In search of new cysteine knot protein (CKP) family members, we found a novel gene called von Willebrand factor C domain-containing protein 2-like (Vwc2l, also known as Brorin-like) and its transcript variants (Vwc2l-1, Vwc2l-2 and Vwc2l-3). Based on the deduced amino acid sequence, Vwc2l-1 has a signal peptide and 2 cysteine-rich (CR) domains, while Vwc2l-2 lacks a part of 2nd CR domain and Vwc2l-3 both CR domains. Although it has been reported that the expression of Brorin-like was predominantly observed in brain, we found that Vwc2l transcript variants were detected in more ubiquitous tissues. In osteoblasts, the induction of Vwc2l expression was observed at matrix mineralization stage. When Vwc2l was stably transfected into osteoblasts, the matrix mineralization was markedly accelerated in Vwc2l-expressing clones compared to that in the control, indicating the modulatory effect of Vwc2l protein on osteoblastic cell function. The mechanistic insight of Vwc2l-modulation was further investigated and we found that the expression of Osterix, one of the key osteogenic markers, was significantly increased by addition of all Vwc2l isoform proteins. Taken together, Vwc2l is a novel secreted protein that promotes matrix mineralization by modulating Osterix expression likely through TGF-β superfamily growth factor signaling pathway. Our data may provide mechanistic insights into the biological functions of this novel CKP member in bone and further suggest a novel approach to enhance osteoblast function, which enables to accerelate bone formation, regeneration and healing.

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.

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