The anterior insula bidirectionally modulates cost-benefit decision-making on a rodent gambling task.

Deficits in cost-benefit decision-making, as assessed in the Iowa Gambling Task (IGT), are commonly observed in neuropsychiatric disorders such as addiction. There is considerable variation in the maximization of rewards on such tasks, both in the general population and in rodent models, suggesting individual differences in decision-making may represent a key endophenotype for vulnerability to neuropsychiatric disorders. Increasing evidence suggests that the insular cortex, which is involved in interoception and emotional processes in humans, may be a key neural locus in the control of decision-making processes. However, the extent to which the insula contributes to individual differences in cost-benefit decision-making remains unknown. Using male Sprague Dawley rats, we first assessed individual differences in the performance over the course of a single session on a rodent analogue of the IGT (rGT). Rats were matched for their ability to maximize reward and received bilateral excitotoxic or sham lesions of the anterior insula cortex (AIC). Animals were subsequently challenged on a second rGT session with altered contingencies. Finally, animals were also assessed for instrumental conditioning and reversal learning. AIC lesions produced bidirectional alterations on rGT performance; rats that had performed optimally prior to surgery subsequently showed impairments, and animals that had performed poorly showed improvements in comparison with sham-operated controls. These bidirectional effects were not attributable to alterations in behavioural flexibility or in motivation. These data suggest that the recruitment of the AIC during decision-making may be state-dependent and help guide response selection towards subjectively favourable options.

From impulses to maladaptive actions: the insula is a neurobiological gate for the development of compulsive behavior.

Impulsivity is an endophenotype of vulnerability for compulsive behaviors. However, the neural mechanisms whereby impulsivity facilitates the development of compulsive disorders, such as addiction or obsessive compulsive disorder, remain unknown. We first investigated, in rats, anatomical and functional correlates of impulsivity in the anterior insular (AI) cortex by measuring both the thickness of, and cellular plasticity markers in, the AI with magnetic resonance imaging and in situ hybridization of the immediate early gene zif268, respectively. We then investigated the influence of bilateral AI cortex lesions on the high impulsivity trait, as measured in the five-choice serial reaction time task (5-CSRTT), and the associated propensity to develop compulsivity as measured by high drinking levels in a schedule-induced polydipsia procedure (SIP). We demonstrate that the AI cortex causally contributes to individual vulnerability to impulsive-compulsive behavior in rats. Motor impulsivity, as measured by premature responses in the 5-CSRTT, was shown to correlate with the thinness of the anterior region of the insular cortex, in which highly impulsive (HI) rats expressed lower zif268 mRNA levels. Lesions of AI reduced impulsive behavior in HI rats, which were also highly susceptible to develop compulsive behavior as measured in a SIP procedure. AI lesions also attenuated both the development and the expression of SIP. This study thus identifies the AI as a novel neural substrate of maladaptive impulse control mechanisms that may facilitate the development of compulsive disorders.

Plasminogen activator-specific inhibitors produced by human monocytes/macrophages.

Human monocytes/macrophages produce plasminogen activator-specific inhibitors (PAIs) that form covalent complexes with urokinase-type plasminogen activator (uPA). We have characterized two functionally and antigenically related forms of PAIs produced by resting and phorbol myristate acetate (PMA)-treated U 937 cells: an Mr 40,000 form, presumably nonglycosylated, with a pI of 5.2, that is constitutively synthetized by these cells and that remains predominantly intracellular; a PMA-induced form of heterogeneous Mr (50,000-65,000) with a pI of 4.7, that is preferentially secreted; this PAI is glycosylated with terminal sialic acid residue(s). Biosynthetic labeling experiments demonstrated that both PAIs are synthetized by U 937 cells. They are inactivated upon treatment with propanol, heat, and acid; the covalent and equimolar complexes formed between these PAIs and 125I-uPA are dissociated by ammonium hydroxide, suggesting that the PAIs are linked to uPA via an ester bond. Human peripheral blood monocytes/macrophages also produce the two forms of PAI. These PAIs are clearly different from the main plasma protease inhibitors and they are both antigenically related to the PAI-2 characterized in human placenta.

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

Gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors.

Exposure of mouse resident and thioglycollate-elicited peritoneal macrophages to IFN-gamma leads to a marked increase in the TNF-alpha (tumor necrosis factor/cachectin), IL-1 and u-PA (urokinase-type plasminogen activator) mRNA levels. Nuclear run-on experiments show that IFN-gamma acts by enhancing the transcription of these three genes. Transcription of these three genes is also rapidly and transiently induced by cycloheximide, an inhibitor of protein synthesis, indicating that they are under the control of short-lived repressors.

Expression of a tumor necrosis factor alpha transgene in murine pancreatic beta cells results in severe and permanent insulitis without evolution towards diabetes.

Mice bearing a tumor necrosis factor (TNF) alpha transgene controlled by an insulin promoter developed an increasingly severe lymphocytic insulitis, apparently resulting from the induction of endothelial changes with features similar to those observed in other places of intense lymphocytic traffic. This was accompanied by dissociation of the endocrine tissue (without marked decrease in its total mass), islet fibrosis, and the development of intraislet ductules containing, by places, beta cells in their walls, suggesting a regenerative capacity. Islet disorganization and fibrosis did not result from lymphocytic infiltration, since they were also observed in SCID mice bearing the transgene. Diabetes never developed, even though a number of potentially inducing conditions were used, including the prolonged perfusion of interferon gamma and the permanent expression of a nontolerogenic viral protein on beta cells (obtained by using mice bearing two transgenes). It is concluded that (a) a slow process of TNF release in pancreatic islets induces insulitis, and may be instrumental in the insulitis resulting from local cell-mediated immune reactions, but (b) that insulitis per se is not diabetogenic, lymphocyte stimulation by cells other than beta cells being necessary to trigger extensive beta cell damage. This provides an explanation for the discrepancy between the occurrence of insulitis and that of clinical disease in autoimmune diabetes.

