Denny L. Cottle

Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy

Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography-tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle.

Four and a Half LIM Protein 1 Binds Myosin-binding Protein C and Regulates Myosin Filament Formation and Sarcomere Assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced “sac-like” myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.

Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy.

Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.

FHL3 Is an Actin-binding Protein That Regulates α-Actinin-mediated Actin Bundling FHL3 LOCALIZES TO ACTIN STRESS FIBERS AND ENHANCES CELL SPREADING AND STRESS FIBER DISASSEMBLY

Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited α-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts.

Four and a half LIM protein 1 gene mutations cause four distinct human myopathies: A comprehensive review of the clinical, histological and pathological features

Mutations in the four and a half LIM protein 1 (FHL1) gene were recently identified as the cause of four distinct skeletal muscle diseases. Since the initial report outlining the first fhl1 mutation in 2008, over 25 different mutations have been identified in patients with reducing body myopathy, X-linked myopathy characterized by postural muscle atrophy, scapuloperoneal myopathy and Emery-Dreifuss muscular dystrophy. Reducing body myopathy was first described four decades ago, its underlying genetic cause was unknown until the discovery of fhl1 mutations. X-linked myopathy characterized by postural muscle atrophy is a novel disease where fhl1 mutations are the only cause. This review will profile each of the FHL1, with a comprehensive analysis of mutations, a comparison of the clinical and histopathological features and will present several hypotheses for the possible disease mechanism(s).

FHL3 binds MyoD and negatively regulates myotube formation

MyoD initiates muscle differentiation and promotes skeletal myogenesis by regulating temporal gene expression. MyoD-interacting proteins induce regulatory effects, and the identification of new MyoD-binding partners may provide mechanistic insights into the regulation of gene expression during myogenesis. FHL3 is one of three members of the FHL protein family that are expressed in skeletal muscle, but its function in myogenesis is unknown. Overexpression of human FHL3 in mouse C2C12 cells retarded myotube formation and decreased the expression of muscle-specific regulatory genes such as myogenin but not MyoD. By contrast, short interfering RNA (siRNA)-mediated FHL3 protein knockdown enhanced myoblast differentiation associated with increased myogenin, but not MyoD protein expression, early during differentiation. We demonstrate that FHL3 is a MyoD-associated protein by direct binding assays, colocalisation in the nucleus of myoblasts and GST pull-down studies. Moreover, we determined that FHL3 interacts with MyoD, functioning as its potent negative co-transcriptional regulator. Ectopic expression of FHL3 in myoblasts impaired MyoD-mediated transcriptional activity and muscle gene expression. By contrast, siRNA-mediated FHL3 knockdown enhanced MyoD transcriptional activity in a dose-dependent manner. These findings reveal that FHL3 association with MyoD may contribute to the regulation of MyoD-dependent transcription of muscle genes and thereby myogenesis.

Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

INPP5E interacts with AURKA, linking phosphoinositide signaling to primary cilium stability

ABSTRACT Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA.

c-MYC-Induced Sebaceous Gland Differentiation Is Controlled by an Androgen Receptor/p53 Axis

Summary Although the sebaceous gland (SG) plays an important role in skin function, the mechanisms regulating SG differentiation and carcinoma formation are poorly understood. We previously reported that c-MYC overexpression stimulates SG differentiation. We now demonstrate roles for the androgen receptor (AR) and p53. MYC-induced SG differentiation was reduced in mice lacking a functional AR. High levels of MYC triggered a p53-dependent DNA damage response, leading to accumulation of proliferative SG progenitors and inhibition of AR signaling. Conversely, testosterone treatment or p53 deletion activated AR signaling and restored MYC-induced differentiation. Poorly differentiated human sebaceous carcinomas exhibited high p53 and low AR expression. Thus, the consequences of overactivating MYC in the SG depend on whether AR or p53 is activated, as they form a regulatory axis controlling proliferation and differentiation.

Dose and context dependent effects of Myc on epidermal stem cell proliferation and differentiation.

Myc is activated in many tumours, yet, paradoxically, stimulates differentiation in mammalian epidermis. To test whether the epidermis responds differently to different levels of Myc, we treated K14MycER transgenic mice with a range of concentrations of the inducing agent, 4‐hydroxy‐tamoxifen (4OHT). Proliferation was stimulated at all levels of Myc activity; sebocyte differentiation was stimulated at low and intermediate levels; and interfollicular epidermal differentiation at intermediate and high levels. Mutational inactivation of the Myc p21 activated kinase 2 (PAK2) phosphorylation sites increased Myc activity and further enhanced epidermal differentiation. We conclude that Myc induced differentiation acts as a fail‐safe device to prevent uncontrolled proliferation and neoplastic conversion of epidermal stem cells expressing high levels of Myc.