LMP1 regulates osteolineage differentiation of human PDL fibroblasts

LMP-1, a recently discovered intracellular LIM domain protein, strongly induces osteolineage differentiation in bone marrow stromal cells and accelerates posterior thoracic and lumbar spine fusion. However, little is known about the role of LMP1 in periodontal ligament (PDL) cell differentiation and alveolar bone regeneration. Objective:This study sought to determine whether LMP1 is a positive regulator in the differentiation of PDL cells in vitro. Methods:Primary culture of PDL cells were observed over 10d via inducing and in non-inducing conditions to determine expression of LMP-1 and osteoblastic-related gene expression. Full-length cDNA of human LMP1 was acquired by RT-PCR from MG-63 cells, and cloned into a pQCXIN retroviral vector and production of retrovirus via 293T cells. PDL cells were transfected by pQC-hLMP1 or control retrovirus. After Geneticin selection for 10d, survival cells were pooled and induced to osteolineage differentiation. qRT-PCR was utilized to detect the expression of bone-specific marker genes over time. Alizarin Red staining was used to evaluate the mineralization over a course of 3w. Results:LMP1 was highly expressed in the early stages of mineralization induced by PDL cells in vitro. LMP1 was stably overexpressed in PDL cells via retroviral transduction. LMP1-engineered stable cells possessed elevated baseline levels of BMP2 expression, compared to controls. Osteolineage promotion conditions resulted in the induction of several osteoblastic genes including potent expression (~20fold) for osterix in pQC-hLMP1 cells compared to controls. At 3w, Alizarin red staining demonstrated more mineralization nodules were found in pQC-hLMP1 cells compared to controls. Conclusion:LMP1 is involved in the osteolineage differentiation of PDL cells leading to mineralization in vitro. Furthermore, PDL cells driven by LMP1 over-expression displayed enhanced osteo-lineage differentiation, which may be associated with elevated BMP2 expression. These studies suggest LMP-1 may play an important role in regulating periodontal tissue homeostasis and repair. Funded by NIH/NIDCR DE13397 and ITI Foundation.