Nathalie Gaspard

Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling.

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.

Pharmacological properties of Chinese hamster ovary cells coexpressing two vasoactive intestinal peptide receptors (hVPAC1 and hVPAC2)

1 In the light of recent findings that VPAC1 and VPAC2 receptors form homodimers and heterodimers, we have evaluated the function of these receptors coexpressed in the same cells, using whole‐cell and membrane preparations. Cells expressing each receptor alone were used for comparison. 2 The study was performed on Chinese hamster ovary cells stably transfected with both human recombinant receptors and we compared receptor occupancy and adenylate cyclase activation by VIP, Ro 25‐1553 – a VPAC2 selective agonist – and [K15,R16,L27]VIP(1‐7)/GRF(8‐27) – a VPAC1 selective agonist – on membranes prepared from each cell line and on a mixture of membranes from cells expressing each receptor individually. We also studied receptor internalization induced by the three agonists on intact cells expressing both receptors alone or together by fluorescence‐activated cell sorting using monoclonal antibodies and demonstrated by using co‐immunoprecipitation that the two receptors did interact. 3 The results indicated that coexpression of the receptors did not modify the recognition of ligands, nor the capacity of the agonists to stimulate adenylate cyclase activity and, in intact cells, to induce internalization of the receptors. 4 As a consequence, the properties of the selective ligands that were established on cell lines expressing a single population of VIP receptors were valid on cells expressing both receptors. Furthermore, the recently demonstrated VPAC1/VPAC2 receptor heterodimerization did not affect the function of either receptor.

Oleate Abrogates Palmitate-Induced Lipotoxicity and Proinflammatory Response in Human Bone Marrow-Derived Mesenchymal Stem Cells and Osteoblastic Cells.

Osteoporosis is a metabolic bone disease associated with unequilibrated bone remodeling resulting from decreased bone formation and/or increased bone resorption, leading to progressive bone loss. In osteoporotic patients, low bone mass is associated with an increase of bone marrow fat resulting from accumulation of adipocytes within the bone marrow. Marrow adipocytes are active secretory cells, releasing cytokines, adipokines and free fatty acids (FA) that influence the bone marrow microenvironment and alter the biology of neighboring cells. Therefore, we examined the effect of palmitate (Palm) and oleate (Ole), 2 highly prevalent FA in human organism and diet, on the function and survival of human mesenchymal stem cells (MSC) and MSC-derived osteoblastic cells. The saturated FA Palm exerted a cytotoxic action via initiation of endoplasmic reticulum stress and activation of the nuclear factor κB (NF-κB) and ERK pathways. In addition, Palm induced a proinflammatory response, as determined by the up-regulation of Toll-like receptor 4 expression as well as the increase of IL-6 and IL-8 expression and secretion. Moreover, we showed that MSC-derived osteoblastic cells were more sensitive to lipotoxicity than undifferentiated MSC. The monounsaturated FA Ole fully neutralized Palm-induced lipotoxicity by impairing activation of the pathways triggered by the saturated FA. Moreover, Ole promoted Palm detoxification by fostering its esterification into triglycerides and storage in lipid droplets. Altogether, our data showed that physiological concentrations of Palm and Ole differently modulated cell death and function in bone cells. We therefore propose that FA could influence skeletal health.

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells

Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC2 receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A11]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 microM monensin but not by 10 microg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 microM monensin and 10 microg/ml cycloheximide.

Perturbation of glycerol metabolism in hepatocytes from n3-PUFA-depleted rats

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹⁴C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹⁴C]glycerol uptake could be explained by an observed increase in glycerol kinase activity.

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors.

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligands binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.

Cell surface targeting of VPAC1 receptors: Evidence for implication of a quality control system and the proteasome

Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.

Osteonecrosis of the Femoral Head: Lipotoxicity Exacerbation in MSC and Modifications of the Bone Marrow Fluid

Osteonecrosis of the femoral head (ON) is a multifactorial bone disease that can evolve to a progressive destruction of the hip joint. Different pathogenic processes have been proposed, among them, an increase of bone marrow (BM) fat resulting from adipocyte accumulation. Marrow adipocytes are active BM residents that influence the microenvironment by releasing cytokines, adipokines, and free fatty acids (FA). We explored the impact of palmitate (Palm) and oleate on function and survival of BM-derived mesenchymal stromal cells (MSC) of osteonecrotic patients (ONMSC) and healthy volunteers. Moreover, we analyzed the FA profile of the serum and the BM supernatant fluid (BMSF). We demonstrated that exposure to the saturated FA Palm favored MSC differentiation through the adipogenic lineage at the expense of the osteoblastic phenotype. Moreover, adipogenesis was intensified in ONMSC. The susceptibility to Palm toxicity was aggravated in ONMSC concomitantly with a greater activation of the proapoptotic extracellular signal-regulated kinase pathway. Moreover, cellular mechanisms implicated in the protection against lipotoxicity, such as stearoyl-coenzyme A desaturase 1 and carnitine palmitoyl transferase 1 expression, were dysregulated in ONMSC. Palm-induced interleukin (IL)-6 and IL-8 secretion was also exacerbated in ONMSC. Our results established that, in the serum, the FA profiles were comparable in ON and healthy subjects. However, both the concentrations and the FA composition were modified in the BMSF of ON patients, highlighting a drastic change of the BM microenvironment in ON patients. Altogether, our work suggests that marrow adipocyte enlargement could affect the process of bone remodeling and, therefore, play a role in the pathogenesis of ON.

Glycerol Metabolism Alteration in Adipocytes from n3-PUFA-Depleted Rats, an Animal Model for Metabolic Syndrome

Aquaglyceroporin 7 (AQP7) is a glycerol transporter expressed in adipocytes. Its expression has been shown to be modulated in obesity. Metabolic syndrome is characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension. An animal model displaying several features of metabolic syndrome was used to study the AQP7 expression at both mRNA and protein level and glycerol flux in adipocytes. Second generation n3-PUFA depleted female rats is a good animal model for metabolic syndrome as it displays characteristic features such as liver steatosis, visceral obesity, and insulin resistance. Our data show a reduced expression of AQP7 at the protein level in adipose tissue from n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-(14)C]-Glycerol uptake was not modified in adipocytes from n3-PUFA-depleted animals.