Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2.

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6+27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.

Autoregulatory circuit of human rpL3 expression requires hnRNP H1, NPM and KHSRP.

Alternative pre-mRNA splicing (AS) is a major mechanism that allows proteomic variability in eukaryotic cells. However, many AS events result in mRNAs containing a premature termination codon, which are degraded by nonsense-mediated mRNA decay (NMD) pathway. We have previously demonstrated that human rpL3 autoregulates its expression through the association of AS with NMD. In fact, overexpression of rpL3 promotes downregulation of canonical splicing and upregulation of alternative splicing that produces an NMD-targeted mRNA isoform. The result of these events is a decreased production of rpL3. We have also identified heterogeneous nuclear ribonucleoprotein (hnRNP) H1 as a splicing factor involved in the regulation of rpL3 alternative splicing and identified its regulatory cis-elements within intron 3 transcript. Here, we report that NPM and KHSRP are two newly identified proteins involved in the regulation of rpL3 gene expression via AS-NMD. We demonstrate that hnRNP H1, KHSRP and NPM can be found associated, and present also in ribonucleoproteins (RNPs) including rpL3 and intron 3 RNA in vivo, and describe protein-protein and RNA-protein interactions. Moreover, our data provide an insight on the crucial role of hnRNP H1 in the regulation of the alternative splicing of the rpL3 gene.

hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene.

By generating mRNA containing a premature termination codon (PTC), alternative splicing (AS) can quantitatively regulate the expression of genes that are degraded by nonsense-mediated mRNA decay (NMD). We previously demonstrated that AS-induced retention of part of intron 3 of rpL3 pre-mRNA produces an mRNA isoform that contains a PTC and is targeted for decay by NMD. We also demonstrated that overexpression of rpL3 downregulates canonical splicing and upregulates the alternative splicing of its pre-mRNA. We are currently investigating the molecular mechanism underlying rpL3 autoregulation. Here we report that the heterogeneous nuclear ribonucleoprotein (hnRNP) H1 is a transacting factor able to interact in vitro and in vivo with rpL3 and with intron 3 of the rpL3 gene. We investigated the role played by hnRNP H1 in the regulation of splicing of rpL3 pre-mRNA by manipulating its expression level. Depletion of hnRNP H1 reduced the level of the PTC-containing mRNA isoform, whereas its overexpression favored the selection of the cryptic 3' splice site of intron 3. We also identified and characterized the cis-acting regulatory elements involved in hnRNP H1-mediated regulation of splicing. RNA electromobility shift assay demonstrated that hnRNP H1 specifically recognizes and binds directly to the intron 3 region that contains seven copies of G-rich elements. Site-directed mutagenesis analysis and in vivo studies showed that the G3 and G6 elements are required for hnRNP H1-mediated regulation of rpL3 pre-mRNA splicing. We propose a working model in which rpL3 recruits hnRNP H1 and, through cooperation with other splicing factors, promotes selection of the alternative splice site.

Conformational and biochemical characterization of a biologically active rat recombinant Protease Nexin-1 expressed in E. coli.

Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed alpha/beta secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin-rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.

cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins.

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3'-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3'-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3'-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pull-down assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3'-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and alpha-tubulin in vitro and in vivo.

Local administration of WIN 55,212-2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-kappaB activation.

Chronic inflammation is often associated with granuloma formation that is a hallmark of many human diseases. The transcription factor nuclear factor-kappa B (NF-kappaB) plays a central role in this process by regulating the expression of several pro-inflammatory genes. Cannabinoids (CBs) from Cannabis sativa L. exert a large number of biological effects including anti-inflammatory and anti-angiogenic effects. In this study, we investigated the role of CBs on granuloma formation induced by lambda-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB(1)/CB(2) agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-kappaB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB(1) selective agonist, and JWH-015, a CB(2) selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB(1) and CB(2) selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.

The 3'-untranslated region directs ribosomal protein-encoding mRNAs to specific cytoplasmic regions.

