Extracellular Signal-Regulated Kinase 2 and CHOP Restrict the Expression of the Growth Arrest-Specific p20K Lipocalin Gene to G0.

The activation of the growth arrest-specific (gas) p20K gene depends on the interaction of C/EBPβ with two elements of a 48-bp promoter region termed the quiescence-responsive unit (QRU). Here we identify extracellular signal-related kinase 2 (ERK2) as a transcriptional repressor of the p20K QRU in cycling chicken embryo fibroblasts (CEF). ERK2 binds to repeated GAAAG sequences overlapping the C/EBPβ sites of the QRU. The recruitment of ERK2 and C/EBPβ is mutually exclusive and dictates the expression of p20K. C/EBP homologous protein (CHOP) was associated with C/EBPβ under conditions promoting endoplasmic reticulum (ER) stress and, to a lesser extent, in cycling CEF but was not detectable when C/EBPβ was immunoprecipitated from contact-inhibited cells. During ER stress, overexpression of CHOP inhibited p20K, while its downregulation promoted p20K, indicating that CHOP is also a potent inhibitor of p20K. Transcriptome analyses revealed that hypoxia-responsive genes are strongly induced in contact-inhibited but not serum-starved CEF, and elevated levels of nitroreductase activity, a marker of hypoxia, were detected at confluence. Conditions of hypoxia (2% O2) induced growth arrest in subconfluent CEF and markedly stimulated p20K expression, suggesting that the control of proliferation and gas gene expression is closely linked to limiting oxygen concentrations associated with high cell densities.