Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model.
Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least threefold in these cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca2+ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca2+ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT-deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.
Slepak
Tatiana I
TI
The Dr. John T. Macdonald Foundation Center for Medical Genetics, Department of Pediatrics, The Leonard M. Miller School of Medicine, University of Miami, P.O. Box 016820 (D-820), Miami, FL 33101, USA.
Tang
Manshu
M
Slepak
Vladlen Z
VZ
Lai
Kent
K
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005690:Galactose / Q000494:pharmacology; D005693:Galactosemias / Q000201:enzymology* / Q000473:pathology; D005695:Galactosephosphates / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005947:Glucose / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D018832:Molecular Chaperones / Q000378:metabolism; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*
D000091342:Endoplasmic Reticulum Chaperone BiP; D005695:Galactosephosphates; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C029973:galactose-1-phosphate; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D015720:5'-Nucleotidase; D005947:Glucose; D002118:Calcium; D005690:Galactose