Regulation of Endothelial NO Synthase mRNA Stability
RNA-Binding Proteins Crowd on the 3′-Untranslated Region
Poised at the interface between the bloodstream and underlying vascular smooth muscle, the endothelium produces nitric oxide (NO), a free radical with critical roles in regulating multiple vascular cell functions including vascular tone. In addition, NO modulates the interaction between circulating blood elements and the blood vessel wall. The enzyme responsible for NO production in endothelial cells, endothelial NO synthase (eNOS or NOS3), is also expressed outside the vasculature in cell types such as platelets, cardiac myocytes, neurons, and bronchial epithelial cells. Thus, insights gained from understanding the mechanisms regulating eNOS function in endothelial cells are likely to have important implications beyond vascular biology.
Initially referred to as a “constitutive” NOS, levels of eNOS in endothelial cells were thought to be static with enzyme activation achieved via calmodulin binding in response to increased [Ca2+]i. However, following the generation of eNOS-specific antisera and the isolation of cDNAs encoding eNOS, research from many laboratories has painted a new picture of the mechanisms controlling eNOS activity with the identification of multiple additional regulatory steps (reviewed in Reference 11 ), including posttranslational modification (phosphorylation, myristoylation, palmitoylation), intracellular localization (membrane bound versus cytoplasmic, interaction with caveolae), gene transcription, and mRNA stability. A wide variety of signals alter eNOS mRNA levels in endothelial cells including shear stress, oxygen tension, proliferative status, cytokines, estrogens, growth factors, oxidized LDL, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors. Many of these signals regulate eNOS mRNA levels, at least in part, by modulating eNOS mRNA stability. For example, Yoshizumi et al2 observed that incubation of human umbilical vein endothelial cells with tumor necrosis factor-α (TNF-α) decreased eNOS mRNA by decreasing the stability of the mRNA >10-fold. In addition, Laufs et al3 reported HMG CoA reductase inhibitors attenuated the ability of hypoxia to destabilize eNOS mRNA in human saphenous vein endothelial cells. Moreover, Bouloumie et al4 recently reported that vascular endothelial growth factor increases endothelial eNOS mRNA stability.
Steady-state mRNA levels represent the balance between gene transcription and mRNA degradation. The half-lives of mRNAs in mammalian cells can vary over a wide range (from minutes to many hours) and may be modulated in response to extracellular and/or intracellular signals. Although, in general, less completely characterized than mechanisms regulating gene transcription, the mechanisms regulating mRNA stability are multiple and diverse (reviewed in References 5 and 65 6 ). mRNA molecules are stabilized by posttranscriptional modifications including a 7-methylguanosine cap structure at the 5′ end and a poly(A) tail at the 3′ end. In addition, ongoing translation has a critical role in the regulation of mRNA stability with many mRNAs stabilized by inhibitors of translation elongation. On the other hand, premature and aberrant translation termination, as well as impaired translation initiation, can promote mRNA degradation.
The rate of mRNA degradation is also governed, at least in part, by nucleotide sequences, which are typically located in the 3′-untranslated region (UTR) or coding region of the mRNA. These “cis-acting” nucleotide sequences may form secondary stem-loop RNA structures that facilitate binding of regulatory “trans-acting” proteins. Cis-acting mRNA sequences alter mRNA stability by a variety of mechanisms. For example, AU-rich elements (such as those found in c-fos mRNA) can destabilize mRNAs by causing poly(A) shortening followed by decapping of the 5′ end and subsequent RNA degradation via a 5′ to 3′ exonuclease. Alternatively, binding of proteins to other cis-acting elements can alter the susceptibility of mRNAs to endonucleolytic cleavage. For example, in the presence of a low concentration of iron, the transferrin receptor mRNA is stabilized by the binding of iron-regulatory protein interacting with iron-response elements in the 3′-UTR of the mRNA, resulting in protection from cleavage by endonucleases (reviewed in Reference 77 ).
