Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation.

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E(2) and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.

The COG database: new developments in phylogenetic classification of proteins from complete genomes.

The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae (http://www.ncbi.nlm.nih. gov/COG). In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included. The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs).

BACKGROUND: Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi. RESULTS: A. pernix and P. abyssi proteins were assigned to COGs using the COGNITOR program; the results were verified on a case-by-case basis and augmented by additional database searches using the PSI-BLAST and TBLASTN programs. Functions were predicted for over 300 proteins from A. pernix, which could not be assigned a function using conventional methods with a conservative sequence similarity threshold, an approximately 50% increase compared to the original annotation. A. pernix shares most of the conserved core of proteins that were previously identified in the Euryarchaeota. Cluster analysis or distance matrix tree construction based on the co-occurrence of genomes in COGs showed that A. pernix forms a distinct group within the archaea, although grouping with the two species of Pyrococci, indicative of similar repertoires of conserved genes, was observed. No indication of a specific relationship between Crenarchaeota and eukaryotes was obtained in these analyses. Several proteins that are conserved in Euryarchaeota and most bacteria are unexpectedly missing in A. pernix, including the entire set of de novo purine biosynthesis enzymes, the GTPase FtsZ (a key component of the bacterial and euryarchaeal cell-division machinery), and the tRNA-specific pseudouridine synthase, previously considered universal. A. pernix is represented in 48 COGs that do not contain any euryarchaeal members. Many of these proteins are TCA cycle and electron transport chain enzymes, reflecting the aerobic lifestyle of A. pernix. CONCLUSIONS: Special-purpose databases organized on the basis of phylogenetic analysis and carefully curated with respect to known and predicted protein functions provide for a significant improvement in genome annotation. A differential genome display approach helps in a systematic investigation of common and distinct features of gene repertoires and in some cases reveals unexpected connections that may be indicative of functional similarities between phylogenetically distant organisms and of lateral gene exchange.

Cutting edge: a short polypeptide domain of HIV-1-Tat protein mediates pathogenesis.

HIV-1 encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. In this report, we provide evidence that cellular activation by Tat involves a short core domain, Tat21-40, containing only 20 aa including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21-40 of both NF-kappaB-mediated HIV replication and TAR-dependent transactivation of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21-40 possesses potent angiogenic activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20-residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a potential novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease.

Lipopolysaccharide induction of monocyte matrix metalloproteinases is regulated by the tyrosine phosphorylation of cytosolic phospholipase A2.

Activation of human monocytes with lipopolysaccharide (LPS) results in the production of matrix metalloproteinases (MMPs) through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. In this study, the early signaling events involved in this signal transduction pathway were evaluated. Pretreatment of human peripheral blood monocytes with herbimycin A, a tyrosine kinase inhibitor, or arachidonyl trifluoromethyl ketone (AACOCF3), a specific inhibitor of cytosolic phospholipase A2 (cPLA2) inhibited the induction of PGE2 by LPS. This resulted in the inhibition of protein expression of gelatinase B (MMP-9) and interstitial collagenase (MMP-1), two major MMPs secreted by activated monocytes. Addition of arachidonic acid (AA) reversed the inhibitory effect of herbimycin A or AACOCF3 on monocyte MMP production, indicating the importance of tyrosine phosphorylation and the involvement of cPLA2 at an early stage in the signal transduction pathway of MMPs. This finding was further supported by LPS-induced shift in cPLA2 migration and tyrosine phosphorylation based on immunoblotting and immunoprecipitation studies. These results provide evidence that tyrosine phosphorylation of cPLA2 is one of the initial steps needed for the LPS induced MMP production in human monocytes.

Regulation of human monocyte matrix metalloproteinases by SPARC.

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM-40, is a collagen-binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP-9) and interstitial collagenase (MMP-1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)-2 as determined by Western and Northern blot analyses. The increase in PGHS-2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E2 (PGE2) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE2 by PGHS-2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE2 or dibutyryl cAMP (Bt2cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS-2 and the subsequent release of PGE2, a pathway that leads to the production of MMP by monocytes.

Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin.

One of insulin's many biological effects is the increased transcription of AP-1-regulated genes. cJun is the principal component of the AP-1 transcription complex, which is regulated by the newly discovered members of the MAPK superfamily referred to as cJun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs). We show that insulin stimulates a dose- and time-dependent increase in JNK activity in Rat 1 fibroblasts overexpressing human insulin receptors (Rat 1 HIR cells). Using two different polyclonal anti-JNK antibodies, JNK activity was measured after immunoprecipitation from whole cell extracts by phosphorylation of GSTcJun(1-79). Peak activation occurred 15 min after insulin addition, resulting in a 2.5-fold increase in GSTcJun(1-79) phosphorylation over unstimulated controls. Maximal JNK activation correlated with the onset of AP-1 DNA binding activity. Both insulin-stimulated JNK activity and insulin-induced AP-1 transcriptional activity were found to be Ras-dependent. These data suggest that in Rat 1 cells, JNK activation may play a role in insulin-regulated AP-1 transcriptional activity leading to a mitogenic response.

Insulin like growth factor 1 receptor signal transduction to the nucleus.

The mechanism by which IGF-1Rs regulate the growth and maintenance of cells in normal and disease states provides an important setting for studies addressing signal transduction events at the nuclear level. With the identification of c-Jun/AP-1 as a nuclear target of IGF-1 action we are provided with a model system for pursuing the molecular mechanisms triggered by IGF-1 action.

Association of c-myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells.

Oncoprotein c-myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c-myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c-myc protein was detected in high molecular weight protein fractions, in 350-600 Kd range, in gel-exclusion chromatography and sucrose gradient centrifugation. This distribution of c-myc protein coincided with the distribution of DNA polymerase alpha and several other enzymes necessary for DNA replication. The data suggest that c-myc product may be a component of the replitase complex of enzymes involved in nuclear DNA replication.