Maria Andries

Structural Basis of Substrate Discrimination and Integrin Binding by Autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B–like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells

Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.

Separated anterior pituitary cells and their response to hypophysiotropic hormones

Publisher Summary This chapter describes the separated anterior pituitary cells and discusses their response to hypophysiotropic hormones. The mammalian anterior pituitary gland is a neuroendocrine tissue composed of many different cell types, each producing different peptide hormones. The unit gravity sedimentation technique, particularly when different animal models are used, produces subpopulations of pituitary cells in which receptors, stimulus-receptor coupling, and other mechanisms of action of the hypophysiotropic hormones can be reliably studied than in conventional in vitro systems. Freshly separated cells remain functional. They incorporate radioactive amino acids into protein in a linear fashion as a function of time. Pituitary hormone secretion fluctuates under different physiological and experimental conditions as well as during development. The hypothalamic regulatory peptides (releasing and inhibiting factors) and amines (dopamine), and the peripheral hormones feed back into the hypothalamo-pituitary system and are responsible for these changes in pituitary hormone release.

Ivermectin inhibits AMPA receptor-mediated excitotoxicity in cultured motor neurons and extends the life span of a transgenic mouse model of amyotrophic lateral sclerosis.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X4 modulators did not influence the number or Ca2+ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X4 receptor.

Gonadotropin-releasing hormone influences the release of prolactin and growth hormone from intact rat pituitary in vitro during a limited period in neonatal life.

Freshly isolated pituitaries from female rats between the day of birth and 14 days were challenged with GnRH in a perifusion system. At the day of birth GnRH significantly stimulated GH release but had no effect on PRL release, whereas PRL secretion was strongly stimulated by TRH. GnRH-stimulated PRL release was seen from day 3 up to about day 9 after birth, with a maximum in the magnitude of the response on day 5. GH release showed also a maximal response to GnRH on day 5. However, although stimulated PRL release declined rapidly at the end of the GnRH pulse, GH release did not and even increased over a period of about 20 min. The latter secretion dynamics are consistent with previous observations in reaggregate cell cultures that the GnRH effect on GH release is dual (stimulation and inhibition). The poststimulus rise of GH release was most pronounced with pituitaries of 7- and 9-day-old rats. Finally, in pituitaries of 14-day-old rats the effect on GH release was largely diminished. The present data show that GnRH affects the secretion of lactotrophs and somatotrophs in intact rat pituitary glands only during a restricted period of early postnatal life in a cell-type specific manner and shed new light in establishing the role of GnRH in modulating PRL and GH release.

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a chronic, adult‐onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over‐expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over‐expressing the human, mutant SOD1G93A were crossed with mice that ubiquitously over‐expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1G93A/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1G93A littermates. There was no protective effect of hHsp27 over‐expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1G93A/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.

Effect of POMC1–76, its C‐terminal Fragment γ3‐MSH and Anti‐POMC1–76 Antibodies on DNA Replication in Lactotrophs in Aggregate Cell Cultures of Immature Rat Pituitary

Treatment of aggregate cell cultures of 14‐day‐old rat pituitary for 40 h with purified human (h) POMC1–76 dose‐dependently augmented the number of DNA replicating lactotrophs as estimated by autoradiography of []> 3H]‐thymidine (3H‐T) incorporation in cells immunostained for prolactin (PRL). No such effect was seen on the total number of 3H‐T labelled cells (the majority of which did not contain any pituitary hormone in a detectable amount) or on the total number of lactotrophs. The effect of hPOMC1–76 on 3H‐T incorporation in lactotrophs was blocked by concomitant treatment with anti‐hPOMC1–76 monoclonal and polyclonal antibodies cross‐reactive with rat POMC1–74. The latter anti‐hPOMC1–76 antibodies also decreased the number of 3H‐T incorporating lactotrophs in the absence of hPOMC1–76. γ3‐MSH, which is the C‐terminal domain of hPOMC1–76, mimicked the effect of hPOMC1–76 on 3H‐T incorporation in lactotrophs but its potency was lower than that of hPOMC1–76. Other melanocortin (MC) peptides such as α‐ and β‐MSH were also effective but were less potent than γ3‐MSH. The difference in potency was not due to partial degradation of the peptides. hPOMC1–76 did not affect 3H‐T incorporation in other pituitary cell types. In contrast γ3‐MSH also augmented the number of 3H‐T labelled somatotrophs and thyrotrophs. In the embryonic kidney 293 cell line stably transfected with the MC‐3 receptor, γ3‐MSH (10 nM) augmented cAMP formation up to 30 times. In contrast, hPOMC1–76 (100 nM) was inactive in this test system, indicating this peptide is not an agonist at the MC‐3 receptor. The present investigation further supports the role of rat POMC1–74 as a paracrine growth factor in the development of lactotrophs. The active core of POMC1–76 does not seem to be restricted to its C‐terminal domain γ3‐MSH as the latter peptide displays a growth promoting effect that is different from that of POMC1–76: it is less potent, it is not specific for lactotrophs and whereas the effect of γ3‐MSH may be mediated by the MC‐3 receptor that of POMC1–76 is not.

