LIM-only Protein Research Articles

Death-Associated LIM-Only Protein Reduces Cry1Ac Toxicity by Sequestration of Cry1Ac Protoxin and Activated Toxin in Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

FHL2 is a novel target in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): - Longfonds Nederland Background/ Objectives Pulmonary arterial hypertension (PAH) is a fatal disorder characterized by excessive proliferation of endothelial (EC) and smooth muscle cells, and impaired BMP signaling in small lung arteries. The LIM-only protein FHL2 is expressed in the vessel wall in ECs and smooth muscle cells and plays a crucial role in vessel wall homeostasis. However, the molecular mechanisms how FHL2 exerts its effects and its role in pulmonary artery EC function remain unclear. Here, we investigated the effect of FHL2 on EC proliferation and unraveled the underlying mechanisms involved. Methods/Results We found that FHL2 is highly expressed in lungs of PAH rats compared to control rats as evidenced by qPCR and western blot analysis. Human microvascular ECs (MVECs) derived from lungs of PAH patients displayed enhanced expression of FHL2. Ectopic expression of FHL2 in MVECs significantly increased mRNA levels of BMPR2 and Id1. To substantiate these results, luciferase assays were performed using BMP response element reporter construct and found that FHL2 increases Id1 expression. Furthermore, overexpression of FHL2 decreased TNF-α induced expression of pro-inflammatory cytokines. Finally, ectopic expression of FHL2 reduced proliferation of ECs. Conclusion Our data indicate that FHL2 is a novel negative regulator of MVEC proliferation through enhancing BMP signaling and inhibiting inflammation. Modulation of FHL2 may potentially represent a novel therapeutic strategy for the treatment of PAH.

A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID.

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.

Pro-inflammatory cytokine IL-6 regulates LMO4 expression in psoriatic keratinocytes via AKT/STAT3 pathway.

The transcription factor LIM-only protein 4 (LMO4) is overexpressed in the psoriatic epidermis and regulates keratinocyte proliferation and differentiation. High LMO4 expression levels are induced by interleukin-23 (IL-23) to activate the AKT/STAT3 signaling pathway. Interleukin-6 (IL-6) is mainly involved in regulating T cell functions and development in patients with psoriasis. However, whether LMO4 expression is regulated by IL-6 remains unclear. Therefore, the purpose of this study is to explore the role and molecular mechanisms of IL-6 in regulating LMO4 expression. The interleukin-6 (IL-6) levels in human plasma were determined using a chemiluminescence immunoassay system. A psoriasis-like mouse model was established using imiquimod induction. Epidermal keratinocytes (HaCaT) were cultured in defined keratinocyte-serum-free medium and stimulated by IL-6 alone or with inhibitors. The proteins of interest were detected using western blot analysis, immunofluorescence, and immunohistochemistry. The 5-ethynyl-2'-deoxyuridine assay was used to detect cell proliferation. The results revealed that IL-6 levels were markedly increased in the plasma of patients with psoriasis, compared to healthy control. The high expression of LMO4 was consistent with high levels of IL-6, p-AKT, and p-STAT3 in the lesions of both psoriasis patients and imiquimod-induced psoriasis-like mice. IL-6 activates the AKT/STAT3 signaling pathway, followed by LMO4 high-expression in HaCaT cells. IL-6 induces HaCaT proliferation and differentiation via AKT/STAT3 signaling pathway activation. We think that the high expression of LMO4 in psoriatic keratinocytes requires IL-6 to activate the AKT/STAT3 signaling pathway and leads to epidermal keratinocytes abnormal proliferation and differentiation.

