Search

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub., Competing Interests: The authors have declared that no competing interests exist.

Background: Hypertrophic cardiomyopathy (HCM) is a hereditary disease manifested by a thickened ventricular wall. Cysteine and glycine-rich protein 3 (CSRP3), the gene encoding muscle LIM protein, is important for initiating hypertrophic gene expression. The mutation of CSRP3 causes dilated cardiomyopathy or HCM., Methods: In the present study, we enrolled a Chinese family with HCM across three generations. Whole-exome sequencing (WES) was performed in the proband to detect the candidate genes of the family. Sanger sequencing was performed for mutational analysis and confirmation of cosegregation., Results: Through histopathological and imaging examinations, an obvious left ventricular hypertrophy was found in the proband. After WES data filtering, bioinformatic prediction and co-segregation analysis, a nonsense mutation (NM_003476.5:c.364C>T; NP_003467.1:p.Arg122*) of CSRP3 was identified in this family. This variant was predicted to be disease-causing and resulted in a truncated protein., Conclusions: This is the first HCM family case of CSRP3 (p.Arg122*) variation in Asia. The finding here not only contributes to the genetic diagnosis and counseling of the family, but also provides a new case with detailed phenotypes that may be caused by the CSRP3 variant., (© 2021 John Wiley & Sons, Ltd.)

Diabetic retinopathy (DR) is a type of diabetes complication, which can result in loss of vision in adults worldwide. Increasing evidence has revealed that microRNAs (miRs) can regulate DR progression. Thus, the present study was aimed at assessing the possible mechanism of miR-139-5p in high glucose- (HG-) incubated retinal pigment epithelial (ARPE-19) cells. The present results demonstrated that miR-139-5p expression was notably reduced in the serum samples of patients with DR, as well as in ARPE-19 cells treated with HG in a time-dependent manner. Moreover, miR-139-5p was markedly overexpressed by transfection of miR-139-5p mimics into ARPE-19 cells. Overexpression of miR-139-5p markedly induced cell viability and repressed HG-triggered apoptosis. Furthermore, overexpression of miR-139-5p relived HG-enhanced oxidative stress injury. It was found that HG induced malondialdehyde levels but decreased superoxide dismutase and glutathione peroxidase activities in ARPE-19 cells. In addition, overexpression of miR-139-5p could markedly decrease intracellular stress. The results demonstrated that overexpression of miR-139-5p effectively repressed HG-activated inflammation, as indicated by the upregulation of inflammation cytokines, including TNF- α , IL-6, and Cox-2, in ARPE-19 cells. Subsequently, it was identified that LIM-only factor 4 (LMO4) could act as a downstream target for miR-139-5p. LMO4 expression was significantly increased in patients with DR and HG-treated ARPE-19 cells. Mechanistically, knockdown of LMO4 reversed the biological role of miR-139-5p in proliferation, apoptosis, oxidative stress, and release of inflammation factors in vitro . Collectively, these results suggested that miR-139-5p significantly decreased ARPE-19 cell injury caused by HG by inducing proliferation and suppressing cell apoptosis, oxidant stress, and inflammation by modulating LMO4., Competing Interests: The authors declare no financial or commercial conflict of interest., (Copyright © 2021 Kan Shao et al.)

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Advanced cervical cancer carries a particularly poor prognosis, and few treatment options exist. Identification of effective molecular markers is vital to improve the individualisation of treatment. We investigated transcriptional data from cervical carcinomas related to patient survival and recurrence to identify potential molecular drivers for aggressive disease., Methods: Primary tumour RNA-sequencing profiles from 20 patients with recurrence and 53 patients with cured disease were compared. Protein levels and prognostic impact for selected markers were identified by immunohistochemistry in a population-based patient cohort., Results: Comparison of tumours relative to recurrence status revealed 121 differentially expressed genes. From this gene set, a 10-gene signature with high prognostic significance (p = 0.001) was identified and validated in an independent patient cohort (p = 0.004). Protein levels of two signature genes, HLA-DQB1 (n = 389) and LIMCH1 (LIM and calponin homology domain 1) (n = 410), were independent predictors of survival (hazard ratio 2.50, p = 0.007 for HLA-DQB1 and 3.19, p = 0.007 for LIMCH1) when adjusting for established prognostic markers. HLA-DQB1 protein expression associated with programmed death ligand 1 positivity (p < 0.001). In gene set enrichment analyses, HLA-DQB1high tumours associated with immune activation and response to interferon-γ (IFN-γ)., Conclusions: This study revealed a 10-gene signature with high prognostic power in cervical cancer. HLA-DQB1 and LIMCH1 are potential biomarkers guiding cervical cancer treatment.

The four-and-a-half LIM domain protein 1 (FHL1) plays a key role in multiple cancers. Here, we characterized its role in glioblastoma (GBM), the most common and incurable form of brain cancer. Overexpression of FHL1 promotes growth, migration, and invasion of GBM cells in vivo and in vitro. In contrast, FHL1 silencing by RNAi exhibits the opposite effects. FHL1 interacts with the transcription factor SP1 to upregulate epidermal growth factor receptor (EGFR) expression and activate the downstream signaling cascades, including Src, Akt, Erk1/2, and Stat3, leading to GBM malignancy. FHL1 is highly expressed and positively correlated with EGFR levels in human GBM, particularly those of the classical subtype. Our results suggest that the FHL1-SP1-EGFR axis plays a tumor-promoting role, and highlight the translational potential of inhibiting FHL1 for GBM treatment., (© 2020 Federation of European Biochemical Societies.)

