VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.     

Protein tyrosine phosphatase-1B and T-cell protein tyrosine phosphatase regulate IGF-2-induced MCF-7 cell migration

The protein tyrosine phosphatase-1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) have been implicated in down-regulation of tyrosine kinase receptors, conferring anti-oncogenic functions to these PTPases. However, recent work has shown that PTP1B is positively implicated in oncogenic properties of breast cancer cells by regulating the ERK pathway. Here, we studied the function of PTP1B and TC-PTP in IGF-2-induced growth, survival and migration of MCF-7 breast cancer cells. Using siRNA, we showed that reduction in the expression of these PTPases decreased cell growth and ERK phosphorylation. Reduction in the expression of these PTPases did not impair IGF-2 effects on cell survival to acute treatment with 4-OH Tamoxifen. In contrast, IGF-2-induced MCF-7 cell migration was markedly impaired by reduction of PTP1B or TC-PTP expression, independently of the ERK pathway. This novel finding reinforces the potential role of these PTPases as therapeutic targets for treatment of breast cancer.     

Protein-tyrosine phosphatase-1B (PTP1B) mediates the anti-migratory actions of Sprouty

Mammalian Sprouty proteins have been shown to inhibit the proliferation and migration of cells in response to growth factors and serum. In this communication, using HeLa cells, we have examined the possibility that human Sprouty 2 (hSPRY2) mediates its anti-migratory actions by modulating the activity or intracellular localization of protein-tyrosine phosphatases. In HeLa cells, overexpression of hSPRY2 resulted in an increase in protein-tyrosine phosphatase (PTP1B) amount and activity in the soluble (100,000 x g) fraction of cells without an increase in total amount of cellular PTP1B. This increase in the soluble form of PTP1B was accompanied by a decrease in the amount of the enzyme in the particulate fraction. The amounts of PTP-PEST or PTP1D in the soluble fractions were not altered. Consistent with an increase in soluble PTP1B amount and activity, the tyrosine phosphorylation of cellular proteins and p130(Cas) was decreased in hSPRY2-expressing cells. In control cells, overexpression of wild-type (WT) PTP1B, but not its C215S catalytically inactive mutant mimicked the actions of hSPRY2 on tyrosine phosphorylation of cellular proteins and migration. On the other hand, in hSPRY2-expressing cells, the C215S mutant, but not WT PTP1B, increased tyrosine phosphorylation of cellular proteins and attenuated the anti-migratory actions of hSPRY2. Interestingly, neither WT nor C215S mutant forms of PTP1B modulated the anti-mitogenic actions of hSPRY2. Therefore, we conclude that an increase in soluble PTP1B activity contributes to the anti-migratory, but not anti-mitogenic, actions of hSPRY2.     

Regulation of the catalytic activity of PTP1B: roles for cell adhesion, tyrosine residue 66, and proline residues 309 and 310

The reversible phosphorylation of proteins on tyrosine residues is fundamental to a variety of intracellular signaling pathways and is controlled by the actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While much progress has been made in understanding the regulation of PTKs, there is still relatively little known concerning the regulation of PTPs. Using immune complex phosphatase assays, we demonstrated that the enzymatic activity of the nonreceptor type PTP, PTP1B, is regulated by cell adhesion. Placing primary human foreskin fibroblasts (HFFs) in suspension leads to a distinct increase in PTP1B activity, whereas the readhesion of suspended HFFs onto fibronectin or collagen I inhibited activity. To gain insight into the mechanisms involved, we analyzed recombinant forms of PTP1B mutated at potential regulatory sites. Our results indicated that tyrosine residue 66 is essential for maintaining activity at 37 degrees C. We also found that the C-terminal region of PTP1B and localization to the endoplasmic reticulum are not required for the inhibition of activity by cell adhesion. However, analysis of PA-PTP1B, in which alanines are substituted for prolines 309 and 310, revealed an important role for these residues as the catalytic activity of this mutant did not decrease following readhesion onto collagen I. Since the binding of p130cas and Src to PTP1B is dependent upon these proline residues, we assayed the regulation of PTP1B in mouse embryo fibroblasts deficient in these proteins. We found that neither p130cas nor Src is required for the inhibition of PTP1B activity by adhesion to extracellular matrix proteins. Additionally, pretreatment with cytochalasin D did not prevent the reduction of PTP1B activity when cells adhered to collagen I, indicating that cell spreading is not required for this regulation. The control of the catalytic activity of PTP1B by cell adhesion demonstrated in this study is likely to have important implications for growth factor and insulin signaling.     

