Arnaud Besson

CDK Inhibitors: Cell Cycle Regulators and Beyond

First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression.

REGULATION OF THE CYTOSKELETON: AN ONCOGENIC FUNCTION FOR CDK INHIBITORS?

Cyclin-dependent kinase inhibitors (CKIs) are well known inhibitors of cell proliferation. Their activity is disrupted in many tumour types. Recent studies show that some of these proteins have interesting alternative functions, acting in the cytoplasm to regulate Rho signalling and thereby controlling cytoskeletal organization and cell migration. The upregulation of CKIs in the cytoplasm of many cancer cells indicates that although loss of nuclear CKIs is important for cancer cell proliferation, gain of cytoplasmic CKI function might be involved in tumour invasion and metastasis.

Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo.

An acute trauma to the CNS rapidly results in the upregulation of inflammatory cytokines that include interleukin‐1 (IL‐1). We report here that the levels of several matrix metalloproteinases (MMPs) are also elevated following a corticectomy trauma injury to the mouse CNS. The delayed upregulation of MMPs compared to that for IL‐1 suggests the possibility that inflammatory cytokines regulate MMP production in CNS trauma. To resolve this, we developed a method to isolate and maintain highly enriched human fetal neurons or astrocytes in culture and examined the regulation by cytokines of the activity of a subgroup of MMPs, the gelatinases (MMP‐2 and ‐9). While both neuronal and astrocytic cultures displayed comparable MMP‐2 activity, as evidenced by gelatin zymography, levels of MMP‐9 were proportionately higher in neurons compared to astrocytes. Of a variety of cytokines and growth factors tested in vitro, only IL‐1β was effective in increasing the neuronal expression of MMP‐9. Finally, an IL‐1 receptor antagonist attenuated the increase of neuronal MMP‐9 in culture and abolished the injury‐induced increase of MMP‐9 in the mouse brain. These results implicate IL‐1β as a key regulator of neuronal MMP‐9 in culture and of the elevation of MMP‐9 that occurs following mouse CNS trauma. J. Neurosci. Res. 61:212–224, 2000.

Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression.

ABSTRACT Protein kinase C (PKC) plays an important role in the regulation of glioma growth; however, the identity of the specific isoform and mechanism by which PKC fulfills this function remain unknown. In this study, we demonstrate that PKC activation in glioma cells increased their progression through the cell cycle. Of the six PKC isoforms that were present in glioma cells, PKC α was both necessary and sufficient to promote cell cycle progression when stimulated with phorbol 12-myristate 13-acetate. Also, decreased PKC α expression resulted in a marked decrease in cell proliferation. The only cell cycle-regulatory molecule whose expression was rapidly altered and increased by PKC α activity was the cyclin-cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1. Coimmunoprecipitation studies revealed that p21Waf1/Cip1 upregulation was accompanied by an incorporation of p21Waf1/Cip1 into various cyclin-CDK complexes and that the kinase activity of these complexes was increased, thus resulting in cell cycle progression. Furthermore, depletion of p21Waf1/Cip1 by antisense strategy attenuated the PKC-induced cell cycle progression. These results suggest that PKC α activity controls glioma cell cycle progression through the upregulation of p21Waf1/Cip1, which facilitates active cyclin-CDK complex formation.

Coupling Cell Cycle Exit, Neuronal Differentiation and Migration in Cortical Neurogenesis

The generation of new neurons in the cerebral cortex requires that progenitor cells leave the cell cycle and activate specific programs of differentiation and migration. Genetic studies have identified some of the molecules controlling these cellular events, but how the different aspects of neurogenesis are integrated into a coherent developmental program remains unclear. One possible mechanism implicates multifunctional proteins that regulate, both cell cycle exit and cell differentiation 1. A prime example is the cyclin-dependent kinase inhibitor p27Kip1, which has recently been shown to function beyond cell cycle regulation and promote both neuronal differentiation and migration of newborn cortical neurons, through distinct and separable mechanisms. p27Kip1 is therefore part of a machinery that couples the multiple events of neurogenesis in the cerebral cortex.

