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1.  The Drosophila Ral Gtpase Regulates Developmental Cell Shape Changes through the Jun Nh2-Terminal Kinase Pathway 
The Journal of Cell Biology  1999;146(2):361-372.
The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.
PMCID: PMC3206575  PMID: 10427090
bristle; dorsal closure; hair; Jun NH2-terminal kinase; Ral
2.  The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex 
Nature Cell Biology  2009;11(6):739-746.
Sonic hedgehog (Shh) and its main receptor Patched (Ptc) are implicated in both neural development and tumorigenesis1, 2. Beside the classic morphogen activity of Shh, Shh is also a survival factor3, 4. Along this line, Ptc has been shown to function as a dependence receptor, inducing apoptosis in the absence of Shh, while its pro-apoptotic activity is blocked in Shh presence5. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL/FHL2. DRAL/FHL2 is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryo. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, the CARD containing domain proteins TUCAN or NALP1 and the apical caspase-9. Ptc triggers caspase-9 activation and enhances cell death via a caspase-9-dependent mechanism. Thus, we propose that, upon absence of its ligand Shh, the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, a CARD domain containing protein and caspase-9.
PMCID: PMC2844407  PMID: 19465923
Adaptor Proteins, Signal Transducing; genetics; metabolism; Animals; Apoptosis; physiology; Apoptosis Regulatory Proteins; genetics; metabolism; CARD Signaling Adaptor Proteins; genetics; metabolism; Caspase 9; metabolism; Cell Line; Chick Embryo; Hedgehog Proteins; genetics; metabolism; Homeodomain Proteins; genetics; metabolism; Humans; Multiprotein Complexes; metabolism; Muscle Proteins; genetics; metabolism; Neoplasm Proteins; genetics; metabolism; RNA, Small Interfering; genetics; metabolism; Receptors, Cell Surface; genetics; metabolism; Signal Transduction; physiology; Transcription Factors; genetics; metabolism; Two-Hybrid System Techniques
3.  The LIM-Only Protein FHL2 Reduces Vascular Lesion Formation Involving Inhibition of Proliferation and Migration of Smooth Muscle Cells 
PLoS ONE  2014;9(4):e94931.
The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.
PMCID: PMC3988136  PMID: 24736599
4.  The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts 
The Journal of Cell Biology  2002;159(1):113-122.
FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.
PMCID: PMC2173499  PMID: 12370240
FHL2; repression of transcription; DRAL; myogenic differentiation; β-catenin–TCF complex
5.  Molecular cloning and characterization of a human PAX-7 cDNA expressed in normal and neoplastic myocytes. 
Nucleic Acids Research  1994;22(22):4574-4582.
The myogenic basic helix-loop-helix proteins are essential components of the regulatory network controlling vertebrate myogenesis. However, determined myoblasts appear in the limb buds which do not initially express any member of this transcription factor family. In a search for potential novel regulators of myogenesis, a human PAX-7 cDNA was isolated from primary myoblasts. Analysis of the DNA-binding properties of the Pax-7 paired domain revealed that it binds DNA in a sequence-specific manner indistinguishable from that of the paralogous Pax-3 protein. Each of the two proteins also binds to palindromic homeodomain-binding sites by cooperative dimerization. Both Pax-3 and Pax-7, which are known to partially overlap in their expression during development, can also efficiently form heterodimers on these sites and stimulate reporter gene transcription in transient transfection experiments which, in the case of Pax-7, is dependent on the transactivation function encoded by the C-terminal sequences. Thus, the formation of heterodimers might have important consequences for target gene recognition and regulation during development. PAX-7 was found to be weakly expressed in normal human myoblasts, while PAX-3 could not be detected in these cells at all. However, transcripts for either PAX-3 and/or PAX-7 were expressed at elevated levels in tumorigenic rhabdomyosarcoma cell lines. Hence, overexpression of these PAX genes may be involved in the genesis of myogenic tumors.
PMCID: PMC308503  PMID: 7527137
6.  Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification 
BMC Research Notes  2012;5:102.
Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied.
In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues.
The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.
PMCID: PMC3392731  PMID: 22339987
7.  Regulation of Estrogen-Dependent Transcription by the LIM Cofactors CLIM and RLIM in Breast Cancer 
Cancer research  2009;69(1):128-136.
