Toll-like receptors (TLRs) play essential roles in generating innate immune responses, and are evolutionarily conserved across species. In mammals, TLRs specifically recognize the conserved microbial structural motifs referred to as pathogen-associated molecular patterns (PAMPs). Ligand recognition by TLRs activates signaling cascades that culminate in proinflammatory cytokine production and eventual elimination of invading pathogens. Although TLRs in mammals are expressed predominantly in the immune system, certain TLRs with poorly characterized function are also found in neurons. We recently profiled TLR8 expression during mouse brain development and established its localization in neurons and axons. We uncovered a novel role for TLR8 as a suppressor of neurite outgrowth as well as an inducer of neuronal apoptosis, and found that TLR8 functions in neurons through an NFκB-independent mechanism. These findings add a new layer of complexity for TLR signaling, and expand the realm of mammalian TLR function to the central nervous system (CNS) beyond the originally discovered immune context. Herein, we complement our earlier report with additional data, discuss their biological and mechanistic implications in CNS developmental and pathological processes, and thus further our perspective on TLR signaling and potential physiological roles in mammals.
Toll-like receptors; TLR8; MyD88; innate immunity; neuron; axon; neurite outgrowth; apoptosis; NFκB; IκBα; IRAK1; IRAK4
Akt; 14-3-3; TRAIL; PACS-2; cancer; ADPKD
Wnt; lung; trachea; progenitor; endoderm
Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) mediates a plethora of physiological and pathological activities via interactions with a series of high affinity G protein-coupled receptors (GPCR). Both LPA receptor family members and autotaxin (ATX/LysoPLD), the primary LPA-producing enzyme, are aberrantly expressed in many human breast cancers and several other cancer lineages. Using transgenic mice expressing either an LPA receptor or ATX, we recently demonstrated that the ATX-LPA receptor axis plays a causal role in breast tumorigenesis and cancer-related inflammation, further validating the ATX-LPA receptor axis as a rich therapeutic target in cancer.
Breast cancer; ATX; LPA; G protein-coupled receptor; inflammation; cytokines; target therapy
Population geneticists can reconstruct the ancestries of macroscopic populations from polymorphisms in present day individuals. For example, the migration “out of Africa” is recorded in human genome variation in different parts of the world. Here we apply this approach to human colorectal cancer cell populations and polymorphic passenger methylation patterns. By sampling molecular variation from different parts of the same cancer, it should be possible to infer how individual tumors grow because recent clonal expansions should be less diverse than older expansions. Average diversity was different between cancers implying that some cancers are older clonal expansions than others. For individual cancers, methylation pattern diversity was relatively uniform throughout the tumor (right versus left side, superficial versus invasive), which is more consistent with a single, uniform or “flat” clonal expansion than with stepwise sequential progression. Many colorectal cancers appear to invade and expand early, but subsequently stall. Epiallele diversity within individual small cancer gland fragments was high and more consistent with frequent rather than extremely rare cancer stem cells (CSCs). These studies suggest that many human colorectal cancers are relatively old uniform clonal expansions, that cancer cell populations contain frequent long-lived CSC lineages, and that some passenger methylation patterns record somatic cell ancestry.
tumor progression; clonal evolution; cancer stem cells; methylation; gompertzian
The kinase RIP1 wears a coat of many colors during TNF receptor signaling and can regulate both activation of pro-survival NFκB and programmed cell death pathways. In this review, we outline how coating RIP1 with K63-linked ubiquitin chains forms a protective layer that prevents RIP1 from binding apoptotic regulators and serves as an early guard against cell death. Further on, binding of NFκB signaling components to the ubiquitin coat of RIP1 activates long-term pro-survival signaling and forms a more impenetrable suit of armor against cell death. If RIP1 is not decorated with ubiquitin chains it becomes an unstoppable harbinger of bad news: programmed cell death.
