The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Hepatitis C Virus Nonstructural 5A Protein Inhibits Lipopolysaccharide-Mediated Apoptosis of Hepatocytes by Decreasing Expression of Toll-Like Receptor 4.

Background. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis. The aim of this study was to assess the effects of HCV NS5A on apoptosis induced by Toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Methods. Apoptotic responses to TLR4 ligands and the expression of molecules involved in TLR signaling pathways in human hepatocytes were examined with or without expression of HCV NS5A. Results. HCV NS5A protected HepG2 hepatocytes against LPS-induced apoptosis, an effect linked to reduced TLR4 expression. A similar downregulation of TLR4 expression was observed in Huh-7-expressing genotype 1b and 2a. In agreement with these findings, NS5A inhibited the expression of numerous genes encoding for molecules involved in TLR4 signaling, such as CD14, MD-2, myeloid differentiation primary response gene 88, interferon regulatory factor 3, and nuclear factor-κB2. Consistent with a conferred prosurvival advantage, NS5A diminished the poly(adenosine diphosphate-ribose) polymerase cleavage and the activation of caspases 3, 7, 8, and 9 and increased the expression of anti-apoptotic molecules Bcl-2 and c-FLIP. Conclusions. HCV NS5A downregulates TLR4 signaling and LPS-induced apoptotic pathways in human hepatocytes, suggesting that disruption of TLR4-mediated apoptosis may play a role in the pathogenesis of HCV infection.

Peroxisome proliferator-activated receptor (PPAR)-beta as a target for wound healing drugs: what is possible?

Peroxisome proliferator-activated receptor (PPAR) dysfunction has been implicated in the manifestation of many diseases and illnesses, ranging from obesity to cancer. Herein, we discuss the role of PPARbeta, one of the three PPAR isotypes, during wound healing. While PPARbeta expression is undetectable in unchallenged and healthy adult interfollicular mouse skin, it is robustly re-activated in stress situations, such as upon phorbol ester treatment, hair plucking and cutaneous wounding. The inflammatory reaction associated with a skin injury activates the keratinocytes at the edges of the wound. This activation involves PPARbeta, whose expression and activity as transcription factor are up-regulated by pro-inflammatory signals. The re-activation of PPARbeta influences three important properties of the activated keratinocytes that are vital for rapid wound closure, namely, survival, migration and differentiation. The anti-apoptotic and, thus, survival role of PPARbeta is mediated by the up-regulation of expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1. Both kinases are required for the full activation of the Akt1 survival cascade. Therefore, the up-regulation of PPARbeta, early after injury, appears to be important to maintain a sufficient number of viable keratinocytes at the wound edge. At a later stage of wound repair, the stimulation of keratinocyte migration and differentiation by PPARbeta is also likely to be important for the formation of a new epidermis at the wounded area. Consistent with these observations, the entire wound healing process is delayed in PPARbeta +/- mice and wound closure is retarded by 2-3 days. The multiple roles of PPARbeta in the complex keratinocyte response after injury and during skin repair certainly justify a further exploration of its potential as a target for wound healing drugs.

Antibody-Mediated Trapping of Helminth Larvae Requires CD11b and Fcγ Receptor I.

Infections with intestinal helminths severely impact on human and veterinary health, particularly through the damage that these large parasites inflict when migrating through host tissues. Host immunity often targets the motility of tissue-migrating helminth larvae, which ideally should be mimicked by anti-helminth vaccines. However, the mechanisms of larval trapping are still poorly defined. We have recently reported an important role for Abs in the rapid trapping of tissue-migrating larvae of the murine parasite Heligmosomoides polygyrus bakeri. Trapping was mediated by macrophages (MΦ) and involved complement, activating FcRs, and Arginase-1 (Arg1) activity. However, the receptors and Ab isotypes responsible for MΦ adherence and Arg1 induction remained unclear. Using an in vitro coculture assay of H. polygyrus bakeri larvae and bone marrow-derived MΦ, we now identify CD11b as the major complement receptor mediating MΦ adherence to the larval surface. However, larval immobilization was largely independent of CD11b and instead required the activating IgG receptor FcγRI (CD64) both in vitro and during challenge H. polygyrus bakeri infection in vivo. FcγRI signaling also contributed to the upregulation of MΦ Arg1 expression in vitro and in vivo. Finally, IgG2a/c was the major IgG subtype from early immune serum bound by FcγRI on the MΦ surface, and purified IgG2c could trigger larval immobilization and Arg1 expression in MΦ in vitro. Our findings reveal a novel role for IgG2a/c-FcγRI-driven MΦ activation in the efficient trapping of tissue-migrating helminth larvae and thus provide important mechanistic insights vital for anti-helminth vaccine development.