In this issue of Circulation Research, Searles et al8 describe studies directed at understanding the molecular mechanisms responsible for the regulation of eNOS mRNA stability in response to changes in the endothelial cell proliferative state. The authors examined the decrease in eNOS mRNA concentrations in cultured bovine aortic endothelial cells (BAECs) in transition from a proliferative to a postconfluent state, a model of reendothelialization after vascular injury. Whereas eNOS gene transcription did not differ in proliferating and postconfluent BAECs, eNOS mRNA stability was markedly enhanced in proliferating cells. Reasoning that mRNA-binding proteins are likely to have an important role in the modulation of eNOS mRNA stability, the authors used in vitro–transcribed cRNA probes spanning the entire eNOS mRNA to identify sequences that bind to proteins in endothelial cell extracts. Surprisingly, RNA-binding proteins were detected for only a small portion of the eNOS mRNA containing the 3′ end of the coding region and the 5′ end of the 3′-UTR. These sequences bound 51- and 75-kDa proteins in cytoplasmic and nuclear extracts, respectively, and the levels of the former were found to be lower in proliferating than in postconfluent endothelial cells, consistent with a role for the 51-kDa protein in the destabilization of eNOS mRNA. Searles et al8 further delineated the target mRNA sequences to which the 51-kDa protein bound: the proximal 96 nt of the 3′-UTR of the eNOS mRNA were sufficient to bind the 51-kDa protein, and binding could be displaced by cRNA probes containing the proximal 43 nt of the 3′-UTR. To evaluate the role of the proximal 43 nt of the 3′-UTR in the regulation of eNOS mRNA stability, BAECs were “stably transfected” with plasmids directing expression of the mRNA encoding chloramphenicol acetyl transferase fused to the sequences derived from the eNOS 3′-UTR. A fusion mRNA containing the 43-nt sequence was markedly less stable than a fusion mRNA lacking this sequence, suggesting that the proximal 43 nt of the 3′-UTR participate in the modulation of eNOS mRNA stability. This report represents the first identification of cis-acting RNA sequences that regulate eNOS mRNA stability, as well as the initial characterization of a cytoplasmic protein that binds to these “destabilizing” sequences.
A different portion of the 3′-UTR may participate in the eNOS mRNA destabilization associated with exposure of endothelial cells to TNF-α. Alonso and colleagues identified a 129-nt uridine-cytidine-rich region in the middle of the eNOS mRNA 3′-UTR that binds a 60-kDa protein in cytoplasmic extracts of TNF-α–treated BAECs.9 10 Moreover, levels of this eNOS mRNA-binding protein inversely correlated with mRNA stability in endothelial cells after exposure to TNF-α.
Taken together, these studies suggest that the molecular mechanisms responsible for destabilizing eNOS mRNA require different cis-acting sequences and corresponding RNA-binding proteins depending on the mRNA-destabilizing signal. Of note, comparison of the nucleotide sequences of the bovine and human eNOS mRNAs reveals an unexpectedly high degree of sequence homology in the 3′-UTR (≈60% of 430 nt in the bovine 3′-UTR are identical to those in the human sequence), suggesting that the cis-acting sequences in the 3′-UTR, which potentially participate in the regulation of eNOS mRNA stability, have been conserved as the species diverged during evolution.
It is important to note that identification of eNOS mRNA-binding proteins and the mRNA sequences to which they bind is just the first step in the characterization of the molecular mechanisms regulating eNOS mRNA stability. Evidence that levels of mRNA-binding proteins are inversely correlated with mRNA stability, as has been shown for postconfluent and TNF-α–exposed endothelial cells, suggests a role for these proteins in the regulation of eNOS mRNA stability, but it is not conclusive. The observation that mRNA sequences, which bind proteins in extracts of postconfluent endothelial cells, can destabilize fusion mRNAs in “stably transfected” endothelial cells also supports a role for these cis-acting sequences in the regulation of eNOS mRNA stability, but it remains to be determined whether these sequences are responsible for the change in mRNA stability associated with the transition of endothelial cells from a proliferative to a postconfluent state. It will also be important to test whether these “destabilizing” cis-acting sequences function in the context of the eNOS mRNA itself, rather than in a fusion mRNA.
An important caveat is that there is nothing “stable” about “stably transfected” endothelial cells. The process of selection for cells expressing the transgene (and the selectable marker) requires multiple cell divisions, and it well-known that the phenotype of endothelial cells in primary culture changes dramatically as they are passaged. It is possible that differences in the rates of fusion mRNA degradation in actinomycin D–treated, “stably transfected” endothelial cells reflect differences in degree of endothelial cell “de-differentiation” rather than inherent differences in fusion mRNA stability. Studies of cis-acting mRNA sequences in endothelial cells (prior to de-differentiation and, ideally, in vivo) will require other gene transfer methods such as transient-transfection techniques, virus-mediated gene transfer, and transgenic animals.
Ultimately, the identification of cis-acting sequences regulating eNOS mRNA stability by Searles et al8 and others will lead to isolation of cDNA clones encoding the corresponding mRNA-binding proteins. Only with these cDNAs as tools will it be possible to demonstrate conclusively whether or not the encoded RNA-binding proteins participate in the regulation of eNOS mRNA stability. If these RNA-binding proteins destabilize eNOS mRNA, the next step will be to learn how they recognize the cis-acting mRNA sequences (primary and/or secondary structures). Moreover, it will become feasible to characterize whether the RNA-binding proteins destabilize eNOS mRNA on their own, potentially via poly(A) shortening, exonucleolytic, or endonucleolytic activities, or whether they act as “docking ports” for recruiting other proteins with these activities. Hence, the studies of Searles et al8 and others provide a critical foundation for future explorations of what are likely to be multiple regulatory RNA-binding proteins “crowded” on the eNOS mRNA 3′-UTR and may lead to new insights into how endothelial NO production is regulated and, perhaps more fundamentally, how mammalian cells regulate mRNA stability.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 1999 American Heart Association, Inc.
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