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PubMed2002

Adhesion-related kinase repression of gonadotropin-releasing hormone gene expression requires Rac activation of the extracellular signal-regulated kinase pathway.

Recent studies suggest that adhesion-related kinase (Ark) plays a role in gonadotropin-releasing hormone (GnRH) neuronal physiology. Ark promotes migration of GnRH neurons via Rac GTPase and concomitantly suppresses GnRH gene expression via homeodomain and myocyte enhancer factor-2 (MEF2) transcription factors. Here, we investigated the signaling cascade required for Ark inhibition of the GnRH promoter in GT1-7 GnRH neuronal cells. Ark repression was blocked by the MEK/ERK pathway inhibitor, PD98059, and dominant negative MEK1 but was unaffected by dominant negative Ras. Inhibitors of the Rho family GTPases, Clostridium difficile toxin B (Rho/Rac/Cdc42 inhibitor) and Clostridium sordellii lethal toxin (Rac/Cdc42 inhibitor), blocked Ark inhibition of GnRH transcription. Moreover, dominant negative Rac blunted both Ark activation of ERK and repression of the GnRH promoter, demonstrating an essential role for Rac in coupling Ark to ERK activation. Like Ark, a constitutively active mutant of Rac suppressed GnRH transcription in an ERK-dependent manner. Finally, Ark-mediated repression was significantly attenuated by a dominant negative MEF2C, whereas repression induced by constitutively active Rac was unaffected, indicating that MEF2 proteins are not targets of the Ark --> Rac --> MEK --> ERK cascade. The data suggest that Ark suppresses GnRH gene expression via the coordinated activation of a Rac --> ERK signaling pathway and a distinct MEF2- dependent mechanism.

Animals↗
PubMed2002

POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

Aldehyde Oxidoreductases↗
PubMed2002

Estradiol represses human T-cell leukemia virus type 1 Tax activation of tumor necrosis factor-alpha gene transcription.

Adult T-cell leukemia is caused by human T-cell leukemia virus type I (HTLV-I). The HTLV-I Tax protein is essential for clinical manifestations because it activates viral and cellular gene transcription. Tax enhances production of tumor necrosis factor-alpha (TNF-alpha), which may lead to bone and joint destruction. Because estrogens might prevent osteoporosis by repressing TNF-alpha gene transcription, we investigated whether estrogens inhibit the transcriptional effects of Tax on the TNF-alpha promoter. Tax activated the -1044, -163, and -125 TNF-alpha promoters by 9-25-fold but not the -82 promoter, demonstrating that Tax activation requires the -125 to -82 region, known as the TNF response element (TNF-RE). Three copies of the TNF-RE upstream of the minimal thymidine kinase promoter conferred a similar magnitude of activation by Tax. We demonstrated that c-Jun, NFkappaB, p50, and p65 interact with and activate the TNF-RE by using mutational analysis of the TNF-RE, Tax mutants that selectively activate NFkappaB or the cAMP-response element binding protein/activating transcription factor pathway, and gel shift assays with nuclear extracts. Estradiol markedly repressed Tax-activated transcription of the TNF-alpha gene with estrogen receptor (ER) alpha or beta. Nuclear extracts from U2OS cells stably transfected with ER(alpha) demonstrated that ERs interact with the TNF-RE. Our studies provide evidence that ERs repress Tax-activated TNF-alpha transcription by interacting with a c-Jun and NFkappaB platform on the TNF-RE. Estrogens may ameliorate bone and inflammatory joint diseases in patients infected with HTLV-I by repressing transcription of the TNF-alpha gene.

Binding Sites↗
PubMed2003

The protein complex composed of nickel-binding SrnQ and DNA binding motif-bearing SrnR of Streptomyces griseus represses sodF transcription in the presence of nickel.

Nickel-responsive transcriptional repression of sodF, which codes for iron- and zinc-containing superoxide dismutase of Streptomyces griseus, was mediated through an operator (-2 to +15) spanning over the 5' end (+1) of the transcript. Two open reading frames, SrnR (12,343 Da) and SrnQ (12,486 Da), with overlapping stop-start codons were identified downstream from sodF and found responsible for the repression of sodF. The deduced amino acid sequence of SrnR revealed a DNA binding motif and showed homology to the transcriptional regulators of ArsR family, whereas SrnQ did not show any similarity to any known proteins. When srnRQ DNA was maintained in trans in S. griseus on a multicopy plasmid, sodF transcription was highly repressed by nickel, but neither srnR nor srnQ alone showed the effect. Consistently, the sodF transcription of srnR-interrupted mutant was no longer repressed by nickel, which was complemented only with srnRQ DNA. Nickel-dependent binding of SrnR and SrnQ to the sodF operator DNA was observed only when the two proteins were provided together. The maximum protein-DNA interaction was shown when SrnR and SrnQ were present in one-to-one stoichiometric ratio. The two proteins appear to constitute an octamer composed of four subunits of each protein. SrnR directly interacted with SrnQ, and the protein interaction did not require nickel. The conformation of SrnQ was changed upon nickel binding, which was in the ratio of one Ni(2+) ion per protein molecule. A model is proposed in which SrnQ of the protein complex senses nickel and subsequently enhances the DNA binding activity of SrnR through the protein-protein interaction.

