WBSCR22 and TRMT112 synergistically suppress cell proliferation, invasion and tumorigenesis in pancreatic cancer via transcriptional regulation of ISG15.

Pancreatic cancer (PC) is one of the most aggressive and devastating types of cancer owing to its poor prognosis and deadly characteristics. It is well established that aberrations in the expression of key regulatory genes, namely tumor suppressors and oncogenes, predispose patients to progression and metastasis of PC. Upregulation of Williams­Beuren syndrome chromosomal region 22 (WBSCR22) expression, a ribosomal biogenesis factor, has been reported in multiple types of human cancer. However, the role of WBSCR22 and its underlying mechanism in PC have not been well investigated. In the present study, the tumor suppressive role of WBSCR22 was reported in PC for the first time; the results indicated that WBSCR22 overexpression (OE) significantly suppressed cellular proliferation, migration, invasion and tumorigenesis in vivo and in vitro. RNA­sequencing analysis revealed that WBSCR22 negatively regulated the transcription of interferon­stimulated gene 15 (ISG15) downstream, which is a ubiquitin­like modifier protein involved in metabolic and proteasome degradation pathways, while the antitumor function of WBSCR22­OE could be rescued by ISG15 OE. In addition, the oncogenic role of ISG15 was further confirmed in PC; its upregulation promoted the proliferation, migration, invasion and tumorigenesis of PC. Furthermore, WBSCR22 and its cofactor tRNA methyltransferase activator subunit 11­2 (TRMT112) functioned synergistically in PC, and concurrent ectopic OE of WBSCR22 and TRMT112 further promoted the tumor suppressive potential of WBSCR22 in PC. Collectively, the findings indicated that WBSCR22 played an important role in PC development and that the WBSCR22/ISG15 axis may provide a novel therapeutic strategy for PC treatment.

IFNß Treatment Inhibits Nerve Injury-induced Mechanical Allodynia and MAPK Signaling By Activating ISG15 in Mouse Spinal Cord.

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for interferon ß (IFNß) in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNß (dosages up to 5,000 U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein interferon-stimulated gene 15 (ISG15), which is potently induced within the spinal cord following intrathecal delivery of IFNß. Both free and conjugated ISG15 are elevated following IFNß treatment, and this effect is increased in UBP43-/- mice lacking a key deconjugating enzyme. The IFNß-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNß, ISG15, and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities. PERSPECTIVE: Neuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the antinociceptive potential of IFNß and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies.

The effects of proteasome on baseline and methamphetamine-dependent dopamine transmission.

The Ubiquitin Proteasome System (UPS) is a major multi-catalytic machinery, which guarantees cellular proteolysis and turnover. Beyond cytosolic and nuclear cell compartments, the UPS operates at the synapse to modulate neurotransmission and plasticity. In fact, dysregulations of the UPS are linked with early synaptic alterations occurring in a variety of dopamine (DA)-related brain disorders. This is the case of psychiatric conditions such as methamphetamine (METH) addiction. While being an extremely powerful DA releaser, METH impairs UPS activity, which is largely due to DA itself. In turn, pre- and post- synaptic neurons of the DA circuitry show a high vulnerability to UPS inhibition. Thus, alterations of DA transmission and UPS activity are intermingled within a chain of events underlying behavioral alterations produced by METH. These findings, which allow escaping the view of a mere implication of the UPS in protein toxicity-related mechanisms, indicate a more physiological role for the UPS in modulating DA-related behavior. This is seminal for those plasticity mechanisms which underlie overlapping psychiatric disorders such as METH addiction and schizophrenia.

Inhibition of Cyclic GMP-AMP Synthase Using a Novel Antimalarial Drug Derivative in Trex1-Deficient Mice.

