28 research outputs found

    PKA activation enhances SF2/ASF-promoted exclusion of exons 14, 15, and 16 of CaMKIIδ.

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    <p>A, HEK-293FT cells were transfected with pCI/CaMKIIδ<sub>E13–E17</sub> for 40 hrs and then treated with 10 µM forskolin or 20 µM isoproterenol for 8 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated. The Data are presented as mean ± S.D. *<i>p</i><0.05 versus control treatment. B, Proposed mechanism by which abnormalities of β-adrenergic-PKA-pathway dysregulates the alternative splicing of exons 14, 15, and 16 of CaMKIIδ via SF2/ASF.</p

    Alternative splicing of CaMKIIδ exons 14, 15, and 16 generates three splicing variants, corresponding to CaMKIIδ isoforms A, B, and C, respectively.

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    <p>A and B, Schematic diagram of the alternative splicing of exons 14, 15, and 16 of mini -CaMKIIδ-genes, pCI/CaMKIIδ<sub>E12–E17</sub> (A) and pCI/CaMKIIδ<sub>E13–E17</sub> (B). C and D, Three splicing variants was generated from mini-CaMKIIδ gene, pCI/CaMKIIδ<sub>E12–E17</sub> (C) or pCI/CaMKIIδ<sub>E13–E17</sub> (D), after transfection into HEK-293T or COS7 cells, respectively, for 48 hrs. The total RNA was used for measurement of the splicing products with RT-PCR.</p

    PKA promotes exclusion of exons 14, 15, and 16 of CaMKIIδ.

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    <p>A, Forskolin treatment activated PKA. HEK-293T cells were transfected with pCI/CaMKIIδ<sub>E13–E17</sub> for 40 hrs and then treated with 10 µM forskolin for 8 hrs. The cells were subjected to Western blots for detection of PKA activity with anti-phosphorylated CREB at Ser133 and anti-CREB. B, Forskolin treatment promoted the exclusion of exons 14, 15, and 16, resulting in an increase in CaMKIIδC expression. HEK-293T cells were transfected with pCI/CaMKIIδ<sub>E13–E17</sub> for 40 hrs and then treated with 10 µM forskolin for 8 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry, and the percentage of each splicing form was calculated. C, Overexpression of PKA-Cα increased PKA activity. HEK-293T cells were co-transfected with pCI/CaMKIIδ<sub>E12–E17</sub> and pCI/PKA-Cα for 48 hrs. The PKA activity in the cells was measured by phosphorylation of CREB at Ser133 with Western blots. D, Overexpression of PKA-Cα promoted the exclusion of exons 14, 15, and 16 of CaMKII™. HEK-293T cells were cotransfected with pCI/CaMKIIδ<sub>E13–E17</sub> and pCI/PKA-Cα for 48 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry, and the percentage of each splicing form was calculated. The Data are presented as mean ± S.D. *<i>p</i><0.05 versus control treatment.</p

    PKA phosphorylates and interacts with SF2/ASF.

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    <p>A, Recombinant GST-SF2/ASF or GST was incubated with PKA in the presence of [γ-<sup>32</sup>P]ATP at 30°C for 10 min, and the reaction mixture was then separated by SDS-PAGE and visualized with Coommassie blue staining (lower panel) or autoradiograph (upper panel). B, PKA-Cα was pull-down by SF2/ASF. GST-SF2/ASF or GST coupled onto glutathione-Sepharose or glutathione-Sepharose (GSH-beads) was incubated with rat brain extract. After extensively washing, the bound proteins were analyzed by Western blots developed with anti-GST or anti-PKA-Cα. C, PKA-Cα was co-immunoprecipitated by anti-HA. SF2/ASF tagged with HA were expressed in HEK-293FT cells for 48 h. The cell extracts were immunoprecipitated with anti-HA, and the immunoprecipitates were subjected to Western blots developed with anti-HA and anti-PKA-Cα. D, HeLa cells were transfected with pCEP4/SF2/ASF and treated without (Con) or with forskolin (Fors) for 30 min, followed by triple immunofluorescence staining.</p

    SF2/ASF promotes the exclusion of exons 14, 15, and 16 of CaMKIIδ.

