FHL1 is a major host factor for chikungunya virus infection.

Chikungunya virus (CHIKV) is a re-emerging alphavirus that is transmitted to humans by mosquito bites and causes musculoskeletal and joint pain1,2. Despite intensive investigations, the human cellular factors that are critical for CHIKV infection remain unknown, hampering the understanding of viral pathogenesis and the development of anti-CHIKV therapies. Here we identified the four-and-a-half LIM domain protein 1 (FHL1)3 as a host factor that is required for CHIKV permissiveness and pathogenesis in humans and mice. Ablation of FHL1 expression results in the inhibition of infection by several CHIKV strains and o'nyong-nyong virus, but not by other alphaviruses and flaviviruses. Conversely, expression of FHL1 promotes CHIKV infection in cells that do not normally express it. FHL1 interacts directly with the hypervariable domain of the nsP3 protein of CHIKV and is essential for the replication of viral RNA. FHL1 is highly expressed in CHIKV-target cells and is particularly abundant in muscles3,4. Dermal fibroblasts and muscle cells derived from patients with Emery-Dreifuss muscular dystrophy that lack functional FHL15 are resistant to CHIKV infection. Furthermore,  CHIKV infection  is undetectable in Fhl1-knockout mice. Overall, this study shows that FHL1 is a key factor expressed by the host that enables CHIKV infection and identifies the interaction between nsP3 and FHL1 as a promising target for the development of anti-CHIKV therapies.

Knockdown of the Non-canonical Wnt Gene Prickle2 Leads to Cerebellar Purkinje Cell Abnormalities While Cerebellar-Mediated Behaviors Remain Intact.

Autism spectrum disorders (ASD) involve brain wide abnormalities that contribute to a constellation of symptoms including behavioral inflexibility, cognitive dysfunction, learning impairments, altered social interactions, and perceptive time difficulties. Although a single genetic variation does not cause ASD, genetic variations such as one involving a non-canonical Wnt signaling gene, Prickle2, has been found in individuals with ASD. Previous work looking into phenotypes of Prickle2 knock-out (Prickle2-/-) and heterozygous mice (Prickle2-/+) suggest patterns of behavior similar to individuals with ASD including altered social interaction and behavioral inflexibility. Growing evidence implicates the cerebellum in ASD. As Prickle2 is expressed in the cerebellum, this animal model presents a unique opportunity to investigate the cerebellar contribution to autism-like phenotypes. Here, we explore cerebellar structural and physiological abnormalities in animals with Prickle2 knockdown using immunohistochemistry, whole-cell patch clamp electrophysiology, and several cerebellar-associated motor and timing tasks, including interval timing and eyeblink conditioning. Histologically, Prickle2-/- mice have significantly more empty spaces or gaps between Purkinje cells in the posterior lobules and a decreased propensity for Purkinje cells to fire action potentials. These structural cerebellar abnormalities did not impair cerebellar-associated behaviors as eyeblink conditioning and interval timing remained intact. Therefore, although Prickle-/- mice show classic phenotypes of ASD, they do not recapitulate the involvement of the adult cerebellum and may not represent the pathophysiological heterogeneity of the disorder.

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Hic-5 deficiency protects cerulein-induced chronic pancreatitis via down-regulation of the NF-κB (p65)/IL-6 signalling pathway.

Chronic pancreatitis (CP), characterized by pancreatic fibrosis, is a recurrent, progressive and irreversible disease. Activation of the pancreatic stellate cells (PSCs) is considered a core event in pancreatic fibrosis. In this study, we investigated the role of hydrogen peroxide-inducible clone-5 (Hic-5) in CP. Analysis of the human pancreatic tissue samples revealed that Hic-5 was overexpressed in patients with CP and was extremely low in healthy pancreas. Hic-5 was significant up-regulated in the activated primary PSCs independently from transforming growth factor beta stimulation. CP induced by cerulein injection was ameliorated in Hic-5 knockout (KO) mice, as shown by staining of tissue level. Simultaneously, the activation ability of the primary PSCs from Hic-5 KO mice was significantly attenuated. We also found that the Hic-5 up-regulation by cerulein activated the NF-κB (p65)/IL-6 signalling pathway and regulated the downstream extracellular matrix (ECM) genes such as α-SMA and Col1a1. Therefore, we determined whether suppressing NF-κB/p65 alleviated CP by treating mice with the NF-κB/p65 inhibitor triptolide in the cerulein-induced CP model and found that pancreatic fibrosis was alleviated by NF-κB/p65 inhibition. These findings provide evidence for Hic-5 as a therapeutic target that plays a crucial role in regulating PSCs activation and pancreatic fibrosis.

