      • Pandolfi P.P.
      The multiple roles of PTEN in tumor suppression.
      ,
      • Hopkins B.D.
      • Hodakoski C.
      • Barrows D.
      • Mense S.M.
      • Parsons R.E.
      PTEN function: the long and the short of it.
      ). PTEN has been found to participate in multiple developmental and regeneration processes, like prostate development, craniofacial development, T-cell lymphopoiesis, neurogenesis, and neural regeneration (
      • Lokody I.B.
      • Francis J.C.
      • Gardiner J.R.
      • Erler J.T.
      • Swain A.
      Pten regulates epithelial cytodifferentiation during prostate development.
      ,
      • Sun F.
      • Park K.K.
      • Belin S.
      • Wang D.
      • Lu T.
      • Chen G.
      • et al.
      Sustained axon regeneration induced by co-deletion of PTEN and SOCS3.
      ,
      • Yang T.
      • Moore M.
      • He F.
      Pten regulates neural crest proliferation and differentiation during mouse craniofacial development.
      ,
      • Li R.A.
      • Traver D.
      • Matthes T.
      • Bertrand J.Y.
      Ndrg1b and fam49ab modulate the PTEN pathway to control T-cell lymphopoiesis in the zebrafish.
      ,
      • Jo H.S.
      • Kang K.H.
      • Joe C.O.
      • Kim J.W.
      Pten coordinates retinal neurogenesis by regulating Notch signalling.
      ). Previously, PTEN was discovered to be present in the prospective preodontoblasts of dental papilla in human embryonic incisor tooth germs (
      • Kero D.
      • Cigic L.
      • Medvedec Mikic I.
      • Galic T.
      • Cubela M.
      • Vukojevic K.
      • et al.
      Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ - an immunohistochemical study.
      ). However, both the expression pattern and function of PTEN during odontoblast differentiation remains unclear. PTEN has been recognized as a substrate of WWP2 for ubiquitination-dependent degradation (
      • Liu J.
      • Wan L.
      • Liu J.
      • Yuan Z.
      • Zhang J.
      • Guo J.
      • et al.
      Cdh1 inhibits WWP2-mediated ubiquitination of PTEN to suppress tumorigenesis in an APC-independent manner.
      ,
      • Maddika S.
      • Kavela S.
      • Rani N.
      • Palicharla V.R.
      • Pokorny J.L.
      • Sarkaria J.N.
      • et al.
      WWP2 is an E3 ubiquitin ligase for PTEN.
      ,
      • Zhang H.
      • Wei P.
      • Lv W.
      • Han X.
      • Yang J.
      • Qin S.
      Long noncoding RNA lnc-DILC stabilizes PTEN and suppresses clear cell renal cell carcinoma progression.
      ). And a previous study has reported that Wwp2 knockout (Wwp2 KO) mice exhibit a phenotype similar to that of Pten transgenic mice with reduced sizes of the whole body and main organs (
      • Li H.
      • Zhang P.
      • Zhang Q.
      • Li C.
      • Zou W.
      • Chang Z.
      • et al.
      WWP2 is a physiological ubiquitin ligase for phosphatase and tensin homolog (PTEN) in mice.
      ). Nevertheless, the role of PTEN and whether it acts as a substrate of WWP2 during odontoblast differentiation needs further investigation.
      In this study, Wwp2 KO mice were established. On account of the continuous growth of mouse incisors, molars were chosen for further investigation regarding the role of Wwp2 deletion in tooth development because their developmental processes are more similar to human teeth. Besides, the function of PTEN in odontoblastic differentiation of mDPCs and its potential mechanism was investigated. Our study confirmed that WWP2 mediates odontoblast differentiation and dentinogenesis by promoting Dspp and Dmp1 expression through the WWP2–PTEN–KLF5 axis.

      Results

      WWP2 promotes dentin formation in vivo

      The expression pattern of WWP2 during early molar development has been reported in our previous study (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). Here, murine molars at later stages from postnatal 2 days (PN2) to 2 weeks (2W) were subjected to immunohistochemistry (IHC). The results showed that WWP2 was distinctly expressed in the odontoblast layer (Fig. 1A). The negative control of IHC was shown in Fig. S2.
      Figure thumbnail gr1
      Figure 1WWP2 is essential for dentin formation. A, the expression of WWP2 in murine molars at PN2 (a and a’), PN5 (b and b’), and 2W (c and c’) shown by IHC. Red arrows show the positive WWP2 staining in odontoblasts. Red dotted lines manifest the boundaries of odontoblasts. (a–c) are higher magnifications of the rectangles in (a’–c’). B, H&E staining (a–f and a’–f’) and micro-CT analysis (g–j) of molars in WT and Wwp2 KO mice. In (k–m), quantitative analysis shows significant reduction of predentin and dentin thickness as well as root length in molars of Wwp2 KO mice. n = 8; Student’s t test; Error bars represent SD. To precisely calculate the dentin formation changes between WT and Wwp2 KO mice, the widths of predentin and dentin were measured in one H&E-stained section every five consecutive slices. Black dotted lines represent the boundaries of dentin and predentin in (a–d), and the boundaries between enamel and dentin (g–j). (a–f) are higher magnifications of the rectangles in (a’–f’). Red arrows show more severe cusp abrasion in molars of Wwp2 KO mice. Red double arrows mark the roots. C, scanning electron microscopy images show the dentinal tubules of molars at 2W in Wwp2 KO mice are less formed than in WT. (c, d) are higher magnifications of the rectangles in (a, b). The roof of pulp chamber marked by the red rectangle represents the site for scanning electron microscopy (e). The dentinal tubules in WT and Wwp2 KO mice at 2W were quantified (f). In (f), n = 8; Student’s t test; Error bars represent SD. The scale bars represent 5 μm for (C), 20 μm for (A) a, b, (B) a, a’, b, b’; 50 μm for (A) c, (B) c, d; 100 μm for (B) e, f; 200 μm for (A) a’–c’ and (B) c’, d’, g–j, 500 μm for e’, f’. D, dentin; E, enamel; Od, odontoblast; Pd, predentin; Rel., relative.
      The in vivo role of WWP2 in regulating odontoblast differentiation and dentinogenesis was further investigated by Wwp2 KO mice. Knockout of Wwp2 was verified by genotyping, Western blot, and IHC (Fig. S1, BD). Interestingly, hematoxylin and eosin (H&E) staining revealed reduced widths of the mineralized dentin but increased widths of the nonmineralized predentin in Wwp2 KO mice at PN5 and 2W (Fig. 1B a–d, k and l). Meanwhile, ablation of Wwp2 led to more abrasion at the molar cusps (Fig. 1B e and f). Micro–computed tomography verified shorter roots in the molars of Wwp2 KO mice (Fig. 1B g–j and m). Besides, scanning electron microscopy of molars at 2W displayed less numbers of dentinal tubules in Wwp2 KO mice compared with the wildtype (WT) group (Fig. 1C a–f). Heterozygous mice did not show these defects (data not shown).

