Targeted therapy for LIMD1-deficient non-small cell lung cancer subtypes.

An early event in lung oncogenesis is loss of the tumour suppressor gene LIMD1 (LIM domains containing 1); this encodes a scaffold protein, which suppresses tumorigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD1, yet this subtype of NSCLC has been overlooked in preclinical and clinical investigations. Defining therapeutic targets in these LIMD1 loss-of-function patients is difficult due to a lack of 'druggable' targets, thus alternative approaches are required. To this end, we performed the first drug repurposing screen to identify compounds that confer synthetic lethality with LIMD1 loss in NSCLC cells. PF-477736 was shown to selectively target LIMD1-deficient cells in vitro through inhibition of multiple kinases, inducing cell death via apoptosis. Furthermore, PF-477736 was effective in treating LIMD1-/- tumours in subcutaneous xenograft models, with no significant effect in LIMD1+/+ cells. We have identified a novel drug tool with significant preclinical characterisation that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need.

Maternal smoking during pregnancy aggravated muscle phenotype in FHL1-/y offspring mice similar to congenital clubfoot through P2RX7-mediated pyroptosis.

Congenital clubfoot (CCF) is a common birth defect. Maternal smoking during pregnancy increases the risk of CCF. In previous research, we found muscle phenotypes similar to CCF in four and a half LIM domain protein 1 (FHLI) offspring mice (FHL1-/y). However, the role of P2RX7-mediated pyroptosis in the effect of cigarette smoke (CS) on the skeletal muscle of FHL1-/y mice during pregnancy is unclear. In the present study, pregnant mice at 11 days of gestation were exposed to CS and male offspring of wild-type (WT) and FHL1-/y mice were divided into four groups (Control-WT, Control-KO, CS-WT, CS-KO). The histomorphology of lower limb muscles was examined using hematoxylin and eosin (H&E) staining. P2RX7, indicators of pyroptosis (NLRP3, ASC, cleaved-caspase 1, IL-1ß), and cytoskeletal proteins (MYBPC2, LDB3) were also detected using immunoblotting. CS exposure during pregnancy aggravated the muscle phenotype similar to CCF in FHL1-/y offspring mice. FHL1 gene knockout (KO) or CS exposure during pregnancy each activated the expression of P2RX7, cell pyroptosis-related proteins (NLRP3, ASC, cleaved-caspase 1, IL-1ß), a muscle injury marker (MYOD1), and cytoskeletal proteins (MYBPC2, LDB3); these two factors had an additive effect. The results showed maternal smoking during pregnancy aggravated muscle phenotype similar to CCF in FHL1-/y offspring mice through P2RX7-mediated pyroptosis.

Lack of expression of LMO2 clone SP51 identifies MYC rearrangements in aggressive large B-cell lymphomas.

MYC rearrangements (MYC-R) confer unfavorable prognosis to large B-cell lymphomas (LBCL). Because of the low incidence of such genetic alteration, surrogates to screen MYC-R may be useful in daily practice. Previous studies suggested that clone 1A9-1 of LMO2 loss may be a good predictor for the presence of MYC-R in LBCL. The present study examines the utility of LMO2 clone SP51. For this purpose, we have analyzed 20 Burkitt lymphomas and 325 LBCL. Among them, 245 cases were studied prospectively using whole tissue sections, and 100 retrospectively by tissue microarrays. The cohort of CD10-positive prospective cases achieved the best results. Lack of LMO2 SP51 expression predicted the presence of MYC-R with high specificity, accuracy, positive and negative predictive value (PPV/NPV), and positive and negative likelihood ratios (PLR/NLR). Compared with MYC protein expression, LMO2 SP51 obtained significantly higher specificity, accuracy, PPV, and PLR (94%, 91%, 85%, and 14.33 vs 73%, 77%, 56%, and 3.26, respectively), and similar NPV and NLR (92% and 0.22 vs 95% and 0.12). Compared with LMO2 clone 1A9-1, the sensitivity of LMO2 SP51 was lower (79% vs 89%). We conclude that LMO2 SP51 may be a useful marker to screen MYC-R in CD10-positive LBCL.

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.

Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples.