Mammary oncogenesis is profoundly influenced by signaling pathways controlled by Estrogen Receptor-alpha (ERα). Although it is known that ERα exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HDs) is critically regulated by Cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERα cofactors that co-localize and interact with ERα in primary human breast tumors. We show that both cofactors associate with estrogen responsive promoters and regulate the expression of endogenous ERα target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERα and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERα on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs, but enhances transcriptional activation of endogenous ERα target genes. Results from a human breast cancer tissue microarray (TMA) of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/PR positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERα during the development of human breast cancer.
PMCID: PMC2713826  PMID: 19117995
Breast cancer; mammary gland; transcriptional regulation; cofactors; Estrogen; Estrogen receptor α; LIM cofactors; CLIM; RLIM
8.  EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane in Entamoeba histolytica▿  
Eukaryotic Cell  2007;6(9):1646-1655.
The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.
PMCID: PMC2043370  PMID: 17630327
9.  Inelastic neutron scattering study on bioprotectant systems 
We collected inelastic neutron scattering (INS) spectra of homologous disaccharide (C12H22O11)/H2O mixtures at a very low temperature by using indirect geometry time-of-flight spectrometer TOSCA at the ISIS pulse neutron facility (DRAL, UK). The aim of this work is to investigate the vibrational behaviour of trehalose, maltose and sucrose/H2O mixtures with INS in order to characterize the structural changes induced by these disaccharides on the H2O hydrogen-bonded network. A higher degree of ‘crystallinity’ for the trehalose/H2O system is observed in the vibrational region corresponding to the ice bending modes. This feature could justify the better cryptobiotic action of trehalose compared with maltose and sucrose. On the other hand, the better bioprotective effectiveness could be explained by the higher destructuring effect of trehalose, emphasized by the analysis of the librational modes region.
PMCID: PMC1618500  PMID: 16849211
disaccharides; bioprotectants; water mixtures; vibrational properties; neutron scattering
10.  Concentration dependence of vibrational properties of bioprotectant/water mixtures by inelastic neutron scattering 
Neutron scattering has been demonstrated to be a powerful tool for characterizing the structure and dynamics of biological molecules and for investigating the physical and chemical mechanisms of biophysical processes. The aim of the present work is to investigate by inelastic neutron scattering (INS) the vibrational behaviour of a class of bioprotectant systems, such as homologous disaccharides, trehalose, maltose and sucrose, in water mixtures. INS measurements have been performed on trehalose/H2O, maltose/H2O and sucrose/H2O mixtures at very low temperature as a function of concentration by using the thermal original spectrometer with cylindrical analyzers (TOSCA) spectrometer at the ISIS Facility (DRAL, UK).
The findings allow the analyses of the vibrational features of the INS spectra in order to study the effect of disaccharides on the H2O hydrogen-bonded tetrahedral network. The obtained neutron scattering findings point out that disaccharides, and in particular trehalose, have a destructuring effect on the water tetrahedral network, as emphasized by the analysis of the librational modes region from 50 to 130 meV energy transfer. On the other hand, the analysis of the bending modes region (130–225  meV) shows a locally ordered structure in the disaccharide/H2O mixtures.
Finally, the observed experimental evidences are linked to the different bioprotective effectiveness of disaccharides as a function of concentration.
PMCID: PMC2358967  PMID: 17018423
disaccharides; bioprotection; water mixtures; vibrational properties; neutron scattering
11.  Biological Potential of Sixteen Legumes in China 
Phenolic acids have been identified in a variety of legumes including lima bean, broad bean, common bean, pea, jack bean, goa bean, adzuki bean, hyacinth bean, chicking vetch, garbanzo bean, dral, cow bean, rice bean, mung bean and soybean. The present study was carried out with the following aims: (1) to identify and quantify the individual phenolic acid and determine the total phenolic content (TPC); (2) to assess their antioxidant activity, inhibition activities of α-glucosidase, tyrosinase, and formation of advanced glycation endproducts; and (3) to investigate correlations among the phytochemicals and biological activity. Common bean possesses the highest antioxidant activity and advanced glycation endproducts formation inhibition activity. Adzuki bean has the highest α-glucosidase inhibition activity, and mung bean has the highest tyrosinase inhibition activity. There are significant differences in phytochemical content and functional activities among the bean species investigated. Selecting beans can help treat diseases such as dermatological hyperpigmentation illness, type 2 diabetes and associated cardiovascular diseases.