      WWP2 is essential for the differentiation and mineralization ability of odontoblasts

      To verify the in vivo role of WWP2 in odontoblast differentiation, the expression changes of odontoblast markers, DSPP and DMP1, were investigated by immunofluorescence (IF). The results showed that the expression levels of both markers were decreased in Wwp2 KO odontoblast layers compared with the WT group at PN5 and 2W (Fig. 2, A and B). The negative control of IF was shown in Fig. S3.
      Figure thumbnail gr2
      Figure 2WWP2 promotes the differentiation of odontoblasts. A and B, immunofluorescence staining of DSPP and DMP1 in molars of WT and Wwp2 KO mice at PN5 (A) and 2W (B). Red fluorescence shows the positive expression of DSPP or DMP1, while blue fluorescence shows the nucleus. C, the mRNA levels of Dspp and Dmp1 were significantly reduced in differentiation-induced Wwp2 KO mDPCs. n = 3; Student’s t test; Error bars represent SD. D, alizarin red S and alkaline phosphatase staining of WT and Wwp2 KO differentiation-induced mDPCs. For alizarin red S, the mDPCs were cultured in differentiation medium (DM) for 7 days. Ten percent cetylpyridinium chloride was used for harvesting the stained cells, and the absorbance values were measured at 562 nm. For alkaline phosphatase staining, the mDPCs were cultured in DM for 5 days. n = 3; Student’s t test; Error bars represent SD. The scale bars represent 20 μm in (A) and (B). Rel., relative.
      mDPCs from molars in both WT and Wwp2 KO mice were isolated and cultured in differentiation medium. Quantitative real-time PCR experiments showed that the mRNA levels of Dspp and Dmp1 were declined in Wwp2 KO cells (Fig. 2C). The mineralization ability of odontoblastic cells was assessed by alizarin red S (ARS) staining and alkaline phosphatase (ALP) assays (Fig. 2D). As expected, knockout of Wwp2 led to a decrease of mineralized nodule formation. ALP activity was also hampered by Wwp2 deficiency (Fig. 2D).

      PTEN acts as an inhibitor for odontoblastic differentiation

      To observe the expression pattern of PTEN in dental mesenchyme, IHC was performed in murine molars. The results showed that PTEN was positively stained in the dental papilla cells at E13.5 and E16.5 (Fig. 3A a and b). At PN1 and 2W, PTEN was expressed in the odontoblast layer (Fig. 3A c and d). Real-time PCR and ARS experiments showed that knockdown of Pten augmented the mRNA levels of Dspp and Dmp1 (Fig. 3B) as well as the formation of mineralized nodules (Fig. 3C). On the contrary, overexpression of Pten inhibited the formation of mineralized nodules (Fig. 3C). These results demonstrated that PTEN plays a negative role in odontoblastic differentiation.
      Figure thumbnail gr3
      Figure 3PTEN is expressed during tooth development and suppresses odontoblastic differentiation. A, the expression pattern of PTEN during tooth development detected by immunohistochemistry. (a–d) are higher magnifications of the rectangles in (a’–d’). Red arrows point to the positive signals of PTEN. B, the effects of Pten knockdown on the mRNA levels of Dspp and Dmp1. The mDPCs were cultured in DM for 7 days. n = 3; Student’s t test; Error bars represent SD. C, the effects of Pten knockdown or overexpression on the formation of mineralized nodules. The mDPCs were cultured in DM for 7 days and then were stained by alizarin red S. Ten percent cetylpyridinium chloride was used for harvesting the stained cells, and the absorbance values were measured at 562 nm. n = 3; Student’s t test; Error bars represent SD. The scale bars represent 20 μm for (A) a–d and a’, 100 μm for (A) b’–c’, and 500 μm for (A) d’. Am, ameloblast; D, dentin; Ep, epithelium; Mes, mesenchyme; Od, odontoblast; OE, overexpression; P, Pten; Scr, scramble; si, knockdown; Rel., relative.

      WWP2 physically interacts with PTEN in mouse molars

      WWP2 is expressed in the odontoblast layer and the underlying dental papilla cells (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). The identical expression pattern between PTEN and WWP2 suggests that they may have physical interaction. First, coimmunoprecipitation (co-IP) assays were performed. Overexpressed WWP2 and PTEN were shown to interact with each other in Human embryonic kidney 293E cell lines (HEK293E) cells (Fig. 4A). Besides, the interaction between endogenous WWP2 and PTEN in mDPCs was also observed (Fig. 4B). As expected, in situ proximity ligation assays (PLAs) further confirmed their interaction in odontoblasts and dental papilla cells in murine molars (Fig. 4C a and b). Deletion of Wwp2 abolished this interaction effect in vivo (Fig. 4C c and d).
      Figure thumbnail gr4
      Figure 4WWP2 physically interacts with PTEN in mouse molars. A, coimmunoprecipitation assays show the interaction between WWP2 and PTEN in HEK293E cells (overexpressed proteins). The arrow shows the indicated bands. B, coimmunoprecipitation assays show the interaction between WWP2 and PTEN in mDPCs (endogenous proteins). C, in vivo interaction between PTEN and WWP2 in PN5 molars of WT and Wwp2 KO mice revealed by in situ proximity ligation assay. b, d are higher magnifications of the rectangles in a, c. The scale bars represent 100 μm for (C) a and 20 μm for (C) b. Dp, dental papilla; Od, odontoblast.

