Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations.

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Tumor progression is associated with a significant decrease in the expression of the endostatin precursor collagen XVIII in human hepatocellular carcinomas.

Endostatin inhibits angiogenesis and tumor growth in mice. The role of its endogenous precursor collagen XVIII in human cancer is unknown. In normal tissues, two variants of collagen XVIII, namely, the short and long forms regulate tissue specificity, the long form being almost exclusively expressed by hepatocytes in the liver. We analyzed RNA arrays from 57 hepatocellular carcinomas (HCCs) with common and variant-specific probes and investigated the relationships between collagen XVIII expression and angiogenesis by measuring the CD34-positive microvessel density. Low collagen XVIII expression by tumor hepatocytes was associated with large tumor size (r, -0.63; P < 0.001) and replacement of trabeculae with pseudoglandular-solid architecture (chi2, 28; P < 0.001), which indicate tumor progression. Tumors expressing the highest collagen XVIII levels were smaller and had lower microvessel density (P = 0.01) than those expressing moderate levels; and HCCs with the lowest collagen XVIII levels approached a plateau of microvessel density, which indicated that a decrease in collagen XVIII expression is associated with angiogenesis in primary liver cancer. HCCs recurring within 2 years of resection showed 2.2-fold lower collagen XVIII mRNA than nonrecurring ones (P = 0.02). The findings relied on the hepatocyte-specific long form. Thus, the endogenous expression of the endostatin precursor decreases along with tumor progression in HCCs.

Lipoproteins containing apolipoprotein A-IV: composition and relation to cholesterol esterification.

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance.

This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).

Structural domain of apolipoprotein A-I involved in its interaction with cells.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Tangier disease: isolation and characterization of LpA-I, LpA-II, LpA-I: A-II and LpA-IV particles from plasma.

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 +/- 0.14 and 0.14 +/- 0.04 mumol of cholesterol esterified/h/micrograms of protein, and 7 +/- 2.5 and 1.4 +/- 0.3 mumol of cholesteryl ester transferred/h/micrograms of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2-1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 +/- 0.16 and 0.05 +/- 0.01 mumol of cholesterol esterified/h/micrograms of protein and, 41 +/- 3.7 and 1 +/- 0.4 mumol of cholesteryl ester transferred/h/micrograms of protein). The LpA-II particle in TD represented in fact an LpA-II:A-IV complex (75% mol apoA-II and 22% mol apoA-IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I-containing particles and reverse cholesterol transport in IDDM.

Lipoprotein particles containing apoA-I but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.

Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

Expression, location and cross-reactivity of two antigenic sites on the amino terminal region of rabbit and human apolipoprotein A-I.

Rabbit apolipoprotein A-I (apo A-I) of molecular weight 27,612 contained 241 amino acids. In contrast to its human counterpart which has 3 methionine residues, the rabbit protein possesses only one and therefore produces 2 fragments after CNBr cleavage (CNBr I and II, NH2- and COOH-terminal, respectively). From a series of monoclonal antibodies raised against human apo A-I, 2 (A05 and A16) cross-reacted with rabbit apo A-I. In the present study, we show that A05 recognizes the rabbit CNBr I fragment while the integrity of the intermediate region between the 2 CNBr fragments (including the methionine residue) is required for the expression of the A16 antigenic determinant. Competition experiments were performed between human 125I-labelled high density lipoprotein (HDL) and a variety of preparations of human and rabbit apo A-I (including the purified and delipidated protein, complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I, HDL subfractions and whole serum). The A05 antigenic determinant was expressed identically in all these fractions of both species. In contrast the A16 showed poor reactivity with delipidated apo A-I, the apparent affinity constant being about 100 times less than for HDL. These data suggest that phospholipids improve the recognition of apo A-I by the A16 antibody. The similar immunoreactivity of the human and rabbit proteins in the present study is consistent with the view that the NH2-terminal region contains the major portion activating lecithin:cholesterol acyltransferase.

Effects of fenofibrate on lipoprotein metabolism and fatty acid distribution in Zucker rats.