TNF; RIP; IAP; TRAF; apoptosis; necrosis; NFκB; ubiquitin
The specialized cell cycles that characterize various aspects of the differentiation of germ cells provide a unique opportunity to understand heretofore elusive aspects of the in vivo function of cell cycle regulators. Key components of the cell cycle machinery are the regulatory sub-units, the cyclins, and their catalytic partners, the cyclin-dependent kinases. Some of the cyclins exhibit unique patterns of expression in germ cells that suggest possible concomitant distinct functions, predictions that are being explored by targeted mutagenesis in mouse models. A novel, meiosis-specific function has been shown for one of the A-type cyclins, cyclin A1. Embryonic lethality has obviated understanding of the germline functions of cyclin A2 and cyclin B1, while yet other cyclins, although expressed at specific stages of germ cell development, may have less essential function in the male germline.
cell cycle machinery; spermatogenesis; germ cell differentiation; meiosis; male germ cell stem cells; mitosis; cyclin-dependent kinases; apoptosis
Checkpoint activation benefits DNA homologous recombination repair and; therefore, protects cells from ionizing radiation (IR)-induced killing. CHK1 is one of the most important checkpoint regulators in mammalian cells. We recently reported that matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) stabilizes CHK1 through interacting with CHK1, thus protecting cells from IR-induced killing. The purpose of this study is to investigate whether a small peptide that mimics the key domain of MEPE/OF45 could interact with CHK1 and protect cells from IR-induced killing. We showed here that the synthesized peptide with 18 amino acids (aa) could enter human transformed lymphoblasts when it is linked to fatty acid CH3(CH2)8CO. After the 18 aa peptide entered the human cells, it interacted with CHK1, increased the CHK1 level and induced a stronger G2 arrest in the cells following IR. More importantly, the 18 aa peptide could protect the cells from IR-induced killing. Our data indicate that the 18 aa peptide, similar to MEPE/OF45, reduces CHK1 degradation and protects cells from IR-induced killing. We believe that these results provide useful information for drug development in two directions: protect cells from IR-induced damage and sensitize cells to radiation therapy.
MEPE/OF45; CHK1; ionizing radiation
A number of recent papers on the WWP2 E3 ubiquitin ligase together with two novel WWP2 isoforms have revealed important biological insight and disease-specific functions, and also impacted on our understanding of ubiquitin ligases in cell cycle regulation, apoptosis and differentiation. Gene knockout studies suggest a developmental role for WWP2 in chondrogenesis via mechanisms involving cartilage-specific transcription factors. Furthermore, WWP2 isoforms have been shown to selectively target oncogenic signaling pathways linked to both the pTEN tumour suppressor and the TGFβ/Smad signaling pathway. Here, it is suggested that WWP2 isoforms have now emerged as central physiological regulators as well as promising new disease targets, and that the challenge ahead is to now develop highly selective WWP2 inhibitors with utility in cartilage disease such as osteoarthritis and as new anticancer strategies.
Ubiquitin ligase; signal transduction; Smads; TGFβ; cancer; cartilage
The circadian clock regulates biological processes from gene expression to organism behavior in a precise, sustained rhythm that is generated at the unicellular level by coordinated function of interlocked transcriptional feedback loops and post-translational modifications of core clock proteins. CLOCK phosphorylation regulates transcriptional activity, cellular localization and stability; however little is known about the specific residues and enzymes involved. We have identified a conserved cluster of serines that include, Ser431, which is a prerequisite phosphorylation site for the generation of BMAL dependent phospho-primed CLOCK and for the potential GSK-3 phosphorylation at Ser427. Mutational analysis and protein stability assays indicate that this serine cluster functions as a phospho-degron. Through the use of GSK-3 activators/inhibitors and kinase assays, we demonstrate that GSK-3β regulates the degron-site by increasing CLOCK phosphorylation/degradation, which correlates with an increase in the expression of CLOCK responsive promoters. Stabilization of phospho-deficient CLOCK delays the phase of oscillation in synchronized fibroblasts. This investigation begins the characterization of a complex phospho-regulatory site that controls the degradation of CLOCK, a core transcription factor that is essential for circadian behavior.
circadian; CLOCK; GSK-3β; phospho-degron; post-translational modification
Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.
Ceramide; Apoptosis; Casapse-8; JNK; Stress signal pathway; Chronic Myelogenous Leukemia
Although cisplatin is a very effective anticancer agent against several types of cancer including ovarian cancer, the mechanisms of acquired resistance are not fully understood. By chronically exposing cisplatin to ovarian cancer cell lines, we established two cisplatin-resistant cell lines OV433 and tOV112D. Our results indicate that the mechanisms underlying their cisplatin resistance are distinct. In OV433 cells, cisplatin resistance is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). By knocking down MKP-1 expression by siRNA or inhibiting MKP-1 expression by its pharmacological inhibitor triptolide, cisplatin-resistant OV433 cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. In tOV112D cells, on the other hand, acquired cisplatin resistance is associated with increased levels of Bcl-2 protein. By inhibiting the activity of Bcl-2 protein with its pharmacological inhibitor gossypol or knocking down Bcl-2 expression by siRNA, cisplatin-resistant tOV112D cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. therefore, our data suggest that the mechanisms of acquired cisplatin resistance vary among ovarian cancer cells, which involve upregulation of molecules associated with the cell survival pathways.
cisplatin resistance; MKP-1; Bcl-2; apoptosis
Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity.