      WWP2 regulates odontoblastic differentiation by promoting PTEN proteolysis

      Next, the proteolysis effect of WWP2 on PTEN was investigated in vivo and in vitro. IF revealed that ablation of WWP2 decreased degradation of PTEN and stabilized PTEN protein level at the odontoblast layer of mouse molars in vivo (Figs. 5A and S4, A and B). Western blot assay also showed that knockout of Wwp2 upregulated the protein level of PTEN (Fig. 5B). Overexpression of WWP2 led to decreased PTEN level, while overexpression of ligase-dead mutant WWP2 (WWP2C838A, in which cysteine 838 of WWP2 was mutated to alanine) did not show this effect (Figs. 5C and S5, A and A’). The regulation of WWP2 on the transcription level of Pten was excluded (Fig. S6A). Furthermore, it was demonstrated that overexpressed WWP2 accelerated the degradation rate of PTEN by using cycloheximide (a protein synthesis inhibitor) (Figs. 5D and S5, B and B’). The decreased differentiation and mineralization ability of mDPCs due to Wwp2 knockdown can be rescued by Pten knockdown (Fig. 5, E and F). Therefore, WWP2 promotes odontoblastic differentiation through targeting PTEN for degradation.
      Figure thumbnail gr5
      Figure 5WWP2 regulates odontoblastic differentiation by promoting PTEN proteolysis. A, increased PTEN expression in the odontoblast layer of Wwp2 KO mice revealed by immunofluorescence. The mean fluorescence intensity of PTEN was quantified by Image J software. n = 6; Student’s t test; Error bars represent SD. B, elevated PTEN level in differentiation-induced Wwp2 KO mDPCs shown by Western blot. C, changes of PTEN expression levels when Wwp2 or Wwp2 ligase activity–dead mutant was overexpressed. D, cycloheximide (CHX) assay showed that overexpression of Wwp2 accelerated the degradation rate of PTEN. CHX was applied to the transfected mDPCs or the untreated cells for 0, 3, 6, 9 h. The cell lysates were harvested for Western blot analysis. E, Dspp and Dmp1 mRNA levels when Wwp2 and Pten were knocked down separately or simultaneously. The transfected mDPCs were cultured in DM for 7 days, and real-time PCR assays were performed. n = 3; Student’s t test; Error bars represent SD. F, the effect of WWP2 and PTEN on the mineralized nodule formation. The transfected mDPCs were cultured in DM for 7 days. Alizarin red S was applied and the stained cells were harvested using 10% cetylpyridinium chloride. The absorbance values were measured at 562 nm. n = 3; Student’s t test; Error bars represent SD. The scale bars represent 20 μm for (A). D, dentin; fluo., fluorescence; Od, odontoblast; OE, overexpression; P, Pten; Rel., relative; Scr, scramble; si, knockdown; W2, Wwp2; W2mut, Wwp2 ligase activity–dead mutant.

      WWP2 inhibits the suppression effect of PTEN on KLF5 transcriptional activity

      In addition to PTEN, KLF5 as an important transcription factor for odontoblast differentiation is another substrate of WWP2 during odontoblastic differentiation (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ), but whether there is a cross talk between PTEN and KLF5 is not clear. Knockdown and overexpression experiments demonstrated that Pten could not influence the mRNA and protein levels of Klf5 (Fig. 6, A and B). However, co-IP assays illustrated that PTEN bound to KLF5 in HEK293E cells (Fig. 6C) and mDPCs (Fig. 6D). In situ PLA further revealed the in vivo interaction between PTEN and KLF5 in the odontoblast layer of PN5 molars (Fig. 6E). Dual luciferase reporter assay was applied to detect whether PTEN influenced the transcriptional activity of KLF5. The results showed that concomitant overexpression of Pten inhibited the upregulated luciferase activity of both Dspp and Dmp1 promoters by overexpression of Klf5 alone (Fig. 6F). Notably, although WWP2 had no effect on the mRNA or protein level of KLF5 (Figs. 6B and S7, A and B), overexpression of WWP2 was able to rescue the impediment of PTEN on KLF5 transcriptional activity (Fig. 6F).
      Figure thumbnail gr6
      Figure 6WWP2 inhibits the suppression effect of PTEN on KLF5 transcriptional activity. A, the effect of PTEN on Klf5 mRNA levels investigated by real-time PCR. n = 3; Student’s t test; Error bars represent SD. B, the effect of PTEN and WWP2 on KLF5 protein levels revealed by Western blot. C, coimmunoprecipitation assays show the interaction between PTEN and KLF5 in HEK293E cells (overexpressed proteins). D, coimmunoprecipitation assays show the interaction between PTEN and KLF5 in mDPCs (endogenous proteins). E, the interaction between KLF5 and PTEN in vivo shown by in situ proximity ligation assay. b and d are higher magnifications of the rectangles in a and c. The arrows show the positive signals for KLF5-PTEN interaction. F, the function of PTEN and WWP2 on KLF5-mediated Dspp- and Dmp1-luciferase reporter activities by dual luciferase assays. The mDPCs were harvested after 48-h transfection. n = 3; ANOVA; error bars represent SD. The scale bars represent 100 μm for (C) a, c; 20 μm for (C) b, d. fluo., fluorescence; luc, luciferase plasmid; Od, odontoblast; P, Pten; Rel., relative; Scr, scramble; si, knockdown; Ub, ubiquitin; W2, Wwp2.

