Jean-Paul Boissel

Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS I and NOS III)

Nitric oxide synthase (NOS) exists in three established isoforms. NOS I (NOS1, ncNOS) was originally discovered in neurons. This enzyme and splice variants thereof have since been found in many other cells and tissues. NOS II (NOS2, iNOS) was first identified in murine macrophages, but can also be induced in many other cell types. NOS III (NOS3, ecNOS) is expressed mainly in endothelial cells. Whereas NOS II is a transcriptionally regulated enzyme, NOS I and NOS III are considered constitutively expressed proteins. However, evidence generated in recent years indicates that these two isoforms are also subject to expressional regulation. In view of the important biological functions of these isoforms, changes in their expression may have physiological and pathophysiological consequences. This review recapitulates compounds and conditions that modulate the expression of NOS I and NOS III, summarizes transcriptional and posttranscriptional effects that underlie these changes, and—where known—describes the molecular mechanisms leading to changes in transcription, RNA stability, or translation of these enzymes.—Förstermann, U., Boissel, J.‐P., Kleinert, H. Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J. 12, 773–790 (1998)

Structure and Function of Cationic Amino Acid Transporters (CATs)

The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

Regulation of the Expression of Nitric Oxide Synthase Isoforms

Publisher Summary There is a large array of regulatory mechanisms for the expression of different nitric oxide synthases (NOS) isoforms. The high-output NOS II is not only turned on transcriptionally, but the stability of the transcripts and their translation can be regulated dynamically. In addition, the expressional levels of the servoregulatory, low-output enzymes, NOS I and NOS III, can also be adjusted to meet local demand. The original paradigm that nitrogen oxide (NO) is synthesized either by constitutive NO synthases or by inducible NOS II is no longer valid. This adds to the diversity of mechanisms controlling NO production in different cells and tissues. Whereas transcriptional regulation of NOS II has been established since about 1990, no expressional regulation was originally known for the other two isoforms. Recent evidence suggests, however, that the expression of NOS I and NOS III can also be regulated under various conditions. Therefore, the mechanisms of expressional regulation of all three NOS isoforms are decribed.

Involvement of PKC and NF-κB in Nitric Oxide Induced Apoptosis in Human Coronary Artery Smooth Muscle Cells

Apoptosis of vascular smooth muscle cells is critically involved in progression of atherosclerosis and may prevent intimal hyperplasia in restenosis and vascular remodeling. Nitric oxide (NO) is known to induce apoptosis, but the signaling pathways still remain unclear. We investigated p53 accumulation, protein kinase C (PKC) activation and nuclear transcription factor (NF-ĸB) binding activity as possible signaling mechanisms of NO-induced apoptosis. Apoptosis was induced dose-dependently with the NO-donors sodiumnitroprusside (SNP: 232±48%) and SIN-1 (241±90% of actinomycin D induced apoptosis; means ± SEM, * p£0.05 vs. control) in HSMC. Inhibition of PKC significantly attenuated NO-induced apoptosis. Staurosporine reduced SIN-1/SNP-mediated DNA fragmentation by 55.3±13.8% and 38.3±13.9% respectively. Comparable results were obtained for calphostin C. However, NO-mediated induction of apoptosis was not preceded by p53 accumulation. SNP decreased NF-ĸB binding activity in HSMC. These results suggest that induction of apoptosis by exogenous NO in HSMC is not dependent on p53 accumulation but involves protein kinase C signaling and regulation of NF-ĸB binding activity. This opens a new therapeutical approach in preventing restenosis after angioplasty.

Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells

OBJECTIVE The efficient introduction of regulatory genes into vascular smooth muscle cells (SMCs) is one of the most promising options for gene therapy of cardiovascular diseases. Cationic liposome-mediated gene transfer may become a favorable transfection technique with regard to patients safety for in vivo administration. However, this method until now has its limitation in a low transfection efficiency. Therefore, the present study was designed to improve cationic liposome-mediated transfection of rabbit vascular SMCs in vitro and in vivo, in order to enhance transfection efficiency and present an optimized system which may offer a potential therapeutic benefit for in vivo application. METHODS AND RESULTS Optimized lipofection of rabbit SMCs with the mammalian expression vector pE-N1 and the reporter gene green fluorescent protein resulted in a mean transfection efficiency of about 50%. The unique transfection of rabbit SMCs in vitro and in vivo with the inducible isoform of human nitric oxide synthase (NOSII), using the same vector, resulted in a successful transient transcription and translation of a functionally active human NOSII in rabbit SMC, persisting 5-6 days. We could further demonstrate that the transfection procedure and the transgene product did neither induce necrosis nor apoptosis under the conditions chosen and did not result in the induction of endogenous NOSII of transfected SMCs. CONCLUSION(S) These findings indicate potential therapeutic relevance for this nonviral gene transfer system for in vivo gene therapy for cardiovascular diseases.

