Activation of serum response factor by RhoA is mediated by the nuclear factor-kappaB and C/EBP transcription factors.
(17/2395)
The activity of the transcription factor NF-kappaB can be modulated by members of the Rho family of small GTPases (Perona, R., Montaner, S., Saniger, L., Sanchez-Perez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475). Ectopic expression of RhoA, Rac1, and Cdc42Hs proteins induces the translocation of NF-kappaB dimers to the nucleus, triggering the transactivation of the NF-kappaB-dependent promoter from the human immunodeficiency virus. Here, we demonstrate that activation of NF-kappaB by RhoA does not exclusively promote its nuclear translocation and binding to the specific kappaB sequences. NF-kappaB is also involved in the regulation of the transcriptional activity of the c-fos serum response factor (SRF), since the activation of a SRE-dependent promoter by RhoA can be efficiently interfered by the double mutant IkappaBalphaS32A/S36A, an inhibitor of the NF-kappaB activity. We also present evidence that RelA and p50 NF-kappaB subunits cooperate with the transcription factor C/EBPbeta in the transactivation of the 4 x SRE-CAT reporter. Furthermore, RhoA increases the levels of C/EBPbeta protein, facilitating the functional cooperation between NF-kappaB, C/EBPbeta, and SRF proteins. These results strengthen the pivotal importance of the Rho family of small GTPases in signal transduction pathways which modulate gene expression and reveal that NF-kappaB and C/EBPbeta transcription factors are accessory proteins for the RhoA-linked regulation of the activity of the SRF. (+info)
CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription.
(18/2395)
Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene. (+info)
Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response.
(19/2395)
Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site. (+info)
The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell-cycle regulated.
(20/2395)
Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle, we separated MEL cells by elutriation, obtaining fractions >80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters. (+info)
The expression of CCAAT/enhancer binding protein (C/EBP) in the human ovary in vivo: specific increase in C/EBPbeta during epithelial tumour progression.
(21/2395)
The CCAAT/enhancer binding protein (C/EBP) family of transcription factors is involved in metabolism and differentiation of cells, especially in rodent liver cells and adipocytes. Their roles in vivo and in particular during pathophysiological conditions in humans are largely unknown. We have investigated the presence of C/EBPalpha, -beta, -delta and -zeta in normal ovaries and in epithelial ovarian tumours of different stages. Immunohistochemical experiments demonstrated that C/EBPalpha and C/EBPbeta were preferentially expressed in epithelial/tumour cells irrespective of stage or grade of the tumour. C/EBPbeta was located in the nuclei of the cells, in contrast to C/EBPalpha, which was present only in the cytoplasm of these cells. The nuclear localization of C/EBPbeta indicates an active role of this transcription factor in tumour cells, whereas the cytoplasmic distribution suggests a more passive function of C/EBPalpha. C/EBPdelta and -zeta demonstrated a more diverse distribution with predominant localization to epithelial cells, but stromal distribution was also noted. The intracellular distribution was confined to both the nucleus and the cytoplasm for C/EBPdelta and -zeta. Western blotting demonstrated that C/EBPalpha, -beta, -delta and -zeta were present in a majority of the samples. The amount of C/EBPbeta increased markedly with malignancy, i.e. with degree of dedifferentiation, while the other members of the C/EBP family displayed a more constant expression level. These results demonstrate an association between the expression of members of the C/EBP family and the formation of epithelial ovarian tumours, with C/EBPbeta as a potential marker for these tumours. As C/EBPbeta is known to be expressed during proliferation of cells in vitro, it may participate in the proliferative process of ovarian epithelial tumour cells in vivo and play a central role in tumour progression. (+info)
CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts.
(22/2395)
Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species. (+info)
Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks hyperlipidemia resulting from production of lipid-rich VLDL.
(23/2395)
Transgenic mice that overexpress the nuclear form of sterol regulatory element binding protein-1a (SREBP-1a) in liver (TgBP-1a mice) were shown previously to overproduce cholesterol and fatty acids and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol ( approximately 45 mg/dl) and triglycerides ( approximately 55 mg/dl) were not elevated, perhaps owing to degradation of lipid-enriched particles by low-density lipoprotein (LDL) receptors. To test this hypothesis, in the current studies we bred TgBP-1a mice with LDL receptor knockout mice. As reported previously, LDLR-/- mice manifested a moderate elevation in plasma cholesterol ( approximately 215 mg/dl) and triglycerides ( approximately 155 mg/dl). In contrast, the doubly mutant TgBP-1a;LDLR-/- mice exhibited marked increases in plasma cholesterol ( approximately 1,050 mg/dl) and triglycerides ( approximately 900 mg/dl). These lipids were contained predominantly within large very-low-density lipoprotein (VLDL) particles that were relatively enriched in cholesterol and apolipoprotein E. Freshly isolated hepatocytes from TgBP-1a and TgBP-1a;LDLR-/- mice overproduced cholesterol and fatty acids and secreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP-1a mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude that the TgBP-1a mice produce large lipid-rich lipoproteins, but these particles do not accumulate in plasma because they are degraded through the action of LDL receptors. (+info)
Modulation of rat preadipocyte adipose conversion by androgenic status: involvement of C/EBPs transcription factors.
(24/2395)
Androgenic status affects rat preadipocyte adipose conversion from two deep intra-abdominal (epididymal and perirenal) fat depots differently. The aim of this study was to establish whether these site-specific alterations of adipogenesis are related to altered expressions of the transcriptional factors regulating proliferation and differentiation of preadipocytes, c-myc and CCAAT/enhancer binding proteins (C/EBPs: C/EBPalpha and beta). The increased proliferation of epididymal and perirenal preadipocytes from castrated rats was not linked to variations in c-myc mRNA and protein levels. The expression of the early marker of adipogenesis, lipoprotein lipase (LPL), was decreased by androgenic deprivation in epididymal cells but remained insensitive to the androgenic status in perirenal preadipocytes. In contrast, LPL expression increased in subcutaneous preadipocytes from castrated rats, an effect which was partly corrected by testosterone treatment. Expression of C/EBPbeta was unaffected by androgenic status whatever the anatomical origin of the preadipocytes. In contrast, the mRNA and protein levels of C/EBPalpha were greatly decreased by androgenic deprivation in epididymal cells, an alteration which could not be corrected by in vivo testosterone administration. Altogether these results demonstrated that in preadipocytes androgenic deprivation affects site-specifically the expression of LPL, an early marker of adipogenesis and of C/EBPalpha, a master regulator of adipogenesis. These observations contribute to an explanation of why castration induces defective adipose conversion in rat epididymal preadipocytes specifically. (+info)