Identification of the C-terminal region in Amelogenesis Imperfecta causative protein WDR72 required for Golgi localization.

Amelogenesis Imperfecta (AI) represents a group of hereditary conditions that manifest tooth enamel defects. Several causative mutations in the WDR72 gene have been identified and patients with WDR72 mutations have brown (or orange-brown) discolored enamel, rough enamel surface, early loss of enamel after tooth eruption, and severe attrition. Although the molecular function of WDR72 is not yet fully understood, a recent study suggested that WDR72 could be a facilitator of endocytic vesicle trafficking, which appears inconsistent with the previously reported cytoplasmic localization of WDR72. Therefore, the aims of our study were to investigate the tissues and cell lines in which WDR72 was expressed and to further determine the sub-cellular localization of WDR72. The expression of Wdr72 gene was investigated in mouse tissues and cell lines. Endogenous WDR72 protein was detected in the membranous fraction of ameloblast cell lines in addition to the cytosolic fraction. Sub-cellular localization studies supported our fractionation data, showing WDR72 at the Golgi apparatus, and to a lesser extent, in the cytoplasmic area. In contrast, a WDR72 AI mutant form that lacks its C-terminal region was exclusively detected in the cytoplasm. In addition, our studies identified a putative prenylation/CAAX motif within the last four amino acids of human WDR72 and generated a WDR72 variant, called CS mutant, in which the putative motif was ablated by a point mutation. Interestingly, mutation of the putative CAAX motif impaired WDR72 recruitment to the Golgi. Cell fractionation assays confirmed subcellular distribution of wild-type WDR72 in both cytosolic and membranous fractions, while the WDR72 AI mutant and CS mutant forms were predominantly detected in the cytosolic fraction. Our studies provide new insights into the subcellular localization of WDR72 and demonstrate a critical role for the C-terminal CAAX motif in regulating WDR72 recruitment to the Golgi. In accordance with structural modelling studies that classified WDR72 as a potential vesicle transport protein, our findings suggest a role for WDR72 in vesicular Golgi transport that may be key to understanding the underlying cause of AI.

For pulp treatment in primary teeth, rotary canal instrumentation may be modestly time efficient, but otherwise similar to manual canal instrumentation.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: A systematic review and meta-analysis of randomized clinical trials comparing rotary canal instrumentation techniques with manual instrumentation techniques in primary teeth. Manchanda S, Sardana D, Yiu CKY. Int Endod J. 2020;53(3):333-353. SOURCE OF FUNDING: Information not available. TYPE OF STUDY/DESIGN: Systematic review with meta-analysis of data.

VWC2 Increases Bone Formation Through Inhibiting Activin Signaling.

By a bioinformatics approach, we have identified a novel cysteine knot protein member, VWC2 (von Willebrand factor C domain containing 2) previously known as Brorin. Since Brorin has been proposed to function as a bone morphogenetic protein (BMP) antagonist, we investigated the binding of Brorin/VWC2 to several BMPs; however, none of the BMPs tested were bound to VWC2. Instead, the ßA subunit of activin was found as a binding partner among transforming growth factor (TGF)-ß superfamily members. Here, we show that Vwc2 gene expression is temporally upregulated early in osteoblast differentiation, VWC2 protein is present in bone matrix, and localized at osteoblasts/osteocytes. Activin A-induced Smad2 phosphorylation was inhibited in the presence of exogenous VWC2 in MC3T3-E1 osteoblast cell line and primary osteoblasts. The effect of VWC2 on ex vivo cranial bone organ cultures treated with activin A was investigated, and bone morphometric parameters decreased by activin A were restored with VWC2. When we further investigated the biological mechanism how VWC2 inhibited the effects of activin A on bone formation, we found that the effects of activin A on osteoblast cell growth, differentiation, and mineralization were reversed by VWC2. Taken together, a novel secretory protein, VWC2 promotes bone formation by inhibiting Activin-Smad2 signaling pathway.

FAM20A binds to and regulates FAM20C localization.

Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn't require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues.

N-terminal Dentin Sialoprotein fragment induces type I collagen production and upregulates dentinogenesis marker expression in osteoblasts.

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly462-Asp463, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.