Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism.

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Modulation by protein kinase C of the hormonal responsiveness of hepatocytes from lean (Fa/fa?) and obese (fa/fa) Zucker rats.

The effect of phorbol myristate acetate (PMA) on the hormonal responsiveness of hepatocytes from lean and obese Zucker rats was studied. Phenylephrine-stimulated phosphatydylinositol labeling and phosphorylase activation were antagonized by PMA in cells from obese and lean animals; bigger residual effects were observed in cells from obese animals even at high PMA concentrations. Cyclic AMP accumulation induced by isoproterenol, glucagon, forskolin and cholera toxin was higher in cells from lean animals than in those from obese rats. PMA diminished glucagon- and cholera toxin-induced cyclic AMP accumulation; cells from lean animals were more sensitive to PMA. Two groups of isoforms of protein kinase C (PKC) were observed in hepatocytes from Zucker rats using DEAE-cellulose column chromatography: PKC 1 and PKC 2. The PKC 1 isozymes were separated into four peaks using hydroxylapatite: aa, 1a (PKC-beta), 1b (PKC-alpha) and 1c. Short treatment with PMA decreased the activity of PKC 1 (peaks 1b (PKC-alpha) and 1c) and to a lesser extent of PKC 2; cells from lean animals were more sensitive to PMA than those obtained from obese rats. Our results indicate that cells from genetically obese Zucker rats are in general less sensitive to this activator of protein kinase C than those from their lean littermates. The possibility that alterations in the phosphorylation/dephosphorylation cycles, that control metabolism and hormonal responsiveness, may contribute to this obese state is suggested.

Opposite effects of hyperglycemia and insulin deficiency on liver glycogen synthase phosphatase activity in the diabetic rat.

The specific effect of hyperglycemia on the reported decrease in liver glycogen synthase phosphatase activity was studied in STZ-induced diabetic rats with normal fasting insulinemia. Four groups of animals were investigated: control (nondiabetic), diabetic hyperglycemic (STZ), diabetic normoglycemic (STZ followed by 3-day phloridzin treatment), and a diabetic normoglycemic group injected with glucose to reinstate hyperglycemia. None of the treatments significantly altered fasting plasma insulin and glucagon concentrations. We found that hepatic synthase phosphatase activity decreased in STZ-induced diabetic rats and was further markedly reduced when glycemia was normalized in the diabetic animals. This additional decrease in phosphatase activity was almost fully reversed when hyperglycemia was restored by acute glucose infusion of the normoglycemic diabetic rats. In parallel, the levels of liver G6P and F6P were markedly reduced in the diabetic normoglycemic rats and restored with reinstatement of hyperglycemia. In contrast, liver microsomal glucose-6-phosphatase activity was enhanced and glucokinase activity was lowered in all diabetic groups, regardless of glycemia. Our results indicate that hyperglycemia per se counteracts part of the loss of hepatic synthase phosphatase in diabetic animals and provokes the stable conversion of synthase phosphatase from a less active to a more active form.

Activation of the extracellular signal-regulated protein kinase (ERK) cascade by membrane-type-1 matrix metalloproteinase (MT1-MMP).

The mechanisms underlying membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent induction of cell migration were investigated. Overexpression of MT1-MMP induced a marked increase in cell migration, this increase being dependent on the presence of the cytoplasmic domain of the protein. MT1-MMP-dependent migration was inhibited by a mitogen-activated protein kinase kinase 1 inhibitor, suggesting the involvement of the extracellular signal-regulated protein kinase (ERK) cascade in the induction of migration. Accordingly, MT1-MMP overexpression induced the activation of ERK, this process being also dependent on the presence of its cytoplasmic domain. MT1-MMP-induced activation of both migration and ERK required the catalytic activity of the enzyme as well as attachment of the cells to matrix proteins. The MT1-MMP-dependent activation of ERK was correlated with the activation of transcription through the serum response element, whereas other promoters were unaffected. Taken together, these results indicate that MT1-MMP trigger important changes in cellular signal transduction events, leading to cell migration and to gene transcription, and that these signals possibly originate from the cytoplasmic domain of the protein.

Ontogeny of the catalytic subunit and putative glucose-6-phosphate transporter proteins of the rat microsomal liver glucose-6-phosphatase system.

The catalytic subunit (p36) and putative glucose-6-phosphate (G6P) transporter (p46) protein levels of the rat glucose-6-phosphatase (G6Pase) system were studied in relation to G6Pase hydrolytic activity and G6P uptake in liver microsomes during the fetal to neonatal period. The mean G6P hydrolytic activity in liver microsomes increased significantly from the 20th to 21st day of gestation (from 6 to 22 mU/mg protein) and was further enhanced by 3-fold 6 hours after birth, with a maximal activity at 1 day of age (112 mU/mg protein). In contrast, G6P uptake into the vesicles was undetectable before birth, appeared after day 1 (656 pmol/mg protein), and decreased after day 2 (about 330 pmol/mg protein). Immunoblot analysis showed that the mean p36 protein level was low (< 1.6 arbitrary units [AU]) during gestation, increased sharply (to about 4.0 AU) during the first day, and remained stable afterward. Unlike p36, p46 protein was present before birth at values comparable to those postpartum. P46 increased from 3.2 AU at 20 days to 4.6 AU at 21 days of gestation, and decreased transiently after birth. These results show that (1) G6Pase hydrolytic activity before birth can occur without detectable G6P uptake function; (2) the presence of the putative G6P transporter protein is not sufficient to elicit G6P uptake; and (3) full G6Pase activity requires optimal expression of both p36 and p46 proteins. These data are discussed in relation to the function of G6Pase.

