Wnt Pathway Inhibitors are Upregulated in XLH Dental Pulp Stem Cells in Response to Odontogenic Differentiation

Wnt Pathway Inhibitors are Upregulated in XLH Dental Pulp Stem Cells in Response to Odontogenic Differentiation

X-linked hypophosphatemia (XLH) is characterized by loss-of-function mutations in phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) resulting in excess of circulating fibroblast growth factor-23 (FGF23). Overexpression of FGF-23 induced by mutation of PHEX gene suppresses differentiation of osteoblasts and odontoblasts along with defective matrix mineralization. Other matrix proteins such as dentin matrix protein 1 (DMP1) and matrix extracellular phosphoglycoprotein (MEPE) can regulate FGF23 expression in bone. Radiographic clinical findings in XLH patients show skeletal deformities due to dysfunctional bone formation. Interestingly, XLH patients demonstrate a high prevalence of dental abscesses and periodontal abnormalities that form in the absence of dental trauma that can lead to serious infections and ultimately tooth loss. Dentin of XLH teeth has been demonstrated to be defective, and our lab has previously shown that XLH dental pulp stem cells generate an impaired mineralized matrix. Overexpression of disease cells with dentin matrix protein 1 (DMP1) restored proper matrix mineralization function to the XLH dental pulp cells.

Tooth development is a highly regulated process that is controlled by the oscillating interactions between signaling pathways and cellular interactions. The canonical Wnt signaling pathway is tied to the morphological development of teeth through odontoblast differentiation and subsequent dentin mineralization. Previous studies of DMP1 knockout mice have reported expression of canonical Wnt pathway inhibitors, particularly secreted frizzled related protein 4 (sFRP-4). Connections between Wnt family member 5a (WNT5A), sFRP-4 and sclerostin (SOST) expression and altered activity of noncanonical Wnt and bone morphogenic protein (BMP) signaling play concurrent roles in decreased bone formation. The higher levels of SOST expression that have been reported in XLH patients in conjunction with a disorganization of the odontoblast layer suggests a relationship between Wnt signaling and the persistent decline of oral health of XLH patients. Guirado et al. employed RNAseq to elucidate some of the genetic pathways affected by odontogenic differentiation conditions in XLH cells with exogenous expression of DMP1 to explain the increase in matrix mineralization previously observed.

Transcriptomic multi-factorial analyses were conducted on samples of dental pulp cells (Ctrl), XLH dental pulp cells (XLH) and XLH dental pulp cells with constitutive DMP1 expression (XLHDMP1) after 8 hours in differentiation media. Initial analysis of the XLH cell transcriptome demonstrated an increase in expression of various Wnt inhibitors in disease samples with respect to Ctrl cells such as, Wnt family members 5A and 16 (WNT5A and WNT16) and dickkopf 3 (DKK3). Similarly, Gene Ontology biological pathway analysis indicated changes in positive regulation of Wnt signaling pathway (GO: 0030177), and angiogenesis (GO:0001525) in XLHDMP1 cells while negative regulation of Wnt signaling pathway (GO:0030178) was enriched in XLH. Further evaluation identified over 300 differentially expressed genes that presented with a reversal in expression between XLH  and XLHDMP1 samples. Within this gene set, WIF1 and SOST were present in high levels in XLH cells that declined significantly in XLHDMP1 cells.

Quantitative real-time PCR was conducted on WNT5A, DKK3, and WNT16 to evaluate expression patterns of all cell groups over 48 hours with respect to DMP1 overexpression. In XLH cells,  WNT5A and DKK gene expression increased with time, a trend that was counteracted in XLHDMP1 samples where expression of both genes returned to near Ctrl sample levels. Effects of DMP1 waned after 48 hours in disease cells indicated by WNT5A, DKK3, and WNT16 gene expression level decreases which may indicate how Wnt signaling was preserved by the presence of DMP1, while the disease phenotype failed to be full rescued. This area requires further study to evaluate the Wnt inhibitor gene expression time-dependence to devise more precise description of XLH phenotype rescue. Validation of WIF1 and SOST will require additional experimental optimization after being undetectable in all cells at the 8-hour time point to evaluate if these critical proteins are involved with the maintenance of Wnt signaling.

Figure 1. Model of gene expression within Wnt signaling observed in XLH dental pulp stem cells including SOST,DKK3 and B-Catenin.

Interaction plots from transcriptomic data revealed that beta-catenin expression differences among the 3 sample groups was dependent on the presence of DMP1. Beta-catenin is required for downstream activation of Wnt targets. This can be regulated by the presence of E-cadherin, a junction protein that is transcriptionally regulated by Wnt signaling, able to sequester beta-catenin. Protein expression of beta-catenin and E-cadherin was increased and decreased in XLH and XLHDMP1 cells compared to the Ctrl samples. This trend demonstrates the connectivity of the Wnt signaling pathway with induction of odontoblast differentiation as XLH cells have been shown to exhibit higher concentrations of Wnt pathway inhibitor proteins.  

Wnt pathway signaling along with cellular polarity, guided by E-cadherin, are critical in multiple cell types, such as periodontal ligament stem cells, to stimulate proper tooth development and have been presented here to be inversely regulated in Ctrl, XHL and XLHDMP1 samples. Hypophosphatemic conditions are marked by a decrease in blood phosphate concentrations classifying these diseases as metabolic disorders. In XLH, this is compounded by mutations of FGF23 which functions as sodium-phosphate co-transporter. The dysregulation of FGF23 further complicates the pathways associated with the disease phenotype of XLH and highlights the importance of understanding the global interaction of Wnt signaling, DMP1 and mineral metabolism in these patients. Additional significance of these finding may lie in their temporal relationship, which has yet to be explored, but could contribute to further mechanistic characterization of the oral co-morbidities in XLH patients.

Please sign in or register for FREE

If you are a registered user on Nature Portfolio Health Community, please sign in