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.

Neuronal plasticity describes changes in structure, function, and connections of neurons. The hippocampus, in particular, has been shown to exhibit considerable plasticity regarding both physiological and morphological functions. Melatonin, a hormone released by the pineal gland, promotes cell survival and dendrite maturation of neurons in the newborn brain and protects against neurological disorders. In this study, we investigated the effect of exogenous melatonin on neuronal architecture and its possible mechanism in the hippocampus of adult male C57BL/6 mice. Melatonin treatment significantly increased the total length and complexity of dendrites in the apical and basal cornu ammonis (CA) 1 and in the dentate gyrus in mouse hippocampi. Spine density in CA1 apical dendrites was increased, but no significant differences in other subregions were observed. In primary cultured hippocampal neurons, the length and arborization of neurites were significantly augmented by melatonin treatment. Additionally, western blot and immunohistochemical analyses in both in vivo and in vitro systems revealed significant increases in the level of cysteine-rich protein 1 (crp-1) protein, which is known to be involved in dendritic branching in mouse hippocampal neurons after melatonin treatment. Our results suggest that exogenous melatonin leads to significant alterations of neuronal micromorphometry in the adult hippocampus, possibly via crp-1 signaling., (© 2020 Wiley Periodicals LLC.)

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress., Competing Interests: The authors declare no competing interest.

Background: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood., Methods: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes., Results: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation., Conclusions: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments., (Copyright © 2020 by the American Society of Nephrology.)

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.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry. SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines., (Copyright © 2020 the authors.)

Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. Here, we describe a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. We show that the Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, we show that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. We provide evidence that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pk pk or Pk sple predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes., Competing Interests: BC, SS, JA No competing interests declared, (© 2020, Cho et al.)

The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer., (© 2020 Elsevier Inc. All rights reserved.)

Our previous study has indicated that the parathyroid hormone type 1 receptor (PTHR1) may play important roles in development and progression of osteosarcoma (OS) by regulating Wnt, angiogenesis, and inflammation pathway genes. The goal of this study was to further illuminate the roles of PTHR1 in OS by investigating upstream regulation mechanisms (including microRNA [miRNA] and transcription factors [TFs]) of crucial genes. The microarray dataset GSE46861 was downloaded from the Gene Expression Omnibus database, in which six tumors with short hairpin RNA (shRNA) PTHR1 knockdown (PTHR1.358) and six tumors with shRNA control knockdown (Ren.1309) were collected from mice. Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the linear models for microarray data (LIMMA) method, and then the miRNA-TF-mRNA regulatory network was constructed using data from corresponding databases, followed by module analysis, to screen crucial regulatory relationships. OS-related human miRNAs were extracted from the curated Osteosarcoma Database. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. As a result, the miRNA-TF-mRNA regulatory network, including 1049 nodes (516 miRNA, 25 TFs, and 508 DEGs) and 15942 edges (interaction relationships, such as Pparg-Abca1 and miR-590-3p-AXIN2), was constructed, from which three significant modules were extracted and modules 2 and 3 contained interactions between miRNAs/TFs and DEGs such as miR-103-3p- AXIN2 , miR-124-3p- AR - Tgfb1i1 , and miR-27a-3p- PPARG - Abca1 . miR-27a-3p was a known miRNA associated with OS. Abca1 , AR , and miR-124-3p were hub genes in the miRNA-TF-mRNA network. Tgfb1i1 was involved in cell proliferation, Abca1 participated in the cholesterol metabolic process, and AXIN2 was associated with the canonical Wnt signaling pathway. Furthermore, we also confirmed upregulation of miR-590-3p and downregulation of AXIN2 in the mouse OS cell line K7M2-WT transfected with PTHR1 shRNA. In conclusion, PTHR1 may play important roles in progression of OS by activating miR-124-3p- AR - Tgfb1i1 , miR-27a-3p- PPARG - Abca1 , and miR-103/590-3p- AXIN2 axes.

Background: Skeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia., Methods: We analyzed gene expression data from muscle of mice or human patients with diverse muscle pathologies and identified LMCD1 as a gene strongly associated with skeletal muscle function. We transiently expressed or silenced LMCD1 in mouse gastrocnemius muscle or in mouse primary muscle cells and determined muscle/cell size, targeted gene expression, kinase activity with kinase arrays, protein immunoblotting, and protein synthesis levels. To evaluate force, calcium handling, and fatigue, we transduced the flexor digitorum brevis muscle with a LMCD1-expressing adenovirus and measured specific force and sarcoplasmic reticulum Ca 2+ release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling., Results: Here, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease., Conclusions: Our gain- and loss-of-function studies show that LMCD1 controls protein synthesis, muscle fiber size, specific force, Ca 2+ handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.