Stimulation of prolactin secretion from rat pituitary by luteinizing hormone-releasing hormone: evidence against mediation by angiotensin II acting through a (Sar1-Ala8)-angiotensin II-sensitive receptor.

In aggregate cell cultures of 15- to 20-day-old rat pituitary maintained in serum-free medium, luteinizing hormone-releasing hormone (LHRH) (10 nM) stimulated prolactin (PRL) release, confirming our previous results and those of others with serum-supplemented medium. Since angiotensin II (AII) stimulates PRL release and a renin-angiotensin system is expressed in gonadotrophs, LHRH stimulation of PRL release might be mediated by AII. To evaluate this hypothesis, the influence of (Sar1,Ala8)AII and (Sar1,Ile8)AII two peptide AII receptor antagonists, of DUP753, a nonpeptide and stable AII receptor antagonist, of a converting enzyme inhibitor, and of angiotensinogen on LHRH-induced PRL release was tested in various in vitro conditions of 15- to 20-day-old female rat pituitary. In aggregates maintained in serum-free medium with or without dexamethasone (DEX) and triiodothyronine (T3), or maintained in serum-supplemented medium, the effect of LHRH on PRL release was not affected by (Sar1Ala8)AII (0.1 microM), (Sar1,Ile8)AII (10 microM) or DUP753 (10 microM). Only a high dose (10 microM) of (Sar1,Ala8)AII attenuated the LHRH-induced PRL release. The latter attenuation was seen only with aggregates cultured in the DEX/T3 medium and not with aggregates cultured in the presence of serum. A dose of 1 or 10 nM (Sar1,Ala8)AII also failed to block the effect of LHRH used at 1 nM. In contrast, (Sar1,Ala8)AII dose dependently as well as DUP753 (10 microM) abolished the AII-induced PRL release. (Sar1,Ala8)AII also failed to affect the LHRH-induced PRL release in pituitary cell aggregates from 6-week-old male rats. However, in aggregates from both immature and 6-week-old rats, (Sar1,Ala8)AII provoked a small and statistically significant attenuation of the LHRH-induced PRL release when a 100 nM dose of LHRH was used. In freshly isolated hemipituitaries from 5-day-old rats, (Sar1,Ala8)AII (1 or 10 microM) did not affect the LHRH- (10 nM) induced PRL release. In single cells obtained by redispersion of aggregates and mounted in a Biogel P2 column, LHRH still stimulated PRL release. Again this effect could not be blocked by DUP753. Treatment of aggregate cell cultures with the angiotensin-converting enzyme inhibitor captopril or with angiotensinogen did not alter the LHRH-induced PRL release. It is concluded that AII is not the paracrine factor mediating the effect of LHRH at low nanomolar doses on PRL release, at least not through the classical AII receptor. The involvement of AII acting on a non-(Sar1,Ala8)AII-sensitive receptor cannot be excluded and warrants further investigation.