H2AX; ATR; replicative stress; INK4a/ARF; DNA damage
B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients.
In a recent study on 111 cases of pre-B cell-derived human ALL, we found that ALL cells carrying a BCR-ABL1-gene rearrangement lack expression of a functional pre-B cell receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from the pre-B cell receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only “crippled” pre-B cells that fail to express a functional pre-B cell receptor are permissive to transformation by BCR-ABL1.
BCR-ABL1; pre-B cell receptor; acute lymphoblastic leukemia; ikaros; cell cycle arrest
Chk1 is a conserved kinase that comprises the first line of defense against DNA damage and replication blocks. Chk1 consists of two primary domains, the well conserved N-terminal kinase domain and the non-catalytic C-terminal domain that contains the two highly conserved TRF and GD sub-domains. Several studies suggested that the C-terminus of Chk1 acts as an inhibitory domain and that phosphorylation of the C-terminus by ATR serves to activate Chk1 by relieving the inhibitory effect of the C-terminus on the N-terminal catalytic domain. However, work carried out in many systems showed that phosphorylation on ATR sites was necessary but not sufficient to increase Chk1 kinase activity. In a recent manuscript we described a single amino acid substitution at an invariant Leucine in the conserved GD domain of the yeast Chk1 C-terminus (L506R) that led to a Chk1 protein that no longer required ATRMec1 phosphorylation at conserved sites for its function, and relieved the requirement of an upstream mediator, Rad9 (53BP1 homolog), for Chk1 activation. Here we show that this single amino acid substitution in the GD domain also led to constitutive phosphorylation of yeast and human Chk1 on ATRMec1 sites, suggesting that the protein was in a conformation in which it could be readily phosphorylated by ATRMec1. Unlike the phospho-mimetic mutants in earlier studies, the L505R and L449R modifications led to increased Chk1 activity both in vitro and in vivo. Therefore, we have uncovered a conserved mechanism for Chk1 regulation separate from the role of known ATR phosphorylation sites.
DNA damage; checkpoint kinases; S-phase checkpoint; DNA replication; ATR; Rad9; Saccharomyces cerevisiae
The response to osmotic stress in developing zebrafish embryos requires proper apical patterning and trafficking of transmembrane ion transporters in ionocytes, specialized cells of the epidermis. The miR-8 family of miRNAs plays a key role in this process by precisely regulating the activity of NHERF1, a regulator of sodium hydrogen exchange that also serves as an adaptor protein linked to the actin cytoskeleton. We have discovered that NHERF1 activity is also coupled to Planar Cell Polarity (PCP) signaling in the zebrafish epidermis. Loss of NHERF1 in wild type fish disrupts actin organization but the observed defects can be largely restored when combined with mutants in the PCP pathway. We propose that proper apical patterning depends on input and coordination between PCP signaling and the response to stress.
microRNA; NHERF1; planar cell polarity; knypek; miR-200; miR-8; osmotic stress
When replication is blocked by a template lesion or polymerase inhibitor while helicase continues unwinding the DNA, single stranded DNA (ssDNA) accumulates and becomes coated with RPA, which then initiates signals via PCNA mono-ubiquitination to activate trans-lesion polymerases and via ATR and Chk1 to inhibit Cdk2-dependent cell cycle progression. The signals are conveyed by way of a complex network of molecular interactions. To clarify those complexities, we have constructed a molecular interaction map (MIM) using a novel hierarchical assembly procedure. Molecules were arranged on the map in hierarchical levels according to interaction step distance from the DNA region of stalled replication. The hierarchical MIM allows us to disentangle the network’s interlocking pathways and loops and to suggest functionally significant features of network architecture. The MIM shows how parallel pathways and multiple feedback loops can provide failsafe and robust switch-like responses to replication stress. Within the central level of hierarchy ATR and Claspin together appear to function as a nexus that conveys signals from many sources to many destinations. We noted a division of labor between those two molecules, separating enzymatic and structural roles. In addition, the network architecture disclosed by the hierarchical map, suggested a speculative model for how molecular crowding and the granular localization of network components in the cell nucleus can facilitate function.
molecular interaction maps; checkpoints; signaling network; replication stress; helicase-polymerase uncoupling; DNA damage; trans-lesion synthesis; ATR; claspin; RPA
Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).