Background: Chronic obstructive pulmonary disease (COPD) is recognized as a chronic lung disease with incomplete reversible airflow limitation, but its pathophysiology was still not clear. This study aimed at investigating regulatory roles of special miRNA-mRNA axis in COPD development., Methods: Differentially expressed miRNAs and downstream mRNAs were screened from the Gene Expression Omnibus (GEO) dataset by using the LIMMA package in R software. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct a co-expression network for COPD. The correlation of dysregulated miRNA(s) and COPD was analyzed, and miRNAs with significant differences were validated in peripheral blood mononuclear cells (PBMCs) from COPD patients by real-time PCR. Regulatory roles of candidate miRNAs and targeted mRNAs were investigated in vitro study., Results: Thirteen modules of co-expressed miRNAs and mRNAs were constructed from a selected cohort with WGCNA. Turquoise module with 12 differentially expressed miRNAs and 120 mRNAs was significantly correlated with COPD. The expression of hsa-miR-664a-3p, an upregulated miRNA in the module, was increased both in lung tissue and PBMCs from COPD patients, whereas that targeted four and a half LIM domains 1 ( FHL1 ) gene was decreased and positively correlated with forced expiratory volume in 1 sec (FEV1)/forced vital capacity (FVC%) ( r = 0.59, p < 0.01). In vitro, luciferase activity assay revealed FHL1 as a target of hsa-miR-664a-3p and it could be directly downregulated by overexpression of hsa-miR-664a-3p. Furthermore, cigarette smoke extract could increase hsa-miR-664a-3p level and decrease FHL1 level in Beas-2B cells., Conclusion: The present study validated significant upregulation of hsa-miR-664a-3p in COPD patients, and its target gene FHL1 was downregulated and positively correlated with FEV1/FVC%; both hsa-miR-664a-3p and FHL1 could be regulated by cigarette smoke extract. Results of bioinformatic analyses and expanded validation suggest that the axis from hsa-miR-664a-3p to FHL1 might play a key role in cigarette smoke-induced COPD, and the exact mechanism should be confirmed in further studies., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Zhong et al.)

Host encounters with viruses lead to an innate immune response that must be rapid and broadly targeted but also tightly regulated to avoid the detrimental effects of unregulated interferon expression. Viral stimulation of host negative regulatory mechanisms is an alternate method of suppressing the host innate immune response. We examined three key mediators of the innate immune response: NF-KB, STAT1 and STAT2 during HCV infection in order to investigate the paradoxical induction of an innate immune response by HCV despite a multitude of mechanisms combating the host response. During infection, we find that all three are repressed only in HCV infected cells but not in uninfected bystander cells, both in vivo in chimeric mouse livers and in cultured Huh7.5 cells after IFNα treatment. We show here that HCV and Flaviviruses suppress the innate immune response by upregulation of PDLIM2, independent of the host interferon response. We show PDLIM2 is an E3 ubiquitin ligase that also acts to stimulate nuclear degradation of STAT2. Interferon dependent relocalization of STAT1/2 to the nucleus leads to PDLIM2 ubiquitination of STAT2 but not STAT1 and the proteasome-dependent degradation of STAT2, predominantly within the nucleus. CRISPR/Cas9 knockout of PDLIM2 results in increased levels of STAT2 following IFNα treatment, retention of STAT2 within the nucleus of HCV infected cells after IFNα stimulation, increased interferon response, and increased resistance to infection by several flaviviruses, indicating that PDLIM2 is a global regulator of the interferon response., Competing Interests: The authors have declared that no competing interests exist.

T cell acute lymphoblastic leukaemia (T-ALL) cases include subfamilies that overexpress the TAL1/LMO, TLX1/3 and HOXA transcription factor oncogenes. While it has been shown that TAL1/LMO transcription factors induce self-renewal of thymocytes, whether this is true for other transcription factor oncogenes is unknown. To address this, we have studied NUP98-HOXD13-transgenic (NHD13-Tg) mice, which overexpress HOXA transcription factors throughout haematopoiesis and develop both myelodysplastic syndrome (MDS) progressing to acute myeloid leukaemia (AML) as well as T-ALL. We find that thymocytes from preleukaemic NHD13-Tg mice can serially transplant, demonstrating that they have self-renewal capacity. Transcriptome analysis shows that NHD13-Tg thymocytes exhibit a stem cell-like transcriptional programme closely resembling that induced by Lmo2, including Lmo2 itself and its critical cofactor Lyl1. To determine whether Lmo2/Lyl1 are required for NHD13-induced thymocyte self-renewal, NHD13-Tg mice were crossed with Lyl1 knockout mice. This showed that Lyl1 is essential for expression of the stem cell-like gene expression programme in thymocytes and self-renewal. Surprisingly however, NHD13 transgenic mice lacking Lyl1 showed accelerated T-ALL and absence of transformation to AML, associated with a loss of multipotent progenitors in the bone marrow. Thus multiple T cell oncogenes induce thymocyte self-renewal via Lmo2/Lyl1; however, NHD13 can also promote T-ALL via an alternative pathway.

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

LIM domains containing 1 (LIMD1) is a member of the Zyxin family proteins and functions as a tumor suppressor in lung cancer. LIMD1 has been shown to regulate Hippo-YAP signaling activity. Here, we report a novel regulatory mechanism for LIMD1. We found that cyclin-dependent kinase 1 (CDK1) and c-Jun NH2-terminal kinases 1/2 (JNK1/2) phosphorylate LIMD1 in vitro and in cells during anti-tubulin drug-induced mitotic arrest. Phosphorylation also occurs during normal mitosis. S272, S277, S421, and S424 were identified as the main phosphorylation sites in LIMD1. Deletion of LIMD1 resulted in a shortened mitotic cell cycle and phosphorylation of LIMD1 is required for proper mitotic progression. We further showed that the phosphorylation-deficient mutant LIMD1-4A is less active in suppressing cell proliferation, anchorage-independent growth, cell migration, and invasion in lung cancer cells. Together, our findings suggest that LIMD1 is a key regulator of mitotic progression, and that dysregulation of LIMD1 contributes to tumorigenesis., (© 2019 Federation of European Biochemical Societies.)

