Role of transglutaminase 2 in liver injury via cross-linking and silencing of transcription factor Sp1.

BACKGROUND & AIMS: Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. METHODS: Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2-/- mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. RESULTS: Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2-/- mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas-induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. CONCLUSIONS: TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Identification of galactose-1-phosphate uridyl transferase gene common mutations in dried blood spots.

BACKGROUND: The California newborn screening program uses newborns' dried blood spots (DBS) to screen for more than 45 genetic disorders. Deficiency of galactose-1-phosphate uridyl transferase (GALT) is one of the metabolic genetic disorders screened using newborn DBS. During follow-up tests, common mutations of the GALT gene have been identified using whole blood samples. To avoid the stress of drawing an additional blood sample from newborns who are identified as presumptive positive for galactosemia, we developed a method to test common mutations in the GALT gene using blood spots. METHODS: This method involves DNA extraction from DBS, followed by polymerase chain reaction (PCR), and single nucleotide extension (SNE). SNE products were detected by capillary electrophoresis. RESULTS: In a double-blind study, GALT gene common mutations/variants: IVS2-2A>G, p.S135L, p.T138M, p.Q188R, p.L195P, p.Y209C, p.L218L, p.K285N, and p.N314D were detected in seventy-three DBS which had previously been screened and confirmed as positive in the California Newborn Screening Program. Mutations found using blood spots gave 100% concordance with mutations from previously genotyped whole blood samples. CONCLUSIONS: This blood spot method decreases the genomic test turnaround time of GALT screened positive patients and potentially reduces emotional stress on families required to provide an additional blood draw.