Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

Abstract

Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients. Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group. Spinal ligament fibroblasts were observed 7-10days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72h after RNA interference targeting PERK was performed on the cells from OPLL patients. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.