PMCID: PMC3211026  PMID: 22072935
legumes; antioxidant; α-glucosidase inhibition; advanced glycation endproducts; tyrosinase inhibition
12.  Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity 
PLoS Medicine  2006;3(10):e422.
Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity.
Methods and Findings
Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization.
X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier.
Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.
Expression profiling can discriminate between groups of patients with and without severe late radiotherapy toxicity but not (yet) predict individual responses.
Editors' Summary
More than half the people who develop cancer receive radiotherapy as part of their treatment. That is, tumor cells are destroyed by exposing them to a source of ionizing radiation such as X-rays. Ionizing radiation damages the genetic material of cancer cells so that they can no longer divide. Unfortunately, it also damages nearby normal cells, although they are less sensitive to radiation than the cancer cells. Radiotherapists minimize how much radiation hits normal tissues by carefully aiming the X-rays at the tumor. Even so, patients often develop side effects such as sore skin or digestive problems during or soon after radiotherapy; the exact nature of the side effects depends on the part of the body exposed to the X-rays. In addition, a few patients develop severe late radiation toxicity, months or years after their treatment. Like early toxicity, late toxicity occurs in the normal tissues near the tumor site. For example, in prostate cancer—a tumor that forms in a gland in the male reproductive system that lies between the bladder and the end of the gut (the rectum)—late radiation toxicity affects rectal, bladder, and sexual function in 5%–10% of patients.
Why Was This Study Done?
It is not known why some patients develop late radiation toxicity, and it is impossible to predict before treatment which patients will have long-term health problems after radiotherapy. It would be useful to know this, because radiation levels might be reduced in those patients, while larger doses of radiation could be given to patients at low risk of late complications to ensure a complete eradication of their cancer. One theory is that some patients are genetically predisposed to develop severe late radiation toxicity. In other words, their genetic make-up makes it more likely that their tissues develop long-term complications after radiation damage. In this study, the researchers looked for markers of a genetic predisposition for late radiation toxicity by comparing radiation-induced changes in the pattern of cellular proteins in patients who had late radiation toxicity after radiotherapy with the changes seen in patients who did not develop such complications.
What Did the Researchers Do and Find?
The researchers recruited 38 patients who had been treated successfully with radiotherapy for prostate cancer two years previously. Of these, 21 had developed severe late radiation toxicity. They isolated lymphocytes (a type of immune system cell) from the patients' blood, stimulated the lymphocytes to divide, exposed them to X-rays, and analyzed the pattern of genes active in these cells—their gene expression profile—before and after irradiation. The researchers found that irradiation induced the expression of numerous genes in the lymphocytes, including many well-known radiation-responsive genes. They then used an analytical process called “random cross-validation” to look for a gene expression profile (or molecular signature) that was associated with late radiation toxicity. They report that a signature based on the radiation response of 50 individual genes correctly classified 63% of the patient population in terms of whether the patient had developed late radiation toxicity. A signature based on the radiation response of gene sets containing genes linked by function or cellular localization correctly classified 86% of the patient population.
What Do These Findings Mean?
Gene expression profiling identified groups of patients who had had severe late radiation toxicity pretty well, particularly when sets of related genes were used to classify the patients. The approach was not so good, however, at identifying individual patients who had had problems, being correct and certain only half the time. Additional studies are needed, therefore, before this promising approach can be used clinically to predict patient responses to radiotherapy. Overall, the study supports the idea that some patients are genetically predisposed to develop late radiation toxicity, and it also provides clues about which cellular pathways help to determine late radiation toxicity. Most of the genes and gene sets that discriminated between the patients with and without late radiation toxicity are involved in protein metabolism, apoptosis (a special sort of cell death), and stress signaling networks (pathways that protect cells from damage). This information, if confirmed, might help researchers to develop therapeutic interventions to minimize late radiation toxicity in vulnerable individuals.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute patient information on radiotherapy and on prostate cancer
American Cancer Society information on radiation therapy
Cancer Research UK patient information on radiotherapy
Wikipedia pages on radiotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1626552  PMID: 17076557
13.  Genome-Wide Analysis of LIM Gene Family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa 
In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: αLIM1, βLIM1, γLIM2, and δLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function.
PMCID: PMC2779900  PMID: 17573466
poplar; Arabidopsis; rice; LIM domain protein; tension wood
14.  A microRNA network functioning in the regulation of radiobiological effects 
Journal of Radiation Research  2014;55(Suppl 1):i57-i58.