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. We have analyzed the subcellular distribution of mRNAs encoding human cytosolic and mitochondrial ribosomal proteins. Biochemical fractionation experiments showed that the transcripts for cytosolic ribosomal proteins associate preferentially with the cytoskeleton via actin microfilaments. Transfection in HeLa cells of a GFP reporter construct containing the cytosolic ribosomal protein L4 3'-UTR showed that the 3'-UTR is necessary for the association of the transcript to the cytoskeleton. Using confocal analysis we demonstrate that the chimeric transcript is specifically associated with the perinuclear cytoskeleton. We also show that mRNA for mitochondrial ribosomal protein S12 is asymmetrically distributed in the cytoplasm. In fact, this transcript was localized mainly in the proximity of mitochondria, and the localization was 3'-UTR-dependent. In summary, ribosomal protein mRNAs constitute a new class of localized transcripts that share a common localization mechanism.

Alternative splicing and nonsense-mediated mRNA decay regulate mammalian ribosomal gene expression.

Messenger RNAs containing premature stop codons are generally targeted for degradation through nonsense-mediated mRNA decay (NMD). This mechanism degrades aberrant transcripts derived from mutant genes containing nonsense or frameshift mutations. Wild-type genes also give rise to transcripts targeted by NMD. For example, some wild-type genes give rise to alternatively spliced transcripts that are targeted for decay by NMD. In Caenorhabditis elegans, the ribosomal protein (rp) L12 gene generates a nonsense codon-bearing alternatively spliced transcript that is induced in an autoregulatory manner by the rpL12 protein. By pharmacologically blocking the NMD pathway, we identified alternatively spliced mRNA transcripts derived from the human rpL3 and rpL12 genes that are natural targets of NMD. The deduced protein sequence of these alternatively spliced transcripts suggests that they are unlikely to encode functional ribosomal proteins. Overexpression of rpL3 increased the level of the alternatively spliced rpL3 mRNA and decreased the normally expressed rpL3. This indicates that rpL3 regulates its own production by a negative feedback loop and suggests the possibility that NMD participates in this regulatory loop by degrading the non-functional alternatively spliced transcript.

Ribosomal protein L7a binds RNA through two distinct RNA-binding domains.

The human ribosomal protein L7a is a component of the major ribosomal subunit. We previously identified three nuclear-localization-competent domains within L7a, and demonstrated that the domain defined by aa (amino acids) 52-100 is necessary, although not sufficient, to target the L7a protein to the nucleoli. We now demonstrate that L7a interacts in vitro with a presumably G-rich RNA structure, which has yet to be defined. We also demonstrate that the L7a protein contains two RNA-binding domains: one encompassing aa 52-100 (RNAB1) and the other encompassing aa 101-161 (RNAB2). RNAB1 does not contain any known nucleic-acid-binding motif, and may thus represent a new class of such motifs. On the other hand, a specific region of RNAB2 is highly conserved in several other protein components of the ribonucleoprotein complex. We have investigated the topology of the L7a-RNA complex using a recombinant form of the protein domain that encompasses residues 101-161 and a 30mer poly(G) oligonucleotide. Limited proteolysis and cross-linking experiments, and mass spectral analyses of the recombinant protein domain and its complex with poly(G) revealed the RNA-binding region.

Inhibition of granuloma-associated angiogenesis by controlling mast cell mediator release: role of mast cell protease-5.

