Protein tyrosine phosphatase deficiency…
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Figure 7.- Effect of PTP1B deficiency in stress and survival signaling in hepatocytes.
PTP1B
+/+
and PTP1B
-/-
immortalized hepatocytes were treated with various doses of APAP for 8 h. Total cell
lysates were analyzed by Western blot with the antibodies against phospho-JNK1/2 and JNK1/2
(A),
phosphor-IGFIR, IGFIR, IRS1, phospho-Akt and Akt
(B)
and
BclxL and β-actin as a loading
control
(C).
Representative autoradiograms corresponding to three independent experiments are
shown.
D.
PTP1B
-/-
immortalized hepatocytes were reconstituted with PTP1B by retroviral gene
transfer. PTP1B
-/-
and PTP1B
-/-
Rec cells were treated with various doses of APAP for 16 h. Total cell
lysates were analyzed by Western blot with the indicated antibodies. Representative
autoradiograms corresponding to three independent experiments are shown.
Our next step was to study the effect of PTP1B reconstitution of PTP1B-
deficient hepatocytes in the signaling pathways modulated by APAP. For this goal,
we reconstituted PTP1B expression in deficient cells by retroviral gene transfer as
described in Materials and Methods. As a control, hepatocytes were infected with
an empty retroviral construct. Figure 7D shows the re-expression of PTP1B in
PTP1B
-/-
immortalized neonatal hepatocytes (refrerred as PTP1B
-/-
Rec).
Importantly, re-expression of recombinant PTP1B in PTP1B
-/-
neonatal
hepatocytes induced a decline of IGF-IR phosphorylation after APAP treatment
similar to the effects observed in wild-type cells. In addition, APAP treatment
activated JNK phosporylation in PTP1B
-/-
Rec neonatal hepatocytes as in wild-type
cells.
We have recently reported that APAP preotected against liver injury
induced by a toxic dose of APAP (300 mg/kg) in mice (33). To reinforce this
finding, we injected both PTP1B
+/+
and PTP1B
-/-
mice with a higher dose of APAP
(500 mg/kg) and analyzed liver toxicity. Serum ALT was higher in APAP-injected
wild-type than in PTP1B
-/-
mice (Figure 8A). Moreover, less injury was observed in
