Hepatic posttranscriptional network comprised of CCR4-NOT deadenylase and FGF21 maintains systemic metabolic homeostasis.

Whole-body metabolic homeostasis is tightly controlled by hormone-like factors with systemic or paracrine effects that are derived from nonendocrine organs, including adipose tissue (adipokines) and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is a hormone-like protein, which is emerging as a major regulator of whole-body metabolism and has therapeutic potential for treating metabolic syndrome. However, the mechanisms that control FGF21 levels are not fully understood. Herein, we demonstrate that FGF21 production in the liver is regulated via a posttranscriptional network consisting of the CCR4-NOT deadenylase complex and RNA-binding protein tristetraprolin (TTP). In response to nutrient uptake, CCR4-NOT cooperates with TTP to degrade AU-rich mRNAs that encode pivotal metabolic regulators, including FGF21. Disruption of CCR4-NOT activity in the liver, by deletion of the catalytic subunit CNOT6L, increases serum FGF21 levels, which ameliorates diet-induced metabolic disorders and enhances energy expenditure without disrupting bone homeostasis. Taken together, our study describes a hepatic CCR4-NOT/FGF21 axis as a hitherto unrecognized systemic regulator of metabolism and suggests that hepatic CCR4-NOT may serve as a target for devising therapeutic strategies in metabolic syndrome and related morbidities.

SUMOylated αNAC potentiates transcriptional repression by FIAT.

The transcriptional coregulator αNAC (Nascent polypeptide associated complex And Coregulator alpha) and the transcriptional repressor FIAT (Factor Inhibiting ATF4-mediated Transcription) interact but the biological relevance of this interaction remains unclear. The activity of αNAC is extensively modulated by post-translational modifications (PTMs). We identified a novel αNAC PTM through covalent attachment of the Small Ubiquitin-like MOdifier (SUMO1). Recombinant αNAC was a SUMO1 target in in vitro SUMOylation assays and we confirmed that αNAC is conjugated to SUMO1 in cultured osteoblasts and in calvarial tissue. The amino acid sequence of αNAC contains one copy of the composite "phospho-sumoyl switch" motif that couples sequential phosphorylation and SUMOylation. We found that αNAC is selectively SUMOylated at lysine residue 127 within the motif and that SUMOylation is enhanced when a phosphomimetic mutation is introduced at the nearby serine residue 132. SUMOylation did not alter the DNA-binding capacity of αNAC. The S132D, hyper-SUMOylated αNAC mutant specifically interacted with histone deacetylase-2 (HDAC2) and enhanced the inhibitory activity of FIAT on ATF4-mediated transcription from the Osteocalcin gene promoter. This effect required binding of SUMOylated αNAC to the target promoter. We propose that maximal transcriptional repression by FIAT requires its interaction with SUMOylated, HDAC2-interacting αNAC.

Control of Fiat (factor inhibiting ATF4-mediated transcription) expression by Sp family transcription factors in osteoblasts.

FIAT (factor inhibiting ATF4-mediated transcription) represses Osteocalcin gene transcription and inhibits osteoblast activity by heterodimerizing with ATF4 to prevent it from binding DNA. It thus appears important to identify and characterize the molecular mechanisms that control Fiat gene expression in osteoblasts. In silico sequence analysis identified a canonical GC-box within a 1,400 bp region of the proximal Fiat gene promoter. Electrophoretic mobility shift assays (EMSA) with MC3T3-E1 osteoblastic cells nuclear extracts indicated that the transcription factors Sp1 and Sp3, but not Sp7/OSTERIX, bound this proximal GC-box. Chromatin immunoprecipitation confirmed interaction of the two transcription factors with the Fiat promoter GC-element in living osteoblasts. Transient transfection studies showed that Sp1 dose-dependently activated the expression of a Fiat-luciferase reporter construct while both the long or short isoforms of Sp3 dose-dependently inhibited transcription from the Fiat reporter construct. Transfection of an Sp7/OSTERIX expression vector did not affect expression of the Fiat-luciferase reporter. Co-transfection of increasing amounts of the Sp3 expression vector in the context of maximal Sp1-dependent Fiat-luciferase activation led to dose-dependent repression of the expression of the reporter. Using RNA knockdown, we measured a reduction in steady-state Fiat expression when Sp1 was inhibited, and a reciprocal increase upon Sp3 knockdown. In parallel, treatment of osteoblasts with WP631, which prevents Sp1/DNA interactions, strongly inhibited the expression of Fiat and reduced the occupancy of the Fiat promoter proximal GC-box by Sp1. Taken together, our results suggest an interplay between Sp1 and Sp3 as a mechanism involved in the control of Fiat gene expression in osteoblasts.

Transplantation to study satellite cell heterogeneity in skeletal muscle.

