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S R Price

Publications and source records attributed to S R Price.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of epithelial sodium channels by the ubiquitin-proteasome proteolytic pathway.

Amiloride-sensitive epithelial Na+ channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends on a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits: alpha, beta, and gamma. The COOH-terminal domain of all three subunits is intracellular and contains a proline-rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta- and gamma-subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell-expressed, developmentally downregulated protein (Nedd4-2), to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when Madin-Darby canine kidney cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected renal cells (A6) expressing endogenous ENaC, ENaC is indeed degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by Nedd4-2 activity. In this review, we discuss the role of the ubiquitin conjugation and the alternative degradative pathways (lysosomal or proteasomal) in regulating the rate of ENaC turnover in untransfected renal cells and compare this regulation to that of transfected cell systems.

Animals↗
PubMed2005

Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits.

Amiloride-sensitive epithelial sodium channels (ENaC) are responsible for transepithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, and gamma). In Madin-Darby canine kidney (MDCK) cells and Xenopus laevis oocytes transfected with all three ENaC subunits, neural precursor cell-expressed developmentally downregulated protein (Nedd4-2) promotes ubiquitin conjugation of ENaC. For native proteins in some cells, ubiquitin conjugation is a signal for their degradation by the ubiquitin-proteasome pathway, whereas in other cell types ubiquitin conjugation is a signal for endocytosis and lysosomal protein degradation. When ENaC are transfected into MDCK cells, ubiquitin conjugation leads to lysosomal degradation. In this paper, we characterize the involvement of the ubiquitin-proteasome proteolytic pathway in the regulation of functional ENaC in untransfected renal A6 cells expressing native ENaC subunits. In contrast to transfected cells, we show that total cellular alpha-, beta-, and gamma-ENaC subunits are polyubiquitinated and that ubiquitin conjugation of subunits increases when the cells are treated with a proteasome inhibitor. We show that Nedd4-2 is associated with alpha- and beta-subunits and is associated with the apical membrane. We also show the Nedd4-2 can regulate the number of functional ENaC subunits in the apical membrane. The results reported here suggest that the ubiquitin-proteasome proteolytic pathway is an important determinant of ENaC function in untransfected renal cells expressing endogenous ENaC.

Animals↗
PubMed2003

Increased transcription of ubiquitin-proteasome system components: molecular responses associated with muscle atrophy.

Muscle atrophy is a common consequence of catabolic conditions like kidney failure, cancer, sepsis, and acute diabetes. Loss of muscle protein is due primarily to activation of the ubiquitin-proteasome proteolytic system. The proteolytic responses to catabolic signals include increased levels of mRNA that encode various components of the system. In the case of two genes, the proteasome C3 subunit and ubiquitin UbC, the higher levels of mRNA result from increased transcription but the mechanisms of transactivation differ between them. This review summaries the evidence that cachectic signals activate a program of selective transcriptional responses in muscle that frequently occurs coordinately with increased protein destruction.

Animals↗
PubMed2002

Effects of high doses of glucocorticoids on free amino acids, ribosomes and protein turnover in human muscle.

BACKGROUND: Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. MATERIALS AND METHODS: Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. RESULTS: The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. CONCLUSIONS: These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.

Adult↗
PubMed2001

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.

Amino Acid Sequence↗
PubMed2001

Molecular mechanisms regulating protein turnover in muscle.

Loss of muscle mass is a risk factor for mortality in chronic renal failure (CRF). Catabolic signals (eg, acidosis, glucocorticoids, insulin resistance) present in CRF stimulate the ubiquitin-proteasome proteolytic pathway in muscle but the activation mechanism(s) have been elusive. We have identified distinct mechanisms that may work in concert to increase the degradation of muscle proteins. Glucocorticoids increase the transcription of genes encoding components of the ubiquitin-proteasome pathway, thereby increasing the proteolytic capacity of muscle cells. Another signal could be a decreased response to insulin because acute diabetes is a potent stimulus for protein degradation by the ubiquitin-proteasome pathway and CRF impairs insulin signaling in muscle. Together, these responses increase the breakdown of muscle, contributing to protein malnutrition in CRF.

Animals↗
PubMed2001

Mechanisms activated by kidney disease and the loss of muscle mass.

