Multiple tumor suppressor microRNAs regulate telomerase and TCF7, an important transcriptional regulator of the Wnt pathway.

The human TERT (hTERT) gene encodes the telomerase catalytic subunit which plays a role in telomerase regulation. Telomerase is activated in more than 90% of all human malignancies and understanding how telomerase is regulated is necessary for implementation of successful anti-cancer therapies. microRNAs (miRNAs) are important regulators of gene expression in eukaryotic cells but evidence of their role in telomerase regulation has not been documented. To determine whether hTERT activity is regulated by multiple miRNAs, eight miRNAs which have putative binding sites in the hTERT 3'UTR together with miR-138-5p were evaluated in luciferase assays with a reporter containing the hTERT 3'UTR. Six miRNAs (let-7g*, miR-133a, miR-138-5p, miR-342-5p, miR-491-5p, and miR-541-3p) specifically inhibited the expression of the reporter luciferase-driven constructs and let-7g*, miR-133a, miR-138-5p, and miR-491-5p also downregulated endogenous telomerase activity in cells. Moreover, all six miRNAs significantly inhibited cell proliferation. miRNAs (miR-133a, miR-138-5p, 342-5p, 491-5p, 541-3p) also have predicted binding sites within the 3'UTR of three genes involved in Wnt signaling (TCF7, MSI1, and PAX5). These miRNAs inhibited the expression of the luciferase reporter constructs containing 3'UTRs of these genes and downregulated protein expression of the TCF7 transcription factor, which mediates the canonical Wnt pathway. Together, these results suggest the existence of a miRNA regulatory network involving the hTERT and Wnt pathway.

Alternatively spliced telomerase reverse transcriptase variants lacking telomerase activity stimulate cell proliferation.

Eight human and six chicken novel alternatively spliced (AS) variants of telomerase reverse transcriptase (TERT) were identified, including a human variant (Δ4-13) containing an in-frame deletion which removed exons 4 through 13, encoding the catalytic domain of telomerase. This variant was expressed in telomerase-negative normal cells and tissues as well as in transformed telomerase-positive cell lines and cells which employ an alternative method to maintain telomere length. The overexpression of the Δ4-13 variant significantly elevated the proliferation rates of several cell types without enhancing telomerase activity, while decreasing the endogenous expression of this variant by use of small interfering RNA (siRNA) technology reduced cell proliferation. The expression of the Δ4-13 variant stimulated Wnt signaling. In chicken cells, AS TERT variants containing internal deletions or insertions that eliminated or reduced telomerase activity also enhanced cell proliferation. This is the first report that naturally occurring AS TERT variants which lack telomerase activity stimulate cell proliferation.

Insights into the evolution of mammalian telomerase: platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes.

BACKGROUND: The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT) ortholog, and provide a comparison with genes of other vertebrates. RESULTS: The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in ray-finned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. CONCLUSIONS: OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.

Characterization of ATF2 in Rel/NFκB oncogenesis reveals its role in the regulation of Ras signaling.

The v-Rel oncoprotein is the acutely transforming member of the Rel/NFκB family of transcription factors. v-Rel transforms cells through the inappropriate activation and suppression of genes normally regulated by cellular Rel/NFκB family members. We have recently demonstrated that activation of Ha-Ras by v-Rel contributes to transformation. Characterization of AP-1 family members in v-Rel-mediated transformation revealed ectopic expression of ATF2 inhibited transformation by blocking Ha-Ras activity. This lack of Ha-Ras activity prevented downstream activation of the Raf-MEK-ERK pathway, a critical pathway for v-Rel-mediated transformation. Microarray analysis of cells treated with an inhibitor to the ERK pathway revealed a relatively small number of genes that are specifically regulated by ERK activity in cells expressing v-Rel. These studies suggest the main contribution of ERK activity is to temper the expression of genes in v-Rel transformed cells. The mechanism by which ATF2 regulates Ras-Raf-MEK-ERK signaling appears to be a context dependent event. The ectopic expression of ATF2 in cells that are not expressing v-Rel results in the activation of Ha-Ras. However, activation of downstream Raf-MEK-ERK signaling pathway is blocked, likely through the recruitment of inhibitory 14-3-3 proteins to c-Raf. These results suggest a diverse role for ATF2 in the regulation of the Ras-Raf-MEK-ERK pathway.