      Discussion

      In the cell culture system we have previously found WWP2 can monoubiquitinate KLF5 and promote the transcriptional activity of KLF5 on Dspp and Dmp1. In this study, it was found that depletion of WWP2 impacted odontoblast differentiation and dentin formation. PTEN is a well-known tumor suppressor. It regulates various cellular activities of diverse cell types (
      • Di Cristofano A.
      • Pandolfi P.P.
      The multiple roles of PTEN in tumor suppression.
      ,
      • Hopkins B.D.
      • Hodakoski C.
      • Barrows D.
      • Mense S.M.
      • Parsons R.E.
      PTEN function: the long and the short of it.
      ). In this study, PTEN was found to negatively regulate odontoblastic differentiation. Some studies have shown that the E3 ligase WWP2 is able to target PTEN for ubiquitination-dependent proteolysis (
      • Maddika S.
      • Kavela S.
      • Rani N.
      • Palicharla V.R.
      • Pokorny J.L.
      • Sarkaria J.N.
      • et al.
      WWP2 is an E3 ubiquitin ligase for PTEN.
      ,
      • Zhang H.
      • Wei P.
      • Lv W.
      • Han X.
      • Yang J.
      • Qin S.
      Long noncoding RNA lnc-DILC stabilizes PTEN and suppresses clear cell renal cell carcinoma progression.
      ,
      • Li H.
      • Zhang P.
      • Zhang Q.
      • Li C.
      • Zou W.
      • Chang Z.
      • et al.
      WWP2 is a physiological ubiquitin ligase for phosphatase and tensin homolog (PTEN) in mice.
      ,
      • Wang K.
      • Liu J.
      • Zhao X.
      • Li H.
      • Luo G.
      • Yu Y.
      • et al.
      WWP2 regulates proliferation of gastric cancer cells in a PTEN-dependent manner.
      ). This also applies to the odontoblastic differentiation process. Further investigation revealed that WWP2 bound to and degraded PTEN in odontoblasts, thus relieving the suppression effect of PTEN on the transcriptional activity of KLF5.
      WWP2 is an active player in some developmental processes. A previous study uncovered that WWP2 was responsible for the proper development of cortical neurons, in coordination with its family member WWP1 (
      • Ambrozkiewicz M.C.
      • Schwark M.
      • Kishimoto-Suga M.
      • Borisova E.
      • Hori K.
      • Salazar-Lazaro A.
      • et al.
      Polarity acquisition in cortical neurons is driven by synergistic action of Sox9-regulated Wwp1 and Wwp2 E3 ubiquitin ligases and intronic miR-140.
      ). WWP2 was also revealed to monoubiquitinate RUNX2 and promote osteogenic differentiation (
      • Zhu W.
      • He X.
      • Hua Y.
      • Li Q.
      • Wang J.
      • Gan X.
      The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.
      ). Similarly, in a cell culture system, WWP2 was illustrated to promote odontoblastic differentiation by monoubiquitinating KLF5 and elevating the transcription levels of its downstream genes, Dspp and Dmp1 (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). In the present study, Wwp2 KO mice were generated by the CRISPR-Cas9 technique for further investigation of the in vivo function of WWP2 in odontoblast differentiation and dentinogenesis. Notably, Wwp2 KO mice exhibited the phenotype with thinner dentin layer, widened predentin layer, as well as shorter roots. Meanwhile, the dentinal tubules of molars in Wwp2 KO mice were less formed shown by scanning electron microscopy. Therefore, WWP2 plays a positive role for odontoblast differentiation and dentin formation in mice. Early cusp abrasion in the molars of Wwp2 KO mice suggests that ablation of Wwp2 led to declined mechanical property of teeth. Intriguingly, previous studies have shown that depletion of either Dspp or Dmp1 led to widened predentin and thinner dentin layer (
      • Gibson M.P.
      • Zhu Q.
      • Wang S.
      • Liu Q.
      • Liu Y.
      • Wang X.
      • et al.
      The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.
      ,
      • Rangiani A.
      • Cao Z.G.
      • Liu Y.
      • Voisey Rodgers A.
      • Jiang Y.
      • Qin C.L.
      • et al.
      Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation.
      ,
      • Sreenath T.
      • Thyagarajan T.
      • Hall B.
      • Longenecker G.
      • D'Souza R.
      • Hong S.
      • et al.
      Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III.
      ), which resembled the phenotypes of Wwp2 KO mice in our study. Besides, Dmp1 KO mice showed a decrease in the amount of dentinal tubules (
      • Rangiani A.
      • Cao Z.G.
      • Liu Y.
      • Voisey Rodgers A.
      • Jiang Y.
      • Qin C.L.
      • et al.
      Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation.
      ), which was also similar to Wwp2 KO mice in our study.
      Then the expression levels of DSPP and DMP1 were detected. It was demonstrated that the levels of DSPP and DMP1 proteins were decreased in the molars of Wwp2 KO mice by IF. In cultured mDPCs, Wwp2 deletion led to a reduction of Dspp and Dmp1 mRNA levels, a decrease of mineralized nodule formation and ALP activity. These results were consistent with our previous in vitro study (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ), further demonstrating that WWP2 is indispensable for proper differentiation and mineralization ability of odontoblasts.
      PTEN has been found to mediate multiple cellular processes (
      • Hopkins B.D.
      • Hodakoski C.
      • Barrows D.
      • Mense S.M.
      • Parsons R.E.
      PTEN function: the long and the short of it.
      ), but its expression pattern and function in tooth development remained unclear. In this study, PTEN was found positively stained in murine molars at different developmental stages (E13.5, E16.5, PN1 and 2W), indicating that PTEN may participate in tooth development. The function of PTEN in odontoblastic differentiation was further explored by a series of experiments, which verified that PTEN negatively regulates odontoblastic differentiation. A lot of evidences have revealed that PTEN is able to mediate the differentiation process of many cell types in positive or negative ways. For example, knockout of Pten in neural crest cells stimulated their osteoblastic differentiation (
      • Yang T.
      • Moore M.
      • He F.
      Pten regulates neural crest proliferation and differentiation during mouse craniofacial development.
      ). Knockdown of Pten markedly promoted neuronal cell differentiation (
      • Song Z.
      • Han X.
      • Shen L.
      • Zou H.
      • Zhang B.
      • Liu J.
      • et al.
