E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

Accelerated DNA replication in E2F1- and E2F2-deficient macrophages leads to induction of the DNA damage response and p21(CIP1)-dependent senescence.

E2F1-3 proteins appear to have distinct roles in progenitor cells and in differentiating cells undergoing cell cycle exit. However, the function of these proteins in paradigms of terminal differentiation that involve continued cell division has not been examined. Using compound E2F1/E2F2-deficient mice, we have examined the effects of E2F1 and E2F2 loss on the differentiation and simultaneous proliferation of bone-marrow-derived cells toward the macrophage lineage. We show that E2F1/E2F2 deficiency results in accelerated DNA replication and cellular division during the initial cell division cycles of bone-marrow-derived cells, arguing that E2F1/E2F2 are required to restrain proliferation of pro-monocyte progenitors during their differentiation into macrophages, without promoting their cell cycle exit. Accelerated proliferation is accompanied by early expression of DNA replication and cell cycle regulators. Remarkably, rapid proliferation of E2F1/E2F2 compound mutant cultures is temporally followed by induction of a DNA damage response and the implementation of a p21(CIP1)-dependent senescence. We further show that differentiating E2F1/E2F2-knockout macrophages do not trigger a DNA damage response pathway in the absence of DNA replication. These findings underscore the relevance of E2F1 and E2F2 as suppressors of hematopoietic progenitor expansion. Our data indicate that their absence in differentiating macrophages initiates a senescence program that results from enforcement of a DNA damage response triggered by DNA hyper-replication.

Innate immune response gene expression profiles characterize primary antiphospholipid syndrome.

Primary antiphospholipid syndrome (PAPS) is a systemic autoimmune disorder characterized by thromboembolic episodes and pregnant morbidity with an increasing clinical importance. To gain insight into the pathogenesis of PAPS, we have investigated the gene expression profiles that characterize peripheral blood mononuclear cells derived from PAPS patients. We show that the transcriptional activity of genes involved in innate immune responses, such as toll-like receptor 8 and CD14, as well as downstream genes of this pathway, such as STAT1, OAS2, TNFSF13 and PLSCR1 are significantly increased in PAPS patients. In addition, the expression of monocyte-specific cytokines is also elevated in PAPS mononuclear cells stimulated in vitro with lipopolysaccharide. Taken together, these results reveal a 'response to pathogen' signature in PAPS, which could reflect an altered monocyte activity. Finally, microarray analyses also revealed a reduced expression of genes coding for proteins involved in transcriptional control. Interestingly, a significant proportion of them exhibit E2F-binding sites in their promoter, suggesting that a deregulated RB/E2F activity could play a role in the pathogenesis of antiphospholipid syndrome.

Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity.

E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.

A role for E2F1 in the induction of apoptosis during thymic negative selection.

Thymic negative selection is the process in which maturing thymocytes that express T-cell receptors recognizing self are eliminated by apoptotic cell death. The molecular mechanism by which this occurs is poorly understood. Notably, genes involved in cell death, even thymocyte death, such as Fas, Fas-ligand, p53, caspase-1, caspase-3, and caspase-9, and Bcl-2 have been found to not be required for normal thymic negative selection. We have demonstrated previously that E2F1-deficient mice have a defect in thymocyte apoptosis. Here we show that E2F1 is required for normal thymic negative selection. Furthermore, we observed an E2F1-dependent increase of p53 protein levels during the process of thymic clonal deletion, which suggests that E2F1 regulates activation-induced apoptosis of self-reactive thymocytes by a p53-dependent mechanism. In contrast, other apoptotic pathways operating on developing thymocytes, such as glucocorticoid-induced cell death, are not mediated by E2F1. The T lymphocytes that escape thymic negative selection migrate to the peripheral immune system but do not appear to be autoreactive, indicating that there may exist E2F1-independent mechanisms of peripheral tolerance, which protect mice from developing an autoimmune response. We expect that E2F1-deficient mice will provide a useful tool for understanding the molecular mechanism of and the immunological importance of thymic negative selection.

E2F-1 functions in mice to promote apoptosis and suppress proliferation.

Members of the E2F transcription factor family (E2F-1-E2F-5) are believed to be critical positive regulators of cell cycle progression in eukaryotes although the in vivo functions of the individual E2Fs have not been elucidated. Mice were generated that lack E2F-1 and, surprisingly, these mice develop and reproduce normally. However, E2F-1-/- mice exhibit a defect in T lymphocyte development leading to an excess of mature T cells due to a maturation stage-specific defect in thymocyte apoptosis. As E2F-1-/- mice age they exhibit a second phenotype marked by aberrant cell proliferation. These findings suggest that while certain members of the E2F family may positively regulate cell cycle progression, E2F-1 functions to regulate apoptosis and to suppress cell proliferation.

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.

Interleukin-1 induces c-fos and c-jun gene expression in T helper type II cells through different signal transmission pathways.