Evidence that the transit of glucose into liver microsomes is not required for functional glucose-6-phosphatase.

We show that the production of glucose from glucose-6-phosphate hydrolysis outside microsomes is a function of glucose-6-phosphatase independent of its property to form glucose inside microsomes. Indeed, during development (before 1 day of age), mouse liver microsomes had glucose-6-phosphatase producing glucose solely outside microsomes. Furthermore, in vivo treatment of rats with the glucocorticoid analogue triamcinolone resulted in increased glucose-6-phosphatase activity outside but not inside microsomes and without change in the catalytic subunit 40 kDa glucose-6-phosphatase mRNA abundance or protein level, indicating that other factors induced by triamcinolone (e.g., altered membrane lipid environment and/or a regulatory protein) were responsible for the activity change. Triamcinolone treatment also lessened the inhibition of glucose-6-phosphatase by pyridoxal 5'-phosphate (PLP), but this effect was not due to an interaction of PLP with the active site. Accordingly, reversal of the inhibition was observed after permeabilization of the microsomes. The two distinct orientations of liver microsomal glucose-6-phosphate phosphohydrolase suggest different physiological roles played by this enzyme in the endoplasmic reticulum membrane.

Glucose transport and glucose 6-phosphate hydrolysis in intact rat liver microsomes.

Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors, but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t1/2 values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though it can be accounted for by diffusion only over the accessible range of sugar concentrations.

Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes.

The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate.

Transmembrane topology of glucose-6-phosphatase.

Deficiency of microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, causes glycogen storage disease type 1a, an autosomal recessive disorder. Characterization of the transmembrane topology of G6Pase should facilitate the identification of amino acid residues contributing to the active site and broaden our understanding of the effects of mutations that cause glycogen storage disease type 1a. Using N- and C-terminal tagged G6Pase, we show that in intact microsomes, the N terminus is resistant to protease digestion, whereas the C terminus is sensitive to such treatment. Our results demonstrate that G6Pase possesses an odd number of transmembrane helices, with its N and C termini facing the endoplasmic reticulum lumen and the cytoplasm, respectively. During catalysis, a phosphoryl-enzyme intermediate is formed, and the phosphoryl acceptor in G6Pase is a His residue. Sequence alignment suggests that mammalian G6Pases, lipid phosphatases, acid phosphatases, and a vanadium-containing chloroperoxidase (whose tertiary structure is known) share a conserved phosphatase motif. Active-site alignment of the vanadium-containing chloroperoxidase and G6Pases predicts that Arg-83, His-119, and His-176 in G6Pase contribute to the active site and that His-176 is the residue that covalently binds the phosphoryl moiety during catalysis. This alignment also predicts that Arg-83, His-119, and His-176 reside on the same side of the endoplasmic reticulum membrane, which is supported by the recently predicted nine-transmembrane helical model for G6Pase. We have previously shown that Arg-83 is involved in positioning the phosphate during catalysis and that His-119 is essential for G6Pase activity. Here we demonstrate that substitution of His-176 with structurally similar or dissimilar amino acids inactivates the enzyme, suggesting that His-176 could be the phosphoryl acceptor in G6Pase during catalysis.

Cloning and characterization of cDNAs encoding a candidate glycogen storage disease type 1b protein in rodents.

Glycogen storage disease type 1 (GSD-1) is a group of genetic disorders caused by a deficiency in the activity of the enzyme glucose-6-phosphatase. (G6Pase). GSD-1a and GSD-1b, the two major subgroups, have been confirmed at the molecular genetic level. The gene responsible for GSD-1b maps to human chromosome 11q23 and a candidate human GSD-1b cDNA that encodes a microsomal transmembrane protein has been identified. In this study, we show that this cDNA maps to chromosome 11q23; thus it is a strong candidate for GSD-1b. Furthermore, we isolated and characterized candidate murine and rat GSD-1b cDNAs. Both encode transmembrane proteins sharing 93-95% sequence homology to the human GSD-1b protein. The expression profiles of murine GSD-1b and G6Pase differ both in the liver and in the kidney; the GSD-1b transcript appears before the G6Pase mRNA during development. In addition to G6Pase deficiency, GSD-1b patients suffer neutropenia, neutrophil dysfunction, and recurrent bacterial infections. Interestingly, although the G6Pase mRNA is expressed primarily in the liver, kidney, and intestine, the GSD-1b mRNA is expressed in numerous tissues, including human neutrophils/monocytes.

Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43 kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1 had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.

Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells.

The gene for glycogen-storage disease type 1b maps to chromosome 11q23.

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.