title: Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.
abstract: Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.
authors.affiliation: Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Boylan
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA; Department of Chemistry, Brown University, Providence, RI, USA.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Salomon
authors.affiliation: Division of Genetics, Department of Pathology, Brown University and Women and Infants Hospital, Providence, RI, USA.
authors.forename: Umadevi
authors.initials: U
authors.lastname: Tantravahi
authors.affiliation: Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA; Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA. Electronic address: philip_gruppuso@brown.edu.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Gruppuso
mesh_terms: D000222:Adaptation, Physiological; D020134:Catalytic Domain; D049109:Cell Proliferation; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000502:physiology; D020543:Proteome / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D059467:Transcriptome
chemical_list: D010750:Phosphoproteins; D021122:Protein Subunits; D020543:Proteome; D034741:RNA, Small Interfering; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6
keywords: DNA repair; DNA replication; Gene expression; Hepatic cells; Polyploidy; Protein phosphatase 6; Protein phosphorylation; Signal transduction