ΔNp63α regulates keratinocyte proliferation by controlling PTEN expression and localization.

ΔNp63α, implicated as an oncogene, is upregulated by activated Akt, part of a well-known cell survival pathway. Inhibition of Akt activation by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the presence of putative p63-binding sites in the pten promoter led us to investigate whether ΔNp63α regulates PTEN expression. Knockdown of ΔNp63α led to increases in PTEN levels and loss of activated Akt, while overexpression of ΔNp63α decreased PTEN levels and elevated active Akt. The repression of PTEN by ΔNp63α occurs independently of p53 status, as loss of ΔNp63α increases PTEN expression in cell lines with and without functional p53. In addition, decreased levels of ΔNp63α resulted in an increase in nuclear PTEN. Conversely, in vivo nuclear PTEN was absent in the proliferative basal layer of the epidermis where ΔNp63α expression is highest. Additionally, we show that in keratinocytes a balance between ΔNp63α and PTEN regulates Akt activation and maintains normal proliferation rates. This balance is disrupted in non-melanoma skin cancers through increased ΔNp63α levels, and could enhance proliferation and subsequent neoplastic development. Our studies show that ΔNp63α negatively regulates PTEN, thereby providing a feedback loop between PTEN, Akt and ΔNp63α, which has an integral role in skin cancer development.

ATM-dependent IGF-1 induction regulates secretory clusterin expression after DNA damage and in genetic instability.

Secretory clusterin (sCLU) is a stress-induced, pro-survival glycoprotein elevated in early-stage cancers, in particular in APC/Min-defective colon cancers. sCLU is upregulated after exposure to various cytotoxic agents, including ionizing radiation (IR), leading to a survival advantage. We found that stimulation of insulin-like growth factor-1 (IGF-1) and IGF-1R protein kinase signaling was required for sCLU induction after IR exposure. Here, we show that activation of Ataxia telangiectasia-mutated kinase (ATM) by endogenous or exogenous forms of DNA damage was required to relieve basal repression of IGF-1 transcription by the p53/NF-YA complex, leading to sCLU expression. Although p53 levels were stabilized and elevated after DNA damage, dissociation of NF-YA, and thereby p53, from the IGF-1 promoter resulted in IGF-1 induction, indicating that NF-YA was rate limiting. Cells with elevated endogenous DNA damage (deficient in H2AX, MDC1, NBS1, mTR or hMLH1) or cells exposed to DNA-damaging agents had elevated IGF-1 expression, resulting in activation of IGF-1R signaling and sCLU induction. In contrast, ATM-deficient cells were unable to induce sCLU after DNA damage. Our results integrate DNA damage resulting from genetic instability, IR, or chemotherapeutic agents, to ATM activation and abrogation of p53/NF-YA-mediated IGF-1 transcriptional repression, that induces IGF-1-sCLU expression. Elucidation of this pathway should uncover new mechanisms for cancer progression and reveal new targets for drug development to overcome resistance to therapy.

Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization.

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus.

The Mdm2 oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein. To regulate p53, Mdm2 must gain nuclear entry, and the mechanism that induces this is now identified. Mitogen-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, the Akt/PKB serine-threonine kinase, results in phosphorylation of Mdm2 on serine 166 and serine 186. Phosphorylation on these sites is necessary for translocation of Mdm2 from the cytoplasm into the nucleus. Pharmacological blockade of PI3-kinase/Akt signaling or expression of dominant-negative PI3-kinase or Akt inhibits nuclear entry of Mdm2, increases cellular levels of p53, and augments p53 transcriptional activity. Expression of constitutively active Akt promotes nuclear entry of Mdm2, diminishes cellular levels of p53, and decreases p53 transcriptional activity. Mutation of the Akt phosphorylation sites in Mdm2 produces a mutant protein that is unable to enter the nucleus and increases p53 activity. The demonstration that PI3-kinase/Akt signaling affects Mdm2 localization provides insight into how this pathway, which is inappropriately activated in many malignancies, affects the function of p53.

Vascular endothelial cell growth factor activates CRE-binding protein by signaling through the KDR receptor tyrosine kinase.

Vascular endothelial cell growth factor (VEGF) plays a crucial role in the development of the cardiovascular system and in promoting angiogenesis associated with physiological and pathological processes. Although a great deal is known of the cytoplasmic signaling pathways activated by VEGF, much less is known of the mechanisms through which VEGF communicates with the nucleus and alters the activity of transcription factors. Binding of VEGF to the KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. p38 MAPK/MSK-1 and protein kinase C/p90RSK pathways mediate CREB phosphorylation. Confocal microscopy shows that VEGF-induced phosphorylation of nuclear CREB is blocked by pharmacological inhibition of protein kinase C and p38 mitogen-activated protein kinase signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the nucleus where CREB becomes activated. These results suggest that CREB may play a role in alterations of gene expression important to angiogenesis.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1.

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase.

Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. Here we show that the Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.

mdm-2 gene amplification in 3T3-L1 preadipocytes.

In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.

Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53.

In response to genotoxic stress, the p53 tumor suppressor protein exerts a G1 cell cycle arrest that is dependent on its ability to transactivate downstream target genes. This p53-dependent G1 block is reversed by the binding of Mdm-2 to p53, preventing further transactivation. Interestingly, following DNA damage, the mdm-2 gene is also transcriptionally activated by p53, and therefore, the question of how p53 can continue to transactivate genes in the presence of its own negative regulator has remained unanswered. Here, we provide evidence that phosphorylation of Mdm-2 protein by DNA-dependent protein kinase (DNA-PK) blocks its ability to associate with p53 and regulate p53 transactivation. The data support a model by which DNA-PK activation by DNA damage and phosphorylation of Mdm-2 renders the Mdm-2 protein unable to inhibit p53 transactivation, resulting in cell cycle arrest. Following DNA repair, the loss of DNA-PK activity results in newly synthesized Mdm-2 protein that is unphosphorylated and, therefore, capable of binding to p53, allowing cell cycle progression.

Wild-type p53 protein is unable to activate the mdm-2 gene during F9 cell differentiation.

In this study, we set out to assess whether the p53 protein affects mdm-2 gene expression as F9 embryonal carcinoma cells differentiate into parietal endoderm cells. It was previously reported that F9 cells possess abundant levels of wild-type p53 and upon induction to differentiate, p53 mRNA and protein levels decrease (Oren et al., 1982; Dony et al., 1985). We demonstrate that while p53 mRNA and protein levels decrease as F9 cells differentiate, mdm-2 mRNA and protein expression remains constitutive. Using RNA primer extension assays, we determined that the mdm-2 mRNA expression is not directed by p53 in either F9 embryonal (undifferentiated) or parietal endoderm (differentiated) cells. However, p53 protein does stimulate mdm-2 mRNA expression in response to u.v. irradiation. The inability of p53 to transactivate mdm-2 in undamaged F9 cells was not the result of latent pools as p53 sequence specific DNA binding activity was observed using electrophoretic mobility shift assays. Our results suggest that, in F9 cells, the p53:Mdm-2 autoregulatory loop is confined to pathways governing DNA damage.