Effect of Hyaluronic Acid on Neurite Outgrowth of PC12 cells

Objective: It is well known that hyaluronic acid (HA), one of glycosaminoglycan, shows a variety of biological effects. In the present study, we examined the effects of HA on NGF-induced neurite outgrowth of PC12 cells to clarify the effects of HA on the differentiation of neuronal cells. Methods: After the exposure of PC12 cells with NGF in the absence and presence of HA, we counted neurite positive cells with neurites which were more than double length of shortest diameter of a cell body under phase contrast microscope. We also examined the cell viability by MTT assay, and expression of NF68, one of differentiation markers, phosphorylation of MAP kinases, p38, ERK and JNK, and CREB, one of transcriptional factors during neuronal cell differentiation, by Western blot. Further, pCRE-Luc transfected PC12 cells were treated with HA in the presence of NGF and their luciferase activities were detected. Results: HA did not affect PC12 cell viability. NGF induced the neurite outgrowth of PC12 cells, and expression of NF68, phosphorylation of p38, and ERK, and CREB, but not JNK. We found that the neurite outgrowth of PC12 cells by NGF was suppressed by HA in a dose dependent manner. We also found that HA suppressed expression of NF68 and phosphorylation of p38, ERK and CREB in a dose dependent manner. Further, HA significantly suppressed luciferase activity of pCRE-Luc transfected PC12 cells in the presence of NGF. Conclusion: Our results suggest that HA inhibits NGF-induced neurite outgrowth of PC12 cells through the inhibition of phosphorylation of p38, ERK, and CREB. Supported by Grants (18592094, 18209057) from The Ministry of Education, Science, and Culture of Japan, Tokyo, Japan.