OBJECTIVE: Type I interferon (IFN) is strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE) as well as rare monogenic interferonopathies such as Aicardi-Goutières syndrome (AGS), a disease attributed to mutations in the DNA exonuclease TREX1. The DNA-activated type I IFN pathway cyclic GMP-AMP (cGAMP) synthase (cGAS) is linked to subsets of AGS and lupus. This study was undertaken to identify inhibitors of the DNA-cGAS interaction, and to test the lead candidate drug, X6, in a mouse model of AGS. METHODS: Trex1-/- mice were treated orally from birth with either X6 or hydroxychloroquine (HCQ) for 8 weeks. Expression of IFN-stimulated genes (ISGs) was quantified by quantitative polymerase chain reaction. Multiple reaction monitoring by ultra-performance liquid chromatography coupled with tandem mass spectrometry was used to quantify the production of cGAMP and X6 drug concentrations in the serum and heart tissue of Trex1-/- mice. RESULTS: On the basis of the efficacy-to-toxicity ratio established in vitro, drug X6 was selected as the lead candidate for treatment of Trex1-/- mice. X6 was significantly more effective than HCQ in attenuating ISG expression in mouse spleens (P < 0.01 for Isg15 and Isg20) and hearts (P < 0.05 for Isg15, Mx1, and Ifnb, and P < 0.01 for Cxcl10), and in reducing the production of cGAMP in mouse heart tissue (P < 0.05), thus demonstrating target engagement by the X6 compound. Of note, X6 was also more effective than HCQ in reducing ISG expression in vitro (P < 0.05 for IFI27 and MX1, and P < 0.01 for IFI44L and PKR) in human peripheral blood mononuclear cells from patients with SLE. CONCLUSION: This study demonstrates that X6 is superior to HCQ for the treatment of an experimental autoimmune myocarditis mediated in vivo by the cGAS/stimulator of IFN genes (cGAS/STING) pathway. The findings suggest that drug X6 could be developed as a novel treatment for AGS and/or lupus to inhibit activation of the cGAS/STING pathway.

Computational Analysis and Functional Prediction of Ubiquitin Hypothetical Protein: A Possible Target in Parkinson Disease.

Parkinson's disease (PD) is a high prevalent progressive neurodegenerative disorder characterized by degeneration of dopaminergic neurons and intracytoplasmatic aggregation of α-synuclein called Lewy Bodies. Anomalies in the proteasomal and endosomal ubiquitin related degradation of α-synuclein have been related with PD. Among the different proteins described in ubiquitin pathway, the hypothetical protein CAB55973.1 was identified previously. Here we modeled this hypothetical protein in order to contribute to the understanding of PD pathogenesis that should be served as an input in the future as drug targets. This study predicted a three-dimensional model of the complete sequence of hypothetical protein CAB55973.1 with a high value of identity and a good homology quality. Subcellular localization was found in the cytoplasm, mainly in the endosomal membrane. 36 protein-protein interactions related to PD were found. 11 residues were predicted to interact with target proteins for ubiquitination. Binding site prediction showed that one domain (residues 163 to 238) might be involved in ubiquitination of target proteins. In this ubiquitin domain, residues were distributed similarly to those of the binding site of the ubiquitin interacting with the UIM of Hrs protein (PDB 2D3G). The hypothetical protein was constructed based on the complete sequence alignment, which allowed predicting the structure with a high accuracy. The functional prediction showed that only one domain of the hypothetical protein might be involved in the α- synuclein ubiquitination of the endosomal pathway of the PD.

A highly reproducible rotenone model of Parkinson's disease.

The systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.

Deubiquitinating enzymes as novel anticancer targets.

Tagging proteins with mono- or poly-ubiquitin is now recognized as a multifaceted and universal means of regulating cell growth and physiology. It does so by controlling the cellular lifetime of nearly all eukaryotic proteins and the cellular localization of many critical proteins. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases [DUBs]) ubiquitin tags to or from their target proteins in a selective fashion. Similarly to the kinases and their corresponding phosphatases, ubiquitin ligases and DUBs have become actively studied molecular oncology targets for drug discovery. Approximately 79 functional DUBs exist in the human proteome, suggesting that selective intervention is a reasonable therapeutic objective, with the goal of downregulating or ablating oncogene products or, alternatively, upregulating or sparing tumor suppressors. In the following review, this fascinating class of regulatory enzymes will be described, and specific examples of DUBs that are viable targets for anticancer therapy will be considered.

Kainic acid induces early and transient autophagic stress in mouse hippocampus.

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2-16 h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4-6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/mTOR were elevated from 6 to 16 h, and ratios of phospho-Akt/Akt were elevated at 16 h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus.

Interferon-alpha suppresses proliferation of chronic myelogenous leukemia cells K562 by extending cell cycle S-phase without inducing apoptosis.