Selective expression of a dominant-negative form of peroxisome proliferator-activated receptor in keratinocytes leads to impaired epidermal healing.

Many nuclear hormone receptors are involved in the regulation of skin homeostasis. However, their role in the epithelial compartment of the skin in stress situations, such as skin healing, has not been addressed yet. The healing of a skin wound after an injury involves three major cell types: immune cells, which are recruited to the wound bed; dermal fibroblasts; and epidermal and hair follicle keratinocytes. Our previous studies have revealed important but nonredundant roles of PPARalpha and beta/delta in the reparation of the skin after a mechanical injury in the adult mouse. However, the mesenchymal or epithelial cellular compartment in which PPARalpha and beta/delta play a role could not be determined in the null mice used, which have a germ line PPAR gene invalidation. In the present work, the role of PPARalpha was studied in keratinocytes, using transgenic mice that express a PPARalpha mutant with dominant-negative (dn) activity specifically in keratinocytes. This dn PPARalpha lacks the last 13 C terminus amino acids, binds to a PPARalpha agonist, but is unable to release the nuclear receptor corepressor and to recruit the coactivator p300. When selectively expressed in keratinocytes of transgenic mice, dn PPARalphaDelta13 causes a delay in the healing of skin wounds, accompanied by an exacerbated inflammation. This phenotype, which is similar to that observed in PPARalpha null mice, strongly suggests that during skin healing, PPARalpha is required in keratinocytes rather than in other cell types.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

When genetic load does not correlate with phenotypic spectrum: lessons from the GnRH receptor (GNRHR).

CONTEXT: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. OBJECTIVE: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. SUBJECTS: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. RESULTS: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. CONCLUSIONS: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas.

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.

The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

Studies of the renal effects of angiotensin II receptor blockade: the confounding factor of acute water loading on the action of vasoactive systems.

The acute renal tubular effects of two pharmacologically distinct angiotensin II receptor antagonists have been evaluated in normotensive volunteers on various salt diets. In the first study, the renal response to a single oral dose of losartan (100 mg) was assessed in subjects on a low (50 mmol Na/d) and on a high (200 mmol Na/d) salt intake. In a second protocol, the renal effects of 50 mg irbesartan were investigated in subjects receiving a 100 mmol Na/d diet. Both angiotensin II antagonists induced a significant increase in urinary sodium excretion. With losartan, a modest, transient increase in urinary potassium and a significant increase in uric acid excretion were found. In contrast, no change in potassium and uric acid excretions were observed with irbesartan, suggesting that the effects of losartan on potassium and uric acid are due to the intrinsic pharmacologic properties of losartan rather than to the specific blockade of renal angiotensin II receptors. Assessment of segmental sodium reabsorption using lithium as a marker of proximal tubular reabsorption demonstrated a decreased distal reabsorption of sodium with both antagonists. A direct proximal tubular natriuretic effect of the angiotensin II antagonist could be demonstrated only with irbesartan. This apparent discrepancy allowed us to reveal the importance of acute water loading as a possible confounding factor in renal studies. The results of the present analysis show that acute water loading per se may enhance renal sodium excretion and hence modify the level of activity of the renin-angiotensin system expected from a given sodium diet. Since acute water loading is a common practice in clinical renal studies, this confounding factor should be taken into account when investigating the renal effects of vasoactive systems.