      PTEN silencing enhances neuronal proliferation and differentiation by activating PI3K/Akt/GSK3beta pathway in vitro.
      ). However, there are also some researches showing positive roles of PTEN on cell differentiation. Under BMP2 stimulation, PTEN was upregulated and then facilitated the differentiation of hair follicle stem cells by inducing autophagy (
      • Cai B.
      • Zheng Y.
      • Yan J.
      • Wang J.
      • Liu X.
      • Yin G.
      BMP2-mediated PTEN enhancement promotes differentiation of hair follicle stem cells by inducing autophagy.
      ). And in terms of immune cells, PTEN promoted T helper 17 cell differentiation through inhibiting IL-2 expression (
      • Kim H.S.
      • Jang S.W.
      • Lee W.
      • Kim K.
      • Sohn H.
      • Hwang S.S.
      • et al.
      PTEN drives Th17 cell differentiation by preventing IL-2 production.
      ). Therefore, the function of PTEN in controlling cell differentiation possibly depends on cell types and circumstances.
      Regulation of PTEN, including posttranslational modification by E3 ligases, is significant for the protein level and phosphatase activity of PTEN (
      • Worby C.A.
      • Dixon J.E.
      PTEN.
      ). WWP2 has been identified to ubiquitinate and regulate PTEN in prostate cancer cells (
      • Maddika S.
      • Kavela S.
      • Rani N.
      • Palicharla V.R.
      • Pokorny J.L.
      • Sarkaria J.N.
      • et al.
      WWP2 is an E3 ubiquitin ligase for PTEN.
      ). Therefore, it was wondered whether WWP2 also targeted PTEN for ubiquitination-dependent degradation in odontoblasts. The interaction between WWP2 and PTEN is a prerequisite for degradation of PTEN by WWP2, and their interaction in vitro and in vivo was verified by co-IP and in situ PLA, respectively. Wwp2 deletion did lead to an increase of PTEN protein level in vitro and in vivo. Overexpression of WWP2 prompted the degradation of PTEN protein, while overexpression of the ligase activity–dead WWP2 did not have this effect, suggesting the degradation of PTEN is dependent on the ligase activity of WWP2. Cycloheximide assays further confirmed that WWP2 promoted PTEN proteolysis. Concomitant knockdown of Pten rescued the decreased differentiation of mDPCs caused by Wwp2 knockdown, suggesting that upregulated PTEN accounts for the inhibition effects of Wwp2 knockdown. All these results demonstrated that WWP2 promotes odontoblast differentiation and dentinogenesis through targeting PTEN for degradation.
      Since our previous study found that WWP2 was able to monoubiquitinate KLF5 and activate its transcriptional activity (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ), we wondered whether a cross talk between PTEN and KLF5 existed during odontoblastic differentiation. The regulation of the transcriptional and translational levels of KLF5 by PTEN was first excluded. Interestingly, PTEN was found to bind with KLF5 in both isolated cells and molars. And their interaction increased in the molars of Wwp2 KO mice, which may be attributed to the elevated PTEN protein level by Wwp2 deletion. PTEN was able to inhibit the transcriptional activity of KLF5 on Dspp and Dmp1. As expected, Wwp2 overexpression suppressed the inhibition effects of PTEN on KLF5 transcriptional activity, which can be partially explained by the degradation of PTEN by WWP2. Similarly, PTEN has been found to participate in regulating transcriptional activity of other transcription factors, like Forkhead transcription factors (FOXO) (
      • Emerling B.M.
      • Weinberg F.
      • Liu J.L.
      • Mak T.W.
      • Chandel N.S.
      PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).
      ). PTEN has been reported to exert its function through phosphatidylinositol 3 kinase/ATK-dependent or -independent pathways (
      • Misra S.
      • Ghosh G.
      • Chowdhury S.G.
      • Karmakar P.
      Non-canonical function of nuclear PTEN and its implication on tumorigenesis.
      ,
      • Ho J.
      • Cruise E.S.
      • Dowling R.J.O.
      • Stambolic V.
      PTEN nuclear functions.
      ). Whether PTEN also influences the transcriptional activity of KLF5 through these pathways needs further investigation.
      In summary, molars in Wwp2 KO mice show the phenotype with thinner dentin, widened predentin, and shorter roots. The dentinal tubules of molars in Wwp2 KO mice are less formed and disordered compared with the WT group. PTEN is expressed in dental mesenchymal cells and odontoblasts during tooth development. PTEN can interact with KLF5 and suppress the transcriptional activity of KLF5 on Dspp and Dmp1, thus playing a negative role for odontoblastic differentiation of mDPCs (Fig. 7A). WWP2 can target PTEN for degradation and consequently antagonize the suppression on KLF5 transcriptional activity by PTEN. Besides, WWP2 interacts with and monoubiquitinates KLF5, thus enhancing the transcriptional activity of KLF5 on Dspp and Dmp1. WWP2 mediates odontoblast differentiation and dentinogenesis by promoting the expression levels of Dspp and Dmp1 through the WWP2–PTEN–KLF5 axis (Fig. 7B) (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). The above mechanism can well explain the defects in the differentiation and mineralization ability of Wwp2 KO odontoblasts. Previous studies have reported that mesenchymal cell proliferation is essential for root development size (
      • Ma Y.
      • Jing J.
      • Feng J.
      • Yuan Y.
      • Wen Q.
      • Han X.
      • et al.
      Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development.
      ,
      • Liu Y.
      • Feng J.
      • Li J.
      • Zhao H.
      • Ho T.V.
      • Chai Y.
      An Nfic-hedgehog signaling cascade regulates tooth root development.
      ); whether cell proliferation alteration exists and contributes to shortened roots in Wwp2 KO mice needs further investigation. Although mice are ideal genetic animal models, they are different from humans for having only one dentition; more investigation on two-dentition animal models is needed.
      Figure thumbnail gr7
      Figure 7The schema of WWP2–PTEN–KLF5 interaction, which regulates odontoblast differentiation and dentinogenesis by mediating Dspp and Dmp1 transcription levels. A, PTEN interacts with KLF5 and suppresses the transcriptional activity of KLF5 on Dspp and Dmp1. B, WWP2 promotes the degradation of PTEN and antagonizes the suppression on KLF5 transcriptional activity by PTEN. Meanwhile, WWP2 monoubiquitinates KLF5 and enhances the transcriptional activity of KLF5 on Dspp and Dmp1.

