Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabolic (or atrophic) processes. Previously, no link between the molecular mediators of atrophy and hypertrophy had been reported. We demonstrate a hierarchy between the signals which mediate hypertrophy and those which mediate atrophy: the IGF-1/PI3K/Akt pathway, which has been shown to induce hypertrophy, prevents induction of requisite atrophy mediators, namely the muscle-specific ubiquitin ligases MAFbx and MuRF1. Moreover, the mechanism for this inhibition involves Akt-mediated inhibition of the FoxO family of transcription factors; a mutant form of FOXO1, which prevents Akt phosphorylation, thereby prevents Akt-mediated inhibition of MuRF1 and MAFbx upregulation. Our study thus defines a previously uncharacterized function for Akt, which has important therapeutic relevance: Akt is not only capable of activating prosynthetic pathways, as previously demonstrated, but is simultaneously and dominantly able to suppress catabolic pathways, allowing it to prevent glucocorticoid and denervation-induced muscle atrophy.

Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1(-/-) mice are treated with DEX, the MuRF1(-/-) animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.

Shigellae cause bacillary dysentery, a bloody form of diarrhoea that affects almost 200 million people and causes nearly 2 million deaths per year. Shigella invades the colonic mucosa, where it initiates an acute inflammation, rich in neutrophils, that initially contributes to tissue damage and eventually resolves the infection. Neutrophils are phagocytic cells that kill microorganisms but it is unclear how neutrophils control pathogenic bacteria expressing virulence factors that manipulate host cells. In contrast to other cells, neutrophils prevent the escape of Shigella from phagocytic vacuoles in which the bacteria are killed. Here we identify human neutrophil elastase (NE) as a key host defence protein: NE degrades Shigella virulence factors at a 1,000-fold lower concentration than that needed to degrade other bacterial proteins. In neutrophils in which NE is inactivated pharmacologically or genetically, Shigella escapes from phagosomes, increasing bacterial survival. NE also preferentially cleaves virulence factors of Salmonella and Yersinia. These findings establish NE as the first neutrophil factor that targets bacterial virulence proteins.

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The transcriptional regulation of peroxisome proliferator-activated receptor (PPAR) α by post-translational modification, such as ubiquitin, has not been described. We report here for the first time an ubiquitin ligase (muscle ring finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARα, but not PPARβ/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts showed significant decreases in nuclear PPARα activity and acyl-carnitine intermediates, while MuRF1−/− hearts exhibited increased PPARα activity and acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid oxidation in a proteasome independent manner. We then identified a previously undescribed nuclear export sequence in PPARα, along with three specific lysines (292, 310, 388) required for MuRF1s targeting of nuclear export. These studies identify the role of ubiquitination in regulating cardiac PPARα, including the ubiquitin ligase that may be responsible for this critical regulation of cardiac metabolism in heart failure.

Expression of heat shock proteins (hsps) results in the protection of cells from subsequent stresses. However, hsps are also toxic when present within cells for a prolonged time period. Thus, the expression of hsps should be tightly regulated. In the present study, the expression of Hsp70 after heat shock was compared between thermotolerant cells, which contain a large concentration of Hsp70, and nonthermotolerant cells (naive). Accumulation of Hsp70, assessed by Western blotting, was negligible when thermotolerant cells were heat-shocked a second time. Hsp70 transcription was similar between thermotolerant and naive cells during heat shock. However, Hsp70 transcription was attenuated more rapidly in thermotolerant than naive cells immediately upon return to non-heat shock conditions. In addition, Hsp70 mRNA stability was reduced in thermotolerant cells as compared with naive cells following the stress. New synthesis of Hsp70 and the efficiency of Hsp70 mRNA translation were similar between thermotolerant and naive cells during the post-stress period. These results suggest that thermotolerant cells limit Hsp70 expression by transcriptional and pretranslational mechanisms, perhaps to avoid the potential cytotoxic effect of these proteins.

Cellular communication mediated by gap junctions plays a major role in organ function. Gap junction channels are formed by the organization of polypeptide subunits, termed connexins (Cx), on the cell surface of adjacent cells. One mechanism to regulate gap-junctional communication is by change in Cx expression. In the present study, the promoter region of the rat Cx43 gene was characterized. Nested deletions of the 5' flanking region of the first Cx43 exon were coupled to the human growth hormone gene and transfected into normal rat kidney (NRK) cells, that express this gene constitutively. The minimal region of the Cx43 gene that showed maximal promoter activity was localized within 110 bp upstream of the transcriptional initiation site. One particular subregion that contains a Sp-1 binding site (located within 98--93 bp from the transcriptional initiation site) was found to sustain Cx43 promoter activity to the same extent as that of the 110 bp promoter region. Mutations of this Sp-1 binding site abolished transcriptional activity and DNA-protein interactions. These observations suggest that the Sp-1 binding site plays a major role in the basal transcriptional activity of Cx43 gene in NRK cells.

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