A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase.

The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since Mr 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.

Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos.

To assess in vivo the postulated participation of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in processes involving tissue remodeling and cell migration, we have studied the cellular distribution of u-PA and t-PA mRNAs during mouse oogenesis and embryo implantation. By in situ hybridizations, we detected t-PA mRNA in oocytes and u-PA mRNA in granulosa and thecal cells from preovulatory follicles. These findings are compatible with a role for plasminogen activators in oogenesis and follicular disruption. We demonstrated the presence of u-PA mRNA in the invasive and migrating trophoblast cells of 5.5- and 6.5-d-old embryos. At 7.5 days, u-PA mRNA was predominantly localized to trophoblast cells that had reached the deep layers of the uterine wall, while the peripheral trophoblast cells surrounding the presomite stage embryo were devoid of specific signal. In 8.5-d-old embryos abundant u-PA mRNA expression resumed transiently in the giant trophoblast cells at the periphery of the embryo and in the trophoblast cells of the ectoplacental cone, to become undetectable in 10.5-d-old embryos. These observations establish the in vivo expression of the u-PA gene by invading and migrating trophoblast cells in a biphasic time pattern; they are in agreement with the proposed involvement of the enzyme in the extracellular proteolysis accompanying embryo implantation.

Plasminogen activator and mouse spermatozoa: urokinase synthesis in the male genital tract and binding of the enzyme to the sperm cell surface.

When ejaculated mouse spermatozoa were embedded in a plasminogen-containing insoluble protein substrate, a zone of proteolysis developed progressively, centered around the sperm head region. Lysis did not occur in absence of plasminogen or in presence of antibodies against the urokinase-type plasminogen activator (u-PA). Zymographic and immunological analyses confirmed the presence of u-PA in extracts of ejaculated mouse spermatozoa. In contrast, the u-PA activity of sperm cells obtained from testis or from vas deferens was low, although these cells were able to bind added murine u-PA. The sites of u-PA synthesis were identified by measuring u-PA activity and u-PA mRNA content in protein extracts and in total RNA preparations of various portions of the male genital tract. The highest levels of u-PA activity and of u-PA mRNA were found in vas deferens and seminal vesicles. The cells that synthesize u-PA were localized by hybridizing frozen sections of various portions of the genital tract to a u-PA cRNA probe. In all tissues examined, u-PA mRNA was predominantly located in the epithelial layer, and the strongest signal was observed over that of the vas deferens. Hence, the u-PA associated with ejaculated sperm cells is probably acquired from genital tract secretions. Sperm-bound u-PA may participate in the proteolytic events that accompany capacitation and fertilization.

Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro.

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular endothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly, we find that these agents also induce plasminogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-beta 1 (TGF-beta 1), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by angiogenic agents and TGF-beta 1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards antiproteolysis in response to TGF-beta 1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-beta 1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid endothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteolytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-beta 1.

Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli.

Disulfide bonds are rarely found in cytoplasmic proteins. Mutations were selected for in Escherichia coli that allow disulfide bond formation in the cytoplasm. In the presence of these mutations, export-defective versions of alkaline phosphatase and mouse urokinase were able to fold into their enzymatically active conformations in the cytoplasm because their disulfide bonds were formed. The mutations were mapped to the gene for thioredoxin reductase and diminish or eliminate the activity of this enzyme. Thioredoxin itself was found to be unnecessary for this disulfide bond formation. Thioredoxin reductase, but not thioredoxin, is thus implicated in keeping cysteines reduced in cytoplasmic proteins.

Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes.

Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.

Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney.

Kidneys have long been recognized as a major source of plasminogen activators (PAs). However, neither the sites of synthesis of the enzymes nor their role in renal function have been elucidated. By the combined use of zymographies on tissue sections and in situ hybridizations, we have explored the cellular distribution of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators and of their mRNAs in developing and adult mouse kidneys. In 17.5-d old embryos, renal tubules synthesize u-PA, while S-shaped bodies produce t-PA. In the adult kidney, u-PA is synthesized and released in urine by the epithelial cells lining the straight parts of both proximal and distal tubules. In contrast, t-PA is produced by glomerular cells and by epithelial cells lining the distal part of collecting ducts. The precise segmental distribution of PAs suggests that both enzymes may be implicated in the maintenance of tubular patency, by catalyzing extracellular proteolysis to prevent or circumvent protein precipitation.

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.

Differential protease expression by cutaneous squamous and basal cell carcinomas.

To assess the postulated role of plasminogen activation in tumor invasion, we have investigated the cellular sites of synthesis for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their inhibitors (PAI-1 and PAI-2) in two human cutaneous neoplasia that differ in their metastatic potential. The combined use of zymography on tissue sections and in situ hybridization demonstrates that uPA is produced by malignant cells of squamous cell carcinomas (SCC) but not by basal cell carcinomas (BCC), whereas tPA is detected exclusively in nonmalignant dermal tissue. In addition, we show that SCC neoplastic cells simultaneously produce variable amounts of PAI-1, and that PAI-1 production correlates inversely with uPA enzymatic activity. These observations establish that invasive human malignant cells in vivo can activate plasminogen through uPA production during the early phases of tumor growth; they also demonstrate that the proteolytic activity of tumor cells can be modulated by the concomitant production of PAI-1. Because SCC have a higher invasive and metastatic potential than BCC, our findings lend further support to the involvement of plasminogen activation in malignant behavior.

Extracellular proteolysis in the adult murine brain.

Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.