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ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
Identification and characterization of TNFalpha responsive genes in human breast cancer cells
(2006)
One of the hallmarks of cancer is the escape of the transformed cells from apoptosis. Therefore, the identification of survival genes, allowing cancer cells to circumvent programmed cell death, could provide new diagnostic markers as well as targets for therapeutic intervention. A well known transcription factor regulating the balance between pro- and anti- apoptotic factors is NF-kappaB, which is strongly induced by tumor necrosis factor alpha (TNFalpha). When cells are stimulated by TNFalpha their response is biphasic with an initial NF-kappaB induction of survival genes which is overridden by the subsequent activation of initiator caspases triggering apoptosis. By combining gene trap mutagenesis with site specific recombination a strategy was developed, which enriches for genes induced by TNFalpha in the human breast cancer cell line MCF-7. The strategy relies on a one way gene expression switch based on Cre/loxP mediated recombination, which uncouples the expression of a marker gene from the trapped cellular promoter thereby enabling the recovery of genes that are only transiently induced by TNFalpha. The marker gene used in these experiments was a dominant negative variant of the TNFalpha-receptor associated protein FADD (dnFADD), which blocks the apoptotic branch of the TNFalpha induced signaling pathway. Initial experiments indicated that MCF-7 cells expressing high levels of dnFADD were insensitive to TNFalpha induced apoptosis and therefore suitable for the installment of a one way gene expression switch susceptible to Cre/loxP mediated recombination. A MCF-7 reporter clone harboring the recombinase dependent gene expression switch was infected with the gene trap retrovirus U3Cre, which inserts the Cre recombinase gene into a large collection of chromosomal sites. Insertion of Cre downstream of an active cellular promoter induces dnFADD expression from the gene expression switch enabling the cells to block TNFalpha triggered apoptosis. From a gene trap integration library containing approximately 2000000 unique proviral integrations, 69 unique TNFalpha inducible gene trap insertion sites were recovered in a two step selection procedure. Sequencing of the genomic regions adjacent to the insertion sites, which were obtained by inverse PCR (gene trap sequence tags, GTSTs), and data base analysis revealed that 42% of the GTSTs belonged to annotated genes, 13% to known cDNAs with open reading frames, 17% to Genscan predicted genes, 9% to ESTs, 9% to repetitive sequences and 10% to unannotated genomic sequence. Overall, 44% of the annotated genes recovered in this screen were directly or indirectly related to cancer, indicating that the gene trap strategy developed here is suitable for the identification of cancer relevant genes. Analysis of the expression patterns of the trapped and annotated genes in wild type cells revealed that 19 out of 24 genes were either up- or down- regulated by a factor of at least 1.45 by TNFalpha. A large fraction of the gene trap insertions were located upstream, in introns or in opposite orientation to annotated transcripts, indicating that the strategy efficiently recovers non-coding RNAs (ncRNAs). While the biological significance of these transcripts still needs to be elucidated, they fall into two main categories. The first category includes gene trap insertions upstream of genes, which could either represent regulatory RNAs interacting with promoter elements or transcripts driven by bidirectional promoters. The second includes inverse orientation gene trap insertions in introns of annotated genes suggesting the presence of natural antisense transcripts (NATs). Interestingly, more than 50% of all antisense integrations are located downstream of transcription start sites predicted by different algorithms supporting the existence of RNAs transcribed from the corresponding genomic regions. Intronic integrations on the coding strand could be derived from cryptic splicing, alternative promoter usage or additional, so far uncharacterized transcripts. Preliminary functional analysis of two genes recovered in this screen encoding the transcription factor ZFP67 and the FLJ14451 protein revealed that FLJ14451 but not ZFP67 inhibited anchorage independent growth in soft agar, suggesting that FLJ14451 might have some tumor suppressor functions. In summary, besides identifying a putative tumor suppressor protein, the present experiments have shown that gene trapping is useful in identifying non-coding transcripts in living cells and may turn out to be the method of choice in characterizing these transcripts whose functions are still largely unknown.
Es ist bekannt, dass Curcumin in einer Vielzahl verschiedener Zellarten die Proliferation hemmt und Apoptose induziert. In der Literatur werden Konzentrationen von 10 bis 150 µM (3.7-55 µg/ml) als dafür notwendig beschrieben. Da Curcumin nach oraler Aufnahme, aufgrund seiner schlechten Resorption aus dem Magen-Darm-Trakt, eine geringe Bioverfügbarkeit im Organismus aufweist, sind therapeutische Lösungen erforderlich um Curcumin besser nutzen zu können. Aus diesem Grund wurde im Rahmen der vorliegenden Arbeit die Wirkung von geringen Mengen Curcumin, die allein keine Effekte zur Folge haben, in Zusammenwirkung mit Licht untersucht. Es wurde gezeigt, dass bereits Konzentrationen von 0,2-1 µg/ml in Keratinozyten die Proliferation hemmen, wenn sie mit UVA- oder sichtbarer Licht-Bestrahlung kombiniert werden. Desweiteren wurde belegt, dass diese Behandlung Apoptose in HaCaT-Zellen induziert, wobei der mitochondriale Apoptoseapparat aktiviert wird. Dies belegen die Freisetzung von Cytochrom c und die frühe Spaltung der Caspase-9 wohingegen Caspase-8 zeitverzögert aktiviert wurde. Weiterhin wurde dargelegt, dass Erk1/2, PKB/Akt und PKC durch Curcumin/Licht gehemmt werden, wohingegen p38 durch diese Behandlung eine Aktivierung erfährt. Zusätzlich ergab sich ein inhibierender Einfluss auf den EGF-Rezeptor, einem “upstream”-Regulator all dieser Kinasen, und den Transkriptionsfaktor NF-kappaB. Bei in vivo-Studien an immundefizienten Mäusen mit A431-Xenografts hatte eine Behandlung mit i.p. verabreichtem Curcumin und anschließender Bestrahlung mit sichtbarem Licht einen signifikanten inhibitorischen Effekt auf das Tumorwachstum zur Folge. In diesen Tumoren fand eine Reduktion der Ki-67-Expression sowie eine Induktion von „apoptotic bodies“ statt. Western Blot-Analysen bestätigten die Apoptoseinduktion durch eine verstärkte Aktivierung der Caspase-9. Zusätzlich zeigte sich auch eine Hemmung von Erk1/2 sowie EGF-R nach beschriebener Behandlung in den Tumoren. Die Wirkungsweise des Curcumins in Kombination mit Licht in vitro wie auch in vivo weisen auf einen neuen therapeutischen Ansatz einer photodynamischen Therapie hin. Dabei kann durch die Verwendung von sichtbarem Licht auf den Einsatz der karzinogenen UV-Bestrahlung, wie sie in üblichen Phototherapien angewandt wird, verzichtet werden.