LMO7 Is a Negative Feedback Regulator of Transforming Growth Factor ß Signaling and Fibrosis.

BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.

Muscle death participates in myofibrillar abnormalities in FHL1 knockout mice.

BACKGROUND: Mutations in the four and-a-half LIM domain protein 1 (FHL1) gene or FHL1 protein deletion have been identified as the cause of rare hereditary myopathies or cardiomyopathies. In our previous study, autophagy activation was associated with myofibrillar abnormalities in FHL1 knockout (KO) mice. P2RX7 induces cell death, such as autophagy, pyroptosis or apoptosis via cell-specific downstream signaling; however, the roles of P2RX7 in pyroptosis or apoptosis in myofibrillar abnormalities in FHL1 KO mice have not been well elucidated. METHODS: In this study, skeletal muscle and heart of 2.5 months old WT and FHL1 KO male mice histomorphology were examined by hematoxylin and eosin staining. The indicators for pyroptosis (NLRP3; ASC; cleaved-caspase1; IL-1ß), apoptosis (Apaf-1; Bcl-2; caspase9; cleaved-caspase3), and P2RX7 were detected in the triceps (Tri), tibialis anterior muscles (TA), and heart by western blot and/or immunohistochemistry in WT and FHL1 KO male mice. RESULTS: Indicators for pyroptosis (ASC; cleaved-caspase1; IL-1ß) and apoptosis (Apaf-1 and cleaved-caspase3), as well as P2RX7 were upregulated in Tri, tibialis TA, and heart in FHL1 KO mice, indicating pyroptosis and apoptosis play important roles in myofibrillar abnormalities in FHL1 KO mice. CONCLUSIONS: P2RX7 may participate in myofibrillar abnormalities by activating pyroptosis and apoptosis in FHL1 KO mice. These findings have basic implications for the understanding of myopathies induced by FHL1 deficiency and provide new avenues for the treatment of these hereditary myopathies by modulating P2RX7.

Knockout of Ajuba Attenuates the Growth and Migration of Hepatocellular Carcinoma Cells.

Ajuba has been found to be mutated or aberrantly regulated in several human cancers and plays important roles in cancer progression via different signaling pathways. However, little is known about the role of Ajuba in hepatocellular carcinoma (HCC). Here, we found an upregulation of Ajuba expression in HCC tissues compared with normal liver tissues, while a poor prognosis was observed in HCC patients with high Ajuba expression. Knockout of Ajuba in HCC cells inhibited cell growth in vitro and in vivo, suppressed cell migration, and enhanced the cell apoptosis under stress. Moreover, re-expression of Ajuba in Ajuba-deficient cells could restore the phenotype of Ajuba-deficient cells. In conclusion, these results indicate that Ajuba is upregulated in HCC and promotes cell growth and migration of HCC cells, suggesting that Ajuba could possibly be a new target for HCC diagnosis and treatment.

FHL-1 is not involved in pressure overload-induced maladaptive right ventricular remodeling and dysfunction.

AIMS: The cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) has recently been identified as a novel key player in pulmonary hypertension as well as in left heart diseases. In this regard, FHL-1 has been implicated in dysregulated hypertrophic signaling in pulmonary arterial smooth muscle cells leading to pulmonary hypertension. In mice, FHL-1-deficiency (FHL-1-/-) led to an attenuated hypertrophic signaling associated with a blunted hypertrophic response of the pressure-overloaded left ventricle (LV). However, the role of FHL-1 in right heart hypertrophy has not yet been addressed. METHODS AND RESULTS: We investigated FHL-1 expression in C57Bl/6 mice subjected to chronic biomechanical stress and found it to be enhanced in the right ventricle (RV). Next, we subjected FHL-1-/- and corresponding wild-type mice to pressure overload of the RV by pulmonary arterial banding for various time points. However, in contrast to the previously published study in LV-pressure overload, which was confirmed here, RV hypertrophy and hypertrophic signaling was not diminished in FHL-1-/- mice. In detail, right ventricular pressure overload led to hypertrophy, dilatation and fibrosis of the RV from both FHL-1-/- and wild-type mice. RV remodeling was associated with impaired RV function as evidenced by reduced tricuspid annular plane systolic excursion. Additionally, PAB induced upregulation of natriuretic peptides and slight downregulation of phospholamban and ryanodine receptor 2 in the RV. However, there was no difference between genotypes in the degree of expression change. CONCLUSION: FHL-1 pathway is not involved in the control of adverse remodeling in the pressure overloaded RV.

