Our Science – Johnson Website
Peter F. Johnson, Ph.D.
Function and Regulation of the C/EBP Family of Transcription Factors
Changes in gene transcription underlie many biological processes, including cell growth, differentiation, and tumorigenesis. Our laboratory is investigating the C/EBP (CCAAT/enhancer-binding protein) family of transcription factors and their roles in regulating these physiological processes. The C/EBP family is composed of six related members that belong to the basic-leucine zipper (bZIP) class of DNA-binding proteins. Our research focuses primarily on C/EBPbeta and its involvement in controlling growth and survival of normal and transformed cells, as well as understanding the mechanisms that regulate C/EBPbeta expression and activity.
We have used C/EBPbeta knockout mice and cells derived from these animals to establish an essential function for C/EBPbeta in oncogenic transformation. In collaboration with Dr. Robert Smart (North Carolina State U), we found that C/EBPbeta null mice are completely resistant to the development of skin tumors when subjected to a DMBA/TPA carcinogenesis protocol. The skin tumors that develop in normal animals contain mutations in the Ras protooncogene. Since C/EBPbeta can be post-translationally activated by the Ras signal transduction pathway, we postulate that activation of C/EBPbeta by oncogenic Ras is a necessary event in the development of skin tumor cells. Additional evidence suggests that C/EBPbeta may function in transformed keratinocytes to suppress apoptosis, thus explaining its requirement for skin tumorigenesis. Low levels of C/EBPbeta overexpression stimulate Ras-induced transformation of NIH 3T3 cells and a dominant negative C/EBPbeta protein blocks transformation. These findings demonstrate that C/EBPbeta is an important regulator of neoplastic transformation elicited by the Ras oncogene.
Recently, our laboratory found that bone marrow-derived macrophages infected with a transforming virus carrying the Myc and Raf oncogenes also require C/EBPbeta to become transformed. Macrophages derived from C/EBPbeta knockout mice show a complete block to transformation. We determined that the transformation defect involves the mutant cells' inability to grow and survive in the absence of exogenous hematopoietic growth factors such as M-CSF. Using microarray screening, we identified the gene for insulin-like growth factor I (IGF-I) as a transcriptional target of C/EBPbeta in transformed macrophages. IGF-I acts as an autocrine growth and survival factor in these cells and allows them to become 'self-sufficient' (i.e., independent of exogenous growth factors). In support of this notion, IGF-I-deficient bone marrow cells are also resistant to Myc/Raf transformation and require an exogenous growth factor. We are currently investigating whether C/EBPbeta plays a similar role in regulating IGF-I expression in other kinds of tumor cells and whether it controls serum IGF-I levels, which are known to contribute to the progression and malignancy of many cancers by promoting tumor cell growth and survival.
Because C/EBPbeta is a downstream target of the Ras-Raf pathway we wish to understand the mechanistic basis of its regulation, including identifying post-translational modifications that mediate its functional activation by Ras signaling and elucidating the effector pathways involved. We found that the DNA-binding activity of C/EBPbeta is intrinsically inhibited (auto-repressed) but can be activated by Ras signaling. Using deletion and site-directed mutagenesis, we mapped specific sequences outside the DNA-binding domain that mediate auto-repression. In addition, we have identified several phosphoacceptor sites that are induced by Ras signaling, two of which are cell cycle-regulated and appear to be targets for cyclin dependent kinases (Cdks). Mutation of either of these phosphoacceptor residues to alanine does not affect DNA-binding but blocks the ability of C/EBPbeta to facilitate NIH 3T3 cell transformation. Thus, cell cycle dependent phosphorylation of C/EBPbeta is a critical event in Ras-induced transformation. We are currently investigating other Ras-induced modifications that appear to mediate de-repression of C/EBPbeta DNA-binding activity and we plan to test whether these sites are also important for the 'pro-oncogenic' function of C/EBPbeta.
Our collaborators include Jonathan Keller, SAIC-Frederick; Italo Mocchetti, Georgetown University; Richard Schwartz, Michigan State University; Carl Barrett, NCI; Steve Hursting, NCI; Robert Smart, North Carolina State University; Terry Copeland, NCI-Frederick; and Dhananjaya Kalvakolanu, University of Maryland
This page was last updated on 3/29/2013.