Objective: To explore the role of microRNA-203 in laryngeal cancer and its underlying mechanism and clarify the relationship between microRNA-203 and LASP1. Method: microRNA-203 expression in laryngeal cancer tissues and paracancerous tissues was detected by quantitative real time-polymerase chain reaction（qRT-PCR）. The regulatory effects of microRNA-203 on invasion and apoptosis of laryngeal cancer cells were detected by Transwell assay and flow cytometry, respectively. Dual-luciferase reporter gene assay was performed to access the binding condition of microRNA-203 and LASP1. Both mRNA and protein levels of LASP1 in laryngeal cancer cells were detected after transfection with microRNA-203 mimic or microRNA-203 inhibitor by qRT-PCR and Western blot. Rescue experiments were finally performed to detect whether microRNA-203 regulates laryngeal cancer development via targeting LASP1. Result: microRNA-203 was lowly expressed in laryngeal cancer tissues and cell lines.Knockdown of microRNA-203 in Hep-2 cells can promote the invasiveness and inhibit apoptosis of laryngeal cancer cells. Subsequently,LASP1 was predicted to be the target gene of microRNA-203，which was further verified by dual-luciferase reporter gene assay.LASP1 expression was negatively regulated by microRNA-203. Furthermore，rescue experiments showed that microRNA-203 regulates invasion and apoptosis of laryngeal cancer cells via targeting LASP1. Conclusion： Low expression of microRNA-203 could promote the invasion and inhibit apoptosis of laryngeal cancer cells viainhibiting LASP1. microRNA-203 and LASP1 both play a very important role in the development of laryngeal cancer.., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Purpose: This study was designed to investigate the effects of LASP1 on proliferation, metastasis, invasion, and cycle of oral squamous cell carcinoma cells and analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel., Methods: The correlation between LASP1 and survival rate and prognosis of patients with head and neck cancer were analyzed on the human protein atlas data. RT-PCR and Western blot were used to detect mRNA and protein expression of LASP1 in oral squamous cell carcinoma cell lines. LASP1 silenced HN30 stable transfectant cell line was constructed by lentivirus. CCK-8 assay was used to detect cell proliferation. Plate colony assay was used to detect cell clone formation ability. Transwell assay was used to detect cell migration and invasion ability. Flow cytometry was used to detect cell cycle changes. Oral squamous cell carcinoma metastases were established in nude mouse, the number of metastatic lung nodules was counted and stained with H-E. CCK-8 method was used to analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel. Statistical analysis was performed using SPSS 11.0 software package., Results: LASP1 was closely related to the survival rate and prognosis of head and neck cancer. LASP1 promoted proliferation, colony formation, metastasis and invasion of oral squamous cell carcinoma cell line HN30, promoted G2/M phase transition of cell cycle, and significantly reduced the formation of lung metastasis in nude mice after silencing. There was significant correlation with docetaxel IC50 but no significant impact on cisplatin IC50 and aptatinib IC50., Conclusions: LASP1 enhances cell proliferation, plate cloning, metastasis and invasion, G2/M phase transition of cell cycle, promotes lung metastasis in nude mice and docetaxel resistance of oral squamous cell carcinoma cell line HN30.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1 Beetlejuice (Prickle1 Bj ). The Prickle1 Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1 Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/β-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1 Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

Background: LIM and SH3 protein 1 (LASP1) is upregulated in several types of human cancer and implicated in cancer progression. However, the expression and intrinsic function of LASP1 in glioblastoma (GBM) remains unclear., Method: Oncomine and The Cancer Genome Atlas (TCGA) database was analyzed for the expression and clinical significance of LASP1 in GBM. LASP1 mRNA and protein level were measured by qRT-PCR and western blotting. The effect of LASP1 on GBM proliferation was examined by MTT assay and colony formation assay, the effect of LASP1 on sensitivity of Temozolomide was measured by flow cytometry and subcutaneous tumor model. The association between LASP1 and PI3K/AKT signaling was assessed by western blotting., Results: Oncomine GBM dataset analysis indicated LASP1 is significantly upregulated in GBM tissues compared to normal tissues. GBM dataset from The Cancer Genome Atlas (TCGA) revealed that high LASP1 expression is related to poor overall survival. LASP1 mRNA and protein in clinical specimens and tumor cell lines are frequently overexpressed. LASP1 knockdown dramatically suppressed U87 and U251 cell proliferation. Silencing LASP1 potentiated cell chemosensitivity to temozolomide in vitro, LASP1 knockdown inhibited tumor growth and enhanced the therapeutic effect of temozolomide in vivo. TCGA dataset analysis indicated LASP1 was correlated with PI3K/AKT signaling pathway, and LASP1 deletion inhibited this pathway. Combination treatment with PI3K/AKT pathway inhibitor LY294002 dramatically accelerated the suppression effect of temozolomide., Conclusion: LASP1 may function as an oncogene in GBM and regulate cell proliferation and chemosensitivity in a PI3K/AKT-dependent mechanism. Thus, the LASP1/PI3K/AKT axis is a promising target and therapeutic strategy for GBM treatment.