Activation of the TGF-ß/Smad signaling pathway in oncogenic transformation by v-Rel.

v-rel, encoded by the avian reticuloendotheliosis virus, is an acutely transforming member of the Rel/NF-κB family of transcription factors. Transformation by v-Rel is mediated by the aberrant expression of genes that are normally regulated by Rel/NF-κB. Here, we demonstrate activation of the TGF-ß/Smad signaling pathway in Rel transformation. RNA and protein levels of key TGF-ß and Smad family members (TGF-ß2, -ß3, TGF-ß type II receptor, and Smad3) are upregulated in v-Rel transformed cells with little to no change in c-Rel-expressing cells. Treatment of v-Rel transformed lymphoid cells with kinase inhibitors of the TGF-ß receptor dramatically reduces soft agar colony formation whereas addition of TGF-ß2 further promotes transformation. Moreover, Smad3 but not Smad2, is selectively activated as the downstream mediator of TGF-ß signaling. Blocking Smad3 expression or activity inhibits the oncogenic potential of v-Rel. Overall, TGF-ß/Smad signaling is activated at multiple levels and is required for the transforming ability of v-Rel.

Dynamic evolution of immune system regulators: the history of the interferon regulatory factor family.

This manuscript presents the first extensive phylogenetics analysis of a key family of immune regulators, the interferon regulatory factor (IRF) family. The IRF family encodes transcription factors that play important roles in immune defense, stress responses, reproduction, development, and carcinogenesis. Several times during their evolution, the IRF genes have undergone expansion and diversification. These genes were also completely lost on two separate occasions in large groups of metazoans. The origin of the IRF family coincides with the appearance of multicellularity in animals. IRF genes are present in all principal metazoan groups, including sea sponges, placozoans, comb jellies, cnidarians, and bilaterians. Although the number of IRF family members does not exceed two in sponges and placozoans, this number reached five in cnidarians. At least four additional independent expansions lead up to 11 members in different groups of bilaterians. In contrast, the IRF genes either disappeared or mutated beyond recognition in roundworms and insects, the two groups that include most of the metazoan species. The IRF family separated very early into two branches ultimately leading to vertebrate IRF1 and IRF4 supergroups (SGs). Genes encoding the IRF-SGs are present in all bilaterians and cnidarians. The evolution of vertebrate IRF family members further proceeded with at least two additional steps. First, close to the appearance of the first vertebrate, the IRF family probably expanded to four family members, predecessors of the four vertebrate IRF groups (IRF1, 3, 4, 5 groups). In the second step, 10 vertebrate family members evolved from these four genes, likely as a result of the 2-fold duplication of the entire genome. Interestingly, the IRF family coevolved with the Rel/NF-kappaB family with which it shares some important evolutionary characteristics, including roles in defense responses, metazoan specificity, extensive diversification in vertebrates, and elimination of all family members in nematodes.

Regulation of telomerase activity by interferon regulatory factors 4 and 8 in immune cells.

Telomerase activity is downregulated in somatic cells but is upregulated during the activation of cells of the immune system. The mechanism of this reactivation is not well understood. In this study, we demonstrated that interferon regulatory factor 4 (IRF-4) and, to a lesser extent, IRF-8 induce telomerase activity. The suppression of IRF-4 results in decreased levels of TERT (telomerase reverse transcriptase) mRNA and telomerase activity and reduces cell proliferation. The overexpression of TERT compensates for this proliferation defect, suggesting that telomerase contributes to the regulation of cell proliferation by IRF-4. The induction of telomerase by IRF-4 and IRF-8 correlates with the activation of the TERT promoter. IRF-4 binds the interferon response-stimulated element and the gamma interferon-activated sequence composite binding site in the TERT core promoter region in vivo. Additionally, the binding of Sp1, Sp3, USF-1, USF-2, and c-Myc to the TERT promoter is elevated in cells expressing IRF-4. IRF-4, but not IRF-8, synergistically cooperates with Sp1 and Sp3 in the activation of the TERT promoter. Collectively, these results indicate that IRF-4 and IRF-8, two lymphoid cell-specific transcription factors, increase telomerase activity by activating TERT transcription in immune cells.

Mechanism of telomerase activation by v-Rel and its contribution to transformation.

Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-kappaB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS.

Differential regulation of the inhibitor of apoptosis ch-IAP1 by v-rel and the proto-oncogene c-rel.

