Description: Cyclic adenosine diphosphate ribose is an endogenous Ca2+ mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca2+ increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca2+ release from endoplasmic reticulum, with concomitant Ca2+ influx in Jurkat cells. Ca2+ release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca2+ influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca2+ release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca2+ release from endoplasmic reticulum via RyRs and triggers Ca2+ influx via TRPM2.

Description: Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3′-[35S]phosphoadenosine 5′-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional 1H and 13C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (kcat/Km) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.

Description: Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.

Description: The PRL (phosphatase of regenerating liver) phosphatases are implicated in the control of cell proliferation and invasion. Aberrant PRL expression is associated with progression and metastasis of multiple cancers. However, the specific in vivo function of the PRLs remains elusive. Here we show that deletion of PRL2, the most ubiquitously expressed PRL family member, leads to impaired placental development and retarded growth at both embryonic and adult stages. Ablation of PRL2 inactivates Akt and blocks glycogen cell proliferation, resulting in reduced spongiotrophoblast and decidual layers in the placenta. These structural defects cause placental hypotrophy and insufficiency, leading to fetal growth retardation. We demonstrate that the tumor suppressor PTEN is elevated in PRL2-deficient placenta. Biochemical analyses indicate that PRL2 promotes Akt activation by down-regulating PTEN through the proteasome pathway. This study provides the first evidence that PRL2 is required for extra-embryonic development and associates the oncogenic properties of PRL2 with its ability to negatively regulate PTEN, thereby activating the PI3K-Akt pathway.

Description: The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser83 by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser539, probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.

Description: Most of the nucleotide-binding domain, leucine-rich repeat (NLR) proteins regulate responses to microbial and damage-associated products. Class II transactivator (CIITA) has a distinct function as the master regulator of class II major histocompatibility complex (MHC-II) transcription. Recently, human NLRC5 was found to regulate MHC-I in cell lines; however, a host of conflicting positive and negative functions has been attributed to this protein. To address the function of NLRC5 in a physiologic setting, we generated an Nlrc5−/− strain that contains a deletion in the exon that encodes the nucleotide-binding domain. We have not detected a role for this protein in cytokine induction by pathogen-associated molecular patterns and viruses. However, Nlrc5−/− cells showed a dramatic decrease of classical (H-2K) and nonclassical (Tla) MHC-I expression by T/B lymphocytes, natural killer (NK) cells, and myeloid-monocytic lineages. As a comparison, CIITA did not affect mouse MHC-I expression. Nlrc5−/− splenocytes and bone marrow-derived macrophages were able to up-regulate MHC-I in response to IFN-γ; however, the absolute levels of MHC-I expression were significantly lower than WT controls. Chromatin immunoprecipitation of IFN-γ-treated cells indicates that Nlrc5 reduced the silencing H3K27me3 histone modification, but did not affect the activating AcH3 modification on a MHC-I promoter. In summary, we conclude that Nlrc5 is important in the regulation of MHC-I expression by reducing H3K27me3 on MHC-I promoter and joins CIITA as an NLR subfamily that controls MHC gene transcription.

Description: Aberrant regulation of DNA damage checkpoint function leads to genome instability that in turn can predispose cellular tissues to become cancerous. Previous works from us and others demonstrated the role of Rad17 in either activation or termination of DNA damage checkpoint function. In the current study, we have revealed the unexpected accumulation of Rad17 in various types of breast cancer cell lines as well as human breast cancer tissues. We observed that Rad17 protein turnover rate in breast epithelial cells is much faster than in breast cancer cells, where the turnover of Rad17 is regulated by the Cdh1/APC pathway. We further observed that Rad17-mediated checkpoint function is modulated by proteolysis. Stabilization of Rad17 disrupts cellular response to chemotherapeutic drug-induced DNA damage and enhances cellular transformation. In addition, manipulation of Rad17 by RNA interference or stabilization of Rad17 significantly sensitize breast cancer cell to various chemotherapeutic drugs. Our present results indicate the manipulation of Rad17 proteolysis could be a valuable approach to sensitize breast cancer cell to the chemotherapeutic treatment despite of the critical role in governing DNA damage response and cellular recovery from genotoxic stress.