Myelo-Erythroid Lineage Segregation Is Regulated By the GATA-2 Interactome

Hematopoiesis arises from hematopoietic stem cell differentiating into downstream progenitors through a multistep process of sequential lineage potential restriction leading to the final production of >10 hematopoietic cell type.The regulation of hematopoietic lineage development involves synergistic and antagonistic interactions between transcription factors (TF) with lineage-restricted expression patterns, both through protein-protein interaction and direct transcriptional activation.Eosinophils, basophils and mast cells are key myeloid cell types involved in allergic inflammation and have been proposed to arise from a granulocyte-macrophage progenitor (GMP), that also generates neutrophils and macrophages, with GATA-2 up-regulation as a critical determinant. However, recent work identified a distinct basophil/eosinophil/mast cells progenitor (BEMP) that arises from multi-potent erythroid-primed multipotent progenitors (EMPP) where GATA-2 is already expressed ( Drissen et al., 2016). Here, we show that the GATA-2 interactome, rather than GATA-2 expression, controls myeloid lineage choice in EMPPs. To investigate the molecular mechanism instructing lineage decision in EMPP, we performed gene-expression profiling (RNA-seq) comparing its downstream progeny, early restricted erythroid/megakaryocyte progenitor (preMegE) and BEMP. This analysis identified differential expressed genes, including antagonist gene-expression pattern between the LIM-only protein 2 ( Lmo2) and protein 4 ( Lmo4), both previously shown as GATA-2 interactor. While Lmo2 function in hematopoiesis have been exhaustively studied, the role of Lmo4 remains unknown. In colony forming unit (CFU)-assay, overexpression of Lmo4 in EMPP mediated by lentiviral delivery, resulted in an increased number of mast-colonies associated with a decreased of erythroid/megakaryocyte colonies, while the total colony number was unchanged. In addition, transplantation of hematopoietic lineage-negative cells transduced with Lmo4/empty(Ctrl) lentiviral particles, showed an increased frequency of basophils and eosinophils associated with a decrease of erythroid cells in Lmo4 overexpressing cells compared to Ctrl, in both bone marrow and spleen of recipient mice, 8 weeks after transplantation. Analysis of transduced Lineage-negative Kit + (LK) compartment of recipient mice, also revealed an increased frequency of BEMP associated with a decreased frequency of preMegE in Lmo4overexpressing cells compared to Ctrl. Taken together, these data suggested that Lmo4 overexpression instruct lineage decision of EMPP toward basophil/eosinophil/mast lineages.Over-expression followed by co-immunoprecipitation in HEK-293T cell line revealed that LMO4 compete with LMO2 binding to GATA-2, suggesting a mutually exclusive transcriptional complex. More importantly, lack of interaction between LMO4 and GATA-2 mediated by a point mutation in the zinc-finger 1 domain (ZnF1) of GATA-2 (GATA2-G320D), frequently identified in AML/AEL, resulted in an absence of mast lineage potential of EMPP compared to GATA2 +/+. Moreover, over-expression of Lmo4 in EMPP from GATA2 G320D/G320D mice compared to GATA2 +/+, failed to instruct myeloid fate in vitro. Overall, our data identified a specific molecular mechanism of hematopoietic cell fate decision driven by protein-protein interaction with GATA-2. Perturbation of GATA-2 interactome through over-expression of the cofactor Lmo4 or specific point mutation in GATA2 ZnF1 domain results in lineage fate switch of EMPP toward the erythroid/megakaryocyte or basophil/eosinophil/mast lineages.

Circ_0007385 promotes the proliferation and inhibits the apoptosis of non-small cell lung cancer cells via miR-337-3p-dependent regulation of LMO3.

This study intended to analyze the expression characteristic, functions and underlying mechanism of circular RNA_0007385 (circ_0007385) in non-small cell lung cancer (NSCLC). RNA and protein expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay. Cell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry were applied to analyze cell proliferation ability. Flow cytometry was also conducted to assess cell apoptosis. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the predicted target relationships. Xenograft tumor model was utilized to analyze the function of circ_0007385 in vivo, and immunohistochemistry (IHC) assay was used to analyze protein expression in xenograft tumor tissues. Circ_0007385 expression was notably enhanced in NSCLC tissues and cell lines. Circ_0007385 facilitated the proliferation but suppressed the apoptosis of NSCLC cells. Circ_0007385 acted as a sponge for microRNA-337-3p (miR-337-3p), and circ_0007385 overexpression-mediated effects were largely overturned by the overexpression of miR-337-3p in NSCLC cells. MiR-337-3p interacted with the 3' untranslated region (3'UTR) of LIM-only protein 3 (LMO3). Circ_0007385 up-regulated LMO3 level by absorbing miR-337-3p in NSCLC cells. LMO3 overexpression largely reversed miR-337-3p overexpression-induced influences in NSCLC cells. Circ_0007385 knockdown significantly restrained the growth of xenograft tumors in vivo. Circ_0007385 promoted the proliferation ability and inhibited the apoptosis of NSCLC cells by binding to miR-337-3p to induce LMO3 expression.