Psoriasis is an autoimmune disease characterized by abnormal differentiation and hyperproliferation of epidermal keratinocytes. LIM-domain only protein 4 (LMO4) is a transcription factor coregulator that promotes the assembly of multiprotein complexes to regulate mammary epithelium and keratinocyte differentiation and proliferation during embryogenesis. In this study, LMO4 has been found to be abundantly expressed in psoriatic epidermis. LMO4 expression is increased in human keratinocytes induced to differentiate by calcium ex vivo, and LMO4 overexpression induces spontaneous differentiation and growth acceleration of human keratinocytes in the absence of calcium. IL-23, a cytokine highly expressed in psoriatic skin lesions, induces differentiation and promotes proliferation of human keratinocytes. The IL-23-mediated effects are accompanied by an increase in LMO4 expression mediated by signal transducer and activator of transcription 3 through an IL-23/acutely transforming retrovirus AKT8 in rodent T-cell lymphoma/signal transducer and activator of transcription 3 pathway in keratinocytes. Knockdown of LMO4 effectively inhibits differentiation and growth of keratinocytes both ex vivo and in IL-23-injected ears of mice. LMO4 appears to mediate IL-23-related responses in psoriatic keratinocytes and is a potential therapeutic target in psoriasis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The linearization of the stromal extracellular matrix (ECM) by cancer-associated fibroblasts (CAFs) facilitates tumor cell growth and metastasis. However, the mechanism by which the ECM is remodeled is not fully understood. Hic-5 (TGFβ1i1), a focal adhesion scaffold protein, has previously been reported to be crucial for stromal ECM deposition and remodeling in vivo. Herein we show that CAFs lacking Hic-5 exhibit a significant reduction in the ability to form fibrillar adhesions, a specialized form of focal adhesion that promote fibronectin fibrillogenesis. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1. Furthermore, Src-dependent phosphorylation of Hic-5 facilitated the interaction with tensin1 to prevent β1 integrin internalization and trafficking to the lysosome. The interaction between Hic-5 and tensin1 was mechanosensitive, promoting fibrillar adhesion formation and fibronectin fibrillogenesis in a rigidity-dependent fashion. Importantly, this Src-dependent mechanism was conserved in three-dimensional (3D) ECM environments. Immunohistochemistry of tensin1 showed enrichment in CAFs in vivo, which was abrogated upon deletion of Hic-5. Interestingly, elevated Hic-5 expression correlates with reduced distant metastasis-free survival in patients with basal-like, HER2+ and grade 3 tumors. Thus, we have identified Hic-5 as a crucial regulator of ECM remodeling in CAFs by promoting fibrillar adhesion formation through a novel interaction with tensin1.

Carcinoma-associated fibroblasts (CAFs) influence tumor initiation, progression, and metastasis within the tumor-associated stroma. This suggests that CAFs would be a potential target for tumor therapy. Here we found that Hydrogen peroxide-inducible clone-5 (Hic-5), also named transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was strongly induced in CAFs found in human colorectal cancer. To investigate the role of Hic-5 in CAFs, we isolated CAFs and the control counterpart normal fibroblasts (NFs) from human colorectal cancer and non-cancerous regions, respectively. Hic-5 was highly expressed in isolated human CAFs and strongly induced in NFs in culture by the supernatant from cultured colorectal cancer cells as well as cytokines such as TGF-β, IL-1β and stromal cell-derived factor 1 (SDF-1/CXCL12). Furthermore, tumor growth was inhibited in a co-culture assay with Hic-5 knockdown fibroblasts compared with control fibroblasts. To clarify the function and significance of Hic-5 in colorectal cancer in vivo, we utilized a mouse model of azoxymethane (AOM)-induced colorectal cancer using Hic-5-deficient mice. Lack of Hic-5 in CAFs completely prevented AOM-induced colorectal cancer development in the colon tissues of mice. Mechanistic investigation revealed that Hic-5 promoted the expression of lysyl oxidase and collagen I in human control counterpart fibroblasts. Taken together, these results demonstrate that Hic-5 in CAFs is responsible for orchestrating or generating a tumor-promoting stroma.

Purpose: Tissue closure/fusion is a fundamental process during organogenesis, driven in part by the Wnt/planar cell polarity (Wnt/PCP) pathway. This study explored the spatial and temporal aspects of PCP signaling in eyelid development through analysis of mice lacking Prickle 1, a core PCP component, and the Prickle1-dependent signaling networks underlying eyelid development., Methods: Wild type and Prickle 1 compound mutant mice with a hypomorphic and a null allele were bred and used to study eyelid morphogenesis. The time course of embryonic eyelid fusion and postnatal reopening was examined by light microscopy of tissue sections and scanning electron microscopy. Immunohistochemistry was conducted to monitor cell proliferation, death, and molecular identities through pre- and postnatal eyelid development., Results: Prickle 1 mutant embryos exhibited a profound delay in eyelid closure at embryonic ages, but manifested precocious eyelid reopening postnatally, with ensuing cornea malformation. Mutant embryonic showed downregulation of phosphorylated c-Jun, and upregulation of increased β-catenin in separate cell populations of the eyelid front area. Increased cell death and decreased mesenchymal infiltration was observed in postnatal mutant eyelid prior to eyelid reopening. While broadly expressed in many tissues, Prickle 1 was spatially restricted to the eyelid front at E15.5, a location where c-Jun and β-catenin expression was altered in Prickle 1 mutants., Conclusions: The study demonstrates a spatiotemporal requirement for Prickle 1-mediated PCP signaling during eyelid morphogenesis and homeostasis. The study links Prickle 1-mediated PCP signaling to existing networks, and provides a useful animal model for studying congenital ocular surface diseases.

