Interferon regulatory factor 4 sustains CD8(+) T cell expansion and effector differentiation.

Upon infection, CD8(+) T cells undergo a stepwise process of early activation, expansion, and differentiation into effector cells. How these phases are transcriptionally regulated is incompletely defined. Here, we report that interferon regulatory factor 4 (IRF4), dispensable for early CD8(+) T cell activation, was vital for sustaining the expansion and effector differentiation of CD8(+) T cells. Mechanistically, IRF4 promoted the expression and function of Blimp1 and T-bet, two transcription factors required for CD8(+) T cell effector differentiation, and simultaneously repressed genes that mediate cell cycle arrest and apoptosis. Selective ablation of Irf4 in peripheral CD8(+) T cells impaired antiviral CD8(+) T cell responses, viral clearance, and CD8(+) T cell-mediated host recovery from influenza infection. IRF4 expression was regulated by T cell receptor (TCR) signaling strength via mammalian target of rapamycin (mTOR). Our data reveal that IRF4 translates differential strength of TCR signaling into different quantitative and qualitative CD8(+) T cell responses.

Tumor-suppressive effects of microRNA-181d-5p on non-small-cell lung cancer through the CDKN3-mediated Akt signaling pathway in vivo and in vitro.

The involvement of several microRNAs (miRs) in the initiation and development of tumors through the suppression of the target gene expression has been highlighted. The aberrant expression of miR-181d-5p and cyclin-dependent kinase inhibitor 3 (CDKN3) in non-small-cell lung cancer (NSCLC) was then screened by microarray analysis. In the present study, we performed a series of in vivo and in vitro experiments for the purpose of investigating their roles in NSCLC and the underlying mechanism. There was a high expression of CDKN3, whereas miR-181d-5p was downregulated in NSCLC. Quantitative RT-PCR, Western blot analysis, and dual-luciferase reporter gene assay further identified that CDKN3 could be negatively regulated by miR-181d-5p. Moreover, the upregulation of miR-181d-5p or silencing of CDKN3 could inactivate the Akt signaling pathway. A549 with the lowest miR-181d-5p and H1975 with the highest CDKN3 among the five NSCLC cell lines (H1299, A549, H1975, NCI-H157, and GLC-82) were adopted for in vitro experiments, in which expression of miR-181d-5p and CDKN3 was altered by transfection of miR-181d-5p mimic/inhibitor or siRNA-targeting CDKN3. Afterwards, cell proliferation, apoptosis, invasion, migration, and angiogenesis, as well as epithelial-mesenchymal transition (EMT), were evaluated, and tumorigenicity was assessed. In addition, an elevation in miR-181d-5p or depletion in CDKN3 led to significant reductions in proliferation, invasion, migration, angiogenesis, EMT, and tumorigenicity of NSCLC cells, coupling with increased cell apoptosis. In conclusion, this study highlights the tumor-suppressive effects of miR-181d-5p on NSCLC via Akt signaling pathway inactivation by suppressing CDKN3, thus providing a promising therapeutic strategy for the treatment of NSCLC.

Arabidopsis JAGGED links floral organ patterning to tissue growth by repressing Kip-related cell cycle inhibitors.

Plant morphogenesis requires coordinated cytoplasmic growth, oriented cell wall extension, and cell cycle progression, but it is debated which of these processes are primary drivers for tissue growth and directly targeted by developmental genes. Here, we used ChIP high-throughput sequencing combined with transcriptome analysis to identify global target genes of the Arabidopsis transcription factor JAGGED (JAG), which promotes growth of the distal region of floral organs. Consistent with the roles of JAG during organ initiation and subsequent distal organ growth, we found that JAG directly repressed genes involved in meristem development, such as CLAVATA1 and HANABA TARANU, and genes involved in the development of the basal region of shoot organs, such as BLADE ON PETIOLE 2 and the GROWTH REGULATORY FACTOR pathway. At the same time, JAG regulated genes involved in tissue polarity, cell wall modification, and cell cycle progression. In particular, JAG directly repressed KIP RELATED PROTEIN 4 (KRP4) and KRP2, which control the transition to the DNA synthesis phase (S-phase) of the cell cycle. The krp2 and krp4 mutations suppressed jag defects in organ growth and in the morphology of petal epidermal cells, showing that the interaction between JAG and KRP genes is functionally relevant. Our work reveals that JAG is a direct mediator between genetic pathways involved in organ patterning and cellular functions required for tissue growth, and it shows that a regulatory gene shapes plant organs by releasing a constraint on S-phase entry.

FBF represses the Cip/Kip cell-cycle inhibitor CKI-2 to promote self-renewal of germline stem cells in C. elegans.

