Phosphorylation status of matrix metalloproteinase 2 in myocardial ischaemia-reperfusion injury.

OBJECTIVE: To investigate whether alterations in the phosphorylation status of matrix metalloproteinase 2 (MMP-2) in the heart may be protective in the setting of ischaemia-reperfusion (IR) injury. DESIGN: In-vitro heart function and biochemical research study. SETTING: University basic science laboratory. INTERVENTIONS: Male Sprague-Dawley rats, weighing 250-350 g. Isolated rat hearts were perfused at constant pressure either aerobically for 75 min or subjected to 20 min of global, no-flow ischaemia followed by 30 min of reperfusion. MAIN OUTCOME MEASURES: Heart mechanical function, MMP-2 activity and troponin I levels. RESULTS: The serine/threonine phosphatase inhibitor okadaic acid (OA) improved the recovery of mechanical function compared with control IR hearts and prevented the loss of troponin I. OA significantly reduced protein phosphatase 2A, but not protein phosphatase 1, activity in perfused hearts. IR stimulated the activation and release of MMP-2 into the coronary effluent in the first 2 min of reperfusion. This was accompanied by a decrease in the remaining activity and protein level of MMP-2 in heart tissue determined at the end of the reperfusion. OA did not alter the IR-stimulated release of MMP-2 into the coronary effluent, but reduced the decrease in MMP-2 in reperfused hearts. The immunoprecipitation of heart homogenates using anti-phosphoserine antibody showed that MMP-2 is phosphorylated. The dephosphorylation of MMP-2 by alkaline phosphatase treatment of homogenates prepared from IR hearts treated with OA significantly increased MMP-2 activity. CONCLUSIONS: These results suggest that the phosphorylation status of MMP-2 is important in its contribution to myocardial IR injury.

Regulation of lysophosphatidate signaling by autotaxin and lipid phosphate phosphatases with respect to tumor progression, angiogenesis, metastasis and chemo-resistance.

Evidence from clinical, animal and cell culture studies demonstrates that increased autotaxin (ATX) expression is responsible for enhancing tumor progression, cell migration, metastases, angiogenesis and chemo-resistance. These effects depend mainly on the rapid formation of lysophosphatidate (LPA) by ATX. Circulating LPA has a half-life of about 3 min in mice and it is degraded by the ecto-activities of lipid phosphate phosphatases (LPPs). These enzymes also hydrolyze extracellular sphingosine 1-phosphate (S1P), a potent signal for cell division, survival and angiogenesis. Many aggressive tumor cells express high ATX levels and low LPP activities. This favors the formation of locally high LPA and S1P concentrations. Furthermore, LPPs attenuate signaling downstream of the activation of G-protein coupled receptors and receptor tyrosine kinases. Therefore, we propose that the low expression of LPPs in many tumor cells makes them hypersensitive to growth promoting and survival signals that are provided by LPA, S1P, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). One of the key signaling pathways in this respect appears to be activation of phospholipase D (PLD) and phosphatidate (PA) production. This is required for the transactivations of the EGFR and PDGFR and also for LPA-induced cell migration. PA also increases the activities of ERK, mTOR, myc and sphingosine kinase-1 (SK-1), which provide individual signals for cells division, survival, chemo-resistance and angiogenesis. This review focuses on the balance of signaling by bioactive lipids including LPA, phosphatidylinositol 3,4,5-trisphosphate, PA and S1P versus the action of ceramides. We will discuss how these lipid mediators interact to produce an aggressive neoplastic phenotype.

Antioxidant treatment protects diabetic rats from cardiac dysfunction by preserving contractile protein targets of oxidative stress.

BACKGROUND: Animal studies suggest that reactive oxygen species (ROS) play an important role in the development of diabetic cardiomyopathy. HYPOTHESIS: Matrix metalloproteinase-2 (MMP-2) is activated by ROS and contributes to the acute loss of myocardial contractile function by targeting and cleaving susceptible proteins including troponin I (TnI) and alpha-actinin. METHODS: Using the streptozotocin-induced diabetic rat model, we evaluated the effect of daily in vivo administration of sodium selenate (0.3 mg/kg; DMS group), or a pure omega-3 fish oil with antioxidant vitamin E (omega-3E; 50 mg/kg; DMFA group), which has antioxidant-like effects, for 4 weeks on heart function and on several biochemical parameters related to oxidant stress and MMP-2. RESULTS: Although both treatments prevented the diabetes-induced depression in left ventricular developed pressure (LVDP) as well as the rates of changes in developed pressure (+/-dP/dt) (P<.001), the improvement in LVDP of the DMS group was greater compared to that of the DMFA group (P<.001). Moreover, these treatments reduced the diabetes-induced increase in myocardial oxidized protein sulfhydryl and nitrite concentrations (P<.001). Gelatin zymography and Western blot data indicated that the diabetes-induced changes in myocardial levels of MMP-2 and tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) and the reduction in TnI and alpha-actinin protein levels were improved in both the DMS and DMFA groups (P<.001). CONCLUSIONS: These results suggest that diabetes-induced alterations in MMP-2 and TIMP-4 contribute to myocardial contractile dysfunction by targeting TnI and alpha-actinin and that sodium selenate or omega-3E could have therapeutic benefits in diabetic cardiomyopathy.

