Proteasomal regulation of C/EBP alpha protein and diminished expression of C/EBP alpha in squamous cell carcinomas

Abstract

The CCAAT/enhancer binding proteins (C/EBPs) are members of the basic leucine zipper (bZIP) class of transcription factors. C/EBP alpha is involved in mitotic growth arrest/differentiation and has been implicated as a human tumor suppressor in acute myeloid leukemia. C/EBP alpha is abundantly expressed in mouse keratinocytes. The purpose of this study was to examine the regulation of the C/EBP alpha protein and to determine if C/EBP alpha expression/function is altered in skin squamous cell carcinomas.

We found that C/EBP alpha is a short-lived protein with a half-life of ~ 1 hour and treatment with proteasome inhibitors blocked the degradation of C/EBP alpha protein. Poly-ubiquitinated C/EBP alpha were detected in BALB/MK2 and C/EBP alpha was degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBP alpha kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in; i) an increase in C/EBP alpha protein levels, ii) increased electrophoretic mobility of C/EBP alpha protein and iii) no increase in C/EBP alpha mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBP alpha may target it for proteasomal degradation. However, a mutant C/EBP alpha containing mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl and additional pharmacological inhibitors of GSK3 did not alter C/EBP alpha levels indicating the effects of LiCl on C/EBP alpha are GSK3-independent. LiCl treatment inhibited C/EBP alpha degradation and produced a six-fold increase in the half-life of C/EBP alpha protein. In vitro studies revealed that LiCl inhibited proteasomal degradation of C/EBP alpha. These results demonstrate C/EBP alpha is degraded via a proteasomal pathway and LiCl stabilizes C/EBP alpha through a GSK3 independent pathway involving inhibition of proteasome activity.

The expression of C/EBP alpha was evaluated in mouse skin SCC lines. C/EBP alpha mRNA and protein levels were greatly diminished or undetectable in all seven SCC cell lines compared to normal keratinocytes. Forced expression of C/EBP alpha resulted in the inhibition in SCC cell proliferation. Expression of C/EBP alpha also resulted in the expression of loricrin, a late stage marker of squamous differentiation. Treatment with 5'-aza-deoxycytidine increased C/EBP alpha expression in some SCC cell lines suggesting the C/EBP alpha promoter region may be transcriptionally silenced by hypermethylation. C/EBP alpha expression was negligible in all 14/14 SCC examined compared to normal epidermis. These results suggest the loss of C/EBP alpha expression may contribute to the altered growth and differentiation characteristics of skin SCCs.