TAB182 aggravates progression of esophageal squamous cell carcinoma by enhancing β-catenin nuclear translocation through FHL2 dependent manner

TAB182 (also named TNKS1BP1), a binding protein of tankyrase 1, has been found to participate in DNA repair. Our previous study has revealed the involvement of TAB182 in the radioresistance of esophageal squamous cell carcinoma (ESCC) cells. However, whether TAB182 contributes to the ESCC tumorigenesis and progression remains unclear. In this study, we found that highly expressed TAB182 is closely associated with a poor prognosis of patients with ESCC. TAB182 silencing reduced ESCC cell proliferation and invasion in vitro, tumorigenicity and metastasis in vivo. RNA-seq and IP-MS analysis revealed that TAB182 could affect the β-catenin signaling pathway via interacting with β-catenin. Furthermore, TAB182 prevented β-catenin to be phosphorylated by GSK3β and recruited four and a half of LIM-only protein 2 (FHL2), which thereby promoted β-catenin nucleus translocation to result in activation of the downstream targets transcription in ESCC cells. Our findings demonstrate that TAB182 enhances tumorigenesis of esophageal cancer by promoting the activation of the β-catenin signaling pathway, which provides new insights into the molecular mechanisms by which TAB182 accelerates progression of ESCC.

The Roles of FHL3 in Cancer.

The four and a half LIM domain protein 3, also named the LIM-protein FHL3, belongs to the LIM-only family. Based on the special structure of LIM-only proteins, FHL3 can perform significant functions in muscle proliferation and cardiovascular diseases by regulating cell growth and signal transduction. In recent years, there has been increasing evidence of a relation between FHLs and tumor biology, since FHL3 is often overexpressed or downregulated in different cancers. On the one hand, FHL3 can function as a tumor suppressor and influence the expression of downstream genes. On the other hand, FHL3 can also play a role as an oncoprotein in some cancers to promote tumor progression via phosphorylation. Thus, FHL3 is proposed to have a dual effect on cancer progression, reflecting its complex roles in cancer. This review focuses on the roles of FHL3 in cancer progression and discusses the interaction of FHL3 with other proteins and transcription factors. Finally, the clinical significance of FHL3 for the treatment of cancers is discussed.

Cysteine-Rich LIM-Only Protein 4 (CRP4) Promotes Atherogenesis in the ApoE-/- Mouse Model.

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Cyclic GMP-Dependent Regulation of Vascular Tone and Blood Pressure Involves Cysteine-Rich LIM-Only Protein 4 (CRP4).

The cysteine-rich LIM-only protein 4 (CRP4), a LIM-domain and zinc finger containing adapter protein, has been implicated as a downstream effector of the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) pathway in multiple cell types, including vascular smooth muscle cells (VSMCs). VSMCs and nitric oxide (NO)-induced cGMP signaling through cGMP-dependent protein kinase type I (cGKI) play fundamental roles in the physiological regulation of vascular tone and arterial blood pressure (BP). However, it remains unclear whether the vasorelaxant actions attributed to the NO/cGMP axis require CRP4. This study uses mice with a targeted deletion of the CRP4 gene (CRP4 KO) to elucidate whether cGMP-elevating agents, which are well known for their vasorelaxant properties, affect vessel tone, and thus, BP through CRP4. Cinaciguat, a NO- and heme-independent activator of the NO-sensitive (soluble) guanylyl cyclase (NO-GC) and NO-releasing agents, relaxed both CRP4-proficient and -deficient aortic ring segments pre-contracted with prostaglandin F2α. However, the magnitude of relaxation was slightly, but significantly, increased in vessels lacking CRP4. Accordingly, CRP4 KO mice presented with hypotonia at baseline, as well as a greater drop in systolic BP in response to the acute administration of cinaciguat, sodium nitroprusside, and carbachol. Mechanistically, loss of CRP4 in VSMCs reduced the Ca2+-sensitivity of the contractile apparatus, possibly involving regulatory proteins, such as myosin phosphatase targeting subunit 1 (MYPT1) and the regulatory light chain of myosin (RLC). In conclusion, the present findings confirm that the adapter protein CRP4 interacts with the NO-GC/cGMP/cGKI pathway in the vasculature. CRP4 seems to be part of a negative feedback loop that eventually fine-tunes the NO-GC/cGMP axis in VSMCs to increase myofilament Ca2+ desensitization and thereby the maximal vasorelaxant effects attained by (selected) cGMP-elevating agents.

