Effects of Growth/Differentiation Factor-5 on human dental pulp cell and its signaling
Date Issued
2011
Date
2011
Author(s)
Tseng, Hui-Chun
Abstract
Aim: Growth/Differentiation factor-5 (GDF-5) is a multifunctional protein and close related to the development and repair of multiple tissues, including cartilage, tendon, intervertebral disk, bone and skin. The purpose of our study is to investigate whether GDF-5 influences the morphological changes, cell proliferation and viability, cell differentiation and collagen formation of human dental pulp cells in vitro within the period of 5 or 10 days. Furthermore, the related signal transduction pathways were also been evaluated.
Materials and Methods: Primary-cultured human dental pulp cells were treated with different concentration of GDF-5 (0-500 ng/ml). In some experiments, cells were pretreated with different specific signaling inhibitors 30 minutes before adding GDF-5 for investigating the signaling of GDF-5. Morphology of pulp cells was observed under light microscopy (100X). Cell proliferation was evaluated by MTT assay. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP quantitative assay. Changes in mRNA expression of cell mitosis-related genes (Cyclin B1, CDC2, CDC25C and p21) were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Besides, immunofluorescence assay was used to observe the percentages of binuclear and mononuclear cells.
Results: Human dental pulp cells were spindle with extended cellular processes with/without GDF-5 treatment. Under the treatment by various concentrations of GDF-5: cell viability was up-regulated significantly in dose-dependent manner; Cylin B1 mRNA expression was stimulated, but the expression of p21 mRNA was inhibited; the percentage of binuclear cells was increased. In the inhibitory experiment of MTT assay, SB431542 (ALK-4/5/7 specific inhibitor) could slightly prevent the GDF-5-induced declination in cell proliferation. In cell differentiation: GDF-5 declined the ALP activity of human dental pulp cells; four inhibitors, including Dorsomorphin (ALK-2/3/6 specicic inhibitor)、SB431542、U0126 and SB203580 (p38 inhiibitor) could not reverse the effect of GDF-5 on ALP activity. GDF-5 did not affect the collagen content significantly.
Conclusion: GDF-5 demonstrated proliferative property in human dental pulp cells, but had inhibitory effect on cell differentiation and no effect on collagen formation. Signal transduction of GDF-5 in human dental pulp cells is a complex system. GDF-5 could induce cell proliferation through the activation of ALK-4/5/7 partially, but the signal pathway of GDF-5-induced cell differentiation is still unknown. These events are crucial in the mechanism of pulpal repair and regeneration.
Subjects
cell proliferation and differentiation
human dental pulp cells
GDF-5
Type
thesis
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