John P. Capone

A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1.

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.

Oxysterol Activators of Liver X Receptor and 9-cis-Retinoic Acid Promote Sequential Steps in the Synthesis and Secretion of Tumor Necrosis Factor-α from Human Monocytes

Liver X receptor α (LXRα), is a nuclear hormone receptor that is activated by oxysterols and plays a crucial role in regulating cholesterol and lipid metabolism in liver and cholesterol efflux from lipid-loaded macrophages. Here we show that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22(R)-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α induction by 22(R)-HC and 9cRA occurs by a novel two-step process. The initial step involves 22(R)-HC-dependent induction of TNF-α mRNA, and intracellular accumulation of TNF-α protein, mediated by binding of LXRα/RXRα to an LXR response element at position −879 of the TNF-α promoter. Subsequent cell release of TNF-α protein occurs via a separable 9cRA-dependent, LXRα-independent step that requires de novo transcription and protein synthesis. Our findings reveal a potentially new dimension of the physiological role of LXRα and identify a unique multistep pathway of TNF-α production that may be of consequence to the normal function of LXR in monocyte/macrophages and in disease conditions such as atherosclerosis.

A mitochondrial ketogenic enzyme regulates its gene expression by association with the nuclear hormone receptor PPARα

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHMG‐CoAS) is a key enzyme in ketogenesis, catalyzing the condensation of acetyl‐CoA and acetoacetyl‐CoA to generate HMG‐CoA, which is eventually converted to ketone bodies. Transcription of the nuclear‐encoded gene for mHMG‐CoAS is stimulated by peroxisome proliferator‐activated receptor (PPAR) α, a fatty acid‐activated nuclear hormone receptor. Here we show that the mHMG‐CoAS protein physically interacts with PPARα in vitro, and potentiates PPARα‐dependent transcriptional activation via the cognate PPAR response element of the mHMG‐CoAS gene in vivo. Immunofluorescence of transiently transfected cells demonstrated that in the presence of PPARα, mHMG‐CoAS is translocated into the nucleus. Binding to PPARα, stimulation of PPARα activity and nuclear penetration require the integrity of the sequence LXXLL in mHMG‐CoAS, a motif known to mediate the interaction between nuclear hormone receptors and coactivators. These findings reveal a novel mechanism of gene regulation whereby the product of a PPARα‐responsive gene, normally resident in the mitochondria, directly interacts with this nuclear hormone receptor to autoregulate its own nuclear transcription.

Crosstalk between the thyroid hormone and peroxisome proliferator-activated receptors in regulating peroxisome proliferator-responsive genes

Peroxisome proliferators and thyroid hormones have overlapping metabolic effects and regulate a similar subset of genes involved in maintaining lipid homeostasis. Transcriptional activation by peroxisome proliferators is mediated by peroxisome proliferator-activated receptors (PPARs) that bind to specific peroxisome proliferator-response elements (PPREs) through heterodimerization with retinoid X receptors (RXRs). We examined the effect of thyroid hormone receptor alpha (TR alpha) on DNA binding in vitro and transcriptional activation in vivo by rat PPAR. Gel mobility shift assays using in vitro translated receptors demonstrated that TR alpha was capable of binding on its own and cooperatively with RXR alpha to the rat acyl-CoA oxidase PPRE and of inhibiting the binding of rat PPAR/RXR alpha heterodimers to this element. This inhibition was the result of competition between TR alpha and PPAR for limiting amounts of the heterodimerization partner RXR alpha and for binding to the PPRE. Interestingly, cotransfection of a TR alpha expression plasmid into mammalian cells resulted in potentiation of the peroxisome proliferator- and PPAR/RXR alpha-dependent transcriptional induction of a reporter gene containing the acyl-CoA oxidase PPRE. TR alpha therefore appears to cooperate with RXR and PPAR to positively modulate peroxisome proliferator-dependent transactivation in vivo. Our findings suggest that there is crosstalk between the thyroid hormone and peroxisome proliferator signaling pathways in the regulation of peroxisome proliferator-responsive genes.

