Role of Dlg5/lp-dlg, a Membrane-Associated Guanylate Kinase Family Protein, in Epithelial-Mesenchymal Transition in LLc-PK1 Renal Epithelial Cells

Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT

Report and preliminary results of R/V SONNE Cruise SO251 - Extreme events Archived in the GEologial Record of JAPAN's subduction margins (EAGER-JAPAN)

Leg A SO251-1, Yokohama - Yokohama, 04.10.2016 - 15.10.2016, Leg B SO251-2, Yokohama - Yokohama, 18.10.2016 - 02.11.201

Impact of mediastinal, liver and lung 123I-metaiodobenzylguanidine (123I-MIBG) washout on calculated 123I-MIBG myocardial washout

PURPOSE: In planar (123)I-metaiodobenzylguanidine ((123)I-MIBG) myocardial imaging mediastinum (M) activity is often used as a background correction in calculating "washout" (WO). However, the most likely sources for counts that might produce errors in estimating myocardial (Myo) activity are lung (Lu) and liver (Li), which typically have higher counts/pixel (cpp) than M. The present study investigated the relationship between changes in Lu, Li and Myo activity between early and late planar (123)I-MIBG images, with comparison to M as the best estimator of non-specific background activity. METHODS: Studies on 98 subjects with both early (e) and late (l) planar (123)I-MIBG images were analysed. There were 68 subjects with chronic heart failure (CHF), 14 with hypertension (HTN) but no known heart disease and 16 controls (C). For each image, regions of interest (ROIs) were drawn: an irregular whole Myo, Lu, upper M and Li. For each ROI, WO was calculated as [(cpp(e)-cpp(l:decay corrected))/cpp(e)]x100%. RESULTS: Multivariable forward stepwise regression analysis showed that overall a significant proportion of the variation in Myo WO could be explained by a model containing M WO and Lu WO (37%, p < 0.001). Only in controls was M WO the sole variable explaining a significant proportion of the variation in Myo WO (27%, p = 0.023). CONCLUSION: Although increased Myo WO in CHF subjects reflects disease severity, part of the count differences measured on planar (123)I-MIBG myocardial images likely reflects changes in the adjacent and surrounding Lu tissue. The results for the controls suggest that this is the only group where a mediastinum correction alone may be appropriate for cardiac WO calculation

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts

Expression of Kruppel-Like Factor KLF4 in Mouse Hair Follicle Stem Cells Contributes to Cutaneous Wound Healing

Kruppel-like factor KLF4 is a transcription factor critical for the establishment of the barrier function of the skin. Its function in stem cell biology has been recently recognized. Previous studies have revealed that hair follicle stem cells contribute to cutaneous wound healing. However, expression of KLF4 in hair follicle stem cells and the importance of such expression in cutaneous wound healing have not been investigated.Quantitative real time polymerase chain reaction (RT-PCR) analysis showed higher KLF4 expression in hair follicle stem cell-enriched mouse skin keratinocytes than that in control keratinocytes. We generated KLF4 promoter-driven enhanced green fluorescence protein (KLF4/EGFP) transgenic mice and tamoxifen-inducible KLF4 knockout mice by crossing KLF4 promoter-driven Cre recombinase fused with tamoxifen-inducible estrogen receptor (KLF4/CreER™) transgenic mice with KLF4(flox) mice. KLF4/EGFP cells purified from dorsal skin keratinocytes of KLF4/EGFP transgenic mice were co-localized with 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells by flow cytometric analysis and immunohistochemistry. Lineage tracing was performed in the context of cutaneous wound healing, using KLF4/CreER™ and Rosa26RLacZ double transgenic mice, to examine the involvement of KLF4 in wound healing. We found that KLF4 expressing cells were likely derived from bulge stem cells. In addition, KLF4 expressing multipotent cells migrated to the wound and contributed to the wound healing. After knocking out KLF4 by tamoxifen induction of KLF4/CreER™ and KLF4(flox) double transgenic mice, we found that the population of bulge stem cell-enriched population was decreased, which was accompanied by significantly delayed cutaneous wound healing. Consistently, KLF4 knockdown by KLF4-specific small hairpin RNA in human A431 epidermoid carcinoma cells decreased the stem cell population and was accompanied by compromised cell migration.KLF4 expression in mouse hair bulge stem cells plays an important role in cutaneous wound healing. These findings may enable future development of KLF4-based therapeutic strategies aimed at accelerating cutaneous wound closure

Autosomal-dominant hypotrichosis with woolly hair : novel gene locus on chromosome 4q35.1-q35.2

Hypotrichosis simplex (HS) with and without woolly hair (WH) comprises a group of rare, monogenic disorders of hair loss. Patients present with a diffuse loss of scalp and/or body hair, which usually begins in early childhood and progresses into adulthood. Some of the patients also show hair that is tightly curled. Approximately 10 genes for autosomal recessive and autosomal dominant forms of HS have been identified in the last decade, among them five genes for the dominant form. We collected blood and buccal samples from 17 individuals of a large British family with HS and WH. After having sequenced all known dominant genes for HS in this family without the identification of any disease causing mutation, we performed a genome-wide scan, using the HumanLinkage-24 BeadChip, followed by a classical linkage analysis; and whole exome-sequencing (WES). Evidence for linkage was found for a region on chromosome 4q35.1-q35.2 with a maximum LOD score of 3.61. WES led to the identification of a mutation in the gene SORBS2, encoding sorbin and SH3 domain containing 2. Unfortunately, we could not find an additional mutation in any other patient/family with HS; and in cell culture, we could not observe any difference between cloned wildtype and mutant SORBS2 using western blotting and immunofluorescence analyses. Therefore, at present, SORBS2 cannot be considered a definite disease gene for this phenotype. However, the locus on chromosome 4q is a robust and novel finding for hypotrichosis with woolly hair. Further fine mapping and sequencing efforts are therefore warranted in order to confirm SORBS2 as a plausible HS disease gene