Macrophage-released ADAMTS1 promotes muscle stem cell activation.

Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.

A glucocorticoid- and diet-responsive pathway toggles adipocyte precursor cell activity in vivo.

Obesity is driven by excess caloric intake, which leads to the expansion of adipose tissue by hypertrophy and hyperplasia. Adipose tissue hyperplasia results from the differentiation of adipocyte precursor cells (APCs) that reside in adipose depots. Investigation into this process has elucidated a network of mostly transcription factors that drive APCs through the differentiation process. Using in vitro and in vivo approaches, our study revealed a signaling pathway that inhibited the initiation of the adipocyte differentiation program. Mouse adipocytes secreted the extracellular protease ADAMTS1, which triggered the production of the cytokine pleiotrophin (PTN) through the Wnt/ß-catenin pathway, and promoted proliferation rather than differentiation of APCs. Glucocorticoid exposure in vitro or in vivo reduced ADAMTS1 abundance in adipocytes. In addition, mice fed a high-fat diet showed decreased Adamts1 expression in the visceral perigonadal adipose depot, which expanded by adipogenesis in response to the diet, and increased Adamts1 expression in the subcutaneous inguinal adipose depot, which did not induce adipogenesis. Similar to what occurred in mouse subcutaneous adipose tissue, diet-induced weight gain increased the expression of ADAMTS1, PTN, and certain Wnt target genes in the subcutaneous adipose depot of human volunteers, suggesting the relevance of this pathway to physiological adipose tissue homeostasis and the pathogenesis of obesity. Thus, this pathway functions as a toggle on APCs, regulating a decision between differentiation and proliferation and coordinating the response of adipose tissue to systemic cues.

Characterization of Cre recombinase activity for in vivo targeting of adipocyte precursor cells.

The increased incidence of obesity and metabolic disease underscores the importance of elucidating the biology of adipose tissue development. The recent discovery of cell surface markers for prospective identification of adipose precursor cells (APCs) in vivo will greatly facilitate these studies, yet tools for specifically targeting these cells in vivo have not been identified. Here, we survey three transgenic mouse lines, Fabp4-Cre, PdgfRα-Cre, and Prx1-Cre, precisely assessing Cre-mediated recombination in adipose stromal populations and mature tissues. Our data provide key insights into the utility of these tools to modulate gene expression in adipose tissues. In particular, Fabp4-Cre is not effective to target APCs, nor is its activity restricted to these cells. PdgfRα-Cre directs recombination in the vast majority of APCs, but also targets other populations. In contrast, adipose expression of Prx1-Cre is chiefly limited to subcutaneous inguinal APCs, which will be valuable for dissection of APC functions among adipose depots.

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy.

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

[Cloning of AE1-c-end cDNA and construction of its expression plasmid for yeast two-hybrid system].

In this study, about 350 bp cDNA fragment was amplified by PCR. After being sequenced, the AE1-c-end gene fragment was cloned into EcoR I-Pst I site of pGADT7 to form AD ends in the yeast two-hybrid system. The recombinant plasmid was transformed into yeast AH109, and the expression in the yeast was observed. The results demonstrate that AE1-c-end was obtained. pGADT7-AE1-c-end has no toxic effect on the yeast. It can serve as a target gene of yeast two-hybrid system.