The short term effects of fenofibrate (150 mg/kg per day), administered by gavage, on lipoprotein and fatty acid distribution have been investigated in an hypertriglyceridemic model, the Zucker rat. Lean rats were compared to control obese and treated obese rats, and control obese animals to treated obese littermates. Classically, plasma cholesterol and triacylglycerol increased by 1.8- and 7.9-fold, respectively, in control obese versus lean rats. Treatment of the Zucker obese rats with fenofibrate reduced their plasma cholesterol by 10% and raised triacylglycerol by 47% (P less than 0.001) in comparison to untreated control obese rats. These effects were accompanied by a change in the composition of all plasma lipoproteins. The cholesterol/triacylglycerol ratio in VLDL rose by 32% while that in LDL and HDL fell by 43 and 47%, respectively. Drug therapy altered the fatty acid profile in both plasma and liver; the percentage of polyunsaturated fatty acids fell while monounsaturated fatty acids increased. The increased proportion of monounsaturated fatty acids in plasma suggests that the fatty acid composition of circulating lipoproteins is modified, particularly in VLDL. This, in association with the altered lipid distribution in VLDL may reflect an abnormal metabolism of this lipoprotein. In view of these abnormalities, we conclude that this rat is not an appropriate model for the short-term study of clofibrate analogues.

Comparison between fat intake assessed by a 3-day food record and phospholipid fatty acid composition of red blood cells: results from the Monitoring of Cardiovascular Disease-Lille Study.

We investigated the relationship between assessment of fatty acid intake by a 3-day food record and by capillary gas chromatography of erythrocyte phospholipid fatty acid. The study was performed in a sample of 244 men aged 45 to 66 years from the general population who were participating in the Monitoring of Cardiovascular Disease (MONICA)-Lille survey. The relationship between each nutrient and food item and erythrocyte phospholipid fatty acid was investigated by a regression model on proportion including each food item and nutrient as a dependent variable and percentage of fatty acid and covariables (nonalcoholic energy intake, age, alcohol intake, and smoking) as independent variables. Polyunsaturated fat and linoleic acid intake were positively correlated with linoleic acid content of erythrocytes (beta = 0.641 and 0.604, respectively, P < .001). Monounsaturated and saturated fat intake were correlated with oleic acid (beta = 0.375 and 0.373, respectively, P < .01). Fish intake correlated positively with docosahexaenoic acid (DHA) (beta = 0.383, P < .001) and negatively with arachidonic acid (beta = -0.509, P < .01). These data confirm, on a group level, a good relationship between assessment of polyunsaturated fat intake by a 3-day record and linoleic acid content of erythrocyte membranes. These data suggest that erythrocyte oleic acid content is a marker of both saturated and monounsaturated fat intake.

The relationship between the phospholipid fatty acid composition of red blood cells, plasma lipids, and apolipoproteins.

This study examined the relationship between the fatty acid composition of red blood cell phospholipids and lipid markers of atherosclerotic risk in an urban male population aged 45 to 66 years. There was a surprisingly significant positive association between the docosahexaenoic acid ([DHA] 22:6n-3) content of erythrocyte phospholipids and the following risk markers: plasma cholesterol (P < .01), low-density lipoprotein (LDL) cholesterol (P < .01), apolipoprotein (apo) B (P < .05), and apo B-containing lipoprotein particles (P < .05) recognized by a monoclonal antibody (LpBL3). On the other hand, phospholipid alpha-linolenate was positively correlated with apo A-I and high-density lipoprotein (HDL) cholesterol levels (P < .05), while arachidonate showed an inverse relationship with plasma cholesterol (P < .05). There was a negative association between palmitoleic acid and apo B (P < .01) and LpBL3 (P < .001); the latter showed a negative association with stearic acid (P < .001). These interesting findings emphasize the beneficial effect on atherosclerotic risk markers of dietary n-6 polyunsaturated and monounsaturated fatty acids, and suggest that long-chain n-3 polyunsaturated fatty acids (DHA) could have an adverse effect on some of the lipid risk markers.

