Gene expression profile during osteoblaslic differentiation of human mesenchymal cells

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Ossification of the posterior longitudinal ligament of the spine (OPLL) is the leading cause of myelopathy in Japan, and is diagnosed by ectopic bone formation in the paravertebral ligament OPLL is a systemic high bone mass disease with a strong genetic background To detect genes relevant to the pathogenesis of OPLL, we performed cDNA microarray analysis of systematic gene expression profiles during osteoblastic differentiation of ligament cells from OPLL patienis (OPLL cells), ossification of yellow ligament patients, and non-OPLL controls, and human mesenchymal stem cells (hMSCs) after stimulating with osteogenic differentiation medium (OS). Twenty-nine genes were up-regulated during osteoblastic differentiation in OPLL cells. Zinc finger protein 145 (PLZF) was one of the highly expressed genes during osteoblastic differentiation in all the cells examined. We investigated the roles of PLZF in the regulation of osteoblastic differentiation ofhMSCs and C2C12 cells. siRNA mediated gene-silencing of PLZF resulted in the reduction of osteoblast-specific genes such as alkaline phosphatase (ALP), collagen lAl (COL1A1), Runx2/cbfa1 (CBFA 1), and osteocalcin (OCN), even in the presence of OS in hMSCs. The expression of PLZF was unaffected by addition of bone morphogenetic protein 2 (BMP-2) and the expression of BMP-2 was not affected by PLZF in hMSCs. In C2C12 cells, overexpression of PLZF increased the expression of Cbfal and Collal, on the other hand, overexpression of CBFA1 did not affect the expression of Plzf. These findings indicate that PLZE plays important roles in early osteoblastic differentiation as an upstream regulator of CBFA1 and thereby might pailicipate in promoting ossification of spinal ligament cells in OPLL patients.