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Piwien Pilipuk, G, Galigniana, MD and Schwartz, J (2003) Subnuclear localization of C/EBP beta is regulated by growth hormone and dependent on MAPK. J. Biol. Chem. 278:35668-77
Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP beta, the effect of GH on the subcellular localization of C/EBP beta was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP beta is diffusely distributed in nuclei of quiescent cells. Within 5 min of GH treatment, the diffuse pattern dramatically becomes punctate. The relocalization of C/EBP beta coincides with DAPI staining of heterochromatin. Further, C/EBP beta and heterochromatin protein (HP)-1 alpha colocalize in the nucleus, consistent with localization of C/EBP beta to pericentromeric heterochromatin. In contrast, C/EBP delta exhibits a diffuse distribution in the nucleus that is not modified by GH treatment. C/EBP beta is rapidly and transiently phosphorylated on a conserved MAPK consensus site in response to GH (Piwien-Pilipuk, G., MacDougald, O. A., and Schwartz, J. (2002) J. Biol. Chem. 277, 44557-44565). Indirect immunofluorescence using antibodies specific for C/EBP beta phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP beta. In addition, phosphorylated C/EBP beta colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP beta, C/EBP delta, and C/EBP alpha (p42 and p30 forms) can bind to satellite DNA as homo- or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP beta from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP beta to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP beta. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GH-mediated phosphorylation and nuclear redistribution of C/EBP beta may be coordinated to achieve spatial-temporal control of gene expression.
3T3 Cells; Adipocytes/cytology; Adipocytes/physiology; Animals; Binding Sites; CCAAT-Enhancer-Binding Protein-beta/metabolism; Cell Cycle/physiology; Cell Differentiation; Cell Nucleus/physiology; DNA, Satellite/metabolism; Heterochromatin/physiology; Human Growth Hormone/physiology; Mice; Mitogen-Activated Protein Kinases/metabolism; Phosphorylation
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