Assessing matrix metalloproteinase expression and activity in hepatocellular carcinomas.

The matrix metalloproteinases (MMPs) constitute a large family of zincand calcium-dependent endopeptidases that cleave extracellular matrix components (1). Hence, MMPs are classified according to their substrate specificities: interstitial collagenases, stromelysins, gelatinases, membrane-type matrix metalloproteinases (MT-MMPs), and elastase (Table 1). The regulation of MMP activity involves gene expression, proteolytic processing of the propeptides to active forms, and inhibition by specific tissue inhibitors of matrix metalloproteinase (TIMPs). MMPs are involved in situations that require extracellular matrix remodeling, including wound healing, development, inflammation, fibrosis angiogenesis, and tumor invasion (2-5). Several complementary methods have provided an insightful description of the expression levels of MMPs and their pathological correlates. These include immunohistochemistry and Northern, Western, and dot blots. Additionally, the activity of MMPs is evaluated by gel substrate analysis. This approach has demonstrated that an increase in the expression of MMP2 (6,7), MT1-MMP (7), TIMP1, and TIMP2 (8-10) is associated with liver fibrosis. Similarly, in hepatocellular carcinomas, a high expression of MMP2, MMP9, MT1-MMP, and matrilysin is related to tumor aggressiveness (11-14). Consistently, by gel substrate analysis, MMP2 activity is increased in primary and secondary liver cancers (13,15,16). By in situ hybridization, the sources of.

MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells.

Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix components. In fibrotic livers, there is a markedly increased activity of matrix metalloproteinase 2 (MMP2), a major enzyme involved in extracellular matrix remodeling. We have previously shown that hepatic stellate cells secrete latent MMP2 and that MMP2 activation occurs in coculture of hepatic stellate cells and hepatocytes concomitantly with matrix deposition. In the present work we investigated the effects of various extracellular matrix components and concanavalin A, an inducer of immune-mediated liver injuries, on MMP2 activation in cultured human hepatic stellate cells. Collagen I induced a dose-dependent MMP2 activation, which was not blocked by both actinomycin and cycloheximide. Collagen VI, laminin, and a reconstituted basement membrane (matrigel) were ineffective in inducing activation. Specific antibodies against the subunits of alpha2beta1 integrins, the major collagen I receptor, induced partial inhibition of MMP2 activation. Treatment of cells with concanavalin A resulted in a marked activation of MMP2 that correlated with the proteolytic processing of MT1-MMP, the MMP2 activator, from a Mr=60 kd toward a Mr=43 kd polypeptide. Actinomycin and cycloheximide inhibited the MMP2 activation induced by concanavalin A. Recombinant tissue inhibitor of metalloproteinase-2 and the MMP inhibitor BB-3103, but not PMSF, blocked MMP2 activation induced by collagen I or concanavalin A, and MT1-MMP processing to its Mr-43 kd form. These results suggest that the accumulation of collagen I may specifically contribute to the remodeling of extracellular matrix in fibrotic livers by inducing MMP2 activation.

Acylgalactosylceramides in developing dysmyelinating mutant mice.

Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which comigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.

Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes.