MicroRNA (miRNA), a small non-coding RNA molecule, is vital in genetic regulation, and miRNA pathway, which regulates gene expression through degradation or translational suppression of their target transcripts, is highly conservative in evolution.
Although profiles of miRNAs are different in various cell types and tissues, miRNAs have been considered as a crucial class of regulators in cellular response to ionizing radiation (IR). By carrying out a series of experiments, we have found that altered transcriptional regulation network composed of radiation-mediated miRNAs regulates the expression of their downstream target genes in most biological processes to control cell growth, cell cycle and apoptosis. For example, the newly identified miR-3928 negatively regulates the expression of Dicer, which has been validated by the luciferase assay and western blotting. Dicer is not only a key participant in responding to radiation, but also a critical factor for the maturation of miRNAs, suggesting that miR-3928 affects on the expression of other miRNAs through regulating Dicer. Among the miRNAs controlled by the Dicer, we reveal that miR-185 and miR-663 can efficiently suppress ATR and TGF-β1 expression, which are both important responders in the process of radiobiological effects. Further experiments reveal that the expression of Dicer is suppressed by miR-3928 induced by IR and consequently, the maturation of other miRNAs including miR-185 and miR-663 is inhibited, resulting in the abundantly enhanced expression of ATR and TGF-β1 respectively. This mechanism to hammer at fixing DNA damage or promote cells to apoptosis caused by IR has important implications in the decision of cell fates.
Moreover, it has been shown that the expression of BTG1 is characterized in response to factors that induce growth arrest and subsequent differentiation both in vivo and in vitro, affecting cellular physiological progresses of angiogenesis, follicular development and myoblast and B cell differentiation, through regulating cell growth, migration, cell cycle, apoptosis and differentiation. BTG1 gene is phylogenetically highly conserved in its coding and 3′-untranslated region (UTR), which is considered as a decisive element involved in regulation of BTG1 expression. We present evidence that BTG1 can be induced by IR and confirm that miR-454-3p, whose gene locates in the intron of Ska2 gene, can regulate BTG1 expression through directly binding to the 3′-UTR of BTG1 mRNA. These results point out that increased expression of BTG1 caused by the down-regulation of miR-454-3p in case that IR modulates endogenous activity of PRMT1, a BTG1-binding partner, which can methylate endogenous transcription factors to change gene expression pattern and reply radiostilumation. An inverse relationship between the levels of expression of BTG1 and miR-454-3p reveals that there exists a new pathway in response to IR stimulation. Furthermore, cell growth will be transiently increased by the knockdown of BTG1 by transfecting BTG1 siRNA or miR-454-3p mimic. However, the apoptotic state of cells can be tested after 2 days. Down-regulation of BTG1 by miR-454-3p increases the sensitivity of human renal cell carcinoma 786-O cells to IR-induced apoptosis, suggesting that BTG1 could serve as a terget for sensitizing renal carcinoma to standard radiotherapy.
Taken together, all these data indicate that alteration of miRNA expression is evident in the cellular response to IR. MiR-3928, miR-185, miR-663 and miR-454-3p may constitute a complex network contributing to the regulation of radiobiological effects. It is apparent that the study of radiation-related miRNAs is beneficial to qualitatively and quantitatively modulating radiobiological effects, and also in favor of the advanced research of miRNA functions.
PMCID: PMC3941529
microRNA; network; Dicer; BTG1; ionizing radiation
15.  Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel Kv1.5 
PLoS ONE  2011;6(10):e26524.
Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G0/G1 phase. Furthermore, low expression of Kv1.5, a voltage-gated potassium channel known to alter myoblast proliferation during the G1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between Kv1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and Kv1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of Kv1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K+ currents when compared to oocytes expressing Kv1.5 only. We here present the first evidence on a biological relevance of FHL1C.
PMCID: PMC3203871  PMID: 22053194
16.  A Family of LIM-Only Transcriptional Coactivators: Tissue-Specific Expression and Selective Activation of CREB and CREM 
Molecular and Cellular Biology  2000;20(22):8613-8622.
Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways. Regulation mediated by members of the CREB family has been linked to various physiological functions. Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP). However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized. We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis). ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP. Here we characterize a family of LIM-only proteins which share common structural organization with ACT. These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression. FHL proteins display different degrees of intrinsic activation potential. They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation. Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties. This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM. The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation.