Skeletal muscle has a remarkable capacity to regenerate throughout life, which is mediated by its resident muscle stem cells, also called satellite cells. Satellite cells, located periphery to the muscle fibers and underneath the basal lamina, are an indispensable cellular source for muscle regeneration. Satellite cell transplantation into regenerating muscle contributes robustly to muscle repair, thereby indicating that satellite cells indeed function as adult muscle stem cells. Moreover, satellite cells are a heterogenous population in adult tissue, with subpopulations that can be distinguished based on gene expression, cell-cycle progression, ability to self-renew, and bi-potential ability. Transplantation assays provide a powerful tool to better understand satellite cell function in vivo enabling the separation of functionally distinct satellite cell subpopulations. In this review, we focus on transplantation strategies to explore satellite cells' functional heterogeneity, approaches targeting the recipient tissue to improve transplantation efficiency, and common strategies to monitor the behaviour of the transplanted cells. Lastly, we discuss some recent approaches to overcome challenges to enhance the transplantation potential of muscle stem cells.

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Crispr-Cas9 engineered osteogenesis imperfecta type V leads to severe skeletal deformities and perinatal lethality in mice.

Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and µCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.

FIAT Deletion Increases Bone Mass But Does Not Prevent High-Fat-Diet-Induced Metabolic Complications.

Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT) interacts with ATF4 to repress its transcriptional activity. We performed a phenotypic analysis of Fiat-deficient male mice (Fiat-/Y) at 8 and 16 weeks of age. Microcomputed tomography analysis of the distal femur demonstrated 46% and 13% age-dependent increases in trabecular bone volume and thickness, respectively, in Fiat-/Y mice. Cortical bone measurements at the femoral midshaft revealed a substantial increase in cortical thickness in older Fiat-/Y mice. Bone gain was related to increased mineral apposition rate and increased osteoblast function. Femoral stiffness and strength were substantially increased in Fiat-/Y compared with wild-type (WT) mice. We also investigated whether FIAT contributes to metabolic function. When fed standard mouse chow, Fiat-/Y animals were glucose-tolerant. However, when fed a high-fat diet (HFD) for 8 weeks, Fiat-/Y mice gained more weight than control mice, with a specific increase in white adipose tissue fat mass. The increase in fat mass was due to reduced energy expenditure, which correlated with reduced fatty acid oxidation and lipolysis in the adipose tissue of mutant mice. The expression of the Scd1 gene, involved in lipogenesis, was upregulated in the subcutaneous adipose tissue of Fiat-/Y mice. Moreover, HFD-fed Fiat-/Y mice exhibited increased circulating leptin and insulin levels relative to WT mice, demonstrating that endocrine abnormalities are associated with the disturbance in energy balance. We conclude that Fiat-/Y mice exhibited an anabolic bone phenotype but displayed increased susceptibility to developing metabolic-related disorders when consuming an HFD.

Altered gene dosage confirms the genetic interaction between FIAT and αNAC.

Factor inhibiting ATF4-mediated transcription (FIAT) interacts with Nascent polypeptide associated complex and coregulator alpha (αNAC). In cultured osteoblastic cells, this interaction contributes to maximal FIAT-mediated inhibition of Osteocalcin (Ocn) gene transcription. We set out to demonstrate the physiological relevance of this interaction by altering gene dosage in compound Fiat and Naca (encoding αNAC) heterozygous mice. Compound Naca(+/-); Fiat(+/-) heterozygous animals were viable, developed normally, and exhibited no significant difference in body weight compared with control littermate genotypes. Animals with a single Fiat allele had reduced Fiat mRNA expression without changes in the expression of related family members. Expression of the osteocyte differentiation marker Dmp1 was elevated in compound heterozygotes. Static histomorphometry parameters were assessed at 8weeks of age using microcomputed tomography (µCT). Trabecular measurements were not different between genotypes. Cortical thickness and area were not affected by gene dosage, but we measured a significant increase in cortical porosity in compound heterozygous mice, without changes in biomechanical parameters. The bone phenotype of compound Naca(+/-); Fiat(+/-) heterozygotes confirms that FIAT and αNAC are part of a common genetic pathway and support a role for the FIAT/αNAC interaction in normal bone physiology.

Combinatorial control of ATF4-dependent gene transcription in osteoblasts.

Osteoblast-specific gene transcription requires interaction between bone cell-specific transcription factors and more widely expressed transcriptional regulators. This is particularly evident for the basic domain-leucine zipper factor activating transcription factor 4 (ATF4), whose activity can be enhanced or inhibited through interaction with other leucine zipper proteins, intermediate filament proteins, components of the basic transcriptional machinery, nuclear matrix attachment molecules, or ubiquitously expressed transcription factors. We discuss the results supporting the relevance of these interactions and present the first evidence of a functional interaction between ATF4, FIAT (factor-inhibiting ATF4-mediated transcription), and αNAC (nascent polypeptide-associated complex and coactivator alpha), three proteins that have been previously shown to associate using various protein-protein interaction assays.