Description: Although the biological role of KRAS is clearly established in carcinogenesis, the molecular mechanisms underlying this phenomenon are not completely understood. In this study, we provide evidence of a novel signaling network regulated by the transcription factor GLI1 mediating KRAS-induced carcinogenesis. Using pancreatic cancer (a disease with high prevalence of KRAS mutations) as a model, we show that loss of GLI1 blocks the progression of KRAS-induced pancreatic preneoplastic lesions in mice with pancreas-specific Cre-activated oncogenic mutant kras. Mice lacking GLI1 develop only low-grade lesions at low frequency, and in most cases, the pancreata are histologically normal. Further characterization of the phenotype showed a decrease in the activation of STAT3 in pancreatic preneoplastic lesions; STAT3 is a transcription factor required for the development of premalignant lesions and their progression into pancreatic cancer. Analysis of the mechanisms revealed a key role for GLI1 in maintaining the levels of activated STAT3 through the modulation of IL-6 signaling. GLI1 binds to the IL-6 mouse promoter and regulates the activity and expression of this cytokine. This newly identified GLI1/IL-6 axis is active in fibroblasts, a known source of IL-6 in the tumor microenvironment. Sonic hedgehog induces GLI1 binding to the IL-6 promoter and increases IL-6 expression in fibroblasts in a paracrine manner. Finally, we demonstrate that mutant KRAS initiates this cascade by inducing the expression of Sonic hedgehog in cancer cells. Collectively, these results define a novel role for GLI1 in carcinogenesis acting as a downstream effector of oncogenic KRAS in the tumor microenvironment.

Description: The role of microRNAs (miRNAs) in mediating adult neurogenesis after stroke has not been extensively studied. The present study investigated the function of the miR17-92 cluster in adult neural progenitor cells after experimental stroke. We found that stroke substantially up-regulated miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of the miR17-92 cluster either in cultured ischemic neural progenitor cells or in the subventricular zone (SVZ) of ischemic animals significantly increased cell proliferation, whereas inhibition of individual members of the miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. The miR17-92 cluster mediated PTEN (phosphatase and tensin homolog) expression, which is a predicted target of the miR17-92 cluster. Addition of Sonic hedgehog (Shh) protein up-regulated miR17-92 expression and elevated c-Myc protein in ischemic neural progenitor cells, whereas blockade of the Shh signaling pathway down-regulated miR17-92 cluster expression and reduced c-Myc levels. Overexpression of c-Myc up-regulated miR17-92 cluster expression. Intraventricular infusion of Shh and a Shh receptor inhibitor, cyclopamine, to ischemic animals further elevated and suppressed, respectively, miR17-92 cluster expression in the SVZ. These data indicate that the miR17-92 cluster plays an important role in mediating neural progenitor cell function and that the Shh signaling pathway is involved in up-regulating miR17-92 cluster expression.

Description: The Hedgehog (Hh) signaling pathway plays evolutionarily conserved roles in controlling embryonic development and tissue homeostasis, and its dysregulation has been implicated in many human diseases including congenital disorder and cancer. The Hh pathway has a unique signal reception system that includes two membrane proteins, the receptor Patched (Ptc) and the transducer Smoothened (Smo). In the Hh signaling cascade, Smo plays a critical role in controlling transduction of Hh gradient signal from the outside into the inside of cells. Although the Smo downstream signal transduction has been intensively studied, the mechanism by which Smo on the plasma membrane is regulated has not been fully understood. As a specific membrane structure of metazoan cells, lipid rafts act as a platform to regulate signal transduction by forming a nanoscale cluster through protein-protein or protein-lipid interactions. However, it remains largely unknown whether lipid rafts are also involved in the regulation of Hh signal transduction. Here, we show that Smo extracellular domain (N terminus) and transmembrane domains form oligomers/higher order clusters in response to Hh signal. Furthermore, we identify that lipid rafts on the plasma membrane are essential for high level activity of Smo during the Hh signal transduction. Finally, our observation suggests that oligomerization/higher order clustering of Smo C-terminal cytoplasmic tail (C-tail) is essential for the transduction of high level Hh signal. Collectively, our data support that in response to Hh gradient signals, Smo transduces high level Hh signal by forming oligomers/higher order clusters in the lipid rafts of cell plasma membrane.