Smpd3 regulates odontoblast differentiation through the Shh-Gli1 pathway

Abstract

Teeth, like other ectodermal organs such as hair, skin, and sweat glands, are complex structures. Specifically, teeth are composed of four principal tissues: enamel, dentin, cementum, and pulp. Among these, dentin is a critical component, synthesized by odontoblasts—specialized cells derived from ectomesenchymal precursors originating in the neural crest. Odontoblasts are uniquely responsible for dentinogenesis, a process essential for tooth development and function. However, the molecular mechanisms regulating odontoblast differentiation remain poorly understood.

In this study, we first analyzed a public single-cell RNA sequencing data set of postnatal (PN1) mouse molars and found Smpd3 as a potential gene of odontoblast differentiation. Then, we investigated the functional role of Smpd3 in odontoblast differentiation using a combination of histological, molecular, and bioinformatics approaches. Knockdown of Smpd3 expression via small interfering RNA (siRNA) significantly impaired odontoblast differentiation of mouse dental papilla cells (mDPCs), as evidenced by reductions in odontoblast-specific markers and mineralization. In contrast, overexpression of Smpd3 enhanced odontogenic differentiation and increased mineralized nodule formation of mDPCs. To elucidate the underlying molecular mechanisms, bulk RNA sequencing was conducted, revealing that Smpd3 is intricately linked to the Sonic Hedgehog (Shh) signaling pathway. In vitro studies and tooth germ culture were applied to validate the mechanism of Smpd3 on odontoblast differentiation through the Shh-Gli1 pathway.

Mechanistically, we show that Smpd3 upregulates dentinogenic markers (Dspp, Dmp1) in a Shh-dependent manner. Smpd3 overexpression increased Shh pathway activity and promoted dentin formation ex vivo. This study highlights the critical role of Smpd3 in tooth development and provides novel insights into the molecular regulation of dentinogenesis, offering potential therapeutic targets for methods that promote dentin regeneration when natural repairs are compromised.