Lokale Medikamentengabe und Gentherapie

ZusammenfassungEines der wichtigsten Probleme der klinischen Kardiologie, die Entwickung einer Restenose nach koronarer Ballonangioplastie, ist bisher noch nicht befriedigend gelöst. Die pathophysiologischen Erkenntnisse über die Mechanismen der Neointimabildung sind noch unvollständig, und zahlreiche Therapiestudien mit systemisch applizierten Pharmaka mit unterschiedlichem Wirkungsmechanismus sind fehlgeschlagen. Mögliche innovative Therapieansätze betreffen die hochdosierte lokale Substanzapplikation an der Dilatationsstelle und lokale gentherapeutische Eingriffe zur Verhinderung der Neointimabildung durch Proliferationshemmung der glatten Gefäßmuskelzellen. Zahlreiche Kathetermodelle sind entwickelt worden, um die lokale hochdosierte Gabe eines Pharmakons oder von DNA zu ermöglichen. Es gibt verschiedene tierexperimentelle Modelle, bei denen durch die Verwendung von Antisense-Oligonukleotiden gegen die RNA von Proteinen oder Peptiden die Expression der entsprechenden Genprodukte vermindert wurde, die regulatorisch in den Zellzyklus eingreifen. Alternativ versucht man, die cDNA für inhibitorische Proteine oder Produkte in die Gefäßwandzellen einzubringen. Allerdings gibt es bisher nur sehr wenige Daten bezüglich einer klinischen Wirksamkeit von gentherapeutischen Eingriffen am Gefäßsystem, die bei Patienten mit peripherer arterieller Verschlußkrankheit erhoben wurden. Trotz des hypothetisch großen Potentials der Gentherapie des Gefäßsystems ist es noch fraglich, ob diese Methoden in der Zukunft einen festen Platz in der klinischen Therapie einnehmen werden.SummaryOne of the most important problems in clinical cardiology is still unresolved, i. e., the development of a restenosis following coronary balloon angioplasty. Our knowlegde about the sequelae of pathophysiologic events occuring during neointima formation is still far from complete (Figure 1) and numerous therapeutic trials using systemic administration of drugs with different mechanisms of action have failed. Possible innovative strategies are the local administration of high doses of drugs into the coronary arteries and local gene therapeutic interventions to inhibit neointima formation by reducing the proliferation of vascular smooth muscle cells. Numerous catheter devices were developed (Figure 2) in order to enable the local application of high doses of a drug or DNA. Additionally, galenic techniques are being developed to guarantee a steady release of locally administered drugs, e. g. from drug containing liposomes or microcarriers (Figure 3). There are already several animal models in which the development of a neointima was reduced by injecting antisense oligonucleotides directed towards the RNA encoding cell cycle regulatory proteins or peptides. Alternatively, the transfer of cDNA encoding proteins or protein products which inhibit the cellular proliferation and migration are being tested in vitro and in vivo with the help of reporter genes (Figure 4). Although, gene transfer techniques are believed to offer great therapeutic options for the future, the clinical data available today regarding this method are very limited and are derived from studies in patients with peripheral arterial disease. Thus, it is still questionable if gene transfer techniques will ever be able to become an integral part of our standard treatment for patients with vascular diseases.One of the most important problems in clinical cardiology is still unresolved, i.e., the development of a restenosis following coronary balloon angioplasty. Our knowledge about the sequelae of pathophysiologic events occurring during neointima formation is still far from complete (Figure 1) and numerous therapeutic trials using systemic administration of drugs with different mechanisms of action have failed. Possible innovative strategies are the local administration of high doses of drugs into the coronary arteries and local gene therapeutic interventions to inhibit neointima formation by reducing the proliferation of vascular smooth muscle cells. Numerous catheter devices were developed (Figure 2) in order to enable the local application of high doses of a drug or DNA. Additionally, galenic techniques are being developed to guarantee a steady release of locally administered drugs, e.g. from drug containing liposomes or microcarriers (Figure 3). There are already several animal models in which the development of a neointima was reduced by injecting antisense oligonucleotides directed towards the RNA encoding cell cycle regulatory proteins or peptides. Alternatively, the transfer of cDNA encoding proteins or protein products which inhibit the cellular proliferation and migration are being tested in vitro and in vivo with the help of reporter genes (Figure 4). Although, gene transfer techniques are believed to offer great therapeutic options for the future, the clinical data available today regarding this method are very limited and are derived from studies in patients with peripheral arterial disease. Thus, it is still questionable if gene transfer techniques will ever be able to become an integral part of our standard treatment for patients with vascular diseases.

Identification of cysteine residues in human cationic amino acid transporter hCAT-2A that are targets for inhibition by N-ethylmaleimide.

Background: The mechanism of N-ethylmaleimide (NEM)-mediated inhibition of cationic amino acid transporters (CATs) was unknown. Results: Cys-33 (cytoplasmic N terminus) and Cys-273 (transmembrane domain VI) in human CAT-2A are the targets of NEM inhibition. Conclusion: The cytoplasmic N terminus and transmembrane domain VI are critically involved in transporter function. Significance: Our results help to understand how these important amino acid transporters are regulated, probably also in vivo. In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y+L (y+LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y+ (most likely CAT-1), but not system y+L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753–766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y+LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y+LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.

A Chimera Carrying the Functional Domain of the Orphan Protein SLC7A14 in the Backbone of SLC7A2 Mediates Trans-stimulated Arginine Transport

Background: The molecular identity of the lysosomal transporter for cationic amino acids, system c, remains unknown. Results: SLC7A14 is a lysosomal localized protein with a functional domain that mediates arginine transport. Conclusion: SLC7A14 may mediate cationic amino acid transport across lysosomal membranes. Significance: As system c represents a salvage pathway in the therapy of cystinosis, characterization of SLC7A14 might help to develop better drugs. In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker®. To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called “functional domain” of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-l-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.