Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray gene expression profiling of kinase-dependent and kinase-independent effects of GRK2

ABSTRACT: The ubiquitously expressed G-protein-coupled receptor kinase 2 (GRK2, ADRBK1) is an indispensable kinase involved in growth, differentiation and development. Exaggerated GRK2 activity plays a major pathophysiological role in the development of cardiovascular diseases such as heart failure and hypertension. GRK2 exerts its functions by kinase-dependent and kinase-independent effects. To assess the differential impact of GRK2 on cellular signalling we established HEK cell clones with over-expression of comparable protein levels of GRK2 or the kinase-deficient GRK2-K220R mutant, respectively. HEK cells were either cultured in vitro or expanded in vivo, in immunodeficient NOD.Scid mice to discriminate between in vitro and in vivo effects of GRK2. Whole genome microarray gene expression profiling was performed of cultured HEK cells and of NOD.Scid mouse-expanded HEK clones. As an additional control, cells were re-cultured in vitro after expansion in NOD.Scid mice. Whole genome microarray gene expression profiling was performed with three different study groups: (i) NOD.Scid mouse-expanded HEK clones expressing GRK2 or GRK2-K220R, (ii) cultured HEK cell clones expressing GRK2 or GRK2-K220R, and (iii) cell clones expressing GRK2 or GRK2-K220R, which were re-cultured after NOD.Scid mouse expansion.

ORGANISM(S): Homo sapiens

SUBMITTER: Ursula Quitterer

PROVIDER: E-GEOD-42771 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. Gene array analysis shows that RPF-1 transcription factor modify the transcriptome of HEK293 cells by regulating a genetic program endowed with developmental programs Comparison of HEK293 cells induced for RPF-1 expression (Tet-) over uninduced control (Tet+) and Mock cells. RPF-1 cDNA was inserted into the pTRE2puro vector to generate stable Tet-inducible KEK293 transfectants. Upon Tet removal from the medium, HEK/RPF-1 transfectants expressed the transcription factor RPF-1. Cells were then seeded in 10 cm dishes, cultured for 48 h either in complete medium supplemented with Tet (+) or in Tet-depleted medium (-), harvested, and spun down before RNA isolation. Comparative analysis between HEK/RPF-1 induced (Tet-) and uninduced (Tet+) transfectants allowed us to identify genes transcriptionally regulated by RPF-1. Residual effects on gene expression due to Tet supplementation into culture media was ruled out by comparison with the Mock transfectant.

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Microarray gene expression profiling of kinase-dependent and kinase-independent effects of GRK2

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