Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into “osteocyte‐like” cells

Abstract

P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, and syncytium formation and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP a protein common to all vertebrate teeth, and crucial for their mineralization. Here we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18 fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN) and β-catenin decreased by 70%, 64% and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an “osteocyte-like” phenotype, an important process in bone biology. The mechanisms involved are presently under study.

Perinatal DNA Methylation at CDKN2A Is Associated With Offspring Bone Mass: Findings From the Southampton Women's Survey

“…There is little evidence in the literature for the specific involvement of the CDKN2A locus in bone metabolism, although p16 INK4a expression has been linked to altered osteoblast morphology and senescence in animal models, and one study identified transitional hypomethylation of CDKN2A in human bone marrow stromal cells in their differentiation toward an osteoblastic lineage . However, the importance of cyclin‐dependent kinases and their inhibitors has been demonstrated by studies of the osteogenic differentiation of adipose‐derived mesenchymal stem cells, in which the promoters of RUNX2 , osteocalcin , and osterix genes are actively demethylated in a process dependent upon growth arrest and DNA‐damage‐inducible protein, GADD45, which is known to interact with both CDK1 and CDKN1A .…”