Walter Wahli

Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

ABSTRACT Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

PPAR: a Key Nuclear Factor in Nutrient / Gene Interactions?

Retinoids as well as steroid and thyroid hormones are small lipophilic molecules that exert an intricate array of combinatorial effects during embryogenesis, cellular differentiation, and homeostasis in the adult organism. Complexity in the signalling pathway of these hormones results from the functional association of low affinity cytoplasmic hormone binding proteins and high affinity nuclear hormone receptors. The latter interact with polymorphic response elements linked to target genes and mediate the hormonal response at the transcriptional level.

Cloning and characterization of synthetic sequences from the Xenopus laevis vitellogenin structural gene

Abstract The mRNA coding for vitellogenin, the yolk protein precursor, has been isolated from the liver of estrogen-stimulated Xenopus laevis. The mRNA has a size of 6.3 kilobases (kb). Optimal conditions were investigated for the synthesis of long complementary DNA (cDNA, referring to DNA synthesized in vitro) copies of the mRNA. Temperature, salt concentration, and enzyme-to-RNA ratio were important factors. Double-stranded cDNA with an average size of 2 to 3 kb was inserted into the vector pMB9 by the poly(dA:dT) method, and the recombinant plasmids were amplified in E. coli. Twenty-one clones with vitellogenin inserts ranging from 1 to 3.7 kb were studied. The regions in the RNA from which these clones had been derived were mapped by R-loop analysis in the electron microscope and by hybridization of the cloned DNAs with specific fractions of mRNA. Slightly more than half of the clones were derived from the 3′-terminal portions of the mRNA while the remaining clones are located internally.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor α (PPARα) of the mouse GRHPR gene in liver. Mice fed with a PPARα ligand or in which PPARα activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARα present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARα ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARα-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARα regulation. Overall, these data indicate a species-specific regulation by PPARα of GRHPR, a key gene of the glyoxylate cycle.

Retinoid X receptor β and peroxisome proliferator-activated receptor activate an estrogen response element

Publisher Summary This chapter discusses a study suggesting that an estrogen-responsive DNA element (ERE) can be specifically activated by cotransfection of the retinoid X receptor (RXR) and peroxisome proliferator-activated receptors (PPAR) in the absence of the estrogen receptor (ER). The specific nucleotide sequence of the ERE differs from the canonical RXR and PPAR element in the orientation and number of nucleotide spaces between half-sites of the element. The cotransfection of RXR and PPAR caused an augmentation of ERE-dependent reporter activity in the presence or absence of either 9-cis-RA or arachidonic acid. Transfection of either RXR or PPAR alone resulted in activation less than that observed when both expression vectors were introduced. The capacity of a single coregulator (RXR) to promote a discriminatory function in DNA site targeting suggested that binding site specificity is conferred both by RXR and the respective nuclear receptor partner. The activation of the ERE-dependent reporter gene by PPAR depends on the RXR as deletion of the DBD of RXR reduces the observed reporter activity. The finding of the experiment explained in the chapter that PPAR and RXR activate an estrogen-responsive element suggests that the two receptors are involved in a mechanism to generate diversity and tissue-specific regulation of estrogen-responsive genes. The concept that receptors other than the ER control estrogen responsive genes is supported by the presence of natural EREs such as in the rat oxytocin promoter, where the ER, TR, RAR and a COUP-like factor have been suggested to control expression of the gene.

Factors promoting the establishment of primary cultures of liver cells fromXenopus larvae

SummaryThe requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated.Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adultXenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165–220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50–60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from theXenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation.In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate.

Peroxisome Proliferator Activated Receptor Alpha Coordinates Intermediary Metabolism During Fasting

In response to repeated and long-lasting food shortages during evolution, humans have evolved with an intricate metabolic control system that allows them to survive prolonged period of food deprivation. One hallmark of this adaptive system is the ability to store large amounts of energy in the form of fat in times of plenty and mobilize this energy under conditions of food shortage such as fasting.