Knockout of multifunction gene cysteine- and glycine-rich protein 3 (CSRP3) in cardiomyocytes (CMs) of mice leads to heart dilation, severely affecting its functions. In humans, CSRP3 mutations are associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM). The absence of the CSRP3 expression produces unknown effects on in vitro neonatal CMs' metabolism. The metabolome changes in culture media conditioned by CSRP3 knockout (KO-CSRP3), and wild type (WT) neonatal cardiomyocytes were investigated under untreated or after metabolic challenging conditions produced by isoproterenol (ISO) stimulation, by in vitro high-resolution proton magnetic resonance spectroscopy (1H-MRS)-based metabolomics. Metabolic differences between neonatal KO-CSRP3 and WT rats' CMs were identified. After 72 h of culture, ISO administration was associated with increased CMs' energy requirements and increased levels of threonine, alanine, and 3-hydroxybutyrate in both neonatal KO-CSRP3 and WT CMs conditioned media. When compared with KO-CSRP3, culture media derived from WT cells presented higher lactate concentrations either under basal or ISO-stimulated conditions. The higher activity of ketogenic biochemical pathways met the elevated energy requirements of the contractile cells. Both cells are considered phenotypically indistinguishable in the neonatal period of animal lives, but the observed metabolic stress responses of KO-CSRP3 and WT CMs to ISO were different. KO-CSRP3 CMs produced less lactate than WT CMs in both basal and stimulated conditions. Mainly, ISO-stimulated conditions produced evidence for lactate overload within KO-CSRP3 CMs, while WT CMs succeeded to manage the metabolic stress. Thus, 1H-MRS-based metabolomics was suitable to identify early inefficient energetic metabolism in neonatal KO-CSRP3 CMs. These results may reflect an apparent lower lactate transport and consumption, in association with protein catabolism.

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.

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.

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.

Alleviation of murine osteoarthritis by deletion of the focal adhesion mechanosensitive adapter, Hic-5.

Excessive mechanical stress is a major cause of knee osteoarthritis. However, the mechanism by which the mechanical stress begets osteoarthritis development remains elusive. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFß1i1), a TGF-ß inducible focal adhesion adaptor, has previously been reported as a mediator of mechanotransduction. In this study, we analyzed the in vivo function of Hic-5 in development of osteoarthritis, and found that mice lacking Hic-5 showed a significant reduction in development of osteoarthritis in the knee. Furthermore, we found reduced expression of catabolic genes, such as metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 in osteoarthritic lesions in mice lacking Hic-5. During osteoarthritis development, Hic-5 is detected in chondrocytes of articular cartilage. To investigate the role of Hic-5 in chondrocytes, we isolated chondrocytes from articular cartilage of wild type and Hic-5-deficient mice. In these primary cultured chondrocytes, Hic-5 deficiency resulted in suppression of catabolic gene expression induced by osteoarthritis-related cytokines such as tumor necrosis factor α and interleukin 1ß. Furthermore, Hic-5 deficiency in chondrocytes suppressed catabolic gene expression induced by mechanical stress. Revealing the regulation of chondrocyte catabolism by Hic-5 contributes to understanding the pathophysiology of osteoarthritis induced by mechanical stress.

MLP-deficient human pluripotent stem cell derived cardiomyocytes develop hypertrophic cardiomyopathy and heart failure phenotypes due to abnormal calcium handling.

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

Yeats4 drives ILC lineage commitment via activation of Lmo4 transcription.

Innate lymphoid cells (ILCs) play critical roles in defending infections and maintaining mucosal homeostasis. All ILCs arise from common lymphoid progenitors (CLPs) in bone marrow. However, how CLPs stratify and differentiate into ILC lineages remains elusive. Here, we showed that Yeats4 is highly expressed in ILCs and their progenitors. Yeats4 conditional KO in the hematopoietic system causes decreased numbers of ILCs and impairs their effector functions. Moreover, Yeats4 regulates α4ß7 + CLP differentiation toward common helper ILC progenitors (CHILPs). Mechanistically, Yeats4 recruits the Dot1l-RNA Pol II complex onto Lmo4 promoter through recognizing H3K27ac modification to initiate Lmo4 transcription in α4ß7 + CLPs. Additionally, Lmo4 deficiency also impairs ILC lineage differentiation and their effector functions. Collectively, the Yeats4-Lmo4 axis is required for ILC lineage commitment.

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.

Sarcomere-based genetic enhancement of systolic cardiac function in a murine model of dilated cardiomyopathy.

Diminished cardiac contractile function is a characteristic feature of dilated cardiomyopathy (DCM) and many other heart failure (HF) causing etiologies. We tested the hypothesis that targeting the sarcomere to increase cardiac contractility can effectively prevent the DCM phenotype in muscle-LIM protein knockout (MLP-/-) mice. The ablation of cardiac myosin binding protein C (MYBPC3-/-) protected the MLP-/- mice from developing the DCM phenotype. We examined the in vivo cardiac function and morphology of the resultant mouse model lacking both MLP and MYBPC3 (DKO) by echocardiography and pressure-volume catheterization and found a significant reduction in hypertrophy, as evidenced by normalized wall thickness and chamber dimensions, and improved systolic function, as evidenced by enhanced ejection fraction (~26% increase compared MLP-/- mice) and rate of pressure development (DKO 7851.0 ±â€¯504.8 vs. MLP-/- 4496.4 ±â€¯196.8 mmHg/s). To investigate the molecular basis for the improved DKO phenotype we performed mechanical experiments in skinned myocardium isolated from WT and the individual KO mice. Skinned myocardium isolated from DKO mice displayed increased Ca2+ sensitivity of force generation, and significantly accelerated rate of cross-bridge detachment (+63% compared to MLP-/-) and rate of XB recruitment (+58% compared to MLP-/-) at submaximal Ca2+ activations. The in vivo and in vitro functional enhancement of DKO mice demonstrates that enhancing the sarcomeric contractility can be cardioprotective in HF characterized by reduced cardiac output, such as in cases of DCM.

