Regulation of transcription by AMP-activated protein kinase: phosphorylation of p300 blocks its interaction with nuclear receptors.

AMP-activated protein kinase (AMP-kinase) modulates many metabolic processes in response to fluctuations in cellular energy status. Although most of its known targets are metabolic enzymes, it has been proposed that AMP-kinase might also regulate gene expression. Here we demonstrate that the transcriptional coactivator p300 is a substrate of AMP-kinase. Phosphorylation of p300 at serine 89 by AMP-kinase dramatically reduced its interaction, in vitro and in vivo, with the nuclear receptors peroxisome proliferator-activated receptor gamma, thyroid receptor, retinoic acid receptor, and retinoid X receptor, but did not affect its interaction with the non-nuclear receptor transcription factors E1a, p53, or GATA4. These findings indicate that the AMP-kinase signaling pathway selectively modulates a subset of p300 activities and represent the first example of a transcriptional component regulated by AMP-kinase. Our results suggest a direct link between cellular energy metabolism and gene expression.

A novel potent antagonist of peroxisome proliferator-activated receptor gamma blocks adipocyte differentiation but does not revert the phenotype of terminally differentiated adipocytes.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Their antihyperglycemic effects seem to be linked to the regulation of PPARgamma-responsive genes. Here, we report the characterization of a specific PPARgamma antagonist that blocks several of the biological activities of the PPARgamma agonist rosiglitazone. PD068235 inhibited rosiglitazone-dependent PPARgamma transcriptional activity with an IC(50) of 0.8 microM and rosiglitazone-stimulated in vitro coactivator association. The role of PPARgamma in the initiation of differentiation is well documented. In this study, we used PD068235 as a tool to evaluate the functional role of PPARgamma in the maintenance of the terminally differentiated state. Treatment of confluent, growth-arrested 3T3-L1 preadipocytes with PD068235 blocked adipocyte differentiation induced by the standard adipogenic hormonal mixture (insulin/dexamethasone/isobutylmethylxanthin) and fully antagonized rosiglitazone-induced adipogenesis. In contrast, long-term treatment of terminally differentiated 3T3-L1 adipocytes with PD068235 did not induce any obvious morphological changes and had no effect on basal lipolysis rates. In addition, in fully differentiated adipocytes PD068235 did not alter the basal expression of PPARgamma target genes aP2 and CAP, but it effectively blocked rosiglitazone-induced expression of both genes. These results suggest that in terminally differentiated adipocytes, the PPARgamma activity is minimal and may not be required for the maintenance of PPARgamma target gene expression.

Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma and exert their antihyperglycemic effects by regulation of PPAR-gamma-responsive genes. We report here that PPAR-gamma activation by troglitazone depends on the experimental setting. Troglitazone acts as a partial agonist for PPAR-gamma in transfected muscle (C2C12) and kidney (HEK 293T) cells, producing a submaximal transcriptional response (1.8- to 2.5-fold activation) compared with rosiglitazone (7.4- to 13-fold activation). Additionally, troglitazone antagonizes rosiglitazone-stimulated PPAR-gamma transcriptional activity. Limited protease digestion of PPAR-gamma suggests conformational differences in the receptor bound to troglitazone versus rosiglitazone. Consistent with this finding, an in vitro coactivator association assay demonstrated that troglitazone-bound PPAR-gamma recruited the transcriptional coactivators p300 and steroid receptor coactivator 1 less efficiently than rosiglitazone-bound receptor. In contrast to these observations, troglitazone behaves as a full agonist of PPAR-gamma in 3T3L1 adipocytes. Two-dimensional protein gel electrophoresis demonstrated that troglitazone and rosiglitazone regulated distinct but overlapping sets of genes in several cell types. Thus, troglitazone may behave as a partial agonist under certain physiological circumstances and as a full agonist in others. These differences could be caused by variations in the amount of specific cofactors, differences in PPAR response elements, or the presence of different isoforms of PPAR-gamma.

PPARgamma activators down-regulate the expression of PPARgamma in 3T3-L1 adipocytes.

Transcriptional activation of PPARgamma by the anti-diabetic compound troglitazone enhances the rate of 3T3-L1 adipocyte differentiation. In this study, we examined the effects of troglitazone, a specific PPARgamma ligand, on the expression of PPARgamma during and after 3T3-L1 adipocyte differentiation. Troglitazone treatment caused a significant decrease in PPARgamma proteins and DNA binding activity. This reduction was associated with a similar decrease in transcription of PPARgamma mRNA. These data suggest that in 3T3-L1 cells, the expression of PPARgamma is auto-regulated.

