Lack of Elk-1 Phosphorylation and Dysregulation of the Extracellular Regulated Kinase Signaling Pathway in Senescent Human Fibroblast

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.