We investigated the role of mast cells in granuloma-associated angiogenesis in rat by using: (i) a mast cell membrane stabilizer, ketotifen; (ii) a mast cell depleting agent, compound 48/80. Moreover, we focused on the role of chymases, which exhibit proinflammatory and pro-angiogenic properties by using: (i) chymostatin, an inhibitor of chymase activity; (ii) a specific antisense oligonucleotide (AS-ODN) designed against rat mast cell protease-5 (rMCP-5), the most abundantly expressed chymase in the skin. The formation of granuloma was evaluated, as wet weight, 96 h after the subcutaneous implant of two lambda-carrageenin (1%)-soaked sponges on the back of male Wistar rats. Angiogenesis was evaluated as haemoglobin content in the granulomatous tissue and as level of tumour necrosis factor-alpha (TNF-alpha) in the exudates. A single injection of ketotifen (1-5-25 mg kg(-1) i.p.) significantly reduced granuloma formation by 31.6, 44.6 and 71.9%, and haemoglobin content by 17.0, 35.0 and 66.2%, suggesting that the release of mediator(s) from mast cells modulates the process. Chymostatin (5-10 nmol(-1) site(-1) day(-1)) reduced granuloma-associated angiogenesis by 57.3 and 70.0%. RT-PCR analysis showed that rMCP-5 mRNA amounts were significantly reduced by rMCP-5 AS-ODN (1.25-2.5-5.0 nmol site(-1)) by 69.5, 72.5 and 81.8%. In parallel experiments, rMCP-5 AS-ODN (1.25, 2.5, 5.0 nmol site(-1)) strongly reduced granuloma weight (26.1, 45.0 and 56.3%) and haemoglobin content (22.2, 50.4, 62.03%), suggesting that the observed effect is mediated through an antisense mechanism. In conclusion, these data suggest that: (i) inhibition of mast cell mediators release may represent a novel strategy to modulate angiogenesis; (ii) among the chymase family, rMCP-5 is a key promoter of angiogenesis in the rat.

The human homologue of the mouse Surf5 gene encodes multiple alternatively spliced transcripts.

Hu-Surf5 is included within the Surfeit locus, a cluster of six genes originally identified in mouse. In the present study, we have cloned and characterized the Hu-Surf5 gene and its mRNA multiple transcripts. Comparison of the most abundant cDNA and genomic sequence shows that the Hu-Surf5 is spread over a region of approximately 7.5 kb and consists of five exons separated by four introns. The nucleotide sequence of the genomic region flanking the 3'-end of the Hu-Surf5 gene revealed the presence of a processed pseudogene of human ribosomal protein L21 followed by Hu-Surf6 gene. Only 110 bp separate the transcription start site of Hu-Surf5 and Hu-Surf3/L7a gene and the transcription direction is divergent. Earlier studies defined the 110 bp region essential for promoter activity of Hu-Surf3/L7a. Here, we show that this region stimulates transcription with a slightly different efficiency in both directions. The bidirectional promoter lacks an identifiable TATA box and is characterized by a CpG island that extends through the first exon into the first intron of both genes. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for Hu-Surf5 expression. Hu-Surf5 encodes three different transcripts, Surf-5a, Surf-5b, and Surf-5c, which result from alternative splicing. Two protein products, SURF-5A and SURF-5B have been characterized. Production of chimaeras between the full-length SURF-5A or SURF-5B and the green fluorescent protein (GFP) allowed to localize both proteins in the cytoplasm.

Human rpL3 induces G1/S arrest or apoptosis by modulating p21 (waf1/cip1) levels in a p53-independent manner.

It is now largely accepted that ribosomal proteins may be implicated in a variety of biological functions besides that of components of the translation machinery. Many evidences show that a subset of ribosomal proteins are involved in the regulation of the cell cycle and apoptosis through modulation of p53 activity. In addition, p53-independent mechanisms of cell cycle arrest in response to alterations of ribosomal proteins availability have been described. Here, we identify human rpL3 as a new regulator of cell cycle and apoptosis through positive regulation of p21 expression in a p53-independent system. We demonstrate that the rpL3-mediated p21 upregulation requires the specific interaction between rpL3 and Sp1. Furthermore, in our experimental system, p21 overexpression leads to a dual outcome, activating the G1/S arrest of the cell cycle or the apoptotic pathway through mitochondria, depending on its intracellular levels. It is noteworthy that depletion of p21 abrogates both effects. Taken together, our findings unravel a novel extraribosomal function of rpL3 and reinforce the proapoptotic role of p21 in addition to its widely reported ability as an inhibitor of cell proliferation.

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.

Stage-specific gene expression in early differentiating oligodendrocytes.

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A(+) mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A(+) mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288.