A functional RNAi screen for Runx2-regulated genes associated with ectopic bone formation in human spinal ligaments

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic ossification in the spinal ligaments, which enlarges with time and compresses the spinal cord, resulting in serious neurological symptoms. We previously reported that Runx2 expression was enhanced in spinal ligament cells from OPLL patients (OPLL cells). To clarify genes regulated by Runx2, Runx2 expression was first enhanced by culturing primary OPLL cells in osteogenic medium (OS induction) and then inhibited by siRNAs targeted to Runx2. DNA microarray demonstrated that in addition to chondrogenic factors such as connective tissue growth factor and cartilage oligomeric matrix protein, angiopoietin-1 was also significantly increased by OS induction and decreased by siRNAs for Runx2 in OPLL cells, suggesting that these genes are regulated by Runx2. However, these changes were not observed in non-OPLL control cells (from cervical spondylotic myelopathy patients). Furthermore, Runx2 was not decreased by siRNAs for angiopoietin-1. OS induction and RNAi inhibition of angiopoietin-1 expression was also observed in osteoblasts. Both siRNAs for Runx2 and angiopoietin-1 completely inhibited aggrecan-1 expression. These results suggest that angiopoietin-1 is downstream of Runx2 in both OPLL primary cells and osteoblasts. Angiopoietin-1 may play an important role in ectopic ossification.