Inhibition of neurite outgrowth by reduced level of NDRG4 protein in antisense transfected PC12 cells.

NDRG4, a member of the new NDRG gene family, was originally cloned as a gene that was expressed predominantly in the early postnatal rat brain. To determine whether the NDRG4 protein contributes to differentiation of neural cells, the effect of lowering the cellular NDRG4 protein level on the nerve growth factor (NGF)-induced neurite formations and transcription factor activations in PC12 cells was examined. An antisense construct of rat NDRG4 cDNA was made and transfected to PC12 cells, which constitutively express a basal level of the NDRG4 protein. Of the stably transfected antisense cell clones that expressed exogenous NDRG4 antisense RNA, six clones showed reduced levels of the NDRG4 protein, but unexpectedly two clones showed quite higher levels of NDRG4 protein than the control cells. The clones having decreased levels of the NDRG4 protein extended shorter neurites than control cells in response to NGF or dibutyryl cAMP. In contrast, the NDRG4 protein-highly expressing clones did not show suppressed neurite outgrowth induced by NGF. NGF-mediated activation of the transcription factor AP-1 was found to be suppressed in the NDRG4 protein-diminished clone and enhanced in the NDRG4 protein-upregulated clone as compared with those in the control cells. These results suggest that NDRG4 plays a role in neurite outgrowth and has an influence on an NGF-stimulated AP-1 activation by an undefined mechanism in PC12 cells.