Mediated Knockdown (mediated + knockdown)

Distribution by Scientific Domains

Selected Abstracts

Neuronal leucine-rich repeat 6 (XlNLRR-6) is required for late lens and retina development in Xenopus laevis

Adam D. Wolfe
Abstract Leucine-rich repeat proteins expressed in the developing vertebrate nervous system comprise a complex, multifamily group, and little is known of their developmental function in vivo. We have identified a novel member of this group in Xenopus laevis, XlNLRR-6, and through sequence and phylogenetic analysis, have placed it within a defined family of vertebrate neuronal leucine-rich repeat proteins (NLRR). XlNLRR-6 is expressed in the developing nervous system and tissues of the eye beginning at the neural plate stage, and expression continues throughout embryonic and larval development. Using antisense morpholino oligonucleotide (MO) -mediated knockdown of XlNLRR-6, we demonstrate that this protein is critical for development of the lens, retina, and cornea. Reciprocal transplantation of presumptive lens ectoderm between MO-treated and untreated embryos demonstrate that XlNLRR-6 plays autonomous roles in the development of both the lens and retina. These findings represent the first in vivo functional analysis of an NLRR family protein and establish a role for this protein during late differentiation of tissues in the developing eye. Developmental Dynamics 235:1027,1041, 2006. 2006 Wiley-Liss, Inc. [source]

Essential role of C/EBP, in G-CSF-induced transcriptional activation and chromatin modification of myeloid-specific genes

GENES TO CELLS, Issue 4 2008
Satoshi Iida
Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation and differentiation of neutrophilic progenitor cells. Here, we investigated the roles of CCAAT/enhancer-binding protein (C/EBP), in the G-CSF-induced transcriptional activation and chromatin modification of the CCR2 and myeloperoxidase (MPO) genes in IL-3-dependent myeloid FDN1.1 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays revealed that G-CSF activates C/EBP, to bind target promoters. ChIP mapping experiments across the CCR2 and MPO genes showed that G-CSF induces histone H3 modifications: the acetylation of Lys9, trimethylation of Lys4 and trimethylation of Lys9. The distribution profile of the trimethylated Lys9 was distinct from that of the two other modifications. All the G-CSF-induced C/EBP, recruitment, transcriptional activation and histone modifications were reversed by re-stimulation with IL-3, and were abolished by short hairpin RNA (shRNA)-mediated knockdown of C/EBP,. These results indicate that C/EBP, is activated by G-CSF to bind target promoters, and plays critical roles in the transcriptional activation and dynamic chromatin modification of target genes during neutrophil differentiation. [source]

The small ubiquitin-like modifier mediates the resistance of prosthesis-loosening fibroblast-like synoviocytes against fas-induced apoptosis

Ingmar Meinecke
Objective To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. Methods Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA,mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. Results SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. Conclusion These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening. [source]

Involvement of protein kinase C, in interleukin-1, induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-,B signaling in primary human osteoarthritic chondrocytes

Priya S. Chockalingam
Objective Protein kinase C, (PKC,), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC, in interleukin-1, (IL-1,),induced NF-,B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC,. Western blot analysis was used to evaluate phosphorylation of PKC, and NF-,B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results Phosphorylation of PKC, and NF-,B was induced by IL-1, treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC, suppressed IL-1,,induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1,,induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC, and NO production. Furthermore, small interfering RNA, or short hairpin RNA,mediated knockdown of PKC, mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKC, is involved in the regulation of IL-1,,induced NF-,B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKC, could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA. [source]