Downregulation of Cypher induces apoptosis in cardiomyocytes via Akt/p38 MAPK signaling pathway.

Background: Dilated cardiomyopathy (DCM) is considered as the most common form of non-ischemic cardiomyopathy with a high mortality worldwide. Cytoskeleton protein Cypher plays an important role in maintaining cardiac function. Genetic studies in human and animal models revealed that Cypher is involved in the development of DCM. However, the underlying molecular mechanism is not fully understood. Accumulating evidences suggest that apoptosis in myocytes may contribute to DCM. Thus, the purpose of this study is to define whether lack of Cypher in cardiomyocytes can elevate apoptosis signaling and lead to DCM eventually. Methods and Results: Cypher-siRNA sufficiently inhibited Cypher expression in cardiomyocytes. TUNEL-positive cardiomyocytes were increased in both Cypher knockdown neonatal rat cardiomyocytes and Cypher knockout mice hearts, which were rare in the control group. Flow cytometry further confirmed that downregulation of Cypher significantly increased myocytes apoptosis in vitro. Cell counting kit-8 assay revealed that Cypher knockdown in H9c2 cells significantly reduced cell viability. Cypher knockdown was found to increase cleaved caspase-3 expression and suppress p21, ratio of bcl-2 to Bax. Cypher-deficiency induced apoptosis was linked to downregulation of Akt activation and elevated p-p38 MAPK accumulation. Pharmacological activation of Akt with SC79 attenuated apoptosis with enhanced phosphorylation of Akt and reduced p-p38 MAPK and Bax expression. Conclusions: Downregulation of Cypher participates in the promotion of cardiomyocytes apoptosis through inhibiting Akt dependent pathway and enhancing p38 MAPK phosphorylation. These findings may provide a new potential therapeutic strategy for the treatment of DCM.

LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage.

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression.

A novel TGFß modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

As some of the most widely utilised intercellular signalling molecules, transforming growth factor ß (TGFß) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFß signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFß signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFß signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFß signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFß signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFß family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFß signalling and thereby embryonic patterning.

Compensatory Actions of Ldb Adaptor Proteins During Corticospinal Motor Neuron Differentiation.

Although many genes that specify neocortical projection neuron subtypes have been identified, the downstream effectors that control differentiation of those subtypes remain largely unknown. Here, we demonstrate that the LIM domain-binding proteins Ldb1 and Ldb2 exhibit dynamic and inversely correlated expression patterns during cerebral cortical development. Ldb1-deficient brains display severe defects in proliferation and changes in regionalization, phenotypes resembling those of Lhx mutants. Ldb2-deficient brains, on the other hand, exhibit striking phenotypes affecting layer 5 pyramidal neurons: Immature neurons have an impaired capacity to segregate into mature callosal and subcerebral projection neurons. The analysis of Ldb2 single-mutant mice reveals a compensatory role of Ldb1 for Ldb2 during corticospinal motor neuron (CSMN) differentiation. Animals lacking both Ldb1 and Ldb2 uncover the requirement for Ldb2 during CSMN differentiation, manifested as incomplete CSMN differentiation, and ultimately leading to a failure of the corticospinal tract.

Hic-5 deficiency attenuates the activation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice.

BACKGROUND & AIM: Hydrogen peroxide-inducible clone-5 (Hic-5), also named as transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was found to be induced by TGF-ß. Previous studies have shown that TGF-ß is a principal mediator of hepatic stellate cell (HSC) activation in liver fibrosis. However, this process remains elusive. In this study, we aimed to define the role of Hic-5 in HSC activation and liver fibrosis. METHODS: We examined the expression levels of Hic-5 during HSCs activation and in fibrotic liver tissues by quantitative real-time reverse transcriptase polymerase chain reaction, Western blot and immunohistochemistry. Hic-5 knockout (KO) and wild-type (WT) mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) injection to induce liver fibrosis. RESULTS: Hic-5 expression was strongly upregulated in activated HSCs of the human fibrotic liver tissue and BDL or CCl4-induced mouse liver fibrosis. Hic-5 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Furthermore, Hic-5 knockdown by siRNA in vivo repressed CCl4-induced liver fibrosis in mice. Mechanistically, the absence of Hic-5 significantly inhibited the TGF-ß/Smad2 signaling pathway, proved by increasing Smad7 expression, resulting in reduced collagen production and α-smooth muscle actin expression in the activated HSCs. CONCLUSION: Hic-5 deficiency attenuates the activation of HSCs and liver fibrosis though reducing the TGF-ß/Smad2 signaling by upregulation of Smad7. Thus, Hic-5 can be regarded as a potential therapeutic target for liver fibrosis.

