TRAF6 signaling in skeletal muscle atrophy and regeneration.

Skeletal muscle is the most abundant tissue in our body that provides a structural framework and regulates important biological processes. It is also a primary reservoir of protein. Skeletal muscle maintains its structural and functional integrity by finely balancing the rates of protein synthesis and degradation. Skeletal muscle also has a very well defined regeneration program to cope with muscle injuries. A disruption in any of these delicately balanced intracellular mechanisms of skeletal muscle results in devastating conditions such as atrophies and chronic injuries. Majority of these debilitating conditions in skeletal muscle eventually lead to morbidity and increased mortality and do not have available therapeutic interventions. The main aim of my research has been focused on understanding the role of an important adapter molecule tumor necrosis factor associated factor 6 (TRAF6) in skeletal muscle wasting and injury-induced regeneration. Using genetic mouse models of TRAF6 muscle-specific knock-out, this study has elucidated the regulatory role of TRAF6 in intracellular signaling pathways in skeletal muscle catabolism. In atrophic conditions, accelerated proteolytic degradation and activation of major catabolic mechanisms of skeletal muscle (p38MAPK, c-Jun N-terminal kinase, AMP activated kinase and NF-KB) cause of loss of skeletal muscle protein content and thus lead to reduced muscle fiber size and contractile ability. Myosin heavy chain, a major contractile protein of skeletal muscle is selectively targeted for degradation in response to different atrophic stimuli. In starvation-induced atrophy, endoplasmic reticulum stress and unfolded protein response were also found to be activated in addition to proteolytic mechanisms. Surprisingly, TRAF6 depletion in skeletal muscle of mice repressed activation of all these mediators of skeletal muscle atrophy and consequently, inhibited skeletal muscle atrophy. Taken together, this study has identified TRAF6 as an important regulator of skeletal muscle catabolic mechanisms in disuse and starvation-induced atrophy. Injury-induced regeneration of skeletal muscle is a highly complex interplay of different signaling networks and effectors. Our results show that TRAF6 activates pro-inflammatory signaling and promotes inflammation and necrosis in skeletal muscle and its depletion reduces inflammation and accelerates skeletal muscle regeneration

Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice

TRAF6 expression is enhanced during muscle atrophy and induces activation of signal transduction cascades that promote muscle wasting

TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle

Skeletal muscle wasting is a major reason for morbidity and mortality in many chronic disease states, disuse conditions and aging. The ubiquitin-proteasome and autophagy-lysosomal systems are the two major proteolytic pathways involved in regulation of both physiological and pathological muscle wasting. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an important adaptor protein involved in receptor-mediated activation of various signaling pathways in response to cytokines and bacterial products. TRAF6 also possesses E3 ubiquitin ligase activity causing lysine-63-linked polyubiquitination of target proteins. We have uncovered a novel role of TRAF6 in regulation of skeletal muscle mass. Muscle-wasting stimuli upregulate the expression, as well as the auto-ubiquitination, of TRAF6 leading to downstream activation of major catabolic pathways in skeletal muscle. Muscle-specific depletion of TRAF6 preserves skeletal muscle mass in a mouse model of cancer cachexia or denervation. Inhibition of TRAF6 also blocks the expression of the components of the ubiquitin-proteasome system (UPS) and autophagosome formation in atrophying skeletal muscle. While more investigations are required to understand its mechanisms of action in skeletal muscle, our results indicate that blocking TRAF6 activity can be used as a therapeutic approach to preserve skeletal muscle mass and function in different disease states and conditions

The Transcription Factor Nfatc2 Regulates β-Cell Proliferation and Genes Associated with Type 2 Diabetes in Mouse and Human Islets

