Understanding insulin-like growth factor 1 actions in otic cells: activation of protein kinase B and reduction of autophagic flux

Abstract

Insulin-like growth factor 1 (IGF-1) deficiency causes sensorineural hearing loss in man and mice. To gain insight into the molecular targets of IGF-1 in otic cells, both sensory and neural, we have used the cell lines HEI-OC1 (derived from the auditory organ of the transgenic mouse Immortomouse™) and Neuro2a (a murine neuroblastoma). The expression of genes of the IGF system has been studied by RT-qPCR in both cell lines. We observed that Igf1, Igf1r and Irs1 transcripts were more abundant in HEI-OC1 than in Neuro2a cells, whilst Irs2 transcripts were highly expressed in Neuro2a. We also studied the actions and signaling of IGF-1 in both lines. Number of cells was determined using crystal violet staining method, apoptosis was studied by flow cytometry, location of IGF1R was determined by immuno-fluorescence and activation of target proteins was measured by Western Blotting. IGF-1 treatment increased the number of cells in both lines, and also protected against apoptosis blocking annexin V exposure and DNA strand breaks in HEI-OC1. IGF1R was translocated from the membrane to a perinuclear area after IGF-1 treatment in Neuro2a. Relative p-AKTSer473 to total AKT levels increased and the autophagic flux decreased after IGF-1 treatment in both cell lines, whereas activated pERK1/2 increase was solely observed in Neuro2a cells. Therefore, both sensory cells and neurons express the IGF system elements and their survival is promoted by IGF-1. Consequences of blockade of the IGF system by both chemical and genetic methods will be discussed.