1α,25-Dihydroxyvitamin D3 Stimulates Activator Protein 1 DNA-Binding Activity by a Phosphatidylinositol 3-Kinase/Ras/MEK/Extracellular Signal Regulated Kinase 1/2 and c-Jun N-Terminal Kinase 1-Dependent Increase in c-Fos, Fra1, and c-Jun Expression in Human Keratinocytes

1α,25-Dihydroxyvitamin D3 added to human keratinocytes increases differentiation through an activation of the transcription factor activator protein 1. We have previously reported that the 1α,25-dihydroxyvitamin D3-induced increase of activator protein 1 DNA binding activity is mediated by a protein kinase C-independent mechanism. The purpose of this study was to investigate further the mechanisms by which 1α,25-dihydroxyvitamin D3 modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Western blotting experiments revealed that 1α,25-dihydroxyvitamin D3 caused a rapid and transient activation of the mitogen-activated protein kinases, extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. 1α,25-Dihydroxyvitamin D3 also enhanced the expression of the activator protein 1 subunits, c-Fos, Fra1, and c-Jun as determined by northern and western blotting. The 1α,25-dihydroxyvitamin D3-induced activator protein 1 DNA binding activity was completely blocked by the MEK inhibitor PD 98059 indicating that the MEK/extracellular signal regulated kinase pathway is involved in the activation of activator protein 1. Transfection experiments showed that 1α,25-dihydroxyvitamin D3 also increased the activator protein 1-dependent transactivation, which was completely blocked by expression of a dominant negative Ras, suggesting that the 1α,25-dihydroxyvitamin D3-induced activator protein 1 activity involves Ras-dependent signaling. Furthermore, preincubation of the keratinocytes with the specific phosphatidylinositol 3-kinase inhibitors, Wortmannin and LY294002, demonstrated that the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 required phosphatidylinositol 3-kinase activity. Finally, preincubation of keratinocytes with a polyclonal antibody against the membrane receptor annexin II, blocked the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. Taken together, our results indicate that 1α,25-dihydroxyvitamin D3, via binding to the membrane receptor annexin II, induces activation of the phos-phatidylinositol 3-kinase/Ras/MEK/extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 signal transduction pathway resulting in increased expression of c-Fos, Fra1, and c-Jun, and subsequently increased activator protein 1 DNA binding activity and gene transcription

Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform

Amino-LNA, Thio-LNA and Alpha-L-Oxy-LNA

Pub. No.: WO/2004/046160 International Application No.: PCT/DK2003/000788 Publication Date: 03.06.2004 International Filing Date: 18.11.2003 Chapter 2 Demand Filed: 08.06.2004 IPC: A61K 31/7088 (2006.01)A novel class of pharmaceuticals which comprises a Locked Nucleic Acid (LNA) which can be used in antisense therapy. These novel oligonucleotides have improved antisense properties. The novel oligonucleotides are composed of at least one LNA selected from beta-D-thio/amino-LNA or alpha-L-oxy/thio/amino-LNA. The oligonucleotides comprising LNA may also include DNA and/or RNA nucleotide

Sense and antisense strand activity of siRNA and 5′ sense end modified siLNA

<p><b>Copyright information:</b></p><p>Taken from "Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality"</p><p>Nucleic Acids Research 2005;33(1):439-447.</p><p>Published online 14 Jan 2005</p><p>PMCID:PMC546170.</p><p>© 2005, the authors © </p> () Schematic representation of the SARS 3 target cloned in the sense (pS3Xs) or the antisense (pS3Xas) direction behind firefly luciferase. Also shown is the parental luciferase plasmid without the SARS 3 target (pGL3). () Activity of siRNA and siLNA against sense (pS3Xs), antisense (pS3Xas) or control target (pGL3-Control). Mean and SD values are from two experiments performed in duplicate