Production of recombinant rat proopiomelanocortin1–74 and characterization of its mitogenic action on pituitary lactotrophs

We report the production of biologically active recombinant rat Gly-2-Ser-1-POMC1-74 (rrPOMC1-74) in a prokaryotic expression system. The polypeptide was produced as a fusion protein with glutathione-S-transferase (GST), using the pGEX-4T-1 vector and subsequently cleaved by thrombin. Amino acid sequencing, up to residue 45, showed a correct primary structure including the two additional amino acids at the N-terminus, Gly and Ser, derived from the thrombin cleavage site. Electrospray ionization mass spectrometry showed a Mr of 8358.5 Da which was 14-16 Da heavier (oxidation or methylation) than the calculated mass. Combined digestion with trypsin and endoproteinase Glu-C followed by MALDI-TOF mass spectrometry and N-terminal sequencing of the separated fragments showed a correct disulphide bridge configuration. In reaggregate cell cultures of immature rat pituitary, rrPOMC1-74 displayed biological activity similar to that of natural human (h) POMC1-76 or rat POMC1-74: it stimulated DNA replication in lactotrophs but not in other pituitary cell types. However, its efficacy was significantly lower than that of the natural product. Gamma3-MSH, a peptide that can be generated from POMC1-74 and a typical ligand of the melanocortin-3 (MC-3) receptor, also stimulated DNA replication in lactotrophs and, in contrast to rrPOMC1-74, also in somatotrophs and thyrotrophs. rrPOMC1-74 increased cAMP levels in 293HEK cells stably transfected with the MC-3 receptor with an intrinsic activity and potency similar to that of gamma3-MSH. However, natural hPOMC1-76 was inactive in the latter test system. These data show that rrPOMC1-74 mimics the selective mitogenic action of natural POMC1-74 on lactotrophs. Since natural POMC1-74 is N- and O-glycosylated and rrPOMC1-74 is not, glycosylation does not seem to determine the selectivity for lactotrophs. In spite of the feature that rrPOMC1-74 is an agonist at the MC-3 receptor and the reported evidence that the MC-3 receptor is expressed in the anterior pituitary, the mitogenic action of rrPOMC1-74 on lactotrophs does not seem to be mediated by the MC-3 receptor.

Interaction of alpha T3-1 cells with lactotropes and somatotropes of normal pituitary in vitro.

A pituitary cell population of 14-day-old female rats, containing lactotropes and somatotropes but deprived of gonadotropes, was prepared by unit gravity sedimentation through a serum albumin gradient and allowed to reaggregate either as such or after mixing this population with cells of the gonadotropic alpha T3-1 cell line. In a perifusion system gonadotropin-releasing hormone (GnRH) had no effect on prolactin (PRL) and growth hormone (GH) release in the former aggregates but stimulated PRL release in the latter. In the alpha T3-1 cell-containing aggregates GnRH showed a biphasic effect on GH release: inhibition during exposure to GnRH followed by a rebound secretion upon removal of the peptide. The aggregation capacity of alpha T3-1 cells with the normal pituitary cells was demonstrated by using an alpha T3-1 cell clone stably transfected with the reporter gene beta-galactosidase. Perifusion of the gonadotrope-deprived aggregates with medium conditioned by alpha T3-1 cell provoked a rapid stimulation of PRL release and a biphasic effect on GH release. Medium conditioned by the corticotropic cell line AtT20 also stimulated PRL release but had no concomitant effect on GH release. Medium conditioned by alpha T3-1 cells, when added for 40 h to aggregates of 14-day-old rat pituitary, provoked an increase in the number of 3H-thymidine (3H-T)-labelled lactotropes and a decreased in the number of 3H-T-labelled somatotropes. The conditioned medium was concentrated on Sep-Pak C18 and ultrafiltrated through an Amicon membrane with 3-kD molecular weight cut-off and the retained molecules separated by reversed-phase HPLC. The material stimulating 3H-T labelling of lactotropes eluted from the column with a different retention time than material inhibiting 3H-T labelling of somatotropes, suggesting that the effect on lactotropes is mediated by (a) molecule(s) different from that affecting somatotropes. The effects of alpha T3-1 cells and their secretion products on lactotropes and somatotropes were comparable to those we previously observed using enriched populations of normal gonadotropes. The HPLC elution profiles of the substances affecting 3H-T incorporation as well as the specificity of these effects were also similar to that of the substances isolated previously from gonadotrope-conditioned medium. The present data, therefore, support previous conclusions on the paracrine control of the lactotrope/somatotrope lineage by the gonadotropes. Further purification and chemical characterization of the growth factors with selective action on lactotropes and somatotropes may lead to a better understanding of the development of the latter lineage.