Stewart H. Lecker

Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy

Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. In atrophying muscles, the ubiquitin ligase, atrogin-1 (MAFbx), is dramatically induced, and this response is necessary for rapid atrophy. Here, we show that in cultured myotubes undergoing atrophy, the activity of the PI3K/AKT pathway decreases, leading to activation of Foxo transcription factors and atrogin-1 induction. IGF-1 treatment or AKT overexpression inhibits Foxo and atrogin-1 expression. Moreover, constitutively active Foxo3 acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When Foxo activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction during starvation and atrophy of myotubes induced by glucocorticoids are prevented. Thus, forkhead factor(s) play a critical role in the development of muscle atrophy, and inhibition of Foxo factors is an attractive approach to combat muscle wasting.

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy.

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin‐induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair‐fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin‐1/MAFbx and MuRF‐1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down‐regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up‐regulated (lipin, metallothionein, AMP deaminase, RNA helicase‐related protein, TG interacting factor) and several growth‐related mRNAs were down‐regulated (P311, JUN, IGF‐1‐BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.—Lecker, S. H., Jagoe, R. T., Gilbert, A., Gomes, M., Baracos, V., Bailey, J., Price, S. R., Mitch, W. E., Goldberg, A. L. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression.—Stewart H. Lecker, R. Thomas Jagoe, Alexander Gilbert, Marcelo Gomes, Vickie Baracos, James Bailey, S. Russ Price, William E. Mitch, Alfred L. Goldberg Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J. 18, 39–51 (2004)

PGC-1α protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription

Maintaining muscle size and fiber composition requires contractile activity. Increased activity stimulates expression of the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which promotes fiber-type switching from glycolytic toward more oxidative fibers. In response to disuse or denervation, but also in fasting and many systemic diseases, muscles undergo marked atrophy through a common set of transcriptional changes. FoxO family transcription factors play a critical role in this loss of cell protein, and when activated, FoxO3 causes expression of the atrophy-related ubiquitin ligases atrogin-1 and MuRF-1 and profound loss of muscle mass. To understand how exercise might retard muscle atrophy, we investigated the possible interplay between PGC-1α and the FoxO family in regulation of muscle size. Rodent muscles showed a large decrease in PGC-1α mRNA during atrophy induced by denervation as well as by cancer cachexia, diabetes, and renal failure. Furthermore, in transgenic mice overexpressing PGC-1α, denervation and fasting caused a much smaller decrease in muscle fiber diameter and a smaller induction of atrogin-1 and MuRF-1 than in control mice. Increased expression of PGC-1α also increased mRNA for several genes involved in energy metabolism whose expression decreases during atrophy. Transfection of PGC-1α into adult fibers reduced the capacity of FoxO3 to cause fiber atrophy and to bind to and transcribe from the atrogin-1 promoter. Thus, the high levels of PGC-1α in dark and exercising muscles can explain their resistance to atrophy, and the rapid fall in PGC-1α during atrophy should enhance the FoxO-dependent loss of muscle mass.

The binding cascade of SecB to SecA to SecY E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases

We previously identified a common set of genes, termed atrogenes, whose expression is coordinately induced or suppressed in muscle during systemic wasting states (fasting, cancer cachexia, renal failure, diabetes). To determine whether this transcriptional program also functions during atrophy resulting from loss of contractile activity and whether atrogene expression correlates with the rate of muscle weight loss, we used cDNA microarrays and RT‐polymerase chain reaction to analyze changes in mRNA from rat gastrocnemius during disuse atrophy induced by denervation or spinal cord isolation. Three days after Den or SI, the rate of muscle weight loss was greatest, and 78% of the atrogenes identified during systemic catabolic states were induced or repressed. Of particular interest were the large inductions of key ubiquitin ligases, atrogin‐1 (35‐to 44‐fold) and MuRF1 (12‐to 22‐fold), and the suppression of PGC‐1α and PGC‐1ᵦ coactivators (15‐fold). When atrophy slowed (day 14), the expression of 92% of these atrogenes returned toward basal levels. At 28 days, the atrophy‐inducing transcription factor, FoxO1, was still induced and may be important in maintaining the “atrophied” state. Thus, 1) the atrophy associated with systemic catabolic states and following disuse involves similar transcriptional adaptations; and 2) disuse atrophy proceeds through multiple phases corresponding to rapidly atrophying and atrophied muscles that involve distinct transcriptional patterns. Sacheck, J. M., Hyatt, J‐P. K., Raffaello, A., Jagoe, R. T., Roy, R. R., Edgerton, V. R., Lecker, S. H., Goldberg, A. L. Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J. 21, 140–155 (2007)

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food‐deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin‐1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap‐independent mRNA translation rose. Significant changes occurred in mRNAs for many growth‐related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy‐specific changes in gene expression.—Jagoe, R. T., Lecker, S. H., Gomes, M., Goldberg, A. L. Patterns of gene expression in atrophying skeletal muscles: response to food deprivation. FASEB J. 16, 1697–1712 (2002)

TNF-α increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

In some inflammatory diseases, TNF‐α is thought to stimulate muscle catabolism via an NF‐κB‐dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF‐α stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF‐κB binding site;NF‐κB binding to this sequence is increased by TNF‐α stimulation. A dominant negative inhibitor of NF‐κB activation blocks both UbcH2 up‐regulation and the increase in ubiquitin‐conjugating activity stimulated by TNF‐α. In extracts from TNF‐α‐treated myotubes, ubiquitin‐conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild‐type UbcH2. Thus, UbcH2 up‐regulation is a novel response to TNF‐α/NF‐κB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.—Y.‐P.Li, S. H.Lecker, Y.Chen, I. D.Waddell, A. L.Goldberg, M. B.Reid TNF‐α increases ubiquitin‐conjugating activity in skeletal muscle by up‐regulating UbcH2/E220k. FASEB J. 17, 1048–1057 (2003)

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli‐‐SecB, trigger factor and GroEL‐‐have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed ‘chaperones’. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation‐ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statin-induced muscle toxicity

Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia. These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1alpha, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle damage induced by statins.