We examined the effects of interferon-alpha (IFN-alpha) treatment on the growth, cell cycle, proliferation, and apoptotic parameters as well as adhesive properties and proteome of chronic myelogenous leukemia (CML)-derived K562 cells. IFN-alpha treatment (200 to 600 U/ml, 24 to 72 h) suppressed growth and caused accumulation of K562 cells in the S-phase of cell cycle (increase in S-phase cells by up to 52% in comparison with the untreated controls) at the expenses of cells in G1-phase. No transition of cells to G0-phase occurred as followed from Ki-67 protein determination. Although the level of chimeric gene product, BCR-ABL mRNA coding for BCR-ABL protein with anti-apoptotic properties, decreased by 30%, apoptosis was not triggered as judged from Annexin-V, APO2.7, and TUNEL assays. Adhesion of K562 cells to fibronectin-coated surfaces increased by up to 52% as determined by calcein assay. The proteomic analysis (2-D electrophoresis in combination with mass spectrometry, MALDI-MS) revealed a single protein, ubiquitine cross-reactive protein (UBCR), whose level markedly increased due to IFN-alpha treatment. The ubiquitination-like directed degradation processes may thus play a role in the mechanism of IFN-alpha antiproliferative effects.

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin. In apoptotic cells, ubiquitin was cleaved from uH2A subsequent to the appearance of plasma membrane blebbing, and deubiquitination of uH2A closely coincided with the onset of nuclear pyknosis and chromatin condensation. Nucleosomal uH2A deubiquitination, poly (ADP-ribose)polymerase (PARP) cleavage and chromatin condensation were prevented in cells challenged with apoptosis inducers by pretreatment with the pan-caspase inhibitor, zVAD-fmk, or by over-expressing anti-apoptotic Bcl-xL protein. These results implicate a connection between caspase cascade activation and nucleosomal uH2A deubiquitination. Transient transfection of 293 cells with the gene encoding Ubp-M, a human deubiquitinating enzyme, promoted uH2A deubiquitination, while an inactive mutated Ubp-M enzyme did not. However, Ubp-M-promoted deubiquitination of uH2A was insufficient to initiate apoptosis in these cells. We conclude that uH2A deubiquitination is a down-stream consequence of procaspase activation and that unscheduled cleavage of ubiquitin from uH2A is a consistent feature of the execution phase of apoptosis rather than a determining or initiating apoptogenic event. Nucleosomal uH2A deubiquitination may function as a cellular sensor of stress in situations like apoptosis through which cells attempt to preserve genomic integrity.

Insights into the stability of native and partially folded states of ubiquitin: effects of cosolvents and denaturants on the thermodynamics of protein folding.

The thermodynamics of the native<-->A state and native<-->unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn.HCl) both separately and in combination. Gdn.HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native<-->unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn.HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same "native" state; however, the thermodynamics of the N<-->U transition change dramatically between alcoholic and Gdn.HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in DeltaC(p) at higher methanol concentrations, as previously reported for the N<-->A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P. A. (1993) J. Mol. Biol. 229, 502-511.). Both the N<-->U and the N<-->A transitions are enthalpy driven to a similar extent. We conclude that under these conditions van der Waals interactions in the packing of the nonpolar protein core, which is common to both the N<-->U and the N<-->A transitions, appear to drive folding in the absence of entropic effects associated with release of ordered solvent (hydrophobic effect). Solvent transfer studies of hydrocarbons into alcoholic solvents, with and without Gdn.HCl, are consistent with a large enthalpic driving force for burial of a nonpolar surface, with a linear dependence of protein stability (DeltaG(N)(<-->)(U)) on cosolvent concentration reflected in a similar linear dependence of hydrocarbon solubility. The data demonstrate that the hydrophobic effect is not a prerequisite for specific stabilization of the native state or the A state and that van der Waals packing of the nonpolar core appears to be the dominant factor in stabilization of the native state.

Ubiquitin-mediated degradation of cellular proteins: why destruction is essential for construction, and how it got from the test tube to the patient's bed.

Between the 1960s and 1980s, the main focus of biological research was nucleic acids and the translation of the coded information into proteins. Protein degradation was a neglected area and regarded by many as a scavenger, non-specific and end process. While it was known that proteins are turning over, the large extent and high specificity of the process--where distinct proteins have half-lives that range from a few minutes to several days--have not been appreciated. The discovery of the lysosome by Dr. Christian de Duve did not change this view significantly, as this organelle is involved mostly in the degradation of extra- and not intracellular proteins, and it was clear that lysosomal proteases, similar to those of the gastrointestinal tract, cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway has changed this view dramatically. It is now clear that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a broad array of basic pathways during cell life and death. With the multitude of substrates targeted and processes involved, it is not surprising that aberrations in the pathway have been recently implicated in the pathogenesis of many diseases, certain malignancies and neurodegeneration among them. Degradation of a protein via the ubiquitin pathway involves two successive steps: a) conjugation of multiple ubiquitin moieties to the substrate, and b) degradation of the tagged protein by the downstream 26S proteasome complex with release of free and re-utilizable ubiquitin. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation and major key problems remain unsolved. Among these are the modes of specific and timed recognition of the myriad substrates of the system and the nature of the mechanisms that underlie aberrations in the system and pathogenesis of diseases.