Interleukin (IL)-1 induces proliferation and expression of several protooncogenes in the T helper 2 cell line D10A. We have analyzed the signal transmission pathways activated by IL-1 in these cells, leading to the expression of c-jun and c-fos. IL-1 induced c-jun gene transcription and mRNA expression by means of a pathway dependent on protein tyrosine kinase activity since tyrphostin, a specific inhibitor of tyrosine kinase, inhibited this induction. This mechanism of transmission signaling was independent of protein kinase C (PKC) and was linked to the 80-kDa IL-1 receptor (IL-1R). In addition, phorbol esters did not induce c-jun mRNA expression, whereas c-fos mRNA expression mediated by IL-1 dependent on PKC; this pathway was linked to a different, still unidentified IL-1R that was functional in the D10A cell line. Accumulation of intracellular cAMP generated by IL-1 through the 80-kDa IL-1R negatively regulated c-fos expression which was induced by IL-1 through PKC activation. We conclude that IL-1 modulates the expression of c-fos in D10A cells by occupying of two independent IL-1R that are linked to different signal transduction pathways.

IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis.

It is well established that T cell maturation and activation are negatively regulated by a mechanism termed apoptosis. We now present evidence that glucocorticoids, known to possess immunosuppressive properties, cause apoptosis in mature Th cells, similarly to what has been reported for thymocytes. Th cells treated with the synthetic glucocorticoid dexamethasone show genome fragmentation into oligonucleosomal fragments, and proliferation of growth factor stimulated Th cells is inhibited by glucocorticoids. We show that IL-4 specifically rescues Th2 cells from dexamethasone-mediated apoptosis, whereas IL-2 and IL-1 are ineffective in these cells. However, IL-2 is the relevant rescue-factor of glucocorticoid-treated Th1 cells. The rescue induced by IL-4 and IL-2 is thought to be mediated by protein kinases (possibly protein kinase C), as evidenced by the fact that the protein kinase inhibitor H7 blocks the action of IL-4 and IL-2 in glucocorticoid-treated cells. Our in vitro data show that mature T cells can be protected by their own growth factors from the deleterious effects of the synthetic glucocorticoid dexamethasone, and suggest that specific interactions occur between lymphokines and naturally produced glucocorticoids in vivo, which may play a role in the regulation of the immune response.

Superinduction of IL-2 gene transcription in the presence of cycloheximide.

Lymphokine production is regulated both at the transcriptional and the posttranscriptional level. To date, it has been shown that the protein synthesis inhibitor cycloheximide (CHX) up-regulates IL-2 expression in T cells by stabilizing its mRNA. In this report we have examined the effect of CHX on IL-2 at the transcriptional level. We have found that CHX has a positive regulatory function in IL-2 transcription, which is dependent on prior activation of this gene. This is not due to posttranslational conversion of inactive NFkB into its active form by CHX, because a clustered mutation in the kB-like sequence in the IL-2 enhancer that abrogates NFkB binding does not affect the up-regulation of IL-2 transcription. These results favor the hypothesis that, in addition to positive factors, negative elements regulate IL-2 transcription. Furthermore, we have tested the effect of CHX on IL-4 and granulocyte-macrophage-CSF transcription of both lymphokines. These results suggest that transcriptional up-regulation by CHX may be specific for IL-2 with respect to lymphokine expression.

Production of IL-1 alpha by activated Th type 2 cells. Its role as an autocrine growth factor.

Autocrine growth of Th type 2 cells has been reported to be mediated by the lymphokine IL-4. In this report we present evidence that in addition to IL-4 Th2 cells also produce IL-1 alpha in its active form in the absence of APC. We have found that this cytokine is an autocrine growth factor, because proliferation of Th2 cells in response to several stimuli is inhibited by anti-IL-1 alpha or anti-IL-1R mAb, or by an IL-1 alpha antisense oligodeoxynucleotide. However, Th1 cells do not produce this cytokine. We have investigated the role of endogenous IL-1 alpha on the induction of c-myc and c-myb, two protooncogenes involved in T cell activation. Here we show that endogenous IL-1 alpha is involved in the activation of both protooncogenes. Our results suggest that a possible function of IL-1 alpha, and perhaps other growth factors, might be to sustain or amplify the initial second messengers derived through the TCR. The possible implications of this finding with respect to interactions between T cell subsets and B cells or macrophages are discussed.

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.

Ultrastructural analysis of the morphology and function of the spermatheca of the pulmonate slug Arion subfuscus.