Muscle hypertrophy as the presenting sign in a patient with a complete FHL1 deletion.

Four and a half LIM protein 1 (FHL1/SLIM1) has recently been identified as the causative gene mutated in four distinct diseases affecting skeletal muscle that have overlapping features, including reducing body myopathy, X-linked myopathy, X-linked dominant scapuloperoneal myopathy and Emery-Dreifuss muscular dystrophy. FHL1 localises to the sarcomere and the sarcolemma and is believed to participate in muscle growth and differentiation as well as in sarcomere assembly. We describe in this case report a boy with a deletion of the entire FHL1 gene who is now 15 years of age and presented with muscle hypertrophy, reduced subcutaneous fat, rigid spine and short stature. This case is the first, to our knowledge, with a complete loss of the FHL1 protein and MAP7D3 in combination. It supports the theory that dominant negative effects (accumulation of cytotoxic-mutated FHL1 protein) worsen the pathogenesis. It extends the phenotype of FHL1-related myopathies and should prompt future testing in undiagnosed patients who present with unexplained muscle hypertrophy, contractures and rigid spine, particularly if male.

Absence of Four-and-a-Half LIM Domain Protein 2 Decreases Atherosclerosis in ApoE-/- Mice.

OBJECTIVE: Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial cells, vascular smooth muscle cells, and leukocytes. It regulates cell survival, migration, and inflammatory response, but its role in atherogenesis is unknown. APPROACH AND RESULTS: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient mice were crossed with ApoE-deficient mice, to generate ApoE/FHL2-/- mice. After high-fat diet, ApoE/FHL2-/- mice had significantly smaller atherosclerotic plaques than ApoE-/- mice in the aortic sinus, the brachiocephalic artery, and the aorta. This was associated with enhanced collagen and smooth muscle cell contents and a 2-fold reduction in macrophage content within the plaques of ApoE/FHL-2-/- versus ApoE-/- mice. This could be explained, in part, by the reduction in aortic ICAM-1 (intracellular adhesion molecule) mRNA and VCAM-1 (vascular cell adhesion molecule) protein expression in the plaque. Aortic gene expression of the chemokines CX3CL1 and CCL5 was increased in ApoE/FHL2-/- versus ApoE-/- mice. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of proinflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- versus ApoE-/- mice. Furthermore, mRNA levels of CX3CR1 were 2-fold higher in monocytes from ApoE/FHL2-/- versus ApoE-/- mice. Finally, we investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. After being irradiated, ApoE-/- or ApoE/FHL2-/- mice were transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After high-fat diet, both chimeric groups developed smaller plaques than ApoE-/- transplanted with ApoE-/- bone marrow. CONCLUSIONS: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting proinflammatory chemokine production, adhesion molecule expression, and proinflammatory monocyte recruitment.

LMO4 is essential for paraventricular hypothalamic neuronal activity and calcium channel expression to prevent hyperphagia.

The dramatic increase in the prevalence of obesity reflects a lack of progress in combating one of the most serious health problems of this century. Recent studies have improved our understanding of the appetitive network by focusing on the paraventricular hypothalamus (PVH), a key region responsible for the homeostatic balance of food intake. Here we show that mice with PVH-specific ablation of LIM domain only 4 (Lmo4) become rapidly obese when fed regular chow due to hyperphagia rather than to reduced energy expenditure. Brain slice recording of LMO4-deficient PVH neurons showed reduced basal cellular excitability together with reduced voltage-activated Ca(2+) currents. Real-time PCR quantification revealed that LMO4 regulates the expression of Ca(2+) channels (Cacna1h, Cacna1e) that underlie neuronal excitability. By increasing neuronal activity using designer receptors exclusively activated by designer drugs technology, we could suppress food intake of PVH-specific LMO4-deficient mice. Together, these results demonstrate that reduced neural activity in LMO4-deficient PVH neurons accounts for hyperphagia. Thus, maintaining PVH activity is important to prevent hyperphagia-induced obesity.

The LIM-domain only protein 4 contributes to lung epithelial cell proliferation but is not essential for tumor progression.