<div><p>Human genome-wide association studies (GWAS) have shown that genetic variation at >130 gene loci is associated with type 2 diabetes (T2D). We asked if the expression of the candidate T2D-associated genes within these loci is regulated by a common locus in pancreatic islets. Using an obese F2 mouse intercross segregating for T2D, we show that the expression of ~40% of the T2D-associated genes is linked to a broad region on mouse chromosome (Chr) 2. As all but 9 of these genes are not physically located on Chr 2, linkage to Chr 2 suggests a genomic factor(s) located on Chr 2 regulates their expression in <i>trans</i>. The transcription factor <i>Nfatc2</i> is physically located on Chr 2 and its expression demonstrates <i>cis</i> linkage; <i>i</i>.<i>e</i>., its expression maps to itself. When conditioned on the expression of <i>Nfatc2</i>, linkage for the T2D-associated genes was greatly diminished, supporting <i>Nfatc2</i> as a driver of their expression. Plasma insulin also showed linkage to the same broad region on Chr 2. Overexpression of a constitutively active (ca) form of <i>Nfatc2</i> induced β-cell proliferation in mouse and human islets, and transcriptionally regulated more than half of the T2D-associated genes. Overexpression of either ca-Nfatc2 or ca-Nfatc1 in mouse islets enhanced insulin secretion, whereas only ca-Nfatc2 was able to promote β-cell proliferation, suggesting distinct molecular pathways mediating insulin secretion <i>vs</i>. β-cell proliferation are regulated by NFAT. Our results suggest that many of the T2D-associated genes are downstream transcriptional targets of NFAT, and may act coordinately in a pathway through which NFAT regulates β-cell proliferation in both mouse and human islets.</p></div

NFAT triggers β-cell proliferation in mouse and human islets.

<p>Adenoviruses (Ad) were used to overexpress constitutively active (ca) <i>Nfatc1</i> or <i>Nfatc2</i> in human and mouse islets; Ad-LacZ was used as the negative control. Cellular proliferation was monitored by incorporation of [³H]-thymidine into islet DNA (<b>A</b>), FACS-based analysis of cell cycle phases (<b>B</b>), and incorporation of BrdU (white arrow heads) into islet cells that were co-stained for insulin or glucagon to identify β-cells and α-cells respectively (<b>C</b>). Thymidine incorporation measurements were conducted on 5 and 3 separate mouse and human islet preparations, respectively. Immunofluorescent images are representative of >30 islets (BrdU) per adenoviral treatment collected from 5 mice, or 3 human islet preparations. FACS analysis of mouse islets was performed on three separate occasions, each using a pool of ~300 islets per mouse (B6) collected from 5 or more mice per adenoviral treatment; analysis of human islets was performed on 4 separate human donor preparations, each with >6000 islets per adenoviral treatment. *, <i>P</i> < 0.05 relative to LacZ for N ≥ 3. Scale bars in C, 25 μm.</p

NFAT transcriptionally regulates a T2D GWAS genes in mouse and human islets.

<p>Heat maps illustrate the change in the expression of T2D-associated GWAS candidate genes in mouse (<b>A</b>, <b>B</b> and <b>C</b>) and human (<b>D</b>) islets in response to the overexpression of ca-Nfatc1, ca-Nfatc2 or GFP. For mouse islets, only those GWAS genes with a posterior probability (<i>PP</i>) > 0.99 of being differentially regulated by one or both of ca-NFATs are shown. For human islets, GWAS genes were selected from those showing robust regulation in mouse islets. Gene expression was determined by RNA-sequencing or qPCR in mouse and human islets respectively. Z-scores were computed from expression values for each gene across all samples (15 for mouse and 9 for human), and are shown relative to GFP (average Z-score for GFP = 0), which ranged from -3 to +3. Blue indicates reduced expression; red, increased expression; white, no change. Mouse genes are grouped according to their differential regulation (<b>A</b>), versus those that showed roughly equivalent suppression (<b>B</b>), or induction (<b>C</b>) in response to the two ca-NFATs. In <b>D</b>, * indicates <i>P</i> < 0.05 for human genes showing differential regulation by ca-Nfatc1 or ca-Nfatc2, relative to GFP.</p

Nfatc1 <i>vs</i>. Nfatc2-mediated gene regulation in mouse islets.

<p>Whole-islet RNA-sequencing was used to profile gene expression 48 hr after overexpression of ca-Nfatc1, ca-Nfatc2 or GFP (negative control). Genes that were differentially expressed (DE) were classified into one of 4 distinct patterns (relative to GFP): <b>A</b>, DE in response to ca-Nfatc1 only (518 genes); <b>B</b>, DE in response to ca-Nfatc2 only (1580 genes); <b>C</b>, equally DE for ca-Nfatc1 and ca-Nfatc2 (2293 genes); and <b>D</b>, unequally DE for ca-Nfatc1 and ca-Nfatc2 (2621 genes). Gene sets enriched with cell cycle regulatory transcripts are highlighted in red. Expression values for all genes and isoforms are contained within <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006466#pgen.1006466.s016" target="_blank">S7 Table</a>.</p