The ubiquitin-proteasome 26s pathway in liver cell protein turnover: effect of ethanol and drugs.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Samuel W. French and R. J. Mayer. The presentations were (1) The ubiquitin-proteasome 26s pathway in liver cell protein turnover: Effect of alcohol and drugs, by Samuel W. French and F. Bardag-Gorce; (2) The role of CYP2E1 phosphorylation and degradation pathway in the induction of the enzyme, by Magnus Ingelman-Sundberg; (3) Role of proteasome in the proteolysis of oxidized proteins in experimental chronic alcoholism, by Helen Rouach; (4) Alcohol, proteolysis and liver cancer, by R. J. Mayer; (5) Effect of ethanol feeding on the ATP-ubiquitin-proteasome pathway in the liver cell, by F. Bardag-Gorce; (6) Novel mechanisms and targets for intracellular transport of CYP2E1, by E. Neve; and (7) Gankyrin, an oncoprotein commonly over expressed in hepatoma, by H. Higashitsuji.

6-Hydroxydopamine increases ubiquitin-conjugates and protein degradation: implications for the pathogenesis of Parkinson's disease.

One of the hallmarks of Parkinson's disease (PD) is pathological structure, termed Lewy body, containing inclusions of ubiquitinated proteins in the dopaminergic neurons in the substantia nigra. The mechanism leading to the formation of these aggregates is unclear, although it has been shown that mutations in alpha-synuclein or in the ubiquitin-related enzyme UCH-L1 might induce such protein aggregation. We, therefore, examined the possible role of 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin used in PD experimental models, in causing protein degradation and its association with the ubiquitin system. Using antiubiquitin antibodies we found that exposure of SH-SY5Y neuroblastoma and PC-12 cell lines to 6-OHDA increased the levels of free ubiquitin and ubiquitin-conjugated proteins, in a dose-dependent manner. Furthermore, metabolic labeling with 35S-methionine, demonstrated that 6-OHDA markedly increased protein degradation, as indicated by the secretion of protein metabolites to the medium. Inhibition of the proteasome activity by the specific inhibitor MG132, attenuated the protein degradation induced by 6-OHDA and potentiated its toxicity. Administration of the antioxidant N-acetylcysteine to the 6-OHDA-treated cells, increased cell survival and reduced protein degradation. In conclusion, our findings suggest that 6-OHDA toxicity is associated with protein degradation and ubiquitin-proteasome system activation.

Responses to peroxynitrite in yeast: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a sensitive intracellular target for nitration and enhancement of chaperone expression and ubiquitination.

Peroxynitrite (ONOO-), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. In S. cerevisiae, a model eukaryotic cell system, ONOO- was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO- in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO- in yeast and in cultured rat astrocytes. Thus, ONOO- is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated.

Interferon-tau and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a functional ubiquitin homolog synthesized by the ruminant endometrium in response to conceptus-derived interferon-tau (IFNtau). Progesterone is required for IFNtau to exert antiluteolytic actions on the endometrium. Therefore, this study was designed to determine whether progesterone is requisite for IFNtau induction of UCRP expression within the ovine uterus. Cyclic ewes were ovariectomized and fitted with intrauterine (i.u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 137.316 (ZK; progesterone receptor antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. In P-treated ewes, roIFNtau increased endometrial UCRP mRNA and protein levels. However, administration of ZK to ewes ablated roIFNtau induction of UCRP. Recombinant ovine IFNtau induced expression of UCRP mRNA in progestinized endometrial luminal (LE) and glandular (GE) epithelium as well as in both stratum compactum and spongiosum layers of the stroma (ST). Progesterone receptor protein was located in endometrial ST, but not in LE and GE from these ewes. Results support the hypothesis that progesterone is required for IFNtau induction of type I IFN-responsive genes, such as UCRP, in the ruminant uterus.

Nitric oxide prevents p21 degradation with the ubiquitin-proteasome pathway in vascular smooth muscle cells.

PURPOSE: We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs. METHODS: p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer. p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with (32)P-labeled myelin basic protein as a substrate. RESULTS: NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose- and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase in both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO. CONCLUSION: These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.