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. Triacylglycerol (TAG)3 plays a key role in metabolic homeostasis, serving as the major energy storage molecule that allows organisms to survive periods of food deprivation. The regulation of TAG storage is important in human disease because both excessive and inadequate fat storage is associated with dyslipidemia, insulin resistance, and diabetes (reviewed in Refs. 1-3). We previously characterized the fatty liver dystrophy mouse, a model of generalized lipodystrophy with impaired TAG storage in adipose tissue, insulin resistance, and increased susceptibility to atherosclerosis (4, 5). Lipodystrophy in the fld mouse results from mutation in the Lpin1 (lipin-1) gene, the founding member of a family of three genes of previously unknown function (6). Genes for lipin-1, lipin-2, and lipin-3 occur in mammals and other vertebrates, whereas a single lipin gene ortholog can be detected in evolutionarily distant organisms including fruit fly, nematode, plants, and yeast (6). This suggests a fundamental function for lipin that is conserved from single celled eukaryotes to mammals.In the mouse, lipin-1 is expressed at high levels in adipose tissue and skeletal muscle, consistent with a role in lipid metabolism in these tissues. Indeed, adipocytes in lipin-1-deficient mice fail to accumulate TAG and do not develop mature adipocyte function (7). By contrast, transgenic mice with enhanced lipin-1 expression in adipocytes accumulate more TAG per cell and are prone to obesity (7-9). Furthermore, lipin-1 expression levels are reduced in adipose tissue of human lipodystrophic patients concomitantly with reduced fat mass (10). A role for lipin-1 in muscle metabolism is suggested by increased energy expenditure and fatty acid oxidation in the muscle of lipin-1-deficient mice and the opposite effects in muscle-specific lipin-1 transgenic mice (8). Thus, alterations in lipin-1 expression levels in either adipose tissue or skeletal muscle produce important physiological effects on energy storage and expenditure.In mammalian cells, the de novo biosynthesis of TAG, PC, and phosphatidylethanolamine is catalyzed mainly through the glycerol phosphate pathway (11). Several enzymes in this pathway have been characterized, but not all of these have been identified at the molecular level. Among those for which a gene has not been isolated is the phosphatidate phosphohydrolase (phosphatase) type-1 that converts the PA formed from glycerol phosphate and lysoPA to DAG (12). There are two main types of PA phosphatase. The first is the type-1 activity (PAP1) that is ...

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.

Background-Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemiareperfusion (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. (Circulation. 2005;112:544-552.)

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.

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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 (1a and 1b), -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-1a or -1b 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-1a or -1b 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-1a not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1a mutant failed to promote

SummaryThree 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.

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 b-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