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

CCAAT/enhancer binding protein alpha;squamous cell carcinoma;proteasome;keratinocyte

Shim, Minsub ; Robert C. Smart, Committee Chair,Shim, Minsub ; Robert C. Smart ; Committee Chair

University:North Carolina State University

关键词: CCAAT/enhancer binding protein alpha;    squamous cell carcinoma;    proteasome;    keratinocyte;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/4591/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

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.