The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). We recently discovered a micropeptide named DWORF (DWarf Open Reading Frame) that enhances SERCA activity by displacing phospholamban (PLN), a potent SERCA inhibitor. Here we show that DWORF has a higher apparent binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA. Additionally, using a well-characterized mouse model of dilated cardiomyopathy (DCM) due to genetic deletion of the muscle-specific LIM domain protein (MLP), we show that DWORF overexpression restores cardiac function and prevents the pathological remodeling and Ca2+ dysregulation classically exhibited by MLP knockout mice. Our results establish DWORF as a potent activator of SERCA within the heart and as an attractive candidate for a heart failure therapeutic.

LDB2 regulates the expression of DLL4 through the formation of oligomeric complexes in endothelial cells.

Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling. [BMB Reports 2018; 51(1): 21-26].

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.

[FHL1 knockdown mediated by lentiviral shRNA promotes the growth of HeLa and HepG2 cells].

OBJECTIVE: To investigate the effect of four-and-a-half LIM domain 1 (FHL1) knockdown by lentiviral-mediated shRNA on the growth of HeLa and HepG2 cells. METHODS: pLenti-H1 FHL1 shRNA was cloned, and then transfered into HEK293T cells. The inhibitory effect of pLenti-H1 FHL1 shRNA on FHL1 gene was detected by Western blotting and real-time quantitative PCR (qRT-PCR). Lentivirus particles were packaged, added to HeLa and HepG2 cells, followed by puromycin treatment for 2-3 weeks to screen stable clones. The knockdown effect on FHL1 expression in these cells was checked by Western blotting and qRT-PCR. Cell growth and colony formation analysis were performed to investigate the effect of FHL1 down-regulation on tumor cell growth. Soft agar analysis was used to analyze its effect on anchorage-independent tumor cell growth. RESULTS: Western blotting and qRT-PCR revealed that the pLenti-H1 FHL1 shRNA apparently inhibited the expression of FHL1 gene. Cell growth and colony formation assay showed that the lentiviral-mediated shRNA for FHL1 gene significantly accelerated the tumor cell growth in HepG2 and HeLa cells. Soft agar analysis demonstrated that FHL1 shRNA increased the anchorage-independent growth of tumor cells. CONCLUSION: pLenti-H1 FHL1 shRNA could significantly accelerate tumor cell growth via inhibiting the expression of FHL1.

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.

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.

Role of Hic-5 in the formation of microvilli-like structures and the monocyte-endothelial interaction that accelerates atherosclerosis.

AIMS: The adhesion of circulating monocytes to endothelial cells (ECs) is an early and critical event in the formation of atherosclerotic plaques. Hydrogen peroxide-inducible clone 5 (Hic-5) serves as an adaptor molecule in cell adhesion complexes. However, the role of endothelial Hic-5 in monocyte-EC interaction and atherogenesis remains unclear. We examined the roles of endothelial Hic-5 in monocyte-EC interaction and atherogenesis using mouse models of atherosclerosis and cultured human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Hic-5 was expressed in ECs, but not in monocytes/macrophages. An ex vivo monocyte adhesion assay revealed that adhesion of THP-1 monocytes to aortas isolated from Apoe(-/-) and LDLR(-/-) mice stimulated by TNF-α or oxidized LDL was suppressed by Hic-5 deficiency. Scanning electron microscopic observations of aortas harvested from Apoe(-/-) mice revealed that TNF-α- or oxidized LDL-induced microvilli-like structures were markedly suppressed by Hic-5 deficiency. Relative Hic-5 deficiency suppressed 60% of the atherosclerotic lesions in aortas from Apoe(-/-) and LDLR(-/-) mice. In contrast, overexpression of Hic-5 in HUVECs promoted induction of microvilli-like structures and adherence of THP-1 cells in an adhesion receptor such as intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-dependent manner. CONCLUSION: Hic-5 in ECs plays an important role in the formation of microvilli-like structures and in the interaction between ECs and monocytes, leading to monocyte recruitment and subsequent development of atherosclerosis.