Amino Acid Motifs↗
PubMed2003

Transforming growth factor-beta inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function.

Transforming growth factor (TGF)-beta is a potent inhibitor of adipocyte differentiation. To identify which adipocyte transcription factors might be targeted by TGF-beta, we overexpressed key adipogenic transcription factors, C/EBPbeta, C/EBPdelta, or peroxisome proliferator-activated receptor (PPAR) gamma in NIH3T3 cells and tested the ability of TGF-beta to block adipogenesis. We show that TGF-beta inhibits adipocyte differentiation driven by either C/EBPbeta or C/EBPdelta without affecting C/EBP protein expression levels, suggesting that these C/EBPs are a direct target of TGF-beta action. Because TGF-beta inhibits adipogenesis by signaling through Smad3, we examined physical and functional interactions of Smad3 and Smad4 with C/EBPbeta, C/EBPdelta, and PPARgamma2. C/EBPbeta and C/EBPdelta were found to physically interact with Smad3 and Smad4, and Smad3 cooperated with Smad4 and TGF-beta signaling to repress the transcriptional activity of C/EBPs. Thus, repression of the activity of C/EBPs by Smad3/4 at C/EBP binding sites inhibited transcription from the PPARgamma2 and leptin promoters. In contrast, PPARgamma interacted only very weakly with Smad3 and its transcriptional activity was not repressed by Smad3/4 or in response to TGF-beta. Smad3/4 did not reduce the ability of C/EBP to bind to its cognate DNA sequence, but repressed transcription by inhibiting the transactivation function of C/EBP.

3T3 Cells↗
PubMed2003

Fusion protein of retinoic acid receptor alpha with promyelocytic leukemia protein or promyelocytic leukemia zinc finger protein recruits N-CoR-TBLR1 corepressor complex to repress transcription in vivo.

Fusion proteins of retinoic acid receptor alpha (RARalpha) with promyelocytic leukemia protein (PML-RARalpha) or with promyelocytic leukemia zinc finger protein (PLZF-RARalpha) are associated with and likely responsible for the development of acute promyelocytic leukemia. These oncoproteins retain the ability to bind DNA and retinoic acid through the RARalpha moiety. This enables them to repress RARalpha target genes in the absence of retinoic acid, but the underlying mechanisms remain to be investigated. Here we use the frog oocyte system to study transcriptional regulation by PML-RARalpha and PLZF-RARalpha in the context of chromatin. We first show that the endogenous corepressor N-CoR forms a complex with TBLR1 (transducin beta-like protein 1-related protein) and that both N-CoR and TBLR1 can interact with unliganded PML-RARalpha and PLZF-RARalpha in vivo. Using chromatin immunoprecipitation, we demonstrate that both oncoproteins recruit TBLR1, as well as N-CoR, to its target promoter, leading to histone deacetylation and transcriptional repression. Furthermore, expression of a dominant negative N-CoR that contains the TBLR1-interacting domain blocks transcription repression by unliganded PML-RARalpha and PLZF-RARalpha. Thus, our studies provide in vivo evidence for targeted recruitment of N-CoR-TBLR1 complexes by PML-RARalpha and PLZF-RARalpha in transcriptional repression in the context of chromatin.

Amino Acid Sequence↗
PubMed2003

Repression of the human adenine nucleotide translocase-2 gene in growth-arrested human diploid cells: the role of nuclear factor-1.

Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide -332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest.

3T3 Cells↗
PubMed2003

Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells.

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3'-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5'-phosphate but not a 3'-hydroxyl group. G.U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3-4 base mismatches and additional G.U mismatches.

3' Untranslated Regions↗
PubMed2003

Fibroblast growth factor-2 represses platelet-derived growth factor receptor-alpha (PDGFR-alpha) transcription via ERK1/2-dependent Sp1 phosphorylation and an atypical cis-acting element in the proximal PDGFR-alpha promoter.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-alpha (PDGFR-alpha) are poorly understood. Here we demonstrate that PDGFR-alpha protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-alpha promoter -61 to -52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-alpha promoter. PDGFR-alpha transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-alpha transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-alpha promoter. Mutation of residues Thr(453) and Thr(739) in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-alpha transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-alpha transcription via the -61/-52 element in the PDGFR-alpha promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-alpha transcription, a finding previously unreported in any Sp1-dependent gene.

Animals↗
PubMed2003

MRGX is a novel transcriptional regulator that exhibits activation or repression of the B-myb promoter in a cell type-dependent manner.

MRGX is a novel transcription factor that is a member of the mortality factor 4 (MORF4)-related gene family. MRG15, a closely related family member, is in a complex with the retinoblastoma tumor suppressor protein Rb and activates the B-myb promoter, which is tightly controlled by Rb/E2F through the E2F binding site. In this study we investigated the effect of MRGX on the B-myb promoter. Interestingly, MRGX repressed the B-myb promoter in EJ cells (human bladder carcinoma cells), which have a functional Rb, but activated B-myb in HeLa cells (human cervical carcinoma cells), which express a lower amount of Rb. This repression and activation was dependent on the helix-loop-helix and leucine zipper regions of the MRGX protein but not the N-terminal region. MRGX interacts with Rb through the helix-loop-helix and leucine zipper regions. Using a treatment of trichostatin A, which is a potent inhibitor of histone deacetylase (HDAC), we determined that the repression of the B-myb promoter by MRGX in EJ cells was dependent on HDAC activity. We confirmed the association of MRGX with HDAC1 by immunoprecipitation/Western analysis and determined that MRGX complexes had HDAC activity. The data indicate that MRGX can repress or activate the B-myb promoter depending on the cell type studied, suggesting that there may be tissue-specific functions of this protein.

Base Sequence↗
PubMed2003

Mi-2 beta associates with BRG1 and RET finger protein at the distinct regions with transcriptional activating and repressing abilities.

Mi-2 beta is the main component of the nucleosome remodeling and deacetylase complex and plays an important role in epigenetic transcriptional repression. Here we show that the amino-terminal and carboxyl-terminal regions of Mi-2 beta have distinct transcriptional activities and bind to BRG1, a component of the SWI/SNF complex, and the RET finger protein (RFP), respectively. Analysis by luciferase reporter assay revealed that the amino-terminal region of Mi-2 beta has a strong transactivating ability, whereas its carboxyl-terminal region has transcriptional repressive activity. Co-localization and association of Mi-2, RFP, and histone deacetylase 1 suggested that these proteins cooperate in transcriptional repression. Furthermore, the functional importance of the association of Mi-2 beta and RFP was confirmed by using Rfp-/- fibroblasts. On the other hand, we demonstrated that Mi-2 and BRG1 were associated with each other and that the bromodomain region of BRG1 strongly suppressed transactivation by the amino-terminal region of Mi-2 beta. The findings that Mi-2 beta interacts with both transactivating and repressing proteins and directly associates with another chromatin remodeling protein, BRG1, provide new insight into the formation of multiprotein supercomplex involved in transcriptional regulation.

Adenosine Triphosphatases↗
PubMed2003

The Escherichia coli transcriptional regulator MarA directly represses transcription of purA and hdeA.

The Escherichia coli MarA protein mediates a response to multiple environmental stresses through the activation or repression in vivo of a large number of chromosomal genes. Transcriptional activation for a number of these genes has been shown to occur via direct interaction of MarA with a 20-bp degenerate asymmetric "marbox" sequence. It was not known whether repression by MarA was also direct. We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA. Transcription and electrophoretic mobility shift experiments in vitro using mutant promoters suggested that the marbox involved in the repression overlapped the -35 promoter motif and was in the "backward" orientation. This organization contrasts with that of the class II promoters activated by MarA, in which the marbox also overlaps the -35 motif but is in the "forward" orientation. We conclude that MarA, a member of the AraC/XylS family, can act directly as a repressor or an activator, depending on the position and orientation of the marbox within a promoter.

Cyclic AMP Response Element-Binding Protein↗
PubMed2004

Structure-function analysis of the estrogen receptor alpha corepressor scaffold attachment factor-B1: identification of a potent transcriptional repression domain.

Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ERalpha)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERalpha activity. SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ERalpha activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.

Animals↗
PubMed2004

Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA.

The Xenopus alpha(fast)-tropomyosin gene contains, at its 3' -end, a composite internal/3' -terminal exon (exon 9A9'), which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles, whereas it is skipped in nonmuscle cells. We have developed an in vivo model based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. We first show that the different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and constitute valuable tools to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in nonmuscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. We show using UV cross-linking and immunoprecipitation experiments that Xenopus PTB (XPTB) interacts with these PTB binding sites. Finally, we show that depletion of endogenous XPTB in Xenopus embryos using a morpholinobased translational inhibition strategy resulted in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' -terminal exon.