PMID: 24621501 CAMSID: cams4371
Bcl-xL; mitosis; PLK1; MAPK14/SAPKp38α; chromosome segregation; spindle-assembly checkpoint; chromosome instability
To better understand the role of E2F1 in tumor formation, we analyzed spontaneous tumorigenesis in p53−/−E2F1+/+ and p53−/−E2F1−/− mice. We show that the combined loss of p53 and E2F1 leads to an increased incidence of sarcomas and carcinomas compared to the loss of p53 alone. E2F1-deficient tumors show wide chromosomal variation, indicative of genomic instability. Consistent with this, p53−/−E2F1−/− primary fibroblasts have a reduced capacity to maintain genomic stability when exposed to S-phase inhibitors or genotoxic drugs. A major mechanism of E2F1’s contribution to genomic integrity lies in mediating stabilization and engagement of the Rb protein.
Rb protein; p53; G2M checkpoint; DNA replication
The D-type cyclins (D1, D2 and D3) are components of the cell cycle machinery and govern progression through G1 phase in response to extracellular signals. Although these proteins are highly homologous and conserved in evolution, they contain distinct structural motifs and are differentially regulated in various cell types. Cyclin D1 appears to play a role in many different types of cancer, whereas cyclins D2 and D3 are less frequently associated with malignancy. In this study, we transiently expressed cyclin D1, D2 or D3 in hepatocytes and analyzed transcriptional networks regulated by each. All three D-type cyclins promoted robust hepatocyte proliferation and marked liver growth, although cyclin D3 stimulated less DNA synthesis than D1 or D2. Accordingly, the three D-type cyclins similarly activated genes associated with cell division. Cyclin D1 regulated transcriptional pathways involved in the metabolism of carbohydrates, lipids, amino acids, and other substrates, whereas cyclin D2 did not regulate these pathways despite having an equivalent effect on proliferation. Comparison of transcriptional profiles following 70% partial hepatectomy and cyclin D1 transduction revealed a highly significant overlap, suggesting that cyclin D1 may regulate diverse cellular processes in the regenerating liver. In summary, these studies provide the first comparative analysis of the transcriptional networks regulated by the D-type cyclins and provide insight into novel functions of these key cell cycle proteins. Further study of the unique targets of cyclin D1 should provide further insight into its prominent role in proliferation, growth and cancer.
cyclin D1; cyclin D2; cyclin D3; liver regeneration
DNA double strand breaks are a particularly toxic form of DNA damage and the mammalian cell has evolved an intricate set of responses to repair this type of DNA lesion. A key early event in the DNA damage response (DDR) is ATM phosphorylation of the histone variant H2AX at serine 139 at the site of the DNA break. Phosphorylated S139 H2AX, or γH2AX, forms a docking site for binding of MDC1, leading to sustained recruitment of other DNA repair factors that mediate the repair of the DNA double strand break. Moreover, recruitment of MDC1 to the break site activates cell cycle checkpoints, protecting the cell from replication of damaged DNA templates. While the molecular events leading to DNA double strand break repair have been well described, the deactivating or homeostatic mechanisms following completion of repair remain largely unexplored. Recent publications by our laboratories and the Medema laboratory shed new light on this issue. Both publications showed that the Wild-type p53-Induced Phosphatase 1 (WIP1) directly dephosphorylates γH2AX. WIP1 migrates to the sites of irradiation-induced foci (IRIF), though at a delayed rate relative to MDC1 and mediates γH2AX dephosphorylation, presumably after DNA repair is complete. This prevents recruitment of other repair factors such as MDC1 and 53BP1 to the DNA damage sites and promotes the dissolution of IRIF. In addition, overexpression of WIP1 has a suppressive effect on DNA double strand break repair. Taken together, these reports further implicate WIP1 as a critical homeostatic regulator of the DDR.
Wip1; PPM1D; γH2AX; MDC1; ATM; ATR; DNA double strand break repair