Hormone-stimulated cell proliferation requires the expression of specific genes, a process effected by the binding of trans-acting protein factors to regulatory gene elements. Given the central role of gene expression in mitogenesis, it is important to understand how hormones induce transcription and how gene products promote proliferation. Toward this end, we have shown that platelet-derived growth factor (PDGF), a potent mitogen for cells of mesenchymal origin, activates the trans-acting factor NF-kappaB in Balb/c-3T3 mouse fibroblasts, NF-kappaB recognizes a 10 bp sequence termed the kappaB site; its activation requires its release from a cytosolic inhibitor protein termed IkappaB and most likely involves phosphorylation of IkappaB. At least four PDGF-inducible genes contain kappaB sites; thus, NF-kappaB is a potential and previously unrecognized mediator of PDGF-stimulated gene expression.
The aim of this proposal is to characterize events leading to and resulting from NF- kappaB activation by PDGF and to evaluate the role of NF-kappaB in fibroblast growth control. As tools for the proposed studies, anti-peptide antibodies to mouse IkappaB (to be cloned) and the 50 kd subunit of mouse NF-kappaB (p50) will be prepared; additional reagents include heterologous promoters containing various kappaB consensus sequences and antisense oligonucleotides to IkappaB. The antibodies will be used tin immunoprecipitation/immunoblotting assays to monitor the phosphorylation of IkappaB as well as the synthesis and degradation of p50 and IkappaB in PDGF-treated cells. Participation of NF-kappaB in PDGF-induced gene expression will be addressed using the heterologous promoters in chloramphenicol transferase assays. Lastly, cells will be transfected with antisense oligonucleotides to ablate IkappaB synthesis and, consequently, specifically activate NF-kappaB; gene expression in the proliferative status of the transfected cells will be determined. It is noted that although NF--kappaB has been extensively studied in lymphocytes, its activation and function in fibroblasts have not been previously addressed. Furthermore, due to lack of specific antibodies and probes, the mechanism of in vivo activation of NF-kappaB has remained obscure.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J et al. (2015) The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates. J Bacteriol 197:2335-49
Laverde Gomez, Jenny A; Bhatty, Minny; Christie, Peter J (2014) PrgK, a multidomain peptidoglycan hydrolase, is essential for conjugative transfer of the pheromone-responsive plasmid pCF10. J Bacteriol 196:527-39
Cascales, Eric; Atmakuri, Krishnamohan; Sarkar, Mayukh K et al. (2013) DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system. J Bacteriol 195:2691-704
Li, Feng; Alvarez-Martinez, Cristina; Chen, Yuqing et al. (2012) Enterococcus faecalis PrgJ, a VirB4-like ATPase, mediates pCF10 conjugative transfer through substrate binding. J Bacteriol 194:4041-51