Enhancement of GDP-Dissociation Inhibitor Gene Expression in Osteoblasts by Low-Level Laser Irradiation

Abstract

Low-level laser irradiation (LLLI) stimulates bone regeneration and may be useful for bone fracture and oral implant therapy. However, the molecular mechanism of LLLI in accelerating bone formation is not well known. In order to understand the mechanism of LLLI in accelerating bone formation, we attempted to identify gene expressions enhanced by LLLI by a subtractive gene cloning and DNA microarray analysis. Osteoblastic cell MC3T3-E1 was treated with 830 nm Ga-Al-As diode laser irradiation and total mRNA was recovered and used to construct a subtractive gene library of genes with transcription enhanced by LLLI. The DNA sequences of subtracted gene was subjected to a homology search using BLASTN program in the National Center for Biotechnology Information (NCBI). Genes with mRNA levels altered by LLLI were also analysed using Affymetrix GeneChip. The mRNA levels were confirmed by RT-PCR and real-time PCR. Subtracted gene clones MCL101 indicated high homology with GDP-dissociation inhibitor beta (GDPIB) genes. Affymetrix GeneChipTM analysis was further analyzed and found that GDI alpha gene expression was also enhanced by LLLI. The increased mRNA level of GDIA and GDIB by LLLI was successfully confirmed by RT-PCR and real-time PCR. Since the up-regulation of GDIA is one of the mechanisms underlying the synergistic boneforming effect of parathyroid hormone and oestrogen. Moreover, GDIA and GDIB involve in the regulation of vesicle-mediated cellular transport and anti-apoptosis, the enhancement of GDIA and GDIB gene expressions by LLLI may be a part of mechanisms in the acceleration of bone formation by LLLI.