The circular RNA circ_GRHPR promotes NSCLC cell proliferation and invasion via interactions with the RNA-binding protein PCBP2.

As the most common malignancy, lung cancer is characterised by high rates of occurrence and mortality. Although circular RNAs (circRNAs) are known to act as important regulators in cancer, their role in lung cancer remains poorly understood. In this study, circ_GRHPR expression was found to be significantly upregulated in the serum of five patients with non‐small cell lung cancer (NSCLC), compared to that in healthy controls. It is expressed at high levels in NSCLC cell lines, as revealed by qRT‐PCR analysis. Functionally, we demonstrated that circ_GRHPR promotes NSCLC proliferation and invasion in vitro and in vivo by cell proliferation, transwell, cell cycle, and tumour‐forming assays. Mechanistically, RNA pull‐down and RNA immunoprecipitation assays showed that circ_GRHPR interacts with the RNA‐binding protein poly(rC)‐binding protein 2 (PCBP2) and regulates its subcellular localisation by forming the circ_GRHPR/PCBP2 complex, localizing PCBP2 mainly in the cytoplasm and reducing the proportion found in the nucleus. Furthermore, we demonstrated that four‐and‐a‐half LIM‐only protein 3 (FHL3) is a tumour‐stimulating factor in NSCLC that interacts with and is influenced by PCBP2. Circ_GRHPR increased FHL3 expression in the nucleus of NSCLC cells by decreasing PCBP2 expression therein and promoting the proliferation and invasion of NSCLC cells. Therefore, our study identified that circ_GRHPR promotes NSCLC proliferation and invasion, providing a possible explanation for its mechanism of action.

The novel six LIM and one PET domain-containing protein Lmpt is involved in the immune response through activation of the NF-κB signalling pathway in the crustacean, Macrobrachium nipponense

The four-and-a-half LIM-only protein family of transcription co-factors participates in various cellular processes, such as cell proliferation, cell differentiation, apoptosis, cell adhesion, migration, transcription and signal transduction. However, the knowledge of the structural characteristics and immune functions of its ancestor Lmpt, which contains six LIM domains at the C-terminus and a PET domain at the N-terminus, is limited in invertebrates, especially in crustaceans. In the present study, a novel Lmpt from oriental river prawn (Macrobrachium nipponense) was identified, and its role in the immune response was investigated. Its full-length cDNA sequence was 6407 bp, which contained a 2595 bp ORF encoding 865 amino acids, exhibiting high similarity to the structure of Lmpt derived from other invertebrates. Tissue distribution analysis revealed that MnLmpt was widely expressed in all examined tissues, and high expression levels were observed in muscle, heart and intestine in M. nipponense. After experimental challenges with bacteria and virus, the transcription levels of MnLmpt significantly fluctuated in gill and hepatopancreas, indicating that it might play a role in the innate immune response in M. nipponense. Silencing of MnLmpt by dsRNA injection in vivo could promote bacterial growth, suggesting that MnLmpt exerted an antibacterial immune function in prawn. Immunocytochemistry assay results demonstrated that MnLmpt was able to translocate from the cytoplasm to the nucleus after being stimulated with pathogens. The expression levels of NF-κB signalling cascade members, such as dorsal, relish, TAK1, TAB1, Ikkβ, and Ikkε, and AMPs, including ALF4, Cru1, and Cru2, exhibited significant downregulation in the MnLmpt silenced group. Similarly, dual-luciferase reporter assays also demonstrated that MnLmpt could stimulate an NF-κB signalling cascade. Meanwhile, all of the LIM domains of MnLmpt could trigger NF-κB signalling; however, their cumulative effect on NF-κB promoter activation was hardly observed. These results showed that MnLmpt might play a crucial role in the innate immune response in M. nipponense, and these findings paved the way for a better understanding of the immune system in crustacean species.

LncRNA SNHG6 promotes LMO3 expression by sponging miR-543 in glioma.