Background: In the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach., Methods: Eight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350mg/kg pyridoxine twice a day for 21days., Results: Phosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus., Conclusion: These results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function., General Significance: Vitamin-B 6 enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Main Conclusion: The plant LIMs comprise two sub-families with one (DA1/DAR) and two (2LIM) LIM domains. This review comprehensively discussed the structure and potential role of this protein family in diverse area of plant biology. The description of first eukaryote lineage-specific plant LIM domain (LIN11, ISL1, and MEC3) proteins was observed in Helianthus long back. The successive study of LIM proteins in diverse plants has shown its vital relation to development, metabolism and defence. This nascent gene family has been worked out for their role in actin dynamics, organ size determination and transcription regulation. On grounds of protein architecture, two sub-families have been delineated as DA1/DAR (one LIM domain) and 2LIMs (two LIM domains). The genomic and expression study guides to the identification of diverse sub-categories. The significance of 2LIMs in regulation of actin dynamics leading to pollen growth and development has prospects to understand the plant reproductive behaviour. Interestingly, new facet of these LIMs as a transcriptional regulator in biological pathway/biosynthesis was also reported. Recently, the cumulative contribution of these features was also recognized for obtaining good quality fibre, thus giving translational outlook to this family. The DA1/DAR proteins are orchestrated with additional domains and provide a key role in regulation of organ size and tolerance to biotic and abiotic stress. This review will focus the journey of plant LIMs till date and will cover details of its structure, type, classification and functional relevance. This will provide insight to identify the potential of this gene family in the improvement of desired crop features.

In the mammalian organ of Corti (OC), the stereocilia on the apical surface of hair cells (HCs) are uniformly organized in a neural to abneural axis (or medial-laterally). This organization is regulated by planar cell polarity (PCP) signaling. Mutations of PCP genes, such as Vangl2, Dvl1/2, Celsr1, and Fzd3/6, affect the formation of HC orientation to varying degrees. Prickle1 is a PCP signaling gene that belongs to the prickle / espinas / testin family. Prickle1 protein is shown to be asymmetrically localized in the HCs of the OC, and this asymmetric localization is associated with loss of PCP in Smurf mutants, implying that Prickle1 is involved in HC PCP development in the OC. A follow-up study found no PCP polarity defects after loss of Prickle1 (Prickle1-/-) in the cochlea. We show here strong Prickle1 mRNA expression in the spiral ganglion by in situ hybridization and β-Gal staining, and weak expression in the OC by β-Gal staining. Consistent with this limited expression in the OC, cochlear HC PCP is unaffected in either Prickle1C251X/C251X mice or Prickle1f/f; Pax2-cre conditional null mice. Meanwhile, type II afferents of apical spiral ganglion neurons (SGN) innervating outer hair cells (OHC) have unusual neurite growth. In addition, afferents from the apex show unusual collaterals in the cochlear nuclei that overlap with basal turn afferents. Our findings argue against the role of Prickle1 in regulating hair cell polarity in the cochlea. Instead, Prickle1 regulates the polarity-related growth of distal and central processes of apical SGNs.

Lineage-specific transcription factors are critical for long-range enhancer interactions, but direct or indirect contributions of architectural proteins such as CCCTC-binding factor (CTCF) to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the β-globin locus through LDB1 self-interaction. We find that an LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping, and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1-deficient cells. Genome-wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression., (Published by Elsevier Inc.)

The broadly expressed transcriptional coregulator LDB1 is essential for β-cell development and glucose homeostasis. However, it is unclear whether LDB1 has metabolic roles beyond the β-cell, especially under metabolic stress. Global Ldb1 deletion results in early embryonic lethality; thus, we used global heterozygous Ldb1+/- and inducible β-cell-specific Ldb1-deficient (Ldb1Δβ-cell) mice. We assessed glucose and insulin tolerance, body composition, feeding, and energy expenditure during high-fat diet exposure. Brown adipose tissue (BAT) biology was evaluated by thermogenic gene expression and LDB1 chromatin immunoprecipitation analysis. We found that partial loss of Ldb1 does not impair the maintenance of glucose homeostasis; rather, we observed improved insulin sensitivity in these mice. Partial loss of Ldb1 also uncovered defects in energy expenditure in lean and diet-induced obese (DIO) mice. This decreased energy expenditure during DIO was associated with significantly altered BAT gene expression, specifically Cidea, Elovl3, Cox7a1, and Dio2. Remarkably, the observed changes in energy balance during DIO were absent in Ldb1Δβ-cell mice, despite a similar reduction in plasma insulin, suggesting a role for LDB1 in BAT. Indeed, LDB1 is expressed in brown adipocytes and occupies a regulatory domain of Elovl3, a gene crucial to normal BAT function. We conclude that LDB1 regulates energy homeostasis, in part through transcriptional modulation of critical regulators in BAT function., (Copyright © 2017 Endocrine Society.)

Podocytes are complex epithelial cells with foot processes that are essential for the integrity and function of the kidney glomerular filters. Podocyte foot processes linked by slit diaphragms constitute signaling platforms that tightly regulate the cell shape and the function of the filtration barrier. Semaphorin (Sema) 3A is a class 3 semaphorin secreted by podocytes that has autocrine and paracrine functions in the kidney. We have shown that Sema3A regulates podocyte shape and that excess Sema3A signaling induces glomerular disease and aggravates diabetic nephropathy. MICAL-1 is an actin-binding protein that mediates Sema3A signals in podocytes. This chapter describes the methods used to examine how Sema3A signaling regulates podocyte shape.

