Structural basis for the synergy of 4'- and 2'-modifications on siRNA nuclease resistance, thermal stability and RNAi activity.

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cß-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and ß-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'ß-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cß-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cß-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'ß-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cß-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.

Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates.

Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.

Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modification on siRNA.

Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.

4'-C-Methoxy-2'-deoxy-2'-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing.

We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.

5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo.

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.

Suppressing Hepatic UGT1A1 Increases Plasma Bilirubin, Lowers Plasma Urobilin, Reorganizes Kinase Signaling Pathways and Lipid Species and Improves Fatty Liver Disease.

Several population studies have observed lower serum bilirubin levels in patients with non-alcoholic fatty liver disease (NAFLD). Yet, treatments to target this metabolic phenotype have not been explored. Therefore, we designed an N-Acetylgalactosamine (GalNAc) labeled RNAi to target the enzyme that clears bilirubin from the blood, the UGT1A1 glucuronyl enzyme (GNUR). In this study, male C57BL/6J mice were fed a high-fat diet (HFD, 60%) for 30 weeks to induce NAFLD and were treated subcutaneously with GNUR or sham (CTRL) once weekly for six weeks while continuing the HFD. The results show that GNUR treatments significantly raised plasma bilirubin levels and reduced plasma levels of the bilirubin catabolized product, urobilin. We show that GNUR decreased liver fat content and ceramide production via lipidomics and lowered fasting blood glucose and insulin levels. We performed extensive kinase activity analyses using our PamGene PamStation kinome technology and found a reorganization of the kinase pathways and a significant decrease in inflammatory mediators with GNUR versus CTRL treatments. These results demonstrate that GNUR increases plasma bilirubin and reduces plasma urobilin, reducing NAFLD and inflammation and improving overall liver health. These data indicate that UGT1A1 antagonism might serve as a treatment for NAFLD and may improve obesity-associated comorbidities.

Antiphospholipid Syndrome Secondary to Lupus Anticoagulant: Case Report for Clinical Anticoagulation Determination.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by venous or arterial thrombosis and/or pregnancy morbidity in the presence of persistent laboratory evidence of antiphospholipid antibodies (APL). APS can occur as a primary condition but can also occur in the presence of systemic lupus erythematosus (SLE) or other systemic autoimmune diseases such as rheumatoid arthritis (RA) or Sjogren's Syndrome. Our case focuses on a 21-year-old female with a history of "going numb and having no ability to speak" with a total of approximately 20 such episodes, with no known triggers for these episodes. A hypercoagulable profile was performed and indicated an elevation in lupus anticoagulant (LA), which was also positive at repeat testing after 12 weeks, meeting the criteria for APS. Oral contraceptive pills (OCP) were stopped immediately, and she was started on daily aspirin. When hematology was consulted and evaluated, the patient reported a history of possible transient ischemic attacks (TIA); however, there was no history of deep vein thrombosis (DVT), pulmonary embolism (PE), or miscarriages. Recommendations from hematology were to continue the daily aspirin but did not recommend the addition of anticoagulation therapy. Additional recommendations included avoiding risk factors for thrombosis such as the use of birth control pills, smoking, and a sedentary lifestyle or obesity. Given the young age of our patient, as well as multiple TIA associated with APS secondary to LA, the patient was started on anticoagulation contrary to hematology's recommendations.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

5'-Morpholino modification of the sense strand of an siRNA makes it a more effective passenger.

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: opposing roles of JNK1/2 and p38alpha MAP kinases.

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Facile Synthesis, Geometry, and 2'-Substituent-Dependent in Vivo Activity of 5'-(E)- and 5'-(Z)-Vinylphosphonate-Modified siRNA Conjugates.

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.

Isolated neonatal rat papillary muscles: a new model to translate neonatal rat myocyte signaling into contractile mechanics.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this in vitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.

p38α MAPK inhibits stretch-induced JNK activation in cardiac myocytes through MKP-1.

Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.

5'-C-Malonyl RNA: Small Interfering RNAs Modified with 5'-Monophosphate Bioisostere Demonstrate Gene Silencing Activity.

5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.

Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis.

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Minimal and inducible regulation of tissue factor pathway inhibitor-2 in human gliomas.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-flanking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, -670 to +1; pTF6, -312 to +1; pTF2, -1511 to +1). Another construct, pTF8 (-81 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (-312 to -81) was deleted, from the [-670 to +1] pTF5 region, also showed no promoter activity. Hence, (-312 to -81) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about -3000 bp upstream of the transcription start site. We also found a strong repressor in the region between -927 to -1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between -1511 to -1181. These positive elements masked the silencer effect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (-312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the -312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines.

Structure, function and biology of tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and remodeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.

Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses.

Interleukin (IL)-21 was recently discovered using a functional cloning approach based on expression of its receptor. It is similar in domain organization and primary sequence to IL-2 and IL-15. Like these cytokines, IL-21 uses the common gamma chain of the IL-2/15 receptor, which forms a heterodimeric receptor complex with IL-21R. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of natural killer cells. The elucidation of the unique biological effects of IL-21 represents an intense area of interest in current cytokine biology.