Activation of matrix metalloproteinase (MMP)-2, the 72-kd collagenase IV/gelatinase A, is involved in extracellular matrix remodeling. It has been suggested that a membrane-type MMP (MT-MMP-1) and the tissue inhibitor of metalloproteinase (TIMP)-2 are involved in MMP-2 processing, but the exact mechanism(s) of its activation remains unclear. We have investigated the role of cell-cell cooperation in the activation of pro-MMP-2 in the liver, using pure cultures and co-cultures of hepatocytes and hepatic stellate cells (HSCs). Northern blot analysis and in situ hybridization showed that, in both pure and co-cultures, HSCs, but not hepatocytes, expressed MMP-2, TIMP-2, and MT-MMP-1 mRNA. Zymography analyses revealed the latent form of MMP-2 in medium from 2-day-old pure HSC cultures with higher amounts in medium from hepatocyte/HSC co-cultures. When hepatocytes were added to 10-day-old HSC cultures, the activated form of MMP-2 was detected, concomitantly with the deposition of an abundant extracellular matrix. Incubation of plasma membrane-enriched fractions from hepatocytes with conditioned medium from pure HSC cultures generated the activated species of MMP-2 (62 and 59 kd). Activation of pro-MMP-2 by hepatocyte membranes was inhibited by EDTA, heat, and trypsin but not by serine proteinase inhibitors. These data show that the co-expression of TIMP-2, MMP-2, and MT-MMP-1 by HSCs does not lead to secretion of the activated form of MMP-2. Hepatocytes, which do not express MMP-2, TIMP-2, or MT-MMP-1, induce MMP-2 activation through a plasma membrane-dependent mechanism(s), thus suggesting that cell-cell interactions are involved in this process in vivo.

Transfer of cholesterol from Ob1771 cells or LDL to reconstituted, defined high density lipoproteins.

We used defined, reconstituted high density lipoproteins (rHDL) to study the effects of structure and composition of these particles on their role as cholesterol acceptors from cell membranes or from low density lipoproteins (LDL). Three discoidal rHDL and one spherical rHDL with distinct apolipoprotein A-I conformations, diameters and compositions were used in conjunction with Ob1771 cells to measure the rate of [3H]cholesterol efflux from the cells, direct binding to the cells, and competition with native HDL3 for binding. In addition, the same rHDL particles were used to study the kinetics of cholesterol mass transfer from LDL. The results show that the rates of cholesterol transfer depend on the nature of the donor (t1/2 11-19 min from LDL, and t1/2 5 h from the cells), on the phosphatidylcholine/cholesterol ratio in the acceptors (the closer this ratio is to the equilibrium value, the slower is the rate), and on the diameter of the acceptors (the smallest particles have the lowest t1/2 for cholesterol uptake from LDL, and are the most effective acceptors of [3H]cholesterol from cells after their phospholipid content is taken into account). The cholesterol uptake by the rHDL, both from the cells and from LDL, is determined mostly by the phospholipid pool available in the acceptors. Binding to the cells was equivalent for all the rHDL (Kd = 38-67 micrograms/ml) and comparable to HDL3, suggesting that the differences in apoA-I conformation have no effect on the binding to cells. Finally we observed that exposure of rHDL to cells may lead to remodeling of some of the lipoprotein particles.

Activation of the envelope proteins by a metalloproteinase enables attachment and entry of the hepatitis B virus into T-lymphocyte.

Previously, we identified an HBV binding factor (HBV-BF), a 50-kDa serum glycoprotein which interacts with HBV envelope proteins and which is also located in the membrane of normal human hepatocyte (A. Budkowska et al. (1993) J. Virol. 67, 4316). Here we show that HBV-BF is a neutral metalloproteinase which shares substrate specificity and properties with a newly described family of membrane type matrix metalloproteinases. HBV-BF treatment of the HBV resulted in the cleavage of the N-terminal part of the middle HBV envelope protein at the pre-S2(136-141) amino acid sequence VRGLYF/L (containing a single arginine cleavage site). HBV-BF affected the reactivity of the large HBV protein with pre-S1-specific MAbs, probably inducing the conformational change of the pre-S1 domain. The HBV-BF-digested virus remained morphologically intact with unchanged S antigenic determinants. The structural modifications of the viral envelope proteins induced by HBV-BF enabled cell membrane attachment and viral entry into the T-lymphocyte. Both processes were blocked by the metalloproteinase inhibitor 1,10 phenanthroline. Thus, the host-dependent proteolytic activation of the envelope proteins seems to be essential for the HBV entry into the cell. HBV-BF under a membrane bound or a secreted form could be (one of) the molecule(s) responsible for the HBV proteolytic activation.

Differential expression and origin of membrane-type 1 and 2 matrix metalloproteinases (MT-MMPs) in association with MMP2 activation in injured human livers.

Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51-58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.