The v-rel oncogene encoded by reticuloendotheliosis virus is the acutely transforming member of the Rel/NF-kappaB family of transcription factors. v-Rel is a truncated and mutated form of c-Rel and transforms cells by inducing the aberrant expression of genes regulated by Rel/NF-kappaB proteins. The expression of ch-IAP1, a member of the inhibitor-of-apoptosis family, is highly elevated in cells expressing v-Rel and contributes to the immortalization of cells transformed by this oncoprotein. In this study we demonstrate that the elevated expression of ch-IAP1 in v-Rel-expressing cells is due to an increased rate of transcription. The ch-IAP1 promoter was isolated, and four Rel/NF-kappaB binding sites were identified upstream of the transcription start site. Two kappaB sites proximal to the transcription start site were required for v-Rel to activate the ch-IAP1 promoter. While c-Rel also utilized these sites, a third more-distal kappaB site was required for its full activation of the ch-IAP1 promoter. Differences in the transactivation domains of v-Rel and c-Rel are responsible for their different abilities to utilize these sites and account for their differential activation of the ch-IAP1 promoter. Although c-Rel was a more potent activator of the ch-IAP1 promoter than v-Rel in transient reporter assays, cells stably overexpressing c-Rel failed to maintain high levels of ch-IAP1 expression. The reduction of ch-IAP1 expression in these cells correlated with the efficient regulation of c-Rel by IkappaBalpha. The ability of v-Rel to escape IkappaBalpha regulation allows for the gradual and sustained elevation of ch-IAP1 expression directly contributing to the transforming properties of v-Rel.

A novel interferon regulatory factor (IRF), IRF-10, has a unique role in immune defense and is induced by the v-Rel oncoprotein.

The cloning and functional characterization of a novel interferon regulatory factor (IRF), IRF-10, are described. IRF-10 is most closely related to IRF-4 but differs in both its constitutive and inducible expression. The expression of IRF-10 is inducible by interferons (IFNs) and by concanavalin A. In contrast to that of other IRFs, the inducible expression of IRF-10 is characterized by delayed kinetics and requires protein synthesis, suggesting a unique role in the later stages of an antiviral defense. Accordingly, IRF-10 is involved in the upregulation of two primary IFN-gamma target genes (major histocompatibility complex [MHC] class I and guanylate-binding protein) and interferes with the induction of the type I IFN target gene for 2',5'-oligo(A) synthetase. IRF-10 binds the interferon-stimulated response element site of the MHC class I promoter. In contrast to that of IRF-1, which has some of the same functional characteristics, the expression of IRF-10 is not cytotoxic for fibroblasts or B cells. The expression of IRF-10 is induced by the oncogene v-rel, the proto-oncogene c-rel, and IRF-4 in a tissue-specific manner. Moreover, v-Rel and IRF-4 synergistically cooperate in the induction of IRF-10 in fibroblasts. The level of IRF-10 induction in lymphoid cell lines by Rel proteins correlates with Rel transformation potential. These results suggest that IRF-10 plays a role in the late stages of an immune defense by regulating the expression some of the IFN-gamma target genes in the absence of a cytotoxic effect. Furthermore, IRF-10 expression is regulated, at least in part, by members of the Rel/NF-kappa B and IRF families.

Mutational analysis of the v-Rel dimerization interface reveals a critical role for v-Rel homodimers in transformation.

The v-rel oncogene encoded by reticuloendotheliosis virus strain T is the acutely transforming member of the Rel/NF-kappaB family of transcription factors. In v-Rel-transformed cells, v-Rel exists as homodimers or heterodimers with the endogenous Rel/NF-kappaB proteins c-Rel, NF-kappaB1, NF-kappaB2, and RelA. To examine the contribution of these complexes to v-Rel-mediated transformation, mutations were introduced into the dimerization interface of v-Rel to generate v-Rel mutants with selective dimerization properties. Nine mutants are described in this study that are defective in homodimer and/or heterodimer formation with specific Rel/NF-kappaB family members. Viruses expressing mutants that failed to homodimerize but were able to form heterodimeric complexes were unable to transform splenic lymphocytes in vitro, indicating that the dimerization of v-Rel with endogenously expressed Rel/NF-kappaB proteins is not in itself sufficient for transformation. In addition, two partially transforming mutants were identified that exhibited an impaired ability to form homodimers. Sequence analysis of the proviral DNA from cells transformed by these mutants revealed the presence of multiple secondary mutations in sequences responsible for dimerization and DNA binding. Two of these mutations either enhanced or restored the ability of these proteins to bind DNA as a homodimer. Viruses expressing these proteins transformed cells at levels comparable to or slightly less than v-Rel, suggesting that a threshold level of DNA binding by v-Rel homodimers is required for transformation.