Protein tyrosine phosphatase deficiency…
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this phosphatase sensitizes neonatal hepatocytes to death signals induced by this
apoptotic stimulus (27). These differential effects resulted from the regulation of
multiple events involving mitochondrial integrity, the Bcl-2 protein family, the
caspases-8,-9 and -3 and the nuclear translocation of Foxo 1 which activates the
death receptor pathway. Thus, these previous observations suggested that levels of
PTB1B may exert a pivotal role in maintaining the balance between survival and
death in hepatocytes.
On that basis,
the main goal
of the present study is to investigate if PTP1B
deficiency is able to protect hepatocytes against the early events during APAP-
induced hepatotoxicity.
2. MATERIALS AND METHODS
2.1. Materials.
Fetal serum (FS) and culture reagents were obtained from Invitrogen. APAP
was purchased from Sigma (Sigma-Aldrich). Anti-phospho-JNK (#9251) (Thr183/
Tyr185) and anti-active caspase 3 (#9661) antibodies were from Cell Signaling
Technology. The antibodies against phospho-Akt (Ser 473) (sc-7985), phospho-
IGF-IR (Tyr 1165/1166) (sc-101704) and total JNK (sc-571) were from Santa Cruz
Biotechnology. The anti-IRS-1 (06-248), anti-IRS-2 (06-248) and anti-mouse
PTP1B (07-088) antibodies were obtained from Upstate (Millipore). Anti-BclxL
(610211) antibody was from BD Pharmingen. Anti-β-actin (A-5441) antibody was
from Sigma. Anti-Cyp2E1 antibody (aB19140) was from Abcam. Total Akt and total
IGF-IR antibodies were gifts from M Birnbaum and MF White, respectively (Joslin
Diabetes Center, USA). Anti GCLc and GCLm antibodies were a gift from T
Kavanagh (University of Washington, USA).
2.2. Human liver biopsies.
Human liver samples were obtained from from five patients intoxicated
with APAP and three healthy subjects. These samples were kindly donated from
Dr Kenneth J. Simpson in the division of Clinical and Surgical Sciences, University
of Edinburgh, Edinburgh EH164TJ, UK. Informed written consent was obtained
from each patient.
2.3. Animal models.
Three months-old male PTP1B
+/+
(wild-type) and PTP1B
-/-
mice maintained
on the C57Bl/6J x 129Sv/J genetic background (28) were used throughout the
study. Animal experimentation was conducted accordingly to the accepted
guidelines for animal care of the Comunidad de Madrid (Spain). Overnight fasted
mice were intraperitoneally (i.p.) injected with 500 mg/kg APAP dissolved in
physiological saline. Mice were sacrificed at 6 h and livers were collected.