Small nucleolar RNA host gene 6 (SNHG6) was a newly discovered long non-coding RNA, which was involved in the occurrence and development of a variety of cancers and was on the rise in human cancers. However, the molecular mechanism of SNHG6 in glioma required further investigation. The levels of SNHG6, microRNA-543 (miR-543) and LIM-only protein 3 (LMO3) were detected in glioma tissues and cells by quantitative real-time polymerase chain reaction. We examined cell proliferation and apoptosis rate by methylthiazolyldiphenyl-tetrazolium bromide and flow cytometry assays, respectively. Transwell assay was used to measure cell migration and invasion. The target relationships were predicted by StarBase v.2.0 and TargetScan and confirmed by dual-luciferase reporter assay. Spearman's test was adopted for expression correlation of SNHG6, miR-543 and LMO3 in tissues. The protein expression level of LMO3 was assessed by western blot. We found that SNHG6 was obviously upregulated in glioma tissues and cells. SNHG6 knockdown significantly repressed glioma cell proliferation, migration and invasion, and induced apoptosis. Additionally, SNHG6 directly targeted miR-543 and their expression was negatively correlated in glioma tissues. And miR-543 targeted LMO3 and their expression was also inversely correlated. We found that silencing LMO3 also inhibited the progression of glioma cells. Importantly, SNHG6 could competitively sponging miR-543 thereby modulating LMO3 in glioma cells. SNHG6 served as an oncogene and played a vital role in glioma development through miR-543/LMO3 axis.

The CDK inhibitor p57Kip2 enhances the activity of the transcriptional coactivator FHL2

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57Kip2 is a member of the Cip/Kip family of CKIs and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and CDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2-regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity by HDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.

LncRNA SNHG14 promotes non-small cell lung cancer progression by sponging miR-382-5p

Long non-coding RNAs (lncRNAs) are involved in development of non-small cell lung cancer (NSCLC) by interacting with microRNAs (miRNAs) and/or mRNAs. However, the function of most lncRNAs in NSCLC remains unclear. Reverse transcription-quantitative PCR (RT-qPCR) and western blot were applied for detection of mRNA/miRNA and protein level. Interaction between LncRNA small nucleolar RNA host gene 14 (SNHG14) and miR-382-5p was validated by dual luciferase reporter assay. Cell proliferation and apoptosis was detected using Cell Counting Kit 8 (CCK8) and Flow cytometry, while cell migration and invasion was detected by scratch assay and Transwell assay. SNHG14 was upregulated in NSCLC tissues and cell lines. SNHG14 silencing reduced proliferation, migration and invasion, and induced apoptosis in A549 cells. SNHG14 was negatively correlated with miR-382-5p in NSCLC tissues, SNHG14 and miR-382-5p negatively regulated each other in A549 cells. SNHG14 promoted expression of miR-382-5p target genes, including LIM-only protein 3 (LMO3) and SET domain containing lysine methyltransferase 8 (SETD8), in A549 cells. miR-382-5p inhibition reversed SNHG14 knockdown-induced increase in apoptosis and decrease in proliferation, migration and invasion. SNHG14 was correlated with SETD8 and LMO3 in NSCLC tissues. Collectively, SNHG14 promoted NSCLC proliferation, migration and invasion, while inhibited apoptosis by sponging miR-382-5p in A549 cells. Abbreviations: CCK8: Cell Counting Kit 8; LMO3: LIM-only protein 3; lncRNAs: long non-coding RNAs; miRNAs: microRNAs; NSCLC: non-small cell lung cancer; RT-qPCR: reverse transcription-quantitative PCR; SNHG14: LncRNA small nucleolar RNA host gene 14; SETD8: SET domain containing lysine methyltransferase 8.

Crystal structure of human LDB1 in complex with SSBP2

The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the β-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. Here we report a crystal structure of the human LDB1/SSBP2 complex at 2.8-Å resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4-helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. Our structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer-promoter interactions.