Phosphatidate degradation: phosphatidate phosphatases (lipins) and lipid phosphate phosphatases.

Three lipid phosphate phosphatases (LPPs) regulate cell signaling by modifying the concentrations of a variety of lipid phosphates versus their dephosphorylated products. In particular, the LPPs are normally considered to regulate signaling by the phospholipase D (PLD) pathway by converting phosphatidate (PA) to diacylglycerol (DAG). LPP activities do modulate the accumulations of PA and DAG following PLD activation, but this could also involve an effect upstream of PLD activation. The active sites of the LPPs are on the exterior surface of plasma membranes, or on the luminal surface of internal membranes. Consequently, the actions of the LPPs in metabolizing PA formed by PLD1 or PLD2 should depend on the access of this substrate to the active site of the LPPs. Alternatively, PA generated on the cytosolic surface of membranes should be readily accessible to the family of specific phosphatidate phosphatases, namely the lipins. Presently, there is only indirect evidence for the lipins participating in cell signaling following PLD activation. So far, we know relatively little about how individual LPPs and specific phosphatidate phosphatases (lipins) modulate cell signaling through controlling the turnover of bioactive lipids that are formed after PLD activation.

The PPARgamma agonist rosiglitazone enhances rat brown adipose tissue lipogenesis from glucose without altering glucose uptake.

We investigated the mechanisms whereby peroxisome proliferator-activated receptor-gamma (PPARgamma) agonism affects glucose and lipid metabolism in brown adipose tissue (BAT) by studying the impact of PPARgamma activation on BAT glucose uptake and metabolism, lipogenesis, and mRNA levels plus activities of enzymes involved in triacylglycerol (TAG) synthesis. Interscapular BAT of rats treated or not with rosiglitazone (15 mg*kg(-1).day(-1), 7 days) was evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (PAP or lipin-1), and diacylglycerol acyltransferase (DGAT). Rosiglitazone increased BAT mass without affecting whole tissue glucose uptake. BAT glycogen content (-80%), its synthesis from glucose (-50%), and mRNA levels of UDP-glucose pyrophosphorylase (-40%), which generates UDP-linked glucose for glycogen synthesis, were all reduced by rosiglitazone. In contrast, BAT TAG-glycerol synthesis in vivo and glucose incorporation into TAG-glycerol in vitro were stimulated by the agonist along with the activities and mRNA levels of glycerol 3-phosphate-generating phosphoenolpyruvate carboxykinase and glycerokinase. Furthermore, rosiglitazone markedly increased the activities of GPAT and DGAT but not those of lipin-1-mediated PAP-1, enzymes involved in the sequential acylation of glycerol 3-phosphate and TAG synthesis. Because an adequate supply of fatty acids is essential for BAT nonshivering thermogenesis, the enhanced ability of BAT to synthesize TAG under PPARgamma activation may constitute an important mechanism by which lipid substrates are stored in preparation for an eventual thermogenic activation.

Depot-specific effects of the PPARgamma agonist rosiglitazone on adipose tissue glucose uptake and metabolism.

We investigated mechanisms whereby peroxisome proliferator-activated receptor gamma (PPARgamma) agonism redistributes lipid from visceral (VF) toward subcutaneous fat (SF) by studying the impact of PPARgamma activation on VF and SF glucose uptake and metabolism, lipogenesis, and enzymes involved in triacylglycerol (TAG) synthesis. VF (retroperitoneal) and SF (inguinal) of rats treated or not for 7 days with rosiglitazone (15 mg/kg/day) were evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (or lipin-1), and diacylglycerol acyltransferase. Rosiglitazone increased SF glucose uptake, GLUT4 mRNA, and insulin-stimulated glucose oxidation, conversion to lactate, glycogen, and the glycerol and fatty acid components of TAG. In VF, only glucose incorporation into TAG-glycerol was stimulated by rosiglitazone and less so than in SF (1.5- vs. 3-fold). mRNA levels of proteins involved in glycolysis, Krebs cycle, glycogen synthesis, and lipogenesis were markedly upregulated by rosiglitazone in SF and again less so in VF. Rosiglitazone activated TAG-glycerol synthesis in vivo (2.8- vs. 1.9-fold) and lipin activity (4.6- vs. 1.5-fold) more strongly in SF than VF, whereas GPAT activity was increased similarly in both depots. The preferential increase in glucose uptake and intracellular metabolism in SF contributes to the PPARgamma-mediated redistribution of TAG from VF to SF, which in turn favors global insulin sensitization.