The Lim domain-binding proteins are key co-factor proteins that assemble with LIM domains of the LMO/LIM-HD family to form functional complexes that regulate cell proliferation and differentiation. Using conditional mutagenesis and comparative phenotypic analysis, we analyze the function of Ldb1 and Ldb2 in mouse retinal development, and demonstrate overlapping and specific functions of both proteins. Ldb1 interacts with Lhx2 in the embryonic retina and both Ldb1 and Ldb2 play a key role in maintaining the pool of retinal progenitor cells. This is accomplished by controlling the expression of the Vsx2 and Rax, and components of the Notch and Hedgehog signaling pathways. Furthermore, the Ldb1/Ldb2-mediated complex is essential for generation of early-born photoreceptors through the regulation of Rax and Crx. Finally, we demonstrate functional redundancy between Ldb1 and Ldb2. Ldb1 can fully compensate the loss of Ldb2 during all phases of retinal development, whereas Ldb2 alone is sufficient to sustain activity of Lhx2 in both early- and late-stage RPCs and in Müller glia. By contrast, loss of Ldb1 disrupts activity of the LIM domain factors in neuronal precursors. An intricate regulatory network exists that is mediated by Ldb1 and Ldb2, and promotes RPC proliferation and multipotency; it also controls specification of mammalian retina cells., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis., Competing Interests: The authors have declared that no competing interests exist.

Objective: To investigate the effect of KyoT2 on the proliferation and migration of airway smooth muscle cells (ASMCs) in mice with asthma., Methods: Ovalbumin (OVA) was used to establish the asthmatic model of airway remodeling in BALB/c mice. ASMCs were isolated and cultured, and primarily cultured ASMCs were used as the control group. The expression of KyoT2 in ASMCs was measured in the control and asthma groups. After the ASMCs from asthmatic mice were transfected with pCMV-Myc (empty vector group) or pCMV-Myc-KyoT2 plasmid with overexpressed KyoT2 (KyoT2 expression group) for 48 hours, RT-PCR and Western blot were used to measure the mRNA and protein expression of KyoT2, the MTT assay and BrdU assay were used to measure the proliferation of ASMCs, and Transwell assay was used to measure the migration of ASMCs. Western blot was used to determine the effect of KyoT2 overexpression on the protein expression of RBP-Jκ, PTEN, and AKT., Results: Compared with the control group, the asthma group had significantly downregulated expression of KyoT2 in ASMCs, and the KyoT2 expression group had significantly upregulated expression of KyoT2 in ASMCs (P<0.05). Compared with the empty vector group, overexpressed KyoT2 significantly inhibited cell proliferation and migration, downregulated the expression of RBP-Jκ and AKT, and upregulated the expression of PTEN., Conclusions: Overexpressed KyoT2 can inhibit the proliferation and migration of ASMCs through the negative regulation of RBP-Jκ/PTEN/AKT signaling pathway.

T-cell acute lymphoblastic leukemia (T-ALL) frequently involves aberrant expression of TAL1 (T-cell acute lymphocytic leukemia 1) and LMO2, oncogenic members of the TAL1 transcriptional complex. Transcriptional activity of the TAL1-complex is thought to have a pivotal role in the transformation of thymocytes and is associated with a differentiation block and self-renewal. The transcription factor Forkhead Box P3 (FOXP3) was recently described to be expressed in a variety of malignancies including T-ALL. Here we show that increased FOXP3 levels negatively correlate with expression of genes regulated by the oncogenic TAL1-complex in human T-ALL patient samples as well as a T-ALL cell line ectopically expressing FOXP3. In these cells, FOXP3 expression results in altered regulation of cell cycle progression and reduced cell viability. Finally, we demonstrate that FOXP3 binds LMO2 in vitro, resulting in decreased interaction between LMO2 and TAL1, providing a molecular mechanism for FOXP3-mediated transcriptional modulation in T-ALL. Collectively, our findings provide initial evidence for a novel role of FOXP3 as a tumor suppressor in T-ALL through modulation of TAL1 transcriptional activity.

Zinc finger (ZNF) proteins, a diverse family of proteins, have multiple biological functions in cancer. Increased expression of ZNF185 has been involved in the regulation of tumor growth and metastasis. However, the function and underlying mechanisms of ZNF185 in the tumorigenesis of lung adenocarcinoma (LAC) remain unclear. The protein expression of ZNF185 was examined in human LAC tissues by immunohistochemical assay. After lentiviral vector-mediated ZNF185 overexpression was infected into the LAC cell lines (A549 and LETPα-2), cell growth and invasive potential were respectively evaluated by MTT and Transwell assays. We found that the protein expression of ZNF185 was significantly downregulated in LAC tissues compared with the adjacent non-cancerous tissues (ANCT) (37.10% vs 58.06%, P=0.015), and was negatively correlated with the lymph node metastasis of the LAC patients (P=0.005). Furthermore, overexpression of ZNF185 reduced cell proliferation and invasion in LAC cells, followed by the downregulation of p-AKT, p-GSK3β, VEGF and MMP-9 expression. Taken together, our findings indicate that the decreased expression of ZNF185 is linked to the tumor metastasis in human LAC patients, and ZNF185 overexpression inhibits the growth and invasion of LAC cells through inhibition of the AKT/GSK3β signaling, suggesting that ZNF185 may represent a potential therapeutic target for the treatment of LAC.