PMCID: PMC102166  PMID: 11046156
17.  Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor. 
Molecular and Cellular Biology  1994;14(5):2985-2993.
Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for binding of a factor which is present in cells of gonadotrope and thyrotrope lineages but not in other cells. Screening of a mouse cDNA library with a DNA probe containing the imperfect palindrome resulted in the isolation of a LIM-homeodomain transcription factor. The cDNA predicts a mouse protein which is 94% identical to the recently described rat LIM-homeodomain protein LH-2. LH-2 contains two zinc fingers (LIM domain) and a consensus homeodomain. Hybridization analysis revealed relatively high expression of LH-2 mRNA in the central nervous system and in pituitary cells of the gonadotrope and thyrotrope lineages. Lower or nondetectable levels of LH-2 mRNA were found in other pituitary cells and tissues, including placental cells. Recombinant LH-2 homeodomain was found to selectively bind to the previously identified imperfect palindrome in the PGBE. Point mutations in the PGBE resulted in parallel losses in the binding of a nuclear factor from a cell line of the gonadotrope lineage and recombinant LH-2-binding activity. Use of an antibody to LH-2 provided evidence that endogenous PGBE-binding activity from cells of the gonadotrope lineage involves a protein which is immunologically related to LH-2. Expression of LH-2 in two heterologous cell types resulted in activation of a reporter gene containing the mouse alpha promoter. These data suggest that the LIM-homeodomain factor LH-2 plays a role in stimulating tissue-specific expression of the mouse glycoprotein hormone alpha subunit. The finding that a LIM-homeodomain protein can stimulate expression of one of the earliest markers of pituitary differentiation raises the possibility that this factor plays a role in cell lineage determination in the pituitary.
PMCID: PMC358666  PMID: 7513049
18.  Angiogenin Promotes U87MG Cell Proliferation by Activating NF-κB Signaling Pathway and Downregulating Its Binding Partner FHL3 
PLoS ONE  2015;10(2):e0116983.
Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.
PMCID: PMC4320115  PMID: 25659096
19.  Requirement of LIM domains for the transient accumulation of paxillin at damaged stress fibres 
Biology Open  2013;2(7):667-674.
Cells recognize and respond to changes in intra- and extracellular mechanical conditions to maintain their mechanical homeostasis. Linear contractile bundles of actin filaments and myosin II known as stress fibres (SFs) mediate mechanical signals. Mechanical cues such as excessive stress driven by myosin II and/or external force may damage SFs and induce the local transient accumulation of SF-repair complexes (zyxin and VASP) at the damaged sites. Using an atomic force microscope mounted on a fluorescence microscope, we applied mechanical damage to cells expressing fluorescently tagged cytoskeletal proteins and recorded the subsequent mobilization of SF-repair complexes. We found that a LIM protein, paxillin, transiently accumulated at the damaged sites earlier than zyxin, while paxillin knockdown did not affect the kinetics of zyxin translocation. The C-terminal half of paxillin, comprising four-tandem LIM domains, can still translocate to damaged sites on SFs, suggesting that the LIM domain is essential for the mechanosensory function of paxillin. Our findings demonstrate a crucial role of the LIM domain in mechanosensing LIM proteins.
PMCID: PMC3711034  PMID: 23862014
Repair of stress fibres; Mechanosensors; LIM domains
20.  An amphioxus LIM-homeobox gene, AmphiLim1/5, expressed early in the invaginating organizer region and later in differentiating cells of the kidney and central nervous system 
A LIM-homeobox gene, AmphiLim1/5, from the Florida amphioxus (Branchiostoma floridae) encodes a protein that phylogenetic analysis positions at the base of a clade comprising vertebrate Lim1 and Lim5. Amphioxus AmphiLim1/5 is expressed in domains that are a composite of those of vertebrate Lim1 and Lim5, which evidently underwent subfunctionalization after duplication of an ancestral protochordate Lim1/5. During amphioxus development, transcription is first detected in the ectoderm of the blastula. Then, in the gastrula, a second expression domain appears in the mesendoderm just within the dorsal lip of the blastopore, a region known to have organizer properties in amphioxus. This mesendodermal expression corresponds to Lim1 expression in the Spemann organizer of vertebrates. At least one of the functions of vertebrate Lim1 in the organizer is to control the transcription of genes involved in cell and tissue movements during gastrulation, and a comparable early function seems likely for AmphiLim1/5 during gastrular invagination of amphioxus. Later embryos and larvae of amphioxus express AmphiLim1/5 in clusters of cells, probably motoneurons, in the anterior part of the central nervous system, in the hindgut, in Hatschek's right diverticulum (a rudiment of the rostral coelom), and in the wall of the first somite on the left side (a precursor of Hatschek's nephridium). In the early larva, expression continues in neural cells, in Hatschek's nephridium, in the wall of the rostral coelom, in the epidermis of the upper lip, and in mesoderm cells near the opening of the second gill slit. The developmental expression in Hatschek's nephridium is especially interesting because it helps support the homology between this amphioxus organ and the vertebrate pronephros.
