Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins

Neurofibromatosis type 1, NF-1, is a common monogenic (NF1) disease, characterized by highly variable clinical presentation and high predisposition for tumors, especially those of astrocytic origin (low- to high-grade gliomas). Unfortunately, very few genotype–phenotype correlations have been possible, and the numerous identified mutations do not offer help for prognosis and patient counselling. Whole gene deletion in animals does not successfully model the disease, as NF-1 cases caused by point mutations could be differentially affected by cell type-specific alternative splice variants of NF1. In this chapter, we will discuss the differential Microtubule-Associated-Protein (MAP) properties of NLS or ΔNLS neurofibromins, produced by the alternatively splicing of exon 51, which also contains a Nuclear Localization Sequence (NLS), in the assembly of the mitotic spindle and in faithful genome transmission. We will also commend on the major theme that emerges about NLS-containing tumor suppressors that function as mitotic MAPs

Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Mitotic spindles are highly organized, microtubule (MT)-based, transient structures that serve the fundamental function of unerring chromosome segregation during cell division and thus of genomic stability during tissue morphogenesis and homeostasis. Hence, a multitude of MT-associated proteins (MAPs) regulates the dynamic assembly of MTs in preparation for mitosis. Some tumor suppressors, normally functioning to prevent tumor development, have now emerged as significant MAPs. Among those, neurofibromin, the product of the Neurofibromatosis-1 gene (NF1), a major Ras GTPase activating protein (RasGAP) in neural cells, controls also the critical function of chromosome congression in astrocytic cellular contexts. Cell type- and development-regulated splicings may lead to the inclusion or exclusion of NF1exon51, which bears a nuclear localization sequence (NLS) for nuclear import at G2; yet the functions of the produced NLS and ΔNLS neurofibromin isoforms have not been previously addressed. By using a lentiviral shRNA system, we have generated glioblastoma SF268 cell lines with conditional knockdown of NLS or ΔNLS transcripts. In dissecting the roles of NLS or ΔNLS neurofibromins, we found that NLS-neurofibromin knockdown led to increased density of cytosolic MTs but loss of MT intersections, anastral spindles featuring large hollows and abnormal chromosome positioning, and finally abnormal chromosome segregation and increased micronuclei frequency. Therefore, we propose that NLS neurofibromin isoforms exert prominent mitotic functions

A functional interaction between the human papillomavirus 16 transcription/replication factor E2 and the DNA damage response protein TopBP1

The human papillomavirus (HPV) transcription/replication factor E2 is essential for the life cycle of HPVs. E2 protein binds to DNA target sequences in the viral long control regions to regulate transcription of the viral genome. It also enhances viral DNA replication by interacting with the viral replication factor E1 and recruiting it to the origin of replication and may also play a more direct role in replication. The cellular proteins with which E2 interacts to carry out these functions are largely unknown. To identify these proteins a yeast two-hybrid screen was carried out with the transcription/replication domain of HPV16 E2. This screen identified several candidate interacting partners for E2 including TopBP1 (topoisomerase IIβ-binding protein 1). TopBP1 has eight BRCA1 carboxyl-terminal domains that are found in proteins regulating the DNA damage response, transcription, and replication. Here we demonstrate that HPV16 E2 and TopBP1 interact in vitro and in vivo and that TopBP1 can enhance the ability of E2 to activate transcription and replication. This is the first time that TopBP1 has been shown to function as a transcriptional coactivator and that E2 interacts with TopBP1. Removal of the amino-terminal domain of TopBP1 abolishes coactivation of transcription and replication. This interaction may have functional consequences upon the viral life cycle

PKCε Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells

<div><p>The protein kinase C (PKC) signaling, a major regulator of chondrocytic differentiation, has been also implicated in pathological extracellular matrix remodeling, and here we investigate the mechanism of PKCε-dependent regulation of the chondrocytic phenotype in human nucleus pulposus (NP) cells derived from herniated disks. NP cells from each donor were successfully propagated for 25+ culture passages, with remarkable tolerance to repeated freeze-and-thaw cycles throughout long-term culturing. More specifically, after an initial downregulation of <i>COL2A1</i>, a stable chondrocytic phenotype was attested by the levels of mRNA expression for aggrecan, biglycan, fibromodulin, and lumican, while higher expression of SOX-trio and Patched-1 witnessed further differentiation potential. NP cells in culture also exhibited a stable molecular profile of PKC isoforms: throughout patient samples and passages, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas β, γ, and θ were not. Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan. More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in <i>ADAMTS5</i> and cleaved aggrecan. Therefore, PKCε activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKCε/ERK/CREB/AP-1-dependent transcriptional program that includes upregulation of both chondrogenic genes and microRNAs. Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.</p> </div

PKCε activation induces aggrecan gene expression, decreases ADAMTS5, and detection of ADAMTS-5-depended cleavage of ECM aggrecan through upregulation of hsa-miR-377 expression.

