Zhi-Xiong Jim Xiao

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1–3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.

IGF-1 induces Pin1 expression in promoting cell cycle S-phase entry

Insulin‐like growth factor I (IGF‐1) is a well‐established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr‐Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1−/−mouse embryo fibroblast (MEF) cells are defective in re‐entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1−/− MEF cells display a delayed cell cycle S‐phase entry in response to IGF stimulation and that IGF‐1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF‐1 is mediated via the phosphatidylinositol 3‐kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF‐1‐induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1−/− MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1−/− MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF‐1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1‐S transition. J. Cell. Biochem. 84: 211–216, 2002.

Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP‐mediated transcription

The retinoblastoma protein (RB) recruits histone deacetylase (HDAC) to repress E2F‐mediated transactivation that plays a critical role in cell cycle regulation. RB is also involved in activation of expression of a number of tissue specific‐ and differentiation‐related genes. In this study, we examined the mechanism by which RB stimulated the expression of a differentiation‐related gene, the surfactant protein D (SP‐D), which plays important roles in innate host defense and the regulation of surfactant homeostasis. We demonstrated that RB specifically stimulated the activity of human SP‐D gene promoter. The RB family member, p107 but not p130, also increased SP‐D promoter activity. Activation by RB was mediated through a NF‐IL6 (C/EBPβ) binding motif in the human SP‐D promoter, and this sequence specifically bound to C/EBPα, C/EBPβ, and C/EBPδ. RB formed stable complexes with all three C/EBP family members. RB small pocket (amino acid residues 379–792), but not the C‐pocket (amino acid residues 792–928), was necessary and sufficient for its interaction with C/EBP proteins. Furthermore, we demonstrated that the complexes containing RB and C/EBP proteins directly interacted with C‐EBP binding site on DNA. These findings indicate that RB plays a positive, selective, and direct role in the C/EBP‐dependent transcriptional regulation of human SP‐D expression. J. Cell. Biochem. 83: 414–425, 2001.

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.

Retinoblastoma protein modulates gankyrin–MDM2 in regulation of p53 stability and chemosensitivity in cancer cells

MDM2 is a key ubiquitin E3 ligase for p53 and its activity is critically regulated by a set of modulators, including ARF, p300, YY1 and recently by gankyrin, an oncoprotein frequently overexpressed in human heptocellular carcinomas. We have previously shown that MDM2 binds to and promotes retinoblastoma protein (Rb) degradation. Here we show that Rb inhibits MDM2 E3 ligase activity resulting in stabilization of p53. In addition, we demonstrated that Rb inhibits MDM2-mediated p53 ubiquitination in a gankyrin-dependent manner and the Rb–gankyrin interaction is critical for Rb-induced p53 stabilization. Furthermore, acute ablation of Rb facilitates gankyrin-mediated p53 destabilization, and desensitizes cancer cells for chemotherapy-induced apoptosis. These results indicate that Rb antagonizes gankyrin to inhibit MDM2-mediate p53 ubiquitination in cancer cells and suggest that the status of both p53 and Rb is important for efficacy of cancer chemotherapy.

ΔNp63α regulates Erk signaling via MKP3 to inhibit cancer metastasis.

Reduced expression of the p53 family member p63 has been suggested to play a causative role in cancer metastasis. Here, we show that ΔNp63α, the predominant p63 isoform, plays a major role in regulation of cell migration, invasion and cancer metastasis. We identified mitogen-activated protein (MAP) kinase phosphatase 3 (MKP3) as a downstream target of ΔNp63α that is required for mediating these effects. We show that ΔNp63α regulates extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) activity via MKP3 in both cancer and non-transformed cells. We further show that exogenous ΔNp63α inhibits cell invasion and is dependent on MKP3 upregulation for repression. Conversely, endogenous pan-p63 ablation results in increased cell migration and invasion, which can be reverted by reintroducing the ΔNp63α isoform alone, but not by other isoforms. Interestingly, these effects require Erk2, but not Erk1 expression, and can be rescued by enforced MKP3 expression. Moreover, MKP3 expression is reduced in invasive cancers, and reduced p63 expression increases metastatic frequency in vivo. Taken together, these results suggest an important role for ΔNp63α in preventing cancer metastasis by inhibition of Erk2 signaling via MKP3.