PMCID: PMC1458433  PMID: 16763670
Spemann organizer; kidney; brain; cephalochordate; lancelet
21.  The maturation of photoreceptors in the avian retina Is stimulated by thyroid hormone 
Neuroscience  2011;178:250-260.
During retinal development, the cell-fate of photoreceptors is committed long before maturation, which entails the expression of opsins and functional transduction of light. The mechanisms that delay the maturation of photoreceptors remain unknown. We have recently reported that immature photoreceptors express the LIM domain transcription factors Islet2 and Lim3, as well as the cell-surface glycoprotein axonin1 (Fischer et al., 2008a). As the photoreceptors mature to form outer segments and express photopigments, the expression of the Islet2, Lim3 and axonin1 is diminished. The purpose of this study was to investigate whether Thyroid Hormone (TH) influences the maturation of photoreceptors. We studied the maturation of photoreceptors across the gradient of maturity that exists in far peripheral regions of the postnatal chicken retina (Ghai et al., 2008). We found that intraocular injections of TH down-regulated Islet2, Lim3 and axonin1 in photoreceptors in far peripheral regions of the retina. By contrast, TH stimulated the up-regulation of red-green opsin, violet opsin, rhodopsin and calbindin in photoreceptors. We found a correlation between the onset of RLIM (RING finger LIM-domain binding protein) and down-regulation of Islet2 and Lim3 in maturing photoreceptors; RLIM is known to interfere with the transcriptional activity of LIM-domain transcription factors. We conclude that TH stimulates the maturation of photoreceptors in the avian retina. We propose that TH inhibits the expression of Islet2 and Lim3, which thereby permits photoreceptor maturation and the onset of photopigment-expression.
PMCID: PMC3048918  PMID: 21256198
retina; photoreceptor; thyroid hormone; Islet2; Lim3; RLIM
22.  The Dictyostelium LIM Domain-containing Protein LIM2 Is Essential for Proper Chemotaxis and Morphogenesis 
Molecular Biology of the Cell  2000;11(4):1275-1291.
We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin–enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell–cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.
PMCID: PMC14846  PMID: 10749929
23.  Functional analysis of the nuclear LIM domain interactor NLI. 
Molecular and Cellular Biology  1997;17(10):5688-5698.
LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions.
PMCID: PMC232417  PMID: 9315627
24.  Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation 
The Journal of Cell Biology  1996;133(1):169-184.
The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5- transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF- alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.
PMCID: PMC2120777  PMID: 8601606
25.  ALP/Enigma PDZ–LIM Domain Proteins in the Heart 
Actinin-associated LIM protein (ALP) and Enigma are two subfamilies of Postsynaptic density 95, discs large and zonula occludens-1 (PDZ)–Lin-11, Isl1 and Mec-3 (LIM) domain containing proteins. ALP family members have one PDZ and one LIM domain, whereas Enigma proteins contain one PDZ and three LIM domains. Four ALP and three Enigma proteins have been identified in mammals, each having multiple splice variants and unique expression patterns. Functionally, these proteins bind through their PDZ domains to α-actinin and bind through their LIM domains or other internal protein interaction domains to other proteins, including signaling molecules. ALP and Enigma proteins have been implicated in cardiac and skeletal muscle structure, function and disease, neuronal function, bipolar disorder, tumor growth, platelet and epithelial cell motility and bone formation. This review will focus on recent advances in the biological roles of ALP/Enigma PDZ–LIM domain proteins in cardiac muscle and provide insights into mechanisms by which mutations in these proteins are related to human cardiac disease.
PMCID: PMC2905065  PMID: 20042479
PDZ; LIM; cypher; ZASP; cardiomyopathy; Z-disc

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