<p>(<b>A</b>) RT-PCR detected the relative expression of ACAN, MKI67, ADAMTS5 after treatment of NP cells with 1 μM ψεRACK (or vehicle) for indicated times and revealed that time-dependent activation of PKCε significantly increased the mRNA expression for aggrecan, while in an opposing manner decreased ADAMTS5; mRNA levels of the proliferation marker Ki67 became barely detectable after 8 hours of PKCε activation. (<b>B</b>) Immunoblotting of ECM proteins isolated from the culture media of NP cells with BC-3, an antibody that detects ADAMTS-5-mediated cleavage of human aggrecan, revealed a drastic decrease of cleaved aggrecan by 40 hours of treatment with ψεRACK, in agreement with the lower levels of ADAMTS5. (<b>C</b>) Levels of expression of hsa-miR-377, predicted to bind at several sites of ADAMTS5 3’-UTR, were significantly induced with PKCε activation, as determined with RT-PCR; † indicates the hsa-miR-377 primers. (<b>D</b>) Quantitation of hsa-miR-377 levels showed early sustained gains with ψεRACK (P < 0.01 at 8 and 18 h), whereas ADAMTS5 decreased gradually (P < 0.01 at 40 h); GAPDH and U6 were used for normalization of ADAMTS5 and hsa-miR-377, respectively. Bars in the graph represent the mean results of four independent experiments ±SEM, in which two different NP lines (NP6 P10 and NP8 P11 were run in parallel (n=8 for each time point), * P < 0.05 and ** P < 0.01 relative to 0 h ψεRACK. </p

Chondrocytic marker expression remains stable over many cell passages of NP cell cultures.

<p>Representative images of RT-PCR results demonstrate expression levels of ACAN; COL2A1; <i>SOX</i> 5,6, and 9; PTCH1; <i>BGN</i>; FMOD; and <i>LUM</i> in NP tissue and in early and late passage of NP cells from different patient samples, and highlight that, under monolayer culture expansion, messages for ECM proteoglycans aggrecan, biglycan, fibromodulin, and lumican were sustained at similar levels throughout passaging. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom panel). </p

PKC isoform expression in human NP cells of early or late passage.

<p>Representative images of RT-PCR results for PKC isoform profiling demonstrate an indistinguishable profile amongst various NP samples and cell passages (NP11, P0; NP10, P23; NP1, at P3 and P13), and AC cells (AC, P17) in culture, with the exception of the ζ isoform, which appears at a slightly different molecular size (402 versus 321 - arrowheads versus arrows). The MPNST 90-8 cell line which expresses all the PKC isoforms but βI, served as a positive control. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom row). The ladder used was the GeneRuler 100 bp (NEB), where the intense band corresponds to the 500 bp size.</p

Cytoskeletal and chondrocyte phenotypic markers in cultured NP cells.

<p>NP cell cultures were fixed, immunostained and imaged with deconvolution microscopy (<b>A</b>) β-tubulin immunostaining (red) reveals elaborate microtubule cytoskeletons in NP cells (arrows), especially in the rare large notochordal-like cells (arrowheads), while phalloidin staining (magenta) shows that F-actin is organized in focal adhesions under the cell body (double arrows); nuclei are stained blue with Hoechst. (<b>B</b>) In contrast, AC cells exhibit polarized cell bodies, lesser microtubule branching (arrows), and F-actin forms intense stress fibers (double arrows). Staining with the BC-3 antibody (red) that recognizes the neoepitope on human aggrecan that is created after cleavage within the sequence TEGE373-₃₇₄ARGS by aggrecanases activity, decorated the cytoplasm of both (<b>C</b>) AC cells and (<b>D</b>) NP cells. In both cell types the staining pattern was consistent with a vesicular distribution (arrows). (<b>E</b>) Aggrecan staining was not detected when chondroitinase was omitted (upper panel) or when the reaction was allowed to proceed at 4° C (asterisk indicate cells at mitosis) (NP6 P10, C7). NP tissue indicate fresh tissue sample, NP numbers indicate patient sample, and P the culture passage; all cells were visualized with a 63x oil immersion lens. </p