Differential activation of ERKs to focal adhesions by PKC ε is required for PMA-induced adhesion and migration of human glioma cells

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of a variety of signals. There is increasing evidence to indicate that specific PKC isoforms are involved in the regulation of distinct cellular processes. In glioma cells, PKC α was found to be a critical regulator of proliferation and cell cycle progression, while PKC ε was found to regulate adhesion and migration. Herein, we report that specific PKC isoforms are able to differentially activate extracellular-signal regulated kinase (ERK) in distinct cellular locations: while PKC α induces the activation of nuclear ERK, PKC ε induces the activation of ERK at focal adhesions. Inhibition of the ERK pathway completely abolished the PKC-induced integrin-mediated adhesion and migration. Thus, we present the first evidence that PKC ε is able to activate ERK at focal adhesions to mediate glioma cell adhesion and motility, providing a molecular mechanism to explain the different biological functions of PKC α and ε in glioma cells.

Astrocytes attenuate oligodendrocyte death in vitro through an α6 integrin-laminin–dependent mechanism

Oligodendrocyte (OL) death occurs in many disorders of the CNS, including multiple sclerosis and brain trauma. Factors reported to induce OL death include deprivation of growth factors, elevation of cytokines, oxidative stress, and glutamate excitotoxicity. Because astrocytes produce a large amount of growth factors and antioxidants and are a major source of glutamate uptake, we tested the hypothesis that astrocytes may have a protective role for OL survival. We report that when OLs from the adult mouse brain were initiated into tissue culture, DNA fragmentation and chromatin condensation resulted, indicative of apoptosis. OL death was significantly reduced in coculture with astrocytes, but not with fibroblasts, which provided a similar monolayer of cells as astrocytes. The protection of OL demise by astrocytes was not reproduced by its conditioned medium and was not accounted for by several neurotrophic factors. In contrast, interference with the α6 integrin subunit, but not the α1, α2, α3, α4, α5, or αv integrin chains, negated astrocyte protection of OLs. Furthermore, a function‐blocking antibody to α6β1 integrin reduced the ability of astrocytes to promote OL survival. The extracellular matrix ligand for α6β1 is laminin, which is expressed by astrocytes. Significantly, neutralizing antibodies to laminin‐2 and laminin‐5 inhibited the astrocyte mediation of OL survival. These results implicate astrocytes in promoting OL survival through a mechanism involving the interaction of α6β1 integrin on OLs with laminin on astrocytes. Enhancing this interaction may provide for OL survival in neurological injury. GLIA 36:281–294, 2001.

p27Kip1 Is a Microtubule-Associated Protein that Promotes Microtubule Polymerization during Neuron Migration

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive, and the present work describes how p27(Kip1) controls cell-cycle-unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27(Kip1) show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, p27(Kip1) is a microtubule-associated protein that promotes polymerization of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27(Kip1) controls actomyosin contractions that drive both forward translocation of the nucleus and growth cone splitting. Thus, p27(Kip1) cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons.

Cytoplasmic p27 is oncogenic and cooperates with Ras both in vivo and in vitro

p27Kip1 (p27) can have opposing roles during malignant transformation depending on cellular context: on one hand it functions as a tumor suppressor by inhibiting cyclin–cyclin-dependent kinase (CDK) activity in the nucleus and on the other it may adopt an oncogenic role that is less well understood. To gain further insight into the roles played by p27 during tumorigenesis, we compared the susceptibility with urethane-induced tumorigenesis of two p27 mouse models, p27−/− and p27CK− knockin, in which p27 cannot bind or inhibit cyclin–CDKs. In this K-Ras-driven tumorigenesis model, p27CK− mice had an increase in both tumor number and aggressiveness compared with p27−/−, indicating a cooperation between p27CK− and activated Ras. In the lung, increased tumorigenesis was associated with cytoplasmic localization of p27CK− and bronchiolaveolar stem cell amplification. The ability of p27CK− to cooperate with other oncogenes was not universal. When c-Myc was used as a transforming agent, p27 status became irrelevant and c-Myc was equally potent in transforming p27+/+, p27−/− and p27CK− cells. In fact, c-Myc induced the degradation of wild-type p27 via the Skp-Cullin-F-box (SCF)–Skp2 pathway. In contrast, p27CK− levels were not affected by c-Myc expression, as p27CK− is insensitive to Skp2-mediated degradation because of its inability to bind cyclin E/CDK2. However, in presence of c-Myc, p27CK− remained mostly nuclear, providing an explanation for its inability to cooperate with Myc during transformation. Thus, we propose that the p27CK− protein needs to be localized in the cytoplasm in order to function as an oncogene, otherwise it just behaves similar to a null allele.

p27Kip1 represses transcription by direct interaction with p130/E2F4 at the promoters of target genes.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27.