      Experimental procedures

      Mice and tissue collection

      All mice were raised in the specific pathogen-free facility with a 12-h light/dark cycle in ventilated cages in groups not surpassing four animals. Mice were fed by standard rodent chow from Huafukang (Cat No: 1032). All animal experiments were approved by the Animal Welfare and Ethics Committee of the School and Hospital of Stomatology at Wuhan University (S07919010A).
      Wwp2 KO mice of C57BL/6J background were established at Gempharmatech (Project NO: GPS00001611). The knockout strategy using the CRISPR-Cas9 technology was described in Fig. S1A. The primers for genotyping of WT and Wwp2 KO mice are listed in Table S1 and were synthesized at Sangon. Other information, such as ages of samples and replicates, is shown in the figure legends and Table S2. The KO mice were viable and fertile. The birth of the KO mice was conformed to Mendel's law of inheritance. In all the animal experiments, both male and female mice were used.
      All mice were sacrificed using carbon dioxide inhalation. The mandibles were isolated. After being fixed with 4% paraformaldehyde at 4 °C overnight, the mandibles were observed by micro–computed tomography. For paraffin slicing, the tissues were decalcified with 10% ethylenediaminetetraacetic acid, then dehydrated and embedded with paraffin.

      Histological staining

      For histological staining, the 5-μm slices were deparaffinized and rehydrated. H&E staining was performed with hematoxylin and eosin dyes. IHC was performed using the HRP-Polymer anti-Rabbit IHC Kit (MaxVision) according to the manufacturer’s instructions. The primary antibodies of WWP2 (Proteintech) and PTEN (Abclonal) were each applied at a 1:200 concentration. After being stained, the slices were mounted by neutral balsam.
      For IF, the concentrations of antibodies applied were as follows: DSPP (1:200; Novus), DMP1 (1:200; Abclonal), PTEN (1:200; Abclonal), and Alexa Fluor Red 594 Donkey anti Rabbit IgG (1:400; Antgene). After being stained, the slices were mounted by DAPI.

      Cell culture and cytologic staining

      The primary mDPCs were isolated from the first mandibular molars of neonatal mice and seeded into cell plates or dishes after 0.25% trypsin digestion for 10 min as described (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). HEK293E and mDPCs were cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (Gibco). mDPCs were induced for odontoblastic cells by differentiation medium supplemented with 10% fetal bovine serum, 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μg/ml ascorbic acid (Sigma-Aldrich). For cytologic staining, cells were fixed with 4% polyoxymethylene at 4 °C for 15 min. Then ALP staining was performed using the BCIP/NBT Alkaline Phosphatase Kit (Beyotime) and ARS staining was applied with 0.5% ARS solution (pH = 4.5) (Aladdin). For ARS, 10% cetylpyridinium chloride was used for harvesting the stained cells, and absorbance values were measured at 562 nm by the spectrophotometer and normalized to the total protein concentration per well.

      In situ PLA

      To histologically visualize the interaction between PTEN and KLF5 (or WWP2) in mouse molars, in situ PLA was performed using the Duolink kit (Sigma-Aldrich) as described in our previous study (
      • Yuan G.
      • Yang G.
      • Zheng Y.
      • Zhu X.
      • Chen Z.
      • Zhang Z.
      • et al.
      The non-canonical BMP and Wnt/beta-catenin signaling pathways orchestrate early tooth development.
      ). In brief, the slices were deparaffinized, rehydrated, and antigen-retrieved, then blocked and incubated with anti-PTEN (mouse monoclonal; Proteintech) antibody together with either anti-KLF5 (rabbit polyclonal; Proteintech) or anti-WWP2 (rabbit polyclonal; Proteintech) antibody at 4 °C overnight. Afterward, the slices were washed and incubated with secondary antibodies conjugated with oligonucleotides. Slices subsequently went through ligation reaction as well as amplification reaction. Finally, slices were mounted with DAPI and observed under fluorescence microscope (Olympus Digital Camera).

      Coimmunoprecipitation

      mDPCs or transfected HEK293E cells were rinsed with PBS and lysed. The cell lysates were collected into the microcentrifuge tubes and shaken gently at 4 °C for 30 min. The supernatant was removed to new microcentrifuge tubes after centrifugation. Co-IP assays were performed as described (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). The primary antibodies or the negative control IgG were as follows: WWP2 (Abcam), KLF5 (Proteintech), PTEN (CST), MYC (Proteintech), HIS (Abcam), IgG (Antgene). After being mixed with the primary antibodies or the negative control IgG, the samples were rotated gently at 4 °C overnight. Then the protein A/G magnetic beads (Bimake) were added to the samples and the mixtures were rotated gently for 1 h. After being washed, the supernatant was discarded and the magnetic beads were resuspended with SDS. The samples were denatured at 95 °C for 15 min and analyzed by Western blotting.

      Small interfering RNA and plasmid transfection

      The siRNAs of Wwp2 and Pten were purchased from Genepharma. The information of overexpression plasmids is listed in Table S3. mDPCs or HEK293E cells were seeded into plates or dishes and were transfected with siRNAs or plasmids with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The negative control scrambled siRNAs and the vector plasmids were transfected as the control groups.

      Western blot and real-time PCR

      The cells were lysed by NP-40 (Beyotime) containing 1/100 protease inhibitor Cocktail (MedChemExpress) for 15 min at 4 °C. After supersonic lysis and centrifugation, the supernatant was obtained and the protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (Thermo). Then the protein samples were mixed with SDS and denatured at 95 °C for 15 min. The samples were fractionated by 10% SDS-PAGE and transferred to Trans-blot membranes (Roche). The membranes were blocked by 5% skim milk for 1 h at room temperature and incubated with the primary antibodies as follows: WWP2 (1:1000; Abcam), KLF5 (1:1000; Proteintech), GAPDH (1:10,000; Proteintech), HIS (1:1000; Abcam), MYC (1:1000; Abcam), and PTEN (1:1000; Abclonal). After being incubated with secondary antibodies, the membranes were detected by ECL solution (Advansta).
      Real-time PCR was carried out using fluorescent quantitation PCR mix from Vazyme in accordance with the manufacturer’s instructions. The primers were synthesized at Sangon. The primer sequences are listed in the Table S4.

      Scanning electron microscopy

      Mandibular molars of Wwp2 KO and WT mice at 2W were fractured vertically from the central fossae and rapidly fixed in 2.5% glutaraldehyde at 4 °C overnight. After being washed, the samples were treated by acid etching by 30% phosphoric acid for 15 s and 5.25% sodium hypochlorite for 4 to 5 min. Then the tissues were washed and dehydrated in an ascending alcohol series. After that, the samples were dried by air and were sputter coated with gold for scanning electron microscopy (TESCAN VEGA3 LMU) (
      • Yuan G.
      • Chen L.
      • Feng J.
      • Yang G.
      • Ni Q.
      • Xu X.
      • et al.
      Dentin sialoprotein is a novel substrate of matrix metalloproteinase 9 in vitro and in vivo.
      ).