c-Jun N-terminal kinase phosphorylates peroxisome proliferator-activated receptor-gamma1 and negatively regulates its transcriptional activity.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) transcription factor plays a pivotal role in adipocyte differentiation and metabolic regulation. The transcriptional activity of PPARgamma is positively modulated by ligand binding and negatively regulated by phosphorylation mediated by the MEK/ERK signaling pathway. The phosphorylation of mouse PPARgamma1 at Ser82 by ERK causes a decrease in both basal and ligand-dependent transcriptional activity. In this report we examined the ability of other mitogen-activated protein kinase family members to phosphorylate PPARgamma1. We demonstrate that in vitro, PPARgamma1 is efficiently phosphorylated by JNK/SAPK (c-Jun N-terminal kinase or stress-activated protein kinase) but only weakly phosphorylated by p38. In transfected 293T cells, PPARgamma1 is phosphorylated at Ser82 in response to known JNK activators such as UV irradiation and anisomycin treatment. This phosphorylation is not blocked by either the specific MEK inhibitor PD98059 or the p38 inhibitor SB203580, indicating that it is independent of the MEK/ERK and p38 signaling pathways. Finally, in transient transfection reporter assays, activation of JNK by anisomycin or by overexpression of MKK4 (the upstream JNK kinase) decreased ligand-dependent PPARgamma1 transcriptional activity. These results suggest that the activation of the JNK/SAPK pathway by extracellular signals, perhaps by inflammatory cytokines such as tumor necrosis factor-alpha, would result in a reduction of PPARgamma transcriptional activity and reduce the effects of PPARgamma ligands.

Thiazolidinediones and insulin resistance: peroxisome proliferatoractivated receptor gamma activation stimulates expression of the CAP gene.

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor gamma (PPARgamma). The stimulation of CAP expression by PPARgamma activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARgamma-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.

Regulation of peroxisome proliferator-activated receptor gamma activity by mitogen-activated protein kinase.

Adipocyte differentiation is regulated both positively and negatively by external growth factors such as insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). A key component of the adipocyte differentiation process is PPARgamma, peroxisomal proliferator-activated receptor gamma. To determine the relationship between PPARgamma activation and growth factor stimulation in adipogenesis, we investigated the effects of PDGF and EGF on PPARgamma1 activity. PDGF treatment decreased ligand-activated PPARgamma1 transcriptional activity in a transient reporter assay. In vivo [32P]orthophosphate labeling experiments demonstrated that PPARgamma1 is a phosphoprotein that undergoes EGF-stimulated MEK/mitogen-activated protein (MAP) kinase-dependent phosphorylation. Purified PPARgamma1 protein was phosphorylated in vitro by recombinant activated MAP kinase. Examination of the PPARgamma1 sequence revealed a single MAP kinase consensus recognition site at Ser82. Mutation of Ser82 to Ala inhibited both in vitro and in vivo phosphorylation and growth factor-mediated transcriptional repression. Therefore, phosphorylation of PPARgamma1 by MAP kinase contributes to the reduction of PPARgamma1 transcriptional activity by growth factor treatment.

Modulation of angiotensin II receptor (AT2) mRNA levels in R3T3 cells.

R3T3 cells, a mouse fibroblast cell line, express the type 2 angiotensin II receptor (AT2), but not the AT1 subtype. We previously reported that expression of AT2 sites in these cells were regulated by various conditions: 1. The number of AT2 sites increased considerably when cells were contact-inhibited; 2. Stimulation of R3T3 cells with various mitogens caused a rapid decline of AT2 binding sites; and 3. Stimulation of cells with angiotensin ligands resulted in upregulation of the AT2 sites. In this study, to determine if altered AT2 expression is under transcriptional, posttranscriptional, or translational control, we examined the level of AT2 mRNA in R3T3 cells in response to various treatments. There was a 200-fold increase in AT2 mRNA levels in quiescent cells as compared to growing cells. Results from nuclear run-on assays suggested that the differences in AT2 mRNA levels were primarily caused by changes in the rate of AT2 gene transcription. Stimulation of cells with fibroblast growth factor caused an approximate threefold reduction of AT2 mRNA levels, and also increased the rate of degradation of AT2 mRNA, which correlated with the decrease in AT2 binding activity seen under these conditions. However, whereas treatment with angiotensin ligands increased AT2 binding activity, the level of AT2 transcripts did not increase. This pattern of expression implies that regulation of AT2 receptors occurs at multiple levels, involving translational and/or posttranslational as well as transcriptional control, and further affords the cell the ability to rapidly modulate the number of AT2 binding sites in response to changing extracellular conditions.

Defective Marek's disease virus DNA contains a gene encoding a potential nuclear DNA binding protein and a HSV a-like sequence.

Four RNA transcripts from chicken embryo fibroblast cells infected with Marek's disease virus (MDV) strain 281Ml/1 hybridized to the 4-kbp MDV replicon DNA. In an attempt to identify open reading frames coding for the four transcripts, we determined the nucleotide sequences of 4-kbp replicon DNA (represents a single monomeric repeat unit of defective MDV genome). Computer analysis indicates that the 4-kbp MDV replicon DNA contains two intact open reading frames (ORFs) with common promoter regulatory elements. ORF-A codes for a putative 204 amino acid protein that shares 21 and 36% amino acid sequence identity to nuclear DNA binding proteins such as the EBNA-1 of Epstein-Barr virus and galline, a chicken sperm histone protein, respectively. ORF-B encodes for a potential 350 amino acid protein, which did not show any significant amino acid sequence identity to known protein sequences within Swiss-Protein data base. ORF-B may, therefore, encode a MDV specific protein. The 5'-region of MDV replicon DNA revealed seven reiterated copies of an 11-bp motif sharing 8 out of 11 nucleotide sequence identity to DR2 elements of the herpes simplex virus strain USA-8 a sequence.

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.