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    <p>A and B, Overexpression of SF2/ASF promoted the exclusion of exons 14, 15, and 16 of pCI/CaMKIIδ<sub>E12–E17</sub> in COS7 cells. PCI/CAMKIIδ<sub>E12–E17</sub> was co-transfected with pCEP4/SF2/ASF into COS7 cells for 48 hr. The splicing products were measured with RT-PCR (A). Each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated (B). C and D, Overexpression of SF2/ASF promoted the exclusion of exons 14, 15, and 16 of pCI/CaMKIIδ<sub>E13–E17</sub> in HEK-239T. PCI/CAMKIIδ<sub>E12–E17</sub> was co-transfected with pCEP4/SF2/ASF into HEK-293T cells for 48 hr. The splicing products were measured with RT-PCR (C). The each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated (D). The Data are presented as mean ± S.D. *<i>p</i><0.05 versus control treatment.</p

    KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

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    <div><p>The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs.</p></div

    KLF4 promotes VEGF-induced tube formation and enhances angiogenesis in vivo.

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    <p><b>A.B.</b> Tube formation assays were performed in KLF4 expressing and knockdown HRMECs, respectively. The angiogenic effect of KLF4 on VEGF induced tube formation was determined by counting nodes and sprouts of tube-like structures from at least three different fields of three independent experiments and normalized to vehicle treated control cells. Significance was compared between KLF4 expressing and control cells with or without VEGF treatment (*p<0.05, **p<0.01, ***p<0.001). Images were presented from one representative experiment. C. Sections of plugs were stained using CD31 antibody and microvessels were counted from 4 sections of each plug and averaged from total 3 plugs. Significance of CD31 positive vessels were compared between sections of KLF4 expressing and control plugs (*p<0.05).</p

    Schematic diagram of KLF4 mediated VEGF signaling pathway.

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    <p>KLF4 binds to the VEGF promoter and induces VEGF expression, subsequently phosphorylates VEGFR2 and activates downstream ERK1/2 and AKT to promote cell proliferation, migration and angiogenesis.</p

    KLF4 transcriptionally activates VEGF expression.

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    <p><b>A.</b> Luciferase reporter assays were performed to assess KLF4 activation of the VEGF promoter. Luciferase activities in KLF4 expressing and control HRMECs were measured at 24h after transfection with 1.5kb and 0.2kbVEGF promoter luciferase constructs in serum-free conditions. Data were presented as the mean ± SE from three independent experiments. Significance of luciferase activity was found between KLF4 expressing and control cells when 1.5Kb VEGF promoter was transfected in both cells (**p<0.01). <b>B.</b> There are three predicted KLF4 binding sites (CACCC) at the VEGFA promoter. The specific binding sites of KLF4 at the VEGFA promoter was detected by ChIP assay and enrichment of KLF4 binding to sites a and b of VEGFA promoter was significant, not c in KLF4 expressing compared to control cells (*p<0.05,**p<0.01) C: VEGF expression in HRMECs was detected by Western Blot at different time points. Significance was compared to 0h from 3, 6 and 12h (*p<0.05, **p<0.01, ***p<0.001). D: VEGF and KLF4 expression was imaged following immunofluorescence staining at 24h following KLF4 induction. E. VEGF released in cell media was detected using VEGF ELISA assay and significance was observed in KLF4 expressing compared to controls at the indicated time points(*p<0.05). F. Cell migration in KLF4 expressing and control HRMECs following VEGF knockdown using VEGF siRNA was examined using transwell migration assay. (*p<0.05, **p<0.01).</p

    KLF4 promotes VEGF induced cell proliferation.

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    <p><b>A, B.</b> Cell proliferation was examined at different time points in KLF4 expressing and knocking down HRMECs. Cells were treated with VEGF or vehicle (Veh) following 24h serum-free media culture before measuring cell proliferation using a MTT assay. Significance was observed between KLF4 expressing and control cells with or without VEGF induction. <b>C</b>. Cell apoptosis was examined using ELISA from HRMECs transduced with lentiviral KLF4shRNA1, 2 and Scramble controls. <b>D.</b> One representative Western blot was shown on the active caspase3 expression at indicated time points in KLF4 knockdown and control cells following 12h serum starvation. Significance was compared between KLF4 knockdown and control cells at the indicated time points. Data are presented as mean ±S.E. from 3 independent experiments, (*p<0.05, **p<0.01).</p
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