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL.

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.

We previously identified mutations in the Lpin1 gene, encoding lipin-1, as the underlying cause of lipodystrophy in the fatty liver dystrophy (fld) mutant mouse. Lipin-1 is normally expressed at high levels in adipose tissue and skeletal muscle, and deficiency in the fld mouse causes impaired adipose tissue development, insulin resistance, and altered energy expenditure. We also identified two additional lipin protein family members of unknown function, lipin-2 and lipin-3. Han et al. (Han, G. S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218) recently demonstrated that the single lipin homolog in yeast, Smp2, exhibits phosphatidate phosphatase type-1 (PAP1) activity, which has a key role in glycerolipid synthesis. Here we demonstrate that lipin-1 accounts for all of the PAP1 activity in white and brown adipose tissue and skeletal muscle. However, livers of lipin-1-deficient mice exhibited normal PAP1 activity, indicating that other members of the lipin protein family could have PAP1 activity. Consistent with this possibility, recombinant lipin-2 and lipin-3 possess PAP1 activity. Each of the three lipin family members showed Mg2+-dependent activity that was specific for phosphatidate under the conditions employed. The different lipins showed distinct tissue expression patterns. Our results establish the three mammalian lipin proteins as PAP1 enzymes and explain the biochemical basis for lipodystrophy in the lipin-1-deficient fld mouse.

Degradation of myosin light chain in isolated rat hearts subjected to ischemia-reperfusion injury: a new intracellular target for matrix metalloproteinase-2.

BACKGROUND: Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP-2 not only remodels the extracellular matrix but also acts intracellularly in I/R by degrading troponin I. Whether other intracellular targets exist for MMP-2 during I/R is unknown. METHODS AND RESULTS: Isolated rat hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The impaired recovery of mechanical function of the heart was attenuated by the MMP inhibitors o-phenanthroline or doxycycline. Quantitative 2D electrophoresis of homogenates of aerobically perfused hearts (control) or those subjected to I/R injury (in the presence or absence of MMP inhibitors) showed 3 low-molecular-weight proteins with levels that were significantly increased upon I/R injury and normalized to control levels by MMP inhibitors. Mass spectrometry analysis identified all 3 proteins as fragments of myosin light chain 1, which possesses theoretical cleavage recognition sequences for MMP-2 and is rapidly degraded by it in vitro. The association of MMP-2 with the thick myofilament in fractions prepared from I/R hearts was observed with immunogold electron microscopy, gelatin zymography for MMP-2 activity, and immunoprecipitation. MMP-2 was found to cleave myosin light chain 1 between tyrosine 189 and glutamine 190 at the C terminus. CONCLUSIONS: Our results demonstrate that myosin light chain 1 is another novel substrate for MMP-2 in the cardiomyocyte and that its degradation may contribute to contractile dysfunction resulting from I/R injury to the heart.

Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro.

Matrix metalloproteinases (MMPs) are traditionally known for their role in extracellular matrix remodeling. Increasing evidence reveals several alternative substrates and novel biological roles for these proteases. Recent evidence showed the intracellular localization of MMP-2 within cardiac myocytes, colocalized with troponin I within myofilaments. Here we investigated the presence of MMP-2 in the nucleus of cardiac myocytes using both immunogold electron microscopy and biochemical assays with nuclear extracts. The gelatinase activity found in both human heart and rat liver nuclear extracts was blocked with MMP inhibitors. In addition, the ability of MMP-2 to cleave poly (ADP-ribose) polymerase (PARP) as a substrate was examined as a possible role for MMP-2 in the nucleus. PARP is a nuclear matrix enzyme involved in the repair of DNA strand breaks, which is known to be inactivated by proteolytic cleavage. PARP was susceptible to cleavage by MMP-2 in vitro in a concentration-dependent manner, yielding novel degradation products of ~66 and <45 kDa. The cleavage of PARP by MMP-2 was also blocked by MMP inhibitors. This is the first characterization of MMP-2 within the nucleus and we hereby suggest its possible role in PARP degradation.