Actins↗
PubMed2004

SUMO modification of repression domains modulates function of nuclear receptor 5A1 (steroidogenic factor-1).

Steroidogenic factor 1 (SF-1 or NR5A1), is a Ftz-F1 member of the nuclear receptor superfamily that plays essential roles in endocrine development, steroidogenesis, and gonad differentiation. We investigated modifications that control SF-1 function and found that SF-1 could be conjugated by SUMO-1 both in vitro and in vivo. SF-1 was modified predominantly at Lys(194) and much less at Lys(119) when free SUMO-1 was supplied. Mutations of Lys(194) and Lys(119) enhanced transcriptional activity of SF-1, although the DNA binding activity of SF-1 was not affected. Sequences around Lys(194) and Lys(119) both repressed transcription intrinsically. The Lys(194) motif repressed transcription more efficiently than the Lys(119) domain, consistent with its ability to be a better substrate for SUMO conjugation. Thus, SUMO modification of SF-1 correlates with transcriptional repression. Wild-type but not conjugation-deficient SF-1 was localized at the nuclear speckles together with SUMO-1. Thus, SUMO-1 conjugation could also target SF-1 into nuclear speckles. Collectively, these results suggest that SUMO modification at the repression domains targets SF-1 to nuclear speckles; this could be an important mechanism by which SF-1 is regulated.

Animals↗
PubMed2004

Redundant mechanisms are used by Ssn6-Tup1 in repressing chromosomal gene transcription in Saccharomyces cerevisiae.

The Ssn6-Tup1 corepressor complex regulates many genes in Saccharomyces cerevisiae. Three mechanisms have been proposed to explain its repression functions: 1) nucleosome positioning by binding histone tails; 2) recruitment of histone deacetylases; and 3) direct interference with the general transcription machinery or activators. It is unclear if Ssn6-Tup1 utilizes each of these mechanisms at a single gene in a redundant manner or each individually at different loci. A systematic analysis of the contribution of each mechanism at a native promoter has not been reported. Here we employed a genetic strategy to analyze the contributions of nucleosome positioning, histone deacetylation, and Mediator interference in the repression of chromosomal Tup1 target genes in vivo. We exploited the fact that Ssn6-Tup1 requires the ISW2 chromatin remodeling complex to establish nucleosome positioning in vivo to disrupt chromatin structure without affecting other Tup1 repression functions. Deleting ISW2, the histone deacetylase gene HDA1, or genes encoding Mediator subunits individually caused slight or no derepression of RNR3 and HUG1. However, when Mediator mutations were combined with Deltaisw2 or Deltahda1 mutations, enhanced transcription was observed, and the strongest level of derepression was observed in triple Deltaisw2/Deltahda1/Mediator mutants. The increased transcription in the mutants was not due to the loss of Tup1 at the promoter and correlated with increased TBP cross-linking to promoters. Thus, Tup1 utilizes multiple redundant mechanisms to repress transcription of native genes, which may be important for it to act as a global corepressor at a wide variety of promoters.

Acetylation↗
PubMed2004

Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase.

The opposing actions of glucagon and insulin on glucose metabolism within the liver are essential mechanisms for maintaining plasma glucose concentrations within narrow limits. Less well studied are the counterregulatory actions of glucagon on protein metabolism. In the present study, the effect of glucagon on amino acid-induced signaling through the mammalian target of rapamycin (mTOR), an important controller of the mRNA binding step in translation initiation, was examined using the perfused rat liver as an experimental model. The results show that amino acids enhance signaling through mTOR resulting in phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP)1, the 70-kDa ribosomal protein (rp)S6 kinase, S6K1, and rpS6. In contrast, glucagon repressed both basal and amino acid-induced signaling through mTOR, as assessed by changes in the phosphorylation of 4E-BP1 and S6K1. The repression was associated with the activation of protein kinase A and enhanced phosphorylation of LKB1 and the AMP-activated protein kinase (AMPK). Surprisingly, the phosphorylation of two S6K1 substrates, rpS6 and eukaryotic initiation factor 4B, was not repressed but instead was increased by glucagon treatment, regardless of the amino acid concentration. The latter finding could be explained by the glucagon-induced phosphorylation of the ERK1 and the 90-kDa rpS6 kinase p90(rsk). Thus, glucagon represses phosphorylation of 4E-BP1 and S6K1 through the activation of a protein kinase A-LKB-AMPK-mTOR signaling pathway, while simultaneously enhancing phosphorylation of other downstream effectors of mTOR through the activation of the extracellular signal-regulated protein kinase 1-p90(rsk) signaling pathway. Amino acids also enhance AMPK phosphorylation, although to a lesser extent than glucagon and amino acids combined.

AMP-Activated Protein Kinase Kinases↗
PubMed2005

Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

Animals↗