The daily turnover of cellular proteins is large, with amounts equivalent to the protein contained in 1.0 to 1.5 kg of muscle. Consequently, even a small, persistent increase in the rate of protein degradation or decrease in protein synthesis will result in substantial loss of muscle mass. Activation of protein degradation in the ubiquitin-proteasome system is the mechanism contributing to loss of muscle mass in kidney disease. Because other catabolic conditions also stimulate this system to cause loss of muscle mass, the identification of activating signals is of interest. A complication of kidney disease, metabolic acidosis, activates this system in muscle by a process that requires glucocorticoids. The influence of inflammatory cytokines on this system in muscle is more complicated, as evidence indicates that cytokines suppress the system, but glucocorticoids block the effect of cytokines to slow protein breakdown in the system. New information identifying mechanisms that activate protein breakdown and the rebuilding of muscle fibers would lead to therapies that successfully prevent the loss of muscle mass in kidney disease and other catabolic illnesses.

Acidosis↗
PubMed2001

Acidification and glucocorticoids independently regulate branched-chain alpha-ketoacid dehydrogenase subunit genes.

Acidification or glucocorticoids increase the maximal activity and subunit mRNA levels of branched chain alpha-ketoacid dehydrogenase (BCKAD) in various cell types. We examined whether these stimuli increase transcription of BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase plasmids containing the mouse E2 or human E1alpha-subunit promoter into LLC-PK(1) cells, which do not express glucocorticoid receptors, or LLC-PK(1)-GR101 cells, which we have engineered to constitutively express the glucocorticoid receptor gene. Dexamethasone or acidification increased luciferase activity in LLC-PK(1)-GR101 cells transfected with the E2 or E1alpha-minigenes; acidification augmented luciferase activity in LLC-PK(1) cells transfected with these minigenes but dexamethasone did not. A pH-responsive element in the E2 subunit promoter was mapped to a region >4.0 kb upstream of the transcription start site. Dexamethasone concurrently stimulated E2 subunit promoter activity and reduced the binding of nuclear factor-kappaB (NF-kappaB) to a site in the E2 promoter. Thus acidification and glucocorticoids independently enhance BCKAD subunit gene expression, and the glucocorticoid response in the E2 subunit involves interference with NF-kappaB, which may act as a transrepressor.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2001

Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down-regulates autocrine insulin-like growth factor I.

We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown was accelerated; inhibiting lysosomal and calcium-activated proteases did not reduce the ang II-induced increase in muscle proteolysis. Circulating IGF-I levels were 33% lower in ang II rats vs. control rats, and this difference was reflected in lower IGF-I messenger RNA levels in the liver. Moreover, IGF-I, IGF-binding protein-3, and IGF-binding protein-5 messenger RNAs in the gastrocnemius were significantly reduced. To investigate whether the reduced circulating IGF-I accounts for the loss in muscle mass, we increased circulating IGF-I by coinfusing ang II and IGF-I, but this did not prevent muscle loss. Our data suggest that ang II causes a loss in skeletal muscle mass by enhancing protein degradation probably via its inhibitory effect on the autocrine IGF-I system.

Angiotensin II↗
PubMed2000

Tools for evaluating ubiquitin (UbC) gene expression: characterization of the rat UbC promoter and use of an unique 3' mRNA sequence.

UbC is one of three members of the ubiquitin gene family. We have cloned the rat UbC promoter and used primer extension analysis to map the UbC site of transcription initiation to 63 bp upstream of the putative first intron. We used a rat UbC promoter-luciferase reporter minigene to transfect H9c2 cardiomyocytes, HepG2 hepatocytes, CaCo2 colon cells, NIH3T3 fibroblasts or L6 myocytes and found the rat UbC promoter has constitutive activity. We also showed that dexamethasone stimulated the UbC promoter in L6 myocytes. Finally, we showed that a UbC-specific sequence at the 3' end of the rat UbC mRNA transcript can be used to selectively and quantitatively measure UbC: (1) mRNA using a RNase protection assay, and (2) transcription using a nuclear run-off assay to measure the rate of transcription of the UbC gene. These findings will be useful in studying the regulation of the UbC gene.

3' Untranslated Regions↗
PubMed2000

Glucocorticoids induce proteasome C3 subunit expression in L6 muscle cells by opposing the suppression of its transcription by NF-kappa B.