BACKGROUND: The lung is constantly exposed to environmental challenges and must rapidly respond to external insults. Mechanisms involved in the repair of the damaged lung involve expansion of different epithelial cells to repopulate the injured cellular compartment. However, factors regulating cell proliferation following lung injury remain poorly understood. Here we studied the role of the transcriptional regulator Lmo4 during lung development, in the regulation of adult lung epithelial cell proliferation following lung damage and in the context of oncogenic transformation. METHODS: To study the role of Lmo4 in embryonic lung development, lung repair and tumorigenesis, we used conditional knock-out mice to delete Lmo4 in lung epithelial cells from the first stages of lung development. The role of Lmo4 in lung repair was evaluated using two experimental models of lung damage involving chemical and viral injury. The role of Lmo4 in lung tumorigenesis was measured using a mouse model of lung adenocarcinoma in which the oncogenic K-Ras protein has been knocked into the K-Ras locus. Overall survival difference between genotypes was tested by log rank test. Difference between means was tested using one-way ANOVA after assuring that assumptions of normality and equality of variance were satisfied. RESULTS: We found that Lmo4 was not required for normal embryonic lung morphogenesis. In the adult lung, loss of Lmo4 reduced epithelial cell proliferation and delayed repair of the lung following naphthalene or flu-mediated injury, suggesting that Lmo4 participates in the regulation of epithelial cell expansion in response to cellular damage. In the context of K-Ras(G12D)-driven lung tumor formation, Lmo4 loss did not alter overall survival but delayed initiation of lung hyperplasia in K-Ras(G12D) mice sensitized by naphthalene injury. Finally, we evaluated the expression of LMO4 in tissue microarrays of early stage non-small cell lung cancer and observed that LMO4 is more highly expressed in lung squamous cell carcinoma compared to adenocarcinoma. CONCLUSIONS: Together these results show that the transcriptional regulator Lmo4 participates in the regulation of lung epithelial cell proliferation in the context of injury and oncogenic transformation but that Lmo4 depletion is not sufficient to prevent lung repair or tumour formation.

LMO7-null mice exhibit phenotypes consistent with emery-dreifuss muscular dystrophy.

INTRODUCTION: Mutations in the inner nuclear envelope protein emerin cause Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by progressive skeletal muscle wasting, cardiac conduction defects, and tendon contractures. We previously showed that emerin binds directly to the transcription regulator Lmo7 and attenuates its activity to regulate the proper temporal expression of important myogenic differentiation genes. METHODS: The skeletal muscle and cardiac phenotypes were analyzed in a newly generated Lmo7-null mouse using histological analysis, echocardiography, and various neuromuscular tests to determine if Lmo7 was important for skeletal muscle and cardiac function. RESULTS: Lmo7-null mice had growth retardation, decreased fiber size, and impaired skeletal muscle and cardiac function. Lmo7-null mice also had lower levels of phosphorylated retinoblastoma (Rb), extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which is consistent with altered Rb and mitogen-activated protein kinase signaling. CONCLUSIONS: These findings demonstrate that loss of Lmo7 in mice causes myopathic phenotypes similar to those seen in other EDMD mouse models.

Lim domain binding 2: a key driver of transendothelial migration of leukocytes and atherosclerosis.

OBJECTIVE: Using a multi-tissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis. APPROACH AND RESULTS: mRNA profiles generated from blood macrophages in patients with CAD were used to infer transcription factor regulatory gene networks; Ldlr(-/-)Apob(100/100) mice were used to study the effects of Ldb2 deficiency on TEML activity and atherogenesis. LDB2 was the most connected gene in a transcription factor regulatory network inferred from TEML and atherosclerosis module genes in CAD macrophages. In Ldlr(-/-)Apob(100/100) mice, loss of Ldb2 increased atherosclerotic lesion size ≈2-fold and decreased plaque stability. The exacerbated atherosclerosis was caused by increased TEML activity, as demonstrated in air-pouch and retinal vasculature models in vivo, by ex vivo perfusion of primary leukocytes, and by leukocyte migration in vitro. In THP1 cells, migration was increased by overexpression and decreased by small interfering RNA inhibition of LDB2. A functional LDB2 variant (rs10939673) was associated with the risk and extent of CAD across several cohorts. CONCLUSIONS: As a key driver of the TEML pathway in CAD macrophages, LDB2 is a novel candidate to target CAD by inhibiting the overall activity of TEML.

The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat.