Purinergic Signaling in Human Pluripotent Stem Cells Is Regulated by the Housekeeping Gene Encoding Hypoxanthine Guanine Phosphoribosyltransferase
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Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase Lina Mastrangeloa, Ji-Eun Kimb, Atsushi Miyanoharaa, Tae Hyuk Kanga, and Theodore Friedmanna,1 aDepartment of Pediatrics, Center for Neural Circuits and Behavior, University of California at San Diego School of Medicine, La Jolla, CA 92093; and bNeurobiology Section, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093 Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved January 19, 2012 (received for review November 3, 2011) Lesch–Nyhan disease (LND) is an X-linked genetic disorder caused purine derivatives in modulating neurotransmitter release and by mutations of the hypoxanthine guanine phosphoribosyltrans- demonstrating the involvement of purinergic receptors in neu- ferase (HPRT) purine biosynthesis gene and characterized by aber- ronal growth and survival (9–13). A role of purinergic receptors rant purine metabolism, deficient basal ganglia dopamine levels, in neuronal differentiation has been reported as well (14, 15). dystonia, and severe neurobehavioral manifestations, including Recent studies also have established roles of nucleotides in compulsive self-injurious behavior. Although available evidence controlling several aspects of neurogenesis, neural differentia- has identified important roles for purinergic signaling in brain tion, and migration in the developing mammalian CNS through development, the mechanisms linking HPRT deficiency, purinergic interactions with two classes of purinergic P2 receptors, the pathways, and neural dysfunction of LND are poorly understood. ionotropic P2X receptors and the P2Y class of G protein-cou- In these studies aimed at characterizing purinergic signaling in pled receptors (GPCRs) (16–18). Other studies have shown that HPRT deficiency, we used a lentivirus vector stably expressing an HPRT fi ATP, acting via P2X and P2Y receptors, stimulates increased shRNA targeted to the gene to produce HPRT-de cient hu- proliferation of embryonic stem cells (19). Moreover, P2Y re- man CVB induced pluripotent stem cells and human HUES11 em- ceptor activation in adult neural progenitor cells occurs at least BIOLOGY bryonic stem cells. Both CVB and HUES11 cells show >99% HPRT in part through effects on nucleotide-induced expression and on DEVELOPMENTAL knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA the function of transcription factors ERK1/2 and CREB phos- phorylation (17). and protein down-regulation by HPRT knockdown is refractory to β activation by the P2Y1 receptor agonist ATP and shows aberrant P2Y receptors also affect the vital Wnt/ -catenin signaling purinergic signaling, as reflected by marked deficiency of the tran- pathway, which includes a major class of growth factors with scription factor pCREB and constitutive activation of the MAP potent effects on stem cells and developmental processes (20, kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells 21). Recent evidence shows that P2Y receptors mediate phos- β also demonstrate marked reduction of phosphorylated β-catenin. phorylation of glycogen synthase kinase (GSK-3 ), a key medi- These results indicate that the housekeeping gene HPRT regulates ator of Wnt signaling and nuclear transport of the transcription purinergic signaling in pluripotent human stem cells, and that this factor β-catenin (22). Furthermore, purinergic signaling has been regulation occurs at least partly through aberrant P2Y1-mediated reported to regulate GSK-3β and nuclear translocation of expression and signaling. We propose that such mechanisms may β-catenin in astrocytes (23). In addition, we recently reported play a role in the neuropathology of HPRT-deficiency LND and may evidence of major disruption of phosphorylated β-catenin point to potential molecular targets for modulation of this intrac- expression and impaired nuclear transport in HPRT-deficient table neurological phenotype. human fibroblasts (24). Our present study on the effect of HPRT deficiency on puri- neurodevelopment | neurotransmitters nergic signaling are based on the working hypothesis that alter- ations in purine pools resulting from defective salvage purine omplete genetic deficiency of the enzyme hypoxanthine biosynthesis might affect the function of purinergic receptors, Cguanine phosphoribosyltransferase (HPRT) causes Lesch– thereby leading to aberrant development of the CNS dopamine- Nyhan disease (LND), an intractable neurobehavioral disorder related pathways and aberrant dopaminergic neurogenesis. We characterized by hyperuricemia, cognitive impairment, dystonia have chosen to study mechanisms associated with that inter- with spasticity choreoathetosis and compulsive self-mutilation action in human induced pluripotent stem (iPS) and embryonic (1). The enzyme HPRT plays a major role in salvage purine stem (ES) cells because of the growing importance of such cells biosynthesis by catalyzing the conversion of hypoxanthine and in delineating the early molecular and cellular events in the guanine to the nucleotides inosine monophosphate (IMP) and defective neural development characteristic of LND and other guanosine monophosphate (1, 2). HPRT deficiency leads to the neurodegenerative and neurodevelopmental diseases (25). A hallmark biochemical defect in LND: greatly increased de novo fi purine synthesis, with resulting elevated levels of oxypurines and more detailed understanding of the effects of HPRT de ciency tissue accumulation of uric acid, gout, and life-threatening during embryogenesis is likely to clarify the developmental out- nephrolithiasis (3, 4). Although treatment with the xanthine oxi- comes of LND and identify potential new therapeutic targets. dase inhibitor allopurinol effectively prevents hyperuricemia and renal damage in LND and thereby markedly extends the lifespan of patients, neither allopurinol nor any other treatment produces Author contributions: L.M. designed research; L.M. and T.H.K. performed research; L.M., lasting improvement in neurological manifestations (5, 6). J.-E.K., A.M., and T.H.K. contributed new reagents/analytic tools; L.M., J.-E.K., A.M., T.H.K., PET scanning and postmortem studies of brains from patients and T.F. analyzed data; and L.M. and T.F. wrote the paper. with LND have demonstrated decreased levels of dopamine, The authors declare no conflict of interest. dopamine biosynthetic enzymes, and dopamine uptake in the This article is a PNAS Direct Submission. basal ganglia (7, 8). However, the mechanisms linking HPRT 1To whom correspondence should be addressed. E-mail: [email protected]. fi de ciency with the neurologic dysfunctions are little understood. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Recently, evidence has accumulated identifying key roles for 1073/pnas.1118067109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118067109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Results expression is both qualitatively and quantitatively reduced in fi HPRT Knockdown in CVB iPS Cells. Expression of the shRNA tar- HPRT-de cient CVB cells. geted to HPRT profoundly reduces HPRT expression but does fi not produce major disturbances in the pluripotency of iPS cells. P2Y1 Expression in HPRT-De cient HUES11 Human ES Cells. To de- A termine whether aberrant P2Y1 expression is specific to CVB Fig. 1 shows mRNA levels of HPRT in CVB iPS cells trans- fi duced with the control anti-luciferase (shLux) and with the anti- iPS cells or is a more general consequence of HPRT de ciency in HPRT vector (shHPRT). HPRT mRNA expression was virtually pluripotent stem cells, we used real-time PCR to examine P2Y1 undetectable in this assay and was reduced by >99% in cells trans- expression in HPRT-knockdown human HUES11 ES cells. We found that in these cells, efficient (>95%) HPRT knockdown duced by shHPRT compared with control luciferase-knockdown A cells. Similarly, HPRT protein expression was markedly reduced with vector shHPRT measured by real-time PCR (Fig. 3 ), Western immunoblot analysis (Fig. 3B), and HPRT enzymatic in the HPRT-knockdown cells (Fig. 1B). Finally, TLC-based activity (Fig. 3C) leads to a significantly (∼60%) reduced ex- HPRT enzyme assays confirmed the virtual complete loss of pression of P2Y1 mRNA levels (Fig. 3D). Our results are con- HPRT enzymatic activity in HPRT-knockdown CVB cells, as in- sistent with changes seen in the HPRT-deficient CVB iPS cells. dicated by absence of IMP in the chromatography assay (Fig. 1C). The process of transduction of cells with the knockdown vectors P2Y1 Regulates CREB Expression and ERK1/2 Phosphorylation in CVB does not lead to major changes in the pluripotency state of the iPS Cells. Recent studies with murine adult neural progenitor cells cells, as measured by expression of the pluripotency markers β D E have shown that the P2Y1 receptor agonists ADP S and UTP Nanog and Oct-4 (Fig. 1 and ). stimulate the expression of phosphorylated CREB and ERK1/2, and that this stimulation is inhibited by the P2Y1 receptor an- Expression of Nucleotide Signaling Components. Transcription of tagonist MRS2179 (17). Because HPRT knockdown markedly nucleotide signaling pathway components was examined by down-regulates P2Y1 expression (Fig. 2 A and B), we examined quantitative PCR assays (Fig. 2). Total mRNA was extracted the effect of HPRT knockdown on phospho-CREB and