      Dual luciferase reporter assay

      The dual luciferase reporter assay was performed as described (
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ). Briefly, HEK293E cells seeded into 24-well plates were cotransfected with overexpression plasmids. The plasmids of each group are listed in the figures. pRL-TK was applied as internal reference. After being transfected for 48 h, the relative fluorescence ratio of the cells was detected using the kit of Dual-Luciferase Reporter Assay System (Promega) and the Glomax Luminometer (Promega).

      Statistical analysis

      Experiments were independently repeated three times. Student’s t test (two tailed) was used to analyze the statistically significant differences of two groups, and one-way analysis of variance (ANOVA) followed by Tukey’s test was applied for three or more groups. All statistical analyses were performed by GraphPad Prism software (version 8.0). The results are represented as means ± standard deviation (means ± SD). p < 0.05 is defined as statistically significant.

      Data availability

      All data in this study are contained within the article and the supplemental materials.

      Supporting information

      This article contains supporting information (
      • Chen Z.
      • Zhang Q.
      • Wang H.
      • Li W.
      • Wang F.
      • Wan C.
      • et al.
      Klf5 mediates odontoblastic differentiation through regulating dentin-specific extracellular matrix gene expression during mouse tooth development.
      ,
      • Fu J.
      • Zheng H.
      • Xue Y.
      • Jin R.
      • Yang G.
      • Chen Z.
      • et al.
      WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This research was supported by grants from the National Natural Science Foundation of China ( 82170914 to Guohua Yuan).

      Author contributions

      J. F. conceptualization; J. F., X. Z., Guobin Yang, and Z. C. methodology; X. Z. software; J. F., X. Z., and H. Z. validation; H. Z. formal analysis; J. F. writing – original draft; H. Z. and Guohua Yuan writing – review & editing; Guobin Yang and Z. C. supervision; Guohua Yuan funding acquisition.

