Regulatory mechanism by BDNF in human cemento-blast-like cells

Objectives: We have reported that brain-derived neurotrophic factor (BDNF) promotes regeneration of periodontal tissue in experimental defect in dogs. Since the reconstruction of cementum on denuded dentin surface is a key to regenerate the functional periodontium; we investigated effects of BDNF on the expression of the cementum (bone)-related proteins and its regulatory mechanism in human cementoblasts.

Methods: Immortalized human cementoblast-like cells (HCEMs), established with transfection of hTERT gene (Kitagawa et al., 2006) were used in the following experiments. The protein expression of tyrosine kinase receptor B (trkB), high affinity for BDNF or nerve growth factor receptor (p75NTR), low affinity receptor in HCEMs was examined by Western blotting and immunofluorescens microscopy. The knockdown of trkB, p75NTR or Elk-1 which is a transcriptional factor was performed by siRNAs. HCEMs were exposed to BDNF at 20 ng/ml in the presence or absence of PD98059 (an inhibitor of ERK1/2) or PDTC (an inhibitor of NF-ƒÛB). The mRNA expressions of osteopontin (OPN), alkaline phosphatase (ALP) and bone morphogenetic protein (BMP)-2 were analyzed by Real-time PCR. The levels of phospho-ERK1/2 and phospho-c-Raf were determined by Western blotting. The expression of phospho-Elk-1 was confirmed by immunofluorescens microscopy.

Results: TrkB and p75NTR were expressed in HCEMs. BDNF increased mRNA levels of OPN, ALP and BMP-2 in HCEMs via trkB, but not p75NTR. PD98059, but not PDTC, inhibited the increase of the mRNA levels. BDNF elevated levels of the phospho-ERK1/2 and phospho-Elk-1. The knockdown of Elk-1 blocked the elevation of the cementum (bone)-related protein mRNA expressions by BDNF. BDNF stimulated the levels of phospho-c-Raf through trkB.

Conclusion: These findings suggest BDNF stimulates the expression of OPN, ALP and BMP-2 in cementoblasts through a trkB-c-Raf-ERKs-Elk-1 signaling pathway.