Key Points: The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase-1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea-pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure., Abstract: Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μm blebbistatin resulted in an ∼3-fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO-1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea-pig model of angiotensin II infusion (400 ng kg(-1)  min(-1) ) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signalling., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Metastasis is a major cause of death in patients with colorectal cancer, and increasing evidence supports the contribution of the epithelial-mesenchymal transition (EMT) to cancer progression. The dissociation of the E-cadherin/β-catenin adhesion complex represents a key step in EMT and promotes cancer invasion and metastasis, but the upstream signaling pathways regulating this interaction are poorly understood. Here, we show that PDLIM1, a member of the PDZ and LIM protein family, was downregulated in highly metastatic colorectal cancer cells and liver metastases from colorectal cancer patients. We found that loss of PDLIM1 promoted the expression of EMT markers and increased the invasive and migratory properties of multiple colorectal cancer cell lines. Furthermore, PDLIM1 knockdown increased colon-derived liver metastasis in an orthotopic colorectal cancer model and promoted distant metastatic colonization in an experimental lung metastasis model. Mechanistic investigations revealed that PDLIM1 interacted with and stabilized the E-cadherin/β-catenin complex, thereby inhibiting the transcriptional activity of β-catenin and preventing EMT. Accordingly, PDLIM1 overexpression attenuated EMT of colorectal cancer cells. Moreover, the downregulation of PDLIM1 in colorectal cancer samples correlated with reduced E-cadherin and membrane β-catenin levels, and was associated with shorter overall survival. In conclusion, our study demonstrates that PDLIM1 suppresses EMT and metastatic potential of colorectal cancer cells by stabilizing β-catenin at cell-cell junctions, and its loss in metastatic tissues may represent a potential prognostic marker of aggressive disease., (©2015 American Association for Cancer Research.)

Lipoma preferred partner (LPP) is a LIM domain protein, which has multiple functions as an actin-binding protein and a transcriptional coactivator, and it has been suggested that LPP has some roles in cell migration or invasion, however, its role in cancer cells remains to be elucidated. Here, we showed that LPP degraded N-cadherin in lung cancer, PC14PE6 cells via regulating the expression of matrix metalloproteinase 15 (MMP-15), and loss-of-LPP increases collective cell migration (CCM) and dissemination consequently. Knockdown of LPP and its functional partner, Etv5, markedly restores the full-length N-cadherin and increases cell-cell adhesion. We investigated the common target of LPP and Etv5, and found that MMP-15 is transcribed as their direct transcriptional target. Furthermore, MMP-15 could directly digest the N-cadherin extracellular domain. LPP knockdown in PC14PE6 cells increases N-cadherin-dependent CCM in the three-dimensional collagen gel invasion assays, and promoted the dissemination of cancer cells when they were orthotopically implanted in nude mice. Immunohistochemistry of lung adenocarcinoma specimens revealed the heterogeneity of LPP intensity and complementary expression of LPP and N-cadherin in the primary tumors. These findings suggest that loss-of-LPP, Etv5 or MMP-15 can be a prognostic marker of increasing malignancy.

During angiogenesis, endothelial cells must coordinate matrix proteolysis with migration. Here, we tested whether the focal adhesion scaffold protein Hic-5 (also known as TGFB1I1) regulated endothelial sprouting in three dimensions. Hic-5 silencing reduced endothelial sprouting and lumen formation, and sprouting defects were rescued by the return of Hic-5 expression. Pro-angiogenic factors enhanced colocalization and complex formation between membrane type-1 matrix metalloproteinase (MT1-MMP, also known as MMP14) and Hic-5, but not between paxillin and MT1-MMP. The LIM2 and LIM3 domains of Hic-5 were necessary and sufficient for Hic-5 to form a complex with MT1-MMP. The degree of interaction between MT1-MMP and Hic-5 and the localization of the complex within detergent-resistant membrane fractions were enhanced during endothelial sprouting, and Hic-5 depletion lowered the surface levels of MT1-MMP. In addition, we observed that loss of Hic-5 partially reduced complex formation between MT1-MMP and focal adhesion kinase (FAK, also known as PTK2), suggesting that Hic-5 bridges MT1-MMP and FAK. Finally, Hic-5 LIM2-LIM3 deletion mutants reduced sprout initiation. Hic-5, MT1-MMP and FAK colocalized in angiogenic vessels during porcine pregnancy, supporting that this complex assembles during angiogenesis in vivo. Collectively, Hic-5 appears to enhance complex formation between MT1-MMP and FAK in activated endothelial cells, which likely coordinates matrix proteolysis and cell motility., (© 2016. Published by The Company of Biologists Ltd.)

How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-β on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-β. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-β. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-β., (© 2016. Published by The Company of Biologists Ltd.)

The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The contractility of vascular smooth muscle cells within the walls of arteries is regulated by mechanical stresses and vasoactive signals. Transduction of these diverse stimuli into a cellular response occurs through many different mechanisms, one being reorganisation of the actin cytoskeleton. In addition to a structural role in maintaining cellular architecture it is now clear that the actin cytoskeleton of contractile vascular smooth muscle cells is a dynamic structure reacting to changes in the cellular environment. Equally clear is that disrupting the cytoskeleton or interfering with its rearrangement, has profound effects on artery contractility. The actin cytoskeleton associates with dense plaques, also called focal adhesions, at the plasma membrane of smooth muscle cells. Vasoconstrictors and mechanical stress induce remodelling of the focal adhesions, concomitant with cytoskeletal reorganisation. Recent work has shown that non-receptor tyrosine kinases and tyrosine phosphorylation of focal adhesion proteins such as paxillin and Hic-5 are important for actin cytoskeleton and focal adhesion remodelling and contraction., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)