      Supporting information

      References

        • Mayor R.
        • Theveneau E.
        The neural crest.
        Development. 2013; 140: 2247-2251
        • Bleicher F.
        Odontoblast physiology.
        Exp. Cell Res. 2014; 325: 65-71
        • Butler W.T.
        Dentin matrix proteins and dentinogenesis.
        Connect. Tissue Res. 1995; 33: 59-65
        • Goldberg M.
        • Kulkarni A.B.
        • Young M.
        • Boskey A.
        Dentin: structure, composition and mineralization.
        Front. Biosci. (Elite Ed.). 2011; 3: 711-735
        • Herhaus L.
        • Dikic I.
        Expanding the ubiquitin code through post-translational modification.
        EMBO Rep. 2015; 16: 1071-1083
        • Rape M.
        Ubiquitylation at the crossroads of development and disease.
        Nat. Rev. Mol. Cell Biol. 2018; 19: 59-70
        • Lorenz S.
        Structural mechanisms of HECT-type ubiquitin ligases.
        Biol. Chem. 2018; 399: 127-145
        • Xu H.M.
        • Liao B.
        • Zhang Q.J.
        • Wang B.B.
        • Li H.
        • Zhong X.M.
        • et al.
        Wwp2, an E3 ubiquitin ligase that targets transcription factor Oct-4 for ubiquitination.
        J. Biol. Chem. 2004; 279: 23495-23503
        • Zhu W.
        • He X.
        • Hua Y.
        • Li Q.
        • Wang J.
        • Gan X.
        The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.
        J. Biol. Chem. 2017; 292: 11178-11188
        • Fang L.
        • Zhang L.
        • Wei W.
        • Jin X.
        • Wang P.
        • Tong Y.
        • et al.
        A methylation-phosphorylation switch determines Sox2 stability and function in ESC maintenance or differentiation.
        Mol. Cell. 2014; 55: 537-551
        • Ambrozkiewicz M.C.
        • Schwark M.
        • Kishimoto-Suga M.
        • Borisova E.
        • Hori K.
        • Salazar-Lazaro A.
        • et al.
        Polarity acquisition in cortical neurons is driven by synergistic action of Sox9-regulated Wwp1 and Wwp2 E3 ubiquitin ligases and intronic miR-140.
        Neuron. 2018; 100: 1097-1115.e1015
        • Nakamura Y.
        • Yamamoto K.
        • He X.
        • Otsuki B.
        • Kim Y.
        • Murao H.
        • et al.
        Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25.
        Nat. Commun. 2011; 2: 251
        • Chen Z.
        • Zhang Q.
        • Wang H.
        • Li W.
        • Wang F.
        • Wan C.
        • et al.
        Klf5 mediates odontoblastic differentiation through regulating dentin-specific extracellular matrix gene expression during mouse tooth development.
        Sci. Rep. 2017; 746746
        • Chen Z.
        • Couble M.L.
        • Mouterfi N.
        • Magloire H.
        • Chen Z.
        • Bleicher F.
        Spatial and temporal expression of KLF4 and KLF5 during murine tooth development.
        Arch. Oral Biol. 2009; 54: 403-411
        • Han N.
        • Chen Z.
        • Zhang Q.
        Expression of KLF5 in odontoblastic differentiation of dental pulp cells during in vitro odontoblastic induction and in vivo dental repair.
        Int. Endod. J. 2017; 50: 676-684
        • Fu J.
        • Zheng H.
        • Xue Y.
        • Jin R.
        • Yang G.
        • Chen Z.
        • et al.
        WWP2 promotes odontoblastic differentiation by monoubiquitinating KLF5.
        J. Dent Res. 2021; 100: 432-439
        • Di Cristofano A.
        • Pandolfi P.P.
        The multiple roles of PTEN in tumor suppression.
        Cell. 2000; 100: 387-390
        • Hopkins B.D.
        • Hodakoski C.
        • Barrows D.
        • Mense S.M.
        • Parsons R.E.
        PTEN function: the long and the short of it.
        Trends Biochem. Sci. 2014; 39: 183-190
        • Lokody I.B.
        • Francis J.C.
        • Gardiner J.R.
        • Erler J.T.
        • Swain A.
        Pten regulates epithelial cytodifferentiation during prostate development.
        PLoS One. 2015; 10e0129470
        • Sun F.
        • Park K.K.
        • Belin S.
        • Wang D.
        • Lu T.
        • Chen G.
        • et al.
        Sustained axon regeneration induced by co-deletion of PTEN and SOCS3.
        Nature. 2011; 480: 372-375
        • Yang T.
        • Moore M.
        • He F.
        Pten regulates neural crest proliferation and differentiation during mouse craniofacial development.
        Dev. Dyn. 2018; 247: 304-314
        • Li R.A.
        • Traver D.
        • Matthes T.
        • Bertrand J.Y.
        Ndrg1b and fam49ab modulate the PTEN pathway to control T-cell lymphopoiesis in the zebrafish.
        Blood. 2016; 128: 3052-3060
        • Jo H.S.
        • Kang K.H.
        • Joe C.O.
        • Kim J.W.
        Pten coordinates retinal neurogenesis by regulating Notch signalling.
        EMBO J. 2012; 31: 817-828
        • Kero D.
        • Cigic L.
        • Medvedec Mikic I.
        • Galic T.
        • Cubela M.
        • Vukojevic K.
        • et al.
        Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ - an immunohistochemical study.
        Organogenesis. 2016; 12: 152-167
        • Liu J.
        • Wan L.
        • Liu J.
        • Yuan Z.
        • Zhang J.
        • Guo J.
        • et al.
        Cdh1 inhibits WWP2-mediated ubiquitination of PTEN to suppress tumorigenesis in an APC-independent manner.
        Cell Discov. 2016; 215044
        • Maddika S.
        • Kavela S.
        • Rani N.
        • Palicharla V.R.
        • Pokorny J.L.
        • Sarkaria J.N.
        • et al.
        WWP2 is an E3 ubiquitin ligase for PTEN.
        Nat. Cell Biol. 2011; 13: 728-733
        • Zhang H.
        • Wei P.
        • Lv W.
        • Han X.
        • Yang J.
        • Qin S.
        Long noncoding RNA lnc-DILC stabilizes PTEN and suppresses clear cell renal cell carcinoma progression.
        Cell Biosci. 2019; 9: 81
        • Li H.
        • Zhang P.
        • Zhang Q.
        • Li C.
        • Zou W.
        • Chang Z.
        • et al.
        WWP2 is a physiological ubiquitin ligase for phosphatase and tensin homolog (PTEN) in mice.
        J. Biol. Chem. 2018; 293: 8886-8899
        • Wang K.
        • Liu J.
        • Zhao X.
        • Li H.
        • Luo G.
        • Yu Y.
        • et al.
        WWP2 regulates proliferation of gastric cancer cells in a PTEN-dependent manner.
        Biochem. Biophys. Res. Commun. 2020; 521: 652-659
        • Gibson M.P.
        • Zhu Q.
        • Wang S.
        • Liu Q.
        • Liu Y.
        • Wang X.
        • et al.
        The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.
        J. Biol. Chem. 2013; 288: 7204-7214
        • Rangiani A.
        • Cao Z.G.
        • Liu Y.
        • Voisey Rodgers A.
        • Jiang Y.
        • Qin C.L.
        • et al.
        Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation.
        Int. J. Oral Sci. 2012; 4: 189-195
        • Sreenath T.
        • Thyagarajan T.
        • Hall B.
        • Longenecker G.
        • D'Souza R.
        • Hong S.
        • et al.
        Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III.
        J. Biol. Chem. 2003; 278: 24874-24880
        • Song Z.
        • Han X.
        • Shen L.
        • Zou H.
        • Zhang B.
        • Liu J.
        • et al.
        PTEN silencing enhances neuronal proliferation and differentiation by activating PI3K/Akt/GSK3beta pathway in vitro.
        Exp. Cell Res. 2018; 363: 179-187
        • Cai B.
        • Zheng Y.
        • Yan J.
        • Wang J.
        • Liu X.
        • Yin G.
        BMP2-mediated PTEN enhancement promotes differentiation of hair follicle stem cells by inducing autophagy.
        Exp. Cell Res. 2019; 385111647
        • Kim H.S.
        • Jang S.W.
        • Lee W.
        • Kim K.
        • Sohn H.
        • Hwang S.S.
        • et al.
        PTEN drives Th17 cell differentiation by preventing IL-2 production.
        J. Exp. Med. 2017; 214: 3381-3398
        • Worby C.A.
        • Dixon J.E.
        PTEN.
        Annu. Rev. Biochem. 2014; 83: 641-669
        • Emerling B.M.
        • Weinberg F.
        • Liu J.L.
        • Mak T.W.
        • Chandel N.S.
        PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).
        Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2622-2627
        • Misra S.
        • Ghosh G.
        • Chowdhury S.G.
        • Karmakar P.
        Non-canonical function of nuclear PTEN and its implication on tumorigenesis.
        DNA Repair (Amst.). 2021; 107103197
        • Ho J.
        • Cruise E.S.
        • Dowling R.J.O.
        • Stambolic V.
        PTEN nuclear functions.
        Cold Spring Harb. Perspect. Med. 2020; 10a036079
        • Ma Y.
        • Jing J.
        • Feng J.
        • Yuan Y.
        • Wen Q.
        • Han X.
        • et al.
        Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development.
        Development. 2021; 148dev196360
        • Liu Y.
        • Feng J.
        • Li J.
        • Zhao H.
        • Ho T.V.
        • Chai Y.
        An Nfic-hedgehog signaling cascade regulates tooth root development.
        Development. 2015; 142: 3374-3382
        • Yuan G.
        • Yang G.
        • Zheng Y.
        • Zhu X.
        • Chen Z.
        • Zhang Z.
        • et al.
        The non-canonical BMP and Wnt/beta-catenin signaling pathways orchestrate early tooth development.
        Development. 2015; 142: 128-139
        • Yuan G.
        • Chen L.
        • Feng J.
        • Yang G.
        • Ni Q.
        • Xu X.
        • et al.
        Dentin sialoprotein is a novel substrate of matrix metalloproteinase 9 in vitro and in vivo.
        Sci. Rep. 2017; 742449