The peroxisome proliferator-activated receptor interacts with the retinoid X receptor in vivo.

The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR). We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo. Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene. Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha. Homodimeric interaction by mPPAR was not detected. These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.

Receptor-interacting protein 140 interacts with and inhibits transactivation by, peroxisome proliferator-activated receptor α and liver-X-receptor α

Abstract Receptor interacting protein 140 (RIP140), a previously identified putative ligand-dependent coactivator of nuclear hormone receptors, was isolated by yeast two-hybrid cloning as a factor that interacts with peroxisome proliferator-activated receptor α (PPAR α ). This interaction in yeast required the integrity of the carboxyl-terminal, ligand-dependent activation domain of PPAR α . However, protein binding studies carried out in vitro showed that full-length RIP140 bound efficiently to PPAR α in the absence of exogenously added ligand. RIP140 also bound strongly to the liver-X-receptor (LXR α ) in the absence of an activator for this receptor. In contrast, a strong interaction of RIP140 with the PPAR α and LXR α heterodimerization partner retinoid-X-receptor α (RXR α ) required the presence of its cognate ligand, 9- cis retinoic acid. Transfection analysis in mammalian cells demonstrated that RIP140 antagonized PPAR α /RXR α - and LXR α /RXR α -mediated signaling. Our findings identify RIP140 as a novel modulator of transcriptional activation mediated by PPAR α and LXR α and indicate that RIP140 can also bind to nuclear hormone receptors in a ligand-independent manner and repress their activity.

The short heterodimer partner receptor differentially modulates peroxisome proliferator-activated receptor alpha-mediated transcription from the peroxisome proliferator-response elements of the genes encoding the peroxisomal beta-oxidation enzymes acyl-CoA oxidase and hydratase-dehydrogenase.

The promoter regions of the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain transcriptional regulatory sequences termed peroxisome proliferator-response elements (PPRE) that are bound by the peroxisome proliferator-activated receptor alpha (PPARalpha) and 9-cis-retinoic acid receptor (RXRalpha) heterodimeric complex. In this study, the role of the short heterodimer partner (SHP) receptor in modulating PPARalpha-mediated gene transcription from the PPREs of the genes encoding AOx and HD was investigated both in vitro and in vivo. In vitro binding assays using glutathione-S-transferase-tagged chimeric receptors for PPARalpha and SHP were used to verify the interaction between PPARalpha and SHP. This interaction was unaffected by the presence of the peroxisome proliferator, Wy-14,643. SHP has been proposed to act as a negative regulator of nuclear hormone receptor activity, and SHP inhibited transcription by PPARalpha/RXRalpha heterodimers from the AOx-PPRE. Surprisingly, SHP potentiated transcription by PPARalpha/RXRalpha heterodimers from the HD-PPRE. This is the first demonstration of positive transcriptional activity attributable to SHP. Together, these results suggest that SHP can modulate PPARalpha/RXRalpha-mediated transcription in a response element-specific manner.

A p56lck Ligand Serves as a Coactivator of an Orphan Nuclear Hormone Receptor

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily, acts as a transcriptional repressor by antagonizing the functions of other nuclear hormone receptors and by actively silencing transcription. However, in certain contexts, COUP-TFII stimulates transcription directly. A cellular factor, isolated by interaction cloning, bound COUP-TFII in vitro and allowed COUP-TFII to function as a transcriptional activator in mammalian cells. This factor is identical to a recently described ligand of the tyrosine kinase signaling molecule p56lck, suggesting that it mediates cross-talk between mitogenic and nuclear hormone receptor signal transduction pathways.

A PPARγ mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

Identification of COUP-TFII as a peroxisome proliferator response element binding factor using genetic selection in yeast: COUP-TFII activates transcription in yeast but anatagonizes PPAR signaling in mammalian cells

Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function. We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs. Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library. Plasmids were isolated from his + transformants. One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily. COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE. Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR. Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.