LIM-only protein FHL2 attenuates inflammation in vascular smooth muscle cells through inhibition of the NFκB pathway

Despite the advent of new-generation drug-eluting stents, in-stent restenosis remains a significant problem in patients with coronary artery disease. In- stent restenosis is defined as the gradual re-narrowing of a stented coronary artery lesion due to arterial damage with subsequent local inflammation of the vessel wall and excessive growth of the vascular smooth muscle cells (vSMCs). Four-and-a-half LIM-domain protein 2 (FHL2) is a scaffold protein involved in regulating vSMC function and inflammation. Previously we have demonstrated that FHL2 prevents vSMC proliferation in a murine carotid artery ligation model. However, the effect of FHL2 on the inflammatory response of the vSMCs is not investigated. Therefore, we studied the inflammatory response in the vessel wall of FHL2-deficient (-KO) mice after carotid artery ligation. We found that circulating cytokines and local macrophage infiltration in the ligated carotid vessels were increased in FHL2-KO mice after carotid artery ligation. Moreover, FHL2-KO vSMCs showed increased secretion of cytokines such as SDF-1α and RANTES, and enhanced activation of the NFκB pathway. Finally, we found that blocking the NFκB signalling pathway abrogated this pro-inflammatory state in FHL2-KO vSMCs. Taken together, our results demonstrate that FHL2 decreases the inflammatory response of vSMCs through inhibition of the NFkB-signalling pathway.

LIM-only protein FHL2 attenuates vascular tissue factor activity, inhibits thrombus formation in mice and FHL2 genetic variation associates with human venous thrombosis.

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans.

MiR-263b Controls Circadian Behavior and the Structural Plasticity of Pacemaker Neurons by Regulating the LIM-Only Protein Beadex.

Circadian clocks drive rhythmic physiology and behavior to allow adaption to daily environmental changes. In Drosophila, the small ventral lateral neurons (sLNvs) are primary pacemakers that control circadian rhythms. Circadian changes are observed in the dorsal axonal projections of the sLNvs, but their physiological importance and the underlying mechanism are unclear. Here, we identified miR-263b as an important regulator of circadian rhythms and structural plasticity of sLNvs in Drosophila. Depletion of miR-263b (miR-263bKO) in flies dramatically impaired locomotor rhythms under constant darkness. Indeed, miR-263b is required for the structural plasticity of sLNvs. miR-263b regulates circadian rhythms through inhibition of expression of the LIM-only protein Beadex (Bx). Consistently, overexpression of Bx or loss-of-function mutation (BxhdpR26) phenocopied miR-263bKO and miR-263b overexpression in behavior and molecular characteristics. In addition, mutating the miR-263b binding sites in the Bx 3′ UTR using CRISPR/Cas9 recapitulated the circadian phenotypes of miR-263bKO flies. Together, these results establish miR-263b as an important regulator of circadian locomotor behavior and structural plasticity.

Overexpression of LIMD2 promotes the progression of non-small cell lung cancer.

LIM domain containing 2 (LIMD2) is a small LIM-only protein that has been demonstrated to promote tumor progression; however, the expression and function of LIMD2 in non-small cell lung cancer (NSCLC) has not previously been reported. In the present study, reverse transcription-quantitative PCR and western blot analysis were conducted to examine the mRNA and protein expression levels of LIMD2. Cell Counting Kit-8, Transwell and wound-healing assays were performed in order to examine cell proliferation, invasion and migration, respectively. The data revealed that the LIMD2 expression levels were significantly increased in NSCLC tissues and cell lines, compared with adjacent non-tumor tissues and normal lung epithelial cells, respectively. In addition, the high expression of LIMD2 was significantly associated with lymph node metastasis, distant metastasis and advanced clinical stage in NSCLC. The patients with NSCLC with a high expression of LIMD2 exhibited shorter survival times than those with low LIMD2 expression. The knockdown of LIMD2 caused remarkable decreases in NSCLC cell proliferation, migration and invasion. Bioinformatics analysis and luciferase reporter gene assay data further confirmed that LIMD2 was a direct target gene of microRNA-124 (miR-124), a well-known tumor suppressor in NSCLC. The expression of LIMD2 was negatively regulated by miR-124 in NSCLC cells. In addition, miR-124 was downregulated in NSCLC tissues compared with adjacent non-tumor tissues, and an inverse correlation was observed between the expression of LIMD2 and miR-124 in NSCLC tissues. In conclusion, the present study demonstrates that LIMD2 serves an oncogenic role in NSCLC, suggesting that it may be used as a potential therapeutic target for the treatment of NSCLC.