AUTOCRINE NERVE GROWTH-FACTOR PROTECTS HUMAN KERATINOCYTES FROM APOPTOSIS THROUGH ITS HIGH-AFFINITY RECEPTOR (TRK) - A ROLE FOR BCL-2

Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low-and the high-affinity NGF receptor, Bec ause NGF has been shown to rescue certain cell types from programmed c ell death, we investigated the role of endogenous NGF in preventing ke ratinocyte apoptosis, We report here that apoptosis is induced in norm al human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibi tor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was a ssessed by DNA laddering, electron microscopy, and in situ nick end la beling technique, In anti-NGF-treated keratinocytes, the apoptotic pro cess starts at 96 h, and is maximal at 120 h. After K252 treatment, ap optosis starts at 48 h and peaks at 120 h, Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measu red the levels of this protein in apoptotic keratinocytes, We found th at both K252 and anti-NGF antibody strikingly downregulate bcl-2 expre ssion, starting at 72 h. Furthermore, HaCat keratinocytes stably trans fected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-a ffinity NGF receptor, possibly by maintaining constant levels of Bcl-2 .