There is evidence of increased PTP1B expression during the progression of non-alcoholic fatty liver disease that concurs with insulin resistance and liver damage.30, 31
However, PTP1B functions in physiological responses regulated by tyrosine kinase signaling, such as hepatocellular survival during drug injury, are poorly understood. Our results have unraveled a central role of PTP1B in the molecular mechanism that mediates APAP-induced liver failure, identifying PTP1B as a potential therapeutic target in the treatment of human APAP hepatotoxicity. This study emerged from the observation of elevated PTP1B expression in livers from individuals suffering from APAP intoxication. Importantly, in APAP-treated human and mouse hepatocytes, upregulation of PTP1B preceded cell death.
Results in mouse hepatocytes demonstrated that PTP1B deficiency attenuates cellular damage in response to APAP. The fact that released LDH activity was reduced in PTP1B−/−
hepatocytes indicates that necrotic cell death was less severe than in wild-type cells. In wild-type hepatocytes, APAP also increased the percentage of sub-G0/G1 cells and release of cytochrome C
from the mitochondria. APAP also induced activation of caspase-3 and decreased expression of antiapoptotic proteins BclxL and Mcl1. These results strongly suggest that APAP also triggered apoptosis in hepatocytes and, again, these effects were ameliorated by PTP1B deficiency. The role of apoptosis in APAP-induced hepatocyte cell death is controversial. However, there are studies that clearly demonstrate both types of cell death following APAP overdose. Circulating apoptotic and necrotic cell death markers have been detected in patients with APAP-induced acute liver injury.32, 33, 34
Cell death via necrosis and apoptosis has been reported in the livers removed during transplantation because of APAP intoxication.35
These data have also been shown in mice during APAP poisoning.36
In light of these results, we and others have shown that PTP1B inhibition protects hepatocytes against activation of mitochondrial (intrinsic) and death receptor (extrinsic)-mediated apoptotic pathways,21, 25
reinforcing the role of this phosphatase in programmed cell death.
As cellular oxidative stress processes (GSH depletion and ROS generation)13
are ameliorated in PTP1B−/−
hepatocytes, we investigated the modulation of oxidative stress by PTP1B. An unknown tyrosine kinase has been proposed to be the upstream activator of GSK3β
by phosphorylation at Tyr216.27, 28
This phenomenon leads to SKF-mediated tyrosine phosphorylation, nuclear export, ubiquitination and degradation of Nrf2 in response to oxidative stress inducers. Our data demonstrated firstly that this signaling pathway is activated by APAP in hepatocytes and secondly that PTP1B deficiency mimics the inhibition of GSK3β
or SKF on the downstream mediators of this pathway. Inhibition of GSK3β
or SKF augmented and prolonged Nrf2 nuclear accumulation, delaying its nuclear exclusion in response to APAP. Importantly, the effect of PTP1B deficiency on this pathway was manifested by delayed Src-Fyn nuclear translocation, Nrf2 tyrosine phosphorylation and its subsequent ubiquitination and degradation, leading to enhanced HO-1 induction. These results suggest that PTP1B might activate this unknown kinase upstream from GSK3β
, probably by dephosphorylation, and thereby triggering the activation of the GSK3β
/Src-Fyn axis that ultimately leads to Nrf2 degradation. Consequently, as demonstrated in hepatocytes, PTP1B deficiency might delay and/or reduce activation of this complex axis, resulting in prolonged nuclear accumulation of Nrf2 and enhanced antioxidant defense.
Besides the novel role of PTP1B in antioxidant defense reported herein, this phosphatase regulates duration and/or intensity of survival signals emerging from the tyrosine kinase receptor family.19, 20, 21
As IGFIR triggers survival responses in hepatocytes,37
the lack of PTP1B prevented the reduction in IGFIR/IRS1/2/Akt-mediated survival signaling in APAP-treated cells. In fact, this is the first report showing degradation of IRS proteins in hepatocytes by APAP, suggesting that high doses of this analgesic may also interfere with hepatic insulin signaling and increase the risk of metabolic diseases such as type 2 diabetes mellitus. Thus, in wild-type hepatocytes, increased ROS-mediated JNK and p38 MAPK phosphorylation by APAP are likely to be responsible for the feedback mechanism on IGFIR-mediated signaling, leading to IRS1/2 serine phosphorylation that precedes degradation.38
In the absence of PTP1B, the decrease in ROS-mediated activation of JNK and p38 MAPK and the increase in IGFIR tyrosine phosphorylation might act in conjunction with the effects on the GSK3β
/Src-Fyn axis, resulting in attenuated oxidative stress and increased cell survival in response to APAP.
Several studies have shown the mechanistic importance of hepatic cytokines in APAP-induced liver injury. In the liver, cytokines are mainly produced by non-parenchymal cells. Our in vivo
data show significant lower expression levels of IL1β
and IL6 mRNAs in the livers of PTP1B−/−
mice upon APAP injection as compared with the elevated levels of the wild-type controls, suggesting that PTP1B also modulates cytokine production by non-parenchymal (i.e. Kupffer) cells in response to liver damage. Cytokines have an essential role as inducers of PTP1B in metabolic diseases. Activation of JNK together with elevated expression of PTP1B in response to IL6 has been reported in skeletal muscle of insulin-resistant mice.39
Likewise, IL4 induces PTP1B to suppress IL4-induced STAT6 signaling in B cells.40
As PTP1B is increased in the livers of APAP-injected wild-type mice, this effect is likely to be due to elevation of IL6 and IL1β
. Therefore, cytokine-mediated increases in PTP1B expression might enhance the GSK3β
/Src-Fyn axis, resulting in increased Nrf2 tyrosine phosphorylation and a rapid nuclear exclusion. Moreover, in the liver of APAP-injected wild-type mice, the negative cross-talk elicited by JNK on IRS proteins and also by PTP1B by direct dephosphorylation of the IGFIR might synergize with the enhancement of the GSK3β
/Src-Fyn axis to reduce survival of hepatocytes in vivo
. This mechanism is supported by lower activation of JNK, higher IGFIR tyrosine phosphorylation and nuclear Nrf2 accumulation in livers of APAP-injected PTP1B−/−
mice as compared with the wild-type controls. As a result, PTP1B-deficient livers are protected against APAP-induced oxidative stress and injury as manifested by decreased GSH depletion favoring NAPQI detoxification and subsequently limiting the formation of APAP–protein adducts. However, the effects of APAP in vivo
are not exclusively mediated by elevation of PTP1B expression induced by proinflammatory cytokines secreted by non-parenchymal cells; our in vitro
data have also demonstrated direct effects of APAP in hepatocytes. Nevertheless, PTP1B deficiency protects against damage in hepatocytes in culture and in whole liver, reinforcing the regulatory role of this phosphatase in multiple molecular mechanisms triggered in response to APAP in distinct liver cells.
As summarized in , we have demonstrated the unique role of PTP1B in the liver as a critical crossroad in the signaling pathways triggered in response to APAP by dual modulation of the Nrf2-mediated antioxidant response through the GSK3β/Src-Fyn kinases axis and the survival signaling through its effects on the IGFIR/IRSs/Akt signaling pathway. Collectively, our data suggest that inhibition of PTP1B would be a suitable therapeutic approach against APAP-induced hepatotoxicity.