The Role of Slc20a2 in Osteogenesis

Abstract

The role of inorganic phosphate transporters at the bone surface remains largely unknown. Prior work has established that Slc20a2, which encodes type III sodium-dependent phosphate transporter PiT-2, plays a crucial anti-calcific role in both vascular and brain tissues and physiologic osteogenesis. This work investigates Slc20a2 expression in bone cells and explores the mechanisms that lead to impaired osteogenesis when Slc20a2 is lost. Tibia samples were collected from C57Bl6/J and C57Bl6/J CD-1 outbred mice carrying the EUCOMM tm1a knockout-first cassette. Whole-tissue X-Gal staining identified Slc20a2 localization to a bone-lining adjacent cell type. Publicly available single-cell RNA-seq (scRNA-seq) datasets were analyzed to corroborate expression signatures across different bone cell populations. To examine gene expression changes in situ, a demineralization protocol was optimized to preserve RNA. Subsequently, spatial transcriptomics (Nanostring GeoMx DSP and 10X Genomics Visium HD) was performed on demineralized bone sections, and immunofluorescence studies further validated the identity of various vascular and bone cell types. Finally, SPiDER-βGal (Dojindo) was employed to sort LacZ-expressing cells from heterozygous and knockout mice, enabling mRNA-seq to confirm their specific identity. Early observations in dental tissues also provided insight into Slc20a2 localization and its effects on mineralization. Key findings indicate that Slc20a2 deficiency impairs osteoblast numbers and function. X-Gal staining localized Slc20a2 to bone-lining cells between marrow vasculature and osteoblasts, suggesting a bridging position between osteoblasts and blood vessels. scRNA-seq data showed Slc20a2 expression is highest in CXCL12-abundant reticular cells, specifically osteo-CAR cells. Additional markers, including Limch1 and KCNK2, co-localized with X-gal staining, validating these observations. Spatial transcriptomic approaches confirmed the cell-specific marker expression in carefully defined regions of interest, and RNA-seq of sorted Slc20a2-deficient LacZ-expressing cells further supported these findings. Moreover, Slc20a2 loss resulted in defective molar mineralization and heightened susceptibility to incisal wear or fracture. Notably, the inner and outer enamel epithelia—especially the stratum intermedium—showed robust Slc20a2 expression. Overall, this work reveals that Slc20a2 is highly expressed in osteo-CAR cells at the endosteal–bone marrow interface, a population that may regulate osteogenesis either by signaling cues or by providing a reservoir of pre-osteoblasts. These insights offer a novel perspective on how inorganic phosphate regulation and Slc20a2 expression could be leveraged in clinical settings to address bone disorders such as osteoporosis.