IGF-1 activates p21 to inhibit UV-induced cell death

The insulin-like growth factor-1 (IGF-1) and its downstream effector Akt have been documented as survival factors in response to a variety of stress signals. In this study, we show that IGF-1 activates p21 protein expression in a p53-dependent manner. Inhibition of PI-3 kinase or ectopic expression of a dominant-negative Akt blocks the effect of IGF-1 on the upregulation of p21 expression. In addition, IGF-1 prevents the UV irradiation-mediated suppression of p21 and MDM2 expression. Furthermore, p21 is important for IGF-1-mediated cell survival upon UV irradiation. Taken together, these data indicate that IGF-1 may activate p21 in executing its survival function upon genotoxic insults.

Nutlin-3 Affects Expression and Function of Retinoblastoma Protein: ROLE OF RETINOBLASTOMA PROTEIN IN CELLULAR RESPONSE TO NUTLIN-3*

The retinoblastoma protein (Rb) plays a pivotal role in regulating cell proliferation and apoptosis. Nutlin-3, a small molecule MDM2 antagonist blocking interaction between MDM2 and p53, activates p53 resulting in cell growth arrest or apoptosis in various cancer cells. However, the molecular basis for the different cellular responses upon nutlin-3 treatment is not fully understood. In this study, we show that nutlin-3 activates p53 resulting in a dramatic increase in MDM2 expression and a marked reduction in total Rb protein levels. Interestingly, nutlin-3 reduces the levels of hypophosphorylated Rb and induces massive apoptosis in SJSA-1 cells, which can be largely rescued by knockdown of MDM2 or by expression of constitutively active Rb. By contrast, nutlin-3 treatment of several human cancer cells, including A549, U2-OS, and HCT116, results in an accumulation of hypophosphorylated Rb and cell cycle arrest but not apoptosis. Furthermore, we show that down-regulation of Rb by nutlin-3 does not lead to E2F1 activation nor does E2F1 play a critical role for nutlin-3-induced apoptosis in SJSA-1 cells. Taken together, these results suggest that Rb plays a critical role in influencing cellular response to activation of p53 pathway by nutlin-3.

TAp63 is a transcriptional target of NF-κB

The p53 homologue p63 encodes multiple protein isoforms either with (TA) or without (ΔN) the N‐terminal transactivation domain. Accumulating evidence indicates that TAp63 plays an important role in various biological processes, including cell proliferation, differentiation, and apoptosis. However, how TAp63 is regulated remains largely unclear. In this study, we demonstrate that NF‐κB induces TAp63 gene expression. The responsible elements for NF‐κB‐mediated TAp63 induction are located within the region from −784 to −296 bp in the TAp63 promoter, which contains two NF‐κB binding sites. Ectopic expression of RelA stimulates TAp63 promoter‐driven reporter activity and increases endogenous TAp63 mRNA levels. Inhibition of NF‐κB by IκBα super‐repressor or with a chemical inhibitor leads to down regulation of TAp63 mRNA expression and activity. In addition, mutations in the critical NF‐κB‐binding sites significantly abolish the effects of NF‐κB on TAp63. Activation of NF‐κB by TNFα enhances p50/RelA binding to the NF‐κB binding sites. Furthermore, we show that an Sp1 site adjacent to the NF‐κB sites plays a role in NF‐κB‐mediated upregulation of TAp63. Taken together, these data reveal that TAp63 is a transcriptional target of NF‐κB, which may play a role in cell proliferation, differentiation and survival upon NF‐κB activation by various stimuli. J. Cell. Biochem. 109: 702–710, 2010.

ARF promotes accumulation of retinoblastoma protein through inhibition of MDM2.

The INK4a/ARF locus, encoding two tumor suppressor proteins, p16INK4a and p14ARF (ARF), plays key roles in many cellular processes including cell proliferation, apoptosis, cellular senescence and differentiation. Inactivation of INK4a/ARF is one of the most frequent events during human cancer development. Although p16INK4a is a critical component in retinoblastoma protein (Rb)-mediated growth regulatory pathway, p14ARF plays a pivotal role in the activation of p53 upon oncogenic stress signals. A body of evidence indicates that ARF also possesses growth suppression functions independent of p53, the mechanism of which is not well understood. We have recently shown that MDM2 interacts with Rb and promotes proteasome-dependent Rb degradation. In this study, we show that ARF disrupts MDM2–Rb interaction resulting in Rb accumulation. Wild-type ARF, but not ARF mutant defective in MDM2 interaction, stabilizes Rb and inhibits colony foci formation independent of p53. In addition, ablation of Rb impairs ARF function in growth suppression. Thus, this study demonstrates that ARF plays a direct role in regulation of Rb and suggests that inactivation of ARF may lead to defects in both p53 and Rb pathways in human cancer development.