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.     

Regulation of ROS signal transduction by NADPH oxidase 4 localization

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch

Transforming growth factor-β (TGF-β) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-β induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-β caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-β-induced apoptosis. However, vNox4, but not TGF-β, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-β enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-β-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.     

Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.     

NADPH oxidase subunit 4 mediates cycling hypoxia-promoted radiation resistance in glioblastoma multiforme

Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve reactive oxygen species (ROS) production. However, details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of an in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence-activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels, and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma; hence, targeting Nox4 may be an attractive therapeutic strategy for blocking cycling hypoxia-mediated radioresistance.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Nox1-dependent superoxide production controls colon adenocarcinoma cell migration

Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C delta inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C delta, and controls cell migration by affecting integrin alpha 2 subunit turn-over.     

Tyrosine phosphatase PTP1B modulates store-operated calcium influx

We have studied modulation of “store-operated calcium influx” by tyrosine phosphatases in the pancreatic acinar cell line AR42J and in HEK 293 cells. We show that inhibition of tyrosine phosphatases by bis-(N,N-dimethyl-hydroxamido) hydrooxovanadate (DMHV) leads to an increase in Ca²⁺ release-activated Ca²⁺ (CRAC) entry. This effect can be blocked in the presence of 2-aminoethyldiphenyl borate (2-APB). Furthermore, transfection of HEK 293 cells with the human wild-type tyrosine phosphatase PTP1B leads to inhibition of CRAC influx, whereas transfection with the substrate-trapping mutant of PTP1B (D181A) slightly increases Ca²⁺ influx. It also decreases enzymatic activity of PTP1B as compared to non-transfected cells. Our data suggest that CRAC influx is modulated by tyrosine phosphorylation and dephosphorylation which involves the tyrosine phosphatase PTP1B.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.     

Levels of protein tyrosine phosphatase 1B determine susceptibility to apoptosis in serum-deprived hepatocytes

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. To assess the importance of PTP1B in the balance between death and survival in the liver, we have developed immortalized neonatal hepatocyte cell lines lacking (PTP1B(-/-)) or overexpressing (PTP1B(+/+PTP1B)) PTP1B. Early activation of caspase-3 occurred in PTP1B(+/+PTP1B) hepatocytes but was nearly abolished in PTP1B(-/-) cells. At the molecular level, PTP1B overexpression/deficiency altered the balance of pro-(Bim) and anti-(Bcl-x(L)) apoptotic members of the Bcl-2 family upon serum withdrawal. Likewise, cytosolic cytochrome C increased rapidly in PTP1B(+/+PTP1B) hepatocytes whereas it was retained in the mitochondria of PTP1B(-/-) cells. DNA fragmentation and the increase of apoptotic cells induced by serum withdrawal in wild-type (PTP1B(+/+)) hepatocytes were absent in PTP1B(-/-) cells. Conversely, overexpression of PTP1B accelerated DNA laddering and increased the number of apoptotic cells. In serum-deprived PTP1B(+/+PTP1B) hepatocytes, a rapid entry of Foxo1 into the nucleus and an earlier activation of caspase-8 was observed. However, both events were suppressed in PTP1B(-/-) hepatocytes. Moreover, PTP1B deficiency conferred resistance to apoptosis induced by activation of Fas and constitutively active Foxo1. Rescue of PTP 1B in deficient hepatocytes recovered the phenotype of wild-type cells whereas reduction of PTP1B by siRNA suppressed apoptosis. Our results reveal a unique role for PTP1B as a mediator of the apoptotic pathways triggered by trophic factors withdrawal in hepatocytes. This novel mechanism may represent an important target in the design of therapeutic strategies for human liver regeneration after pathological damage as well as for treatment of hepatocarcinomas.