Activation of the phosphoenolpyruvate carboxykinase gene retinoic acid response element is dependent on a retinoic acid receptor/coregulator complex.

The accessory factor 1 (AF1) element is an upstream transcriptional control region that plays a role in the response of the phosphoenolpyruvate carboxykinase (PEPCK) gene to both glucocorticoids and retinoic acid. We demonstrate here that retinoic acid receptor alpha (RAR alpha) binds to a sequence within the AF1 element, TGACCT (site B), that is a consensus retinoic acid response element (RARE) half-site. A similar DNA sequence, TGGCCG (site C), located 1 bp downstream of site B, is not involved in the binding of RAR alpha monomers or dimers but is required for the constitution of a functional RARE. Site C is also required for the formation of a complex involving RAR alpha and a liver nuclear factor designated CR, for coregulator. Mutational analysis of the AF1 element shows that the RAR alpha/CR complex is the trans-acting unit that mediates the retinoic acid response of the PEPCK gene. Another member of the retinoid receptor family, retinoid X receptor alpha (RXR alpha), can also form a complex with RAR alpha and the AF1 element. Several observations, including the observation that RXR alpha antibody interacts with CR, indicate that RXR alpha and CR are identical or closely related proteins. Through RXR alpha forms a complex with RAR alpha and the AF1 element, we demonstrate that the AF1 element is functionally distinguishable from a retinoid X response element. Taken together, our results show that the AF1 element contains an RARE that mediates a retinoic acid response by binding an RAR alpha/coregulator complex; this coregulator is presumably RXR alpha.

Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes.

The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.

Ontogeny of dopaminergic function in the rat midbrain tegmentum, corpus striatum and frontal cortex.

Ontogenic development of the dopaminergic system in rat brain was investigated. This was accomplished by monitoring changes in postsynaptic dopamine receptor formation and presynaptic dopamine content in the midbrain tegmentum, frontal cortex and corpus striatum from the 18th day of gestation through adulthood. The dopamine antagonist spiperone was used as the binding ligand to quantitate receptor number while dopamine content was measured chromatographically. [3H]Spiperone binding kinetics in adult animals revealed that the maximum number of receptor sites (Bmax) was 160, 900 and 597 fmol/mg protein in midbrain tegmentum, frontal cortex and corpus striatum, respectively, while the corresponding equilibrium constant (Kd) values were 0.15, 0.52 and 0.15 nM. During the course of development, the affinity for spiperone binding in corpus striatum and frontal cortex did not change significantly, while in midbrain tegmentum the binding affinity in younger animals was significantly lower. Results from competitive inhibition experiments using various serotonergic and dopaminergic antagonists suggested that at all ages dopamine D2-receptors were responsible for spiperone binding in corpus striatum and midbrain tegmentum. In frontal cortex, binding properties consistent with D2-receptors were observed in non-adult animals; by the time adulthood was reached, however, spiperone binding characteristics were altered and appeared to correspond to serotonin sites. The developmental patterns of the dopaminergic markers were different in all 3 tissues. Adult receptor levels were achieved very early in midbrain tegmentum, while increases in receptor number continued in corpus striatum and frontal cortex, at different rates, throughout the postnatal period. A marked increase in dopamine in corpus striatum occurred during the second and third postnatal weeks and the transmitter content remained relatively constant after this time. Transient fluctuations in endogenous dopamine during the postnatal period were observed in midbrain tegmentum and frontal cortex. A general feature of the ontogenic pattern in all tissues appeared to be increases in dopamine receptor preceding increases in dopamine synthesis. A hypothesis on the developmental regulation of dopamine neurons was derived.

Choline uptake by glomerular synapses isolated from bovine cerebellar vermis.

[3H]Choline uptake was investigated in a highly enriched preparation of glomerular particles isolated from bovine cerebellar vermis. Kinetic analysis indicates that a high-affinity choline uptake system with a relatively low maximum uptake velocity is present. At a substrate concentration of 10(-7) M, [3H]choline uptake was shown to be sodium-dependent, hemicholinium-sensitive and non-reactive to increasing concentrations of unlabeled choline. These findings support the presence of a high-affinity choline uptake system in cerebellar glomeruli and would seem to be consistent with the suggestion that a small proportion of mossy fibers are cholinergic in this brain region.

Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes.

Trypanosoma cruzi amastigotes present receptors for human fibronectin as indicated by the saturable binding of [125I]fibronectin to this form of the parasite. Scatchard analysis indicates that the number of fibronectin receptors per amastigote was 1.3 x 10(3) with a Kd of approximately 2.3 nM. Addition of physiological concentrations of fibronectin to amastigote-macrophage cocultures significantly increased the binding of amastigotes to murine macrophages. This increase was evidenced in both the number of amastigotes bound to macrophages and the percentage of macrophages containing bound amastigotes. The uptake of amastigotes by either murine macrophages or human blood monocytes was also increased in the presence of exogenous fibronectin. The increase induced by fibronectin was blocked when amastigotes were pretreated with the RGDS tetrapeptide of the fibronectin cell attachment site. Furthermore, the ability of fibronectin to enhance amastigote binding to and uptake by macrophages was inhibited by the F(ab')2 fragment of anti-fibronectin immunoglobulin G (IgG) but not by an irrelevant anti-human IgG F(ab')2 fragment. Pretreatment of either amastigotes or macrophages with fibronectin also resulted in a significant increase in amastigote binding to and uptake by macrophages. These results suggest that fibronectin may play a role in facilitating the attachment and ingestion of T. cruzi amastigotes by macrophages and monocytes in chagasic tissue lesions.

Comparison of the effects of insulin and okadaic acid on phosphoenolpyruvate carboxykinase gene expression.

Many hormones regulate the rate of synthesis of phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that governs the rate-limiting step in gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retinoic acid and cyclic AMP (cAMP) increase PEPCK gene transcription whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant as they prevent cAMP- and glucocorticoid-stimulated PEPCK gene transcription. In contrast, insulin and phorbol esters both stimulate transcription of gene 33 in the same H4IIE cells, with the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibitor, okadaic acid, mimics the action of insulin and phorbol esters on expression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glucocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acetyltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and glucocorticoid-stimulated CAT expression. Desensitization of the protein kinase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not markedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadaic acid appear to repress PEPCK gene expression through a pathway initially distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, insulin and phorbol ester is the same DNA element.

Potential convergence of insulin and cAMP signal transduction systems at the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter through CCAAT/enhancer binding protein (C/EBP).

Adenosine 3',5'-monophosphate (cAMP) stimulates phosphoenolpyruvate carboxykinase (PEPCK) gene transcription, whereas insulin has the opposite effect. In H4IIE cells, the effect of insulin is dominant since it represses cAMP-stimulated transcription. Discrete cis-acting elements in the PEPCK promoter that serve as an insulin response sequence (IRS) and cAMP response element (CRE) have been identified. Here we show that common proteins can bind both elements, since: (i) an almost identical pattern of protein binding is seen when oligonucleotides representing either the IRS or the CRE are used as the labeled probe in a gel retardation assay and (ii) the unlabeled wild-type, but not mutated, CRE oligonucleotide competes for protein binding to the labeled IRS probe, and vice versa. Six homo- and heterodimer complexes interact with these DNA elements; the complexes are composed of three individual protein species: (a) 42-kDa C/EBP alpha, (b) 30-kDa C/EBP alpha, and (c) an unidentified 20-kDa factor termed p20- CRE/IRS Binding Protein (p20-C/IBP). These proteins have a 30-fold greater affinity for the CRE at room temperature, a difference explained by the rapid dissociation rate of protein bound to the IRS, since the association rate of protein binding to both the IRS and CRE is the same. Protease digestion experiments suggest that the proteins bind to the CRE and IRS in different conformations. The IRS and CRE both function in the context of a heterologous promoter to mediate effects of insulin and cAMP, respectively, but, although the PEPCK IRS and CRE bind common proteins, the PEPCK CRE is not a functional IRS and the PEPCK IRS is not a functional CRE.