We created transgenic mice that overexpress WT androgen receptor (AR) exclusively in their skeletal muscle fibers. Unexpectedly, these mice display androgen-dependent muscle weakness and early death, show changes in muscle morphology and gene expression consistent with neurogenic atrophy, and exhibit a loss of motor axons. These features reproduce those seen in models of Kennedy disease, a polyglutamine expansion disorder caused by a CAG repeat expansion in the AR gene. Kennedy disease ͉ neuromuscular ͉ skeletal muscle ͉ spinal and bulbar muscular atrophy ͉ axonopathy A polymorphism in exon 1 of the androgen receptor (AR) gene, consisting of a variable number of glutamine (Q) repeats, affects AR function. Very long polyglutamine repeat (polyQ) tracts are associated with a progressive neuromuscular disease known as Kennedy disease (KD, or spinal bulbar muscular atrophy) (1). The etiological mechanism mediating polyQ toxicity is uncertain, but is generally thought to confer novel toxic functions to the protein, because expansion of polyQ tracts beyond 40 repeats in other proteins also cause neurodegenerative disease, including Huntington's disease (HD), and several autosomal dominant forms of spinocerebellar ataxia (SCA) (2). Histopathological studies of KD patients suggest ''neurogenic'' responses to denervation, and the etiology of this disease is therefore generally thought to begin with motoneuron pathology (3).Explicit mouse models of KD, in which polyQ AR alleles containing 60 CAG repeats or more are expressed, develop a disease phenotype that includes a marked reduction in body weight, kyphosis, and striking deficits in muscle strength and motor coordination (4-8). Androgen dependence, motoneuron and muscle pathology, and/or inclusions containing AR are also observed in these models (4,5,7,8). Our studies of AR in skeletal muscle (9, 10) led us to generate transgenic (Tg) mice in which AR is overexpressed solely in skeletal muscle fibers using an expression cassette containing the human skeletal ␣-actin (HSA) promoter. We discovered a striking phenotypic resemblance between these HSA-AR mice and mouse models of KD. This similarity is surprising given that the Q repeat in this AR transgene comprises only 22 glutamines and is expressed exclusively in skeletal muscle fibers and not in motoneurons. Results HSA Promoter Drives Transgene Expression Exclusively in SkeletalMuscle Fibers. We first validated our HSA expression cassette by generating HSA-LacZ (LacZ ϭ ␤-galactosidase gene) reporter mice [supporting information (SI) Fig. 5A]. Consistent with other expression cassettes containing the HSA promoter (11, 12) these reporter mice express ␤-gal specifically in skeletal muscle fibers, starting at embryonic day 9.5-10.5, with no detectable expression in other tissues, including the heart, viscera, fat or spinal cord ( SI Fig 5 B and C). We also created Tg mice in which a rat WT AR cDNA is driven by this same HSA expression cassette (SI Fig 6A), resulting in selective overexpression of AR in skeletal muscle fiber...

Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline-treatment. This spatiotemporal specificity is achieved using 2 transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline-controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP-LacZ reporter mice, which express β–galactosidase when activated by Cre. We find that expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle and/or interactions between muscle and motoneurons in mice.

Our results demonstrate that AGEs-RAGE signalling inhibits the osteogenic potential of ASCs under osteoinductive conditions by modulating DNA methylation and Wnt signalling. FPS-ZM1 can rescue the negative effects of AGEs and provide a possible treatment for bone tissue regeneration in patients with diabetic osteoporosis.

Testosterone and other androgens are thought to increase lean body mass and reduce fat body mass in men by activating the androgen receptor. However, the clinical potential of androgens for improving body composition is hampered by our limited understanding of the tissues and cells that promote such changes. Here we show that selective overexpression of androgen receptor in muscle cells (myocytes) of transgenic male rats both increases lean mass percentage and reduces fat mass. Similar changes in body composition are observed in human skeletal actin promoter driving expression of androgen receptor (HSA-AR) transgenic mice and result from acute testosterone treatment of transgenic female HSA-AR rats. These shifts in body composition in HSA-AR transgenic male rats are associated with hypertrophy of type IIb myofibers and decreased size of adipocytes. Metabolic analyses of transgenic males show higher activity of mitochondrial enzymes in skeletal muscle and increased O(2) consumption by the rats. These results indicate that androgen signaling in myocytes not only increases muscle mass but also reduces fat body mass, likely via increases in oxidative metabolism.

BackgroundEmerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR.Methodology/Principal FindingsWe performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntington's Disease, and to those common to muscle atrophy from diverse causes.Conclusions/SignificanceBy comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.

Cartilage tissue engineering is an emerging technique for the regeneration of cartilage tissue damaged as a result of trauma or disease. As the propensity for healing and regenerative capabilities of articular cartilage are limited, its repair remains one of the most challenging issues of musculoskeletal medicine. Clinical treatments intended to promote the success and complete repair of partial- and fullthickness articular cartilage defects are still unpredictable. However, one of the most exciting theories is that treatment of damaged articular cartilage can be realized with cartilage tissue engineering. This notion has prompted tissue engineering research involving cells, stimulating factors and scaffolds, either alone or in combination. With these perspectives, this review aims to present a summary of cartilage tissue engineering including development, recent progress, and major steps taken toward the regeneration of functional cartilage tissue. In addition, we discussed the role of stimulating factors, including growth factors, gene therapies, biophysical stimuli, and bioreactors, as well as scaffolds, including natural, synthetic, and nanostructured scaffolds, in cartilage tissue regeneration. Special emphasis was placed on cell source, including chondrocytes, fibroblasts, and stem cells, as an important component of cartilage tissue engineering techniques. In conclusion, continued development of cartilage tissue engineering will support future applications for patients suffering from diseased cartilage tissue problems and osteoarthritis.

Objectives Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose‐derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. Materials and methods Enzyme‐linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5‐aza2′‐deoxycytidine (5‐aza‐dC) and DKK‐1 were used to treat ASCs. Real‐time cell analysis and cell counting kit‐8 were used to monitor the proliferation of ASCs within and without AGEs. Real‐time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/β‐catenin signalling pathway among the different groups. Results The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs‐treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs‐treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including β‐Catenin, Lef1 and P‐GSK‐3β, were inhibited. After treatment with 5‐aza‐dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. Conclusions Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/β‐catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.