Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase

Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3377-82. doi: 10.1073/pnas.1118067109. Epub 2012 Feb 13.

Abstract

Lesch-Nyhan disease (LND) is an X-linked genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated β-catenin. These results indicate that the housekeeping gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Cell Line
  • Fibroblasts / enzymology
  • Gene Knockdown Techniques
  • Genes, Essential
  • Genetic Vectors / genetics
  • Glycogen Synthase Kinase 3 / physiology
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Hypoxanthine Phosphoribosyltransferase / physiology*
  • Lentivirus / genetics
  • Lesch-Nyhan Syndrome / enzymology
  • MAP Kinase Signaling System / physiology
  • Male
  • Mitogen-Activated Protein Kinase 1 / physiology
  • Mitogen-Activated Protein Kinase 3 / physiology
  • Neurogenesis / physiology*
  • Pluripotent Stem Cells / enzymology*
  • Purinergic P2Y Receptor Agonists / pharmacology
  • Purines / metabolism*
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics
  • Receptors, Purinergic P2Y1 / genetics
  • Receptors, Purinergic P2Y1 / physiology
  • beta Catenin / metabolism

Substances

  • CTNNB1 protein, human
  • Purinergic P2Y Receptor Agonists
  • Purines
  • RNA, Messenger
  • RNA, Small Interfering
  • Receptors, Purinergic P2Y1
  • beta Catenin
  • Adenosine Triphosphate
  • Hypoxanthine Phosphoribosyltransferase
  • Glycogen Synthase Kinase 3 beta
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Glycogen Synthase Kinase 3