A study of the histology, histochemistry and ultrastructure of the spermatheca of Arion subfuscus in different stages of its life cycle shows that the spermatheca, an accessory organ of the femal genital tract of pulmonates, is a spherical-shaped organ lined with a columnar and non-ciliated epithelium surrounded by a thin network of connective tissue with some muscle fibres. The narrow epithelial cells possess numerous microvilli that, excepting young specimens, are provided with long, thin and generally curved, membranous process. Golgi apparatus, RER and mitochondria are abundant. Basal infolds are not very deep. In specimens killed two days after-copula, the spermatheca is swollen and its lumen is full of degenerating spermatozoa, mucus masses and spermatophore fragments. The apex of the cell is rich in granules with varied content, including multivesicular bodies. In specimens killed two weeks after-copula there are numerous endocytic vesicles in the apex and big vacuoles containing lipid. It is considered that after mating the excess of exogenous spermatozoa and copulatory seminal fluids are digested in the spermatheca. First, there is extracellular digestion that may be carried out by the enzymes contained in the multivesicular bodies exocytosed to the lumen, as well as by the enzymes secreted in apocrine vesicles. The partially digested materials would then be absorbed by endocytosis and further digested intracellularly. The great accumulation of lipid in the epithelial cells two weeks after mating suggest that the spermatheca could be involved in lipid synthesis, acting as a reserve organ.

IL-1 signal transduction pathways. I. Two functional IL-1 receptors are expressed in T cells.

We have recently characterized a subline of the Th2 cell line D10.G4.1, D10A, which responds to exogenous IL-1 in the absence of cofactors. In this cell line IL-1 activates two distinct transmission signal pathways, one leading to increased levels of intracellular cAMP, and the other to the phosphorylation of an 80-kDa substrate of protein kinase C (PKC). To determine whether both pathways are activated upon occupancy of a single IL-1R, we used a mAb, M15, which blocks binding of IL-1 to the 80-kDa IL-1R, known to be expressed in Th2 cells. Whereas M15 was able to inhibit the accumulation of cAMP induced by IL-1, it had no effect on the IL-1-induced phosphorylation of the 80-kDa substrate of PKC or the c-fos mRNA expression. In addition, we show that IL-1 induces the expression of the two proto-oncogenes c-myb and c-myc through the 80-kDa IL-1R, and that this induction is PKC independent. The proliferation of D10A cells in response to IL-1 is blocked by M15, but is unaffected by H-7, a potent inhibitor of PKC. These results indicate that the IL-1-induced proliferation of D10A cells is independent of PKC. In addition, IL-1 induces c-fos mRNA expression in thymocytes but not in EL-4 cells.

Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation.

CD4+ T helper (Th) clones can be divided into interleukin 2 (IL-2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin inhibit T cell receptor (TCR)-mediated IL-2 production and proliferation in Th1 cells, while the same reagents fail to block IL-4 production and proliferation in Th2 cells. In addition, CT and forskolin differentially influence the proto-oncogene mRNA expression in Th1 vs. Th2 cells after stimulation with Con A. Since both reagents lead to elevated levels of intracellular cAMP, it is likely that Th1 and Th2 cells differ in their sensitivity to an increase in cAMP. Our results indicate that the two Th subsets use different transmission signal pathways upon TCR-mediated activation.

Regulation of IL-4 lymphokine gene expression and cellular proliferation in murine T helper type II cells.

Activation of T cells results in the production of lymphokines and cellular proliferation. Protein kinase C (PKC) plays a key role in this process. It has been shown that this enzyme is essential to elicit a response to Con A or specific antigen in CD4+ T helper type 1 (Th 1) cells that secrete IL-2. We have now explored the signal transduction pathway that leads to transcription of the IL-4 gene and proliferation in murine CD4+ T helper type 2 (Th 2) cells. Surprisingly, we have found in two independently derived Th 2 clones that neither cellular proliferation nor IL-4 lymphokine production is affected by blocking or depletion of PKC. This differential mechanism of signal transmission leading to cellular activation implies a new distinction between murine Th 1 and Th 2 cells.

Regulation of interleukin 6 production in T helper cells.

The cytokine interleukin 6 (IL-6) is a cellular regulatory molecule that is produced by both lymphoid and non-lymphoid cells in response to several stimuli. In this report we present evidence that within the murine T cell compartment T helper type 2 cells (Th2) produce this lymphokine, whereas unprimed CD4+ T cells and a T helper type 1 clone (Th1) do not. Furthermore, IL-6 is not an autocrine growth factor for in vitro cultured Th cells, in contrast to what occurs in freshly isolated CD4+ and CD8+ T cells. We have examined the signal transduction pathways that lead to IL-6 production in activated Th2 cells. We have found that protein kinase C activators, such as PMA, Con A, or IL-1, increase the IL-6 expression in these cells. On the other hand, activation of the cAMP-dependent pathway does not seem to have an effect on the IL-6 production, since forskolin, 8BrcAMP, or TNF-alpha, which in these cells increases the level of intracellular cAMP, do not lead to an accumulation of IL-6 message. These results indicate that the IL-6 gene is more tightly regulated in T cells than in other systems described previously.