WWP2 Promotes Odontoblastic Differentiation by Monoubiquitinating KLF5

Abstract

WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes ( Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5.

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

“…WWP2 is expressed in the odontoblast layer and the underlying dental papilla cells ( 16 ). The identical expression pattern between PTEN and WWP2 suggests that they may have physical interaction.…”

“…In addition to PTEN, KLF5 as an important transcription factor for odontoblast differentiation is another substrate of WWP2 during odontoblastic differentiation ( 16 ), 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.…”

“…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 ( 13 , 14 , 15 ). 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 ( 16 ). However, the in vivo role of WWP2 during odontoblast differentiation and dentinogenesis is still unknown.…”

Dlx3 Ubiquitination by Nuclear Mdm2 Is Essential for Dentinogenesis in Mice

“…Primary mDPCs were isolated as previously described (Fu et al 2021). Briefly, mouse dental papilla were separated from Kunming mice at embryonic day 18.5 (E18.5).…”

The Genes Involved in Dentinogenesis

“… 141 WWP2 Promotes Odontoblastic Differentiation. 142 TANGO1 Severe dentinogenesis imperfecta. 143 Mdm2 Promotes the odontoblast-like differentiation.…”