Muscle wasting in catabolic conditions results from activation of the ubiquitin-proteasome proteolytic pathway by a process that requires glucocorticoids and is generally associated with increased levels of mRNAs encoding components of this proteolytic system. In L6 muscle cells, dexamethasone stimulates proteolysis and increases the amount of the proteasome C3 subunit protein by augmenting its transcription. Transfection studies with human C3 promoter-luciferase reporter genes and electrophoretic mobility shift assays revealed that a NF-kappaB.protein complex containing Rel A is abundant in L6 muscle cell nuclei. Glucocorticoids stimulate C3 subunit expression by antagonizing the interaction of this NF-kappaB protein with an NF-kappaB response element in the C3 subunit promoter region. Dexamethasone also increased the cytosolic amounts of the NF-kappaB p65 subunit and the IkappaBalpha inhibitor proteins in L6 cells. Incubation of L6 cells with a cytokine mixture not only increased the amount of activated NF-kappaB but also decreased C3 promoter activity and lowered endogenous C3 subunit mRNA. Thus, NF-kappaB is a repressor of C3 proteasome subunit transcription in muscle cells, and glucocorticoids stimulate C3 subunit expression by opposing this suppressor action.

Animals↗
PubMed2000

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.

Animals↗
PubMed1999

Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting.

The ubiquitin-proteasome proteolytic system is stimulated in conditions causing muscle atrophy. Signals initiating this response in these conditions are unknown, although glucocorticoids are required but insufficient to stimulate muscle proteolysis in starvation, acidosis, and sepsis. To identify signals that activate this system, we studied acutely diabetic rats that had metabolic acidosis and increased corticosterone production. Protein degradation was increased 52% (P < 0.05), and mRNA levels encoding ubiquitin-proteasome system components, including the ubiquitin-conjugating enzyme E214k, were higher (transcription of the ubiquitin and proteasome subunit C3 genes in muscle was increased by nuclear run-off assay). In diabetic rats, prevention of acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored by administration of a physiological dose of glucocorticoids to adrenalectomized, diabetic rats. Finally, treating diabetic rats with insulin for >/=24 h reversed muscle proteolysis and returned pathway mRNAs to control levels. Thus acidification is not necessary for these responses, but glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system.

Acidosis↗
PubMed1999

Angiotensin II stimulates gene expression of cardiac insulin-like growth factor I and its receptor through effects on blood pressure and food intake.

Angiotensin II (Ang II) is known to act as a growth factor and may be involved in cardiac remodeling. We have shown that insulin-like growth factor-I (IGF-I) is an autocrine mediator of growth responses to Ang II in vascular smooth muscle cells in vitro, and we hypothesized that IGF-I also serves as an important modulator of cardiovascular growth in vivo. To study the effect of Ang II on cardiac IGF-I, we infused rats for 3, 7, or 14 days with Ang II through osmotic minipumps. After 7 days, left ventricular mass normalized for body weight was increased by 20% (P<0.01) in Ang II rats compared with pair-fed control rats that were given a restricted amount of food identical to that eaten by the anorexic, Ang II-infused rats. Ang II increased left ventricular IGF-I mRNA levels by 1.5- to 1.8-fold compared with ad libitum-fed or pair-fed control rats (P<0.05). Cardiac IGF-I protein was increased correspondingly and was localized on the cardiomyocytes. Treatment with hydralazine abolished the induction of IGF-I mRNA, which indicates that Ang II induces cardiac IGF-I mRNA expression through a pressor-mediated mechanism. IGF-I receptor (IGF-IR) mRNA was induced 2.1-fold in Ang II rats compared with ad libitum-fed rats (P<0.01). However, this increase was also observed in pair-fed controls and is thus due to the anorexigenic effect of Ang II. We have recently shown that circulating IGF-I levels are reduced in response to Ang II infusion. Elevation of IGF-I levels by coinfusion of IGF-I and Ang II significantly increased left ventricular index by 16% compared with rats infused with Ang II alone (P<0.05). In conclusion, autocrine upregulation of cardiac IGF-I and IGF-IR mRNA by Ang II occurs through hemodynamic and nonhemodynamic mechanisms, respectively, and may modulate cardiac structural changes that occur in hypertension.

Angiotensin II↗