Using Col2.3GFP transgenic mice expressing GFP in maturing osteoblasts, we isolated Col2.3GFP+ enriched osteoblasts from 3 sources. We performed RNA-sequencing, identified 593 overlapping genes and confirmed these genes are highly enriched in osteoblast differentiation and bone mineralization annotation categories. The top 3 annotations are all associated with endoplasmic reticulum and Golgi vesicle transport. We selected 22 trafficking genes that have not been well characterized in bone for functional validation in MC3T3-E1 pre-osteoblasts. Transient siRNA knockdown of trafficking genes including Sec24d, Gosr2, Rab2a, Stx5a, Bet1, Preb, Arf4, Ramp1, Cog6 and Pacs1 significantly increased mineralized nodule formation and expression of osteoblast markers. Increased mineralized nodule formation was suppressed by concurrent knockdown of P4ha1 and/or P4ha2, encoding collagen prolyl 4-hydroxylase isoenzymes. MC3T3-E1 pre-osteoblasts with knockdown of Cog6, Gosr2, Pacs1 or Arf4 formed more and larger ectopic mineralized bone nodules in vivo, which was attenuated by concurrent knockdown P4ha2. Permanent knockdown of Cog6 and Pacs1 by CRISPR/Cas9 gene editing in MC3T3-E1 pre-osteoblasts recapitulated increased mineralized nodule formation and osteoblast differentiation. In summary, we have identified several vesicle trafficking genes with roles in osteoblast function. Our findings provide potential targets for regulating bone formation.
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