Protein therapy of skeletal muscle atrophy and mechanism by angiogenic factor AGGF1.

BACKGROUND: Skeletal muscle atrophy is a common condition without a pharmacologic therapy. AGGF1 encodes an angiogenic factor that regulates cell differentiation, proliferation, migration, apoptosis, autophagy and endoplasmic reticulum stress, promotes vasculogenesis and angiogenesis and successfully treats cardiovascular diseases. Here, we report the important role of AGGF1 in the pathogenesis of skeletal muscle atrophy and attenuation of muscle atrophy by AGGF1. METHODS: In vivo studies were carried out in impaired leg muscles from patients with lumbar disc herniation, two mouse models for skeletal muscle atrophy (denervation and cancer cachexia) and heterozygous Aggf1+/- mice. Mouse muscle atrophy phenotypes were characterized by body weight and myotube cross-sectional areas (CSA) using H&E staining and immunostaining for dystrophin. Molecular mechanistic studies include co-immunoprecipitation (Co-IP), western blotting, quantitative real-time PCR analysis and immunostaining analysis. RESULTS: Heterozygous Aggf1+/- mice showed exacerbated phenotypes of reduced muscle mass, myotube CSA, MyHC (myosin heavy chain) and α-actin, increased inflammation (macrophage infiltration), apoptosis and fibrosis after denervation and cachexia. Intramuscular and intraperitoneal injection of recombinant AGGF1 protein attenuates atrophy phenotypes in mice with denervation (gastrocnemius weight 81.3 ± 5.7 mg vs. 67.3 ± 5.1 mg for AGGF1 vs. buffer; P < 0.05) and cachexia (133.7 ± 4.7 vs. 124.3 ± 3.2; P < 0.05). AGGF1 expression undergoes remodelling and is up-regulated in gastrocnemius and soleus muscles from atrophy mice and impaired leg muscles from patients with lumbar disc herniation by 50-60% (P < 0.01). Mechanistically, AGGF1 interacts with TWEAK (tumour necrosis factor-like weak inducer of apoptosis), which reduces interaction between TWEAK and its receptor Fn14 (fibroblast growth factor-inducing protein 14). This leads to inhibition of Fn14-induced NF-kappa B (NF-κB) p65 phosphorylation, which reduces expression of muscle-specific E3 ubiquitin ligase MuRF1 (muscle RING finger 1), resulting in increased MyHC and α-actin and partial reversal of atrophy phenotypes. Autophagy is reduced in Aggf1+/- mice due to inhibition of JNK (c-Jun N-terminal kinase) activation in denervated and cachectic muscles, and AGGF1 treatment enhances autophagy in two atrophy models by activating JNK. In impaired leg muscles of patients with lumbar disc herniation, MuRF1 is up-regulated and MyHC and α-actin are down-regulated; these effects are reversed by AGGF1 by 50% (P < 0.01). CONCLUSIONS: These results indicate that AGGF1 is a novel regulator for the pathogenesis of skeletal muscle atrophy and attenuates skeletal muscle atrophy by promoting autophagy and inhibiting MuRF1 expression through a molecular signalling pathway of AGGF1-TWEAK/Fn14-NF-κB. More importantly, the results indicate that AGGF1 protein therapy may be a novel approach to treat patients with skeletal muscle atrophy.

NINJ2 deficiency inhibits preadipocyte differentiation and promotes insulin resistance through regulating insulin signaling.

OBJECTIVE: Genetic variants in ninjurin-2 (NINJ2; nerve injury-induced protein 2) confer risk of ischemic strokes and coronary artery disease as well as endothelial activation and inflammation. However, little is known about NINJ2's in vivo functions and underlying mechanisms. METHODS: The phenotypes of NINJ2 knockout mice were analyzed, and mechanisms of NINJ2 that regulate body weight, insulin resistance, and glucose homeostasis and lipogenesis were investigated in vivo and in vitro. RESULTS: This study found that mice lacking NINJ2 showed impaired adipogenesis, increased insulin resistance, and abnormal glucose homeostasis, all of which are risk factors for strokes and coronary artery disease. Mechanistically, NINJ2 directly interacts with insulin receptor/insulin-like growth factor 1 receptor (INSR/IGF1R), and NINJ2 knockdown can block insulin-induced mitotic clonal expansion during preadipocyte differentiation by inhibiting protein kinase B/extracellular signal-regulated kinase (AKT/ERK) signaling and by decreasing the expression of key adipocyte transcriptional regulators CCAAT/enhancer-binding protein ß (C/EBP-ß), C/EBP-α, and peroxisome proliferator-activated receptor γ (PPAR-γ). Furthermore, the interaction between NINJ2 and INSR/IGF1R is needed for maintaining insulin sensitivity in adipocytes and muscle via AKT and glucose transporter type 4. Notably, adenovirus-mediated NINJ2 overexpression can ameliorate diet-induced insulin resistance in mice. CONCLUSIONS: In conclusion, these findings reveal NINJ2 as an important new facilitator of insulin receptors, and the authors propose a unique regulatory mechanism between insulin signaling, adipogenesis, and insulin resistance.

Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis.

Objective: Angiogenic factor AGGF1 (angiogenic factor with G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results: Using functional blocking studies with neutralizing antibodies, we identified [alpha]5[beta]1 as the receptor for AGGF1 on ECs. AGGF1 interacts with [alpha]5[beta]1 and activates FAK (focal adhesion kinase), Src (proto-oncogene tyrosine-protein kinase), and AKT (protein kinase B). Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from -RDD- to -RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions: We have identified integrin [alpha]5[beta]1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of [alpha]5[beta]1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with [alpha]5[beta]1 and signaling.

Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression.

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. To elucidate new regulatory mechanisms for heptocarcinogenesis, we investigated the regulation of p21, a cyclin-dependent kinase (CDK) inhibitor encoded by CDKN1A, in HCC. The expression level of p21 is decreased with the progression of HCC. Luciferase assays with a luciferase-p21-3' UTR reporter and its serial deletions identified a 15-bp repressor element at the 3'-UTR of CDKN1A, which contains a binding site for miR-95-3p. Mutation of the binding site eliminated the regulatory effect of miR-95-3p on p21 expression. Posttranscriptional regulation of p21 expression by miR-95-3p is mainly on the protein level (suppression of translation). Overexpression of miR-95-3p in two different HCC cell lines, HepG2 and SMMC7721, significantly promoted cell proliferation, cell cycle progression and cell migration, whereas a miR-95-3p specific inhibitor decreased cell proliferation, cell cycle progression and cell migration. The effects of miR-95-3p on cellular functions were rescued by overexpression of p21. Overexpression of miR-95-3p promoted cell proliferation and tumor growth in HCC xenograft mouse models. Expression of miR-95-3p was significantly higher in HCC samples than in adjacent non-cancerous samples. These results demonstrate that miR-95-3p is a potential new marker for HCC and regulates hepatocarcinogenesis by directly targeting CDKN1A/p21 expression.