Although the decision between stem cell self-renewal and differentiation has been linked to cell-cycle modifications, our understanding of cell-cycle regulation in stem cells is very limited. Here, we report that FBF/Pumilio, a conserved RNA-binding protein, promotes self-renewal of germline stem cells by repressing CKI-2(Cip/Kip), a Cyclin E/Cdk2 inhibitor. We have previously shown that repression of CYE-1 (Cyclin E) by another RNA-binding protein, GLD-1/Quaking, promotes germ cell differentiation. Together, these findings suggest that a post-transcriptional regulatory circuit involving FBF and GLD-1 controls the self-renewal versus differentiation decision in the germline by promoting high CYE-1/CDK-2 activity in stem cells, and inhibiting CYE-1/CDK-2 activity in differentiating cells.

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation.

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.

Critical requirement for cell cycle inhibitors in sustaining nonproliferative states.

In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference-mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D-cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle-driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.

Cyclin-dependent kinase inhibitors as potential targeted anticancer agents.

Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Genes involved in cell cycle are frequently mutated in human cancer and deregulated CDK activity represents a hallmark of malignancy. This knowledge provides a rationale for regarding CDKs and their associated molecules as potential targets for new drug development in anticancer research. The present article will review the most relevant CDK inhibitors with emphasis on the newer molecules in clinical development and the biological rationale of this therapeutic approach.

Cyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis.

OBJECTIVE: The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions. MATERIALS AND METHODS: Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA. RESULTS: Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation. CONCLUSION: We conclude that both p21(CIP1) and p27(KIP1) induce partial secretory differentiation of mammary cells in monolayer, but during acinus morphogenesis in 3D culture they have a highly regulated temporal expression pattern.

E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs.

Recently, long noncoding RNAs (lncRNAs) have been shown to have important regulatory roles in human cancer biology. By utilizing publicly available lncRNAs expression profiling data and integrating analyses, we screened out LINC00668, whose expression is significantly increased and correlated with outcomes in gastric cancer (GC). Further experiments revealed that LINC00668 knockdown significantly repressed proliferation, both in vitro and in vivo. Mechanistic investigations showed that LINC00668 was a direct transcriptional target of E2F transcription factor 1 (E2F1). We further demonstrated that LINC00668 was associated with PRC2 and that this association was required for epigenetic repression of cyclin-dependent protein kinase inhibitors (CKIs), including p15, p16, p21, p27 and p57, thus contributing to the regulation of the gastric cancer cell cycle. Our results suggest that E2F1-activated LINC00668, as a cell cycle regulator, enriches the mechanistic link between lncRNA and the E2F1-mediated cell cycle regulation pathway and may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer.

Computational experiments reveal the efficacy of targeting CDK2 and CKIs for significantly lowering cellular senescence bar for potential cancer treatment.

Lowering the threshold of cellular senescence, the process employed by cells to thwart abnormal cell proliferation, though inhibition of CDK2 or Skp2 (regulator of CDK inhibitors) has been recently suggested as a potential avenue for cancer treatment. In this study, we employ a published mathematical model of G1/S transition involving the DNA-damage signal transduction pathway to conduct carefully constructed computational experiments to highlight the effectiveness of manipulating cellular senescence in inhibiting damaged cell proliferation. We first demonstrate the suitability of the mathematical model to explore senescence by highlighting the overlap between senescence pathways and those involved in G1/S transition and DNA damage signal transduction. We then investigate the effect of CDK2 deficiency on senescence in healthy cells, followed by effectiveness of CDK2 deficiency in triggering senescence in DNA damaged cells. For this, we focus on the behaviour of CycE, whose peak response indicates G1/S transition, for several reduced CDK2 levels in healthy as well as two DNA-damage conditions to calculate the probability (ß) or the percentage of CDK2 deficient cells passing G1/S checkpoint ((1-ß) indicates level of senescence). Results show that 50% CDK2 deficiency can cause senescence in all healthy cells in a fairly uniform cell population; whereas, most healthy cells (≈67%) in a heterogeneous population escape senescence. This finding is novel to our study. Under both low- and high-DNA damaged conditions, 50% CDK deficiency can cause 65% increase in senescence in a heterogeneous cell population. Furthermore, the model analyses the relationship between CDK2 and its CKIs (p21, p27) to help search for other effective ways to bring forward cellular senescence. Results show that the degradation rate of p21 and initial concentration of p27 are effective in lowering CDK2 levels to lower the senescence threshold. Specifically, CDK2 and p27 are the most effective in triggering senescence while p21 having a smaller influence. While receiving experimental support, these findings specify in detail the inhibitory effects of CKIs. However, simultaneous variation of CDK2 and CKIs produces a dramatic reduction of damage cells passing the G1/S with CDK2&p27 combination causing senescence in almost all damaged cells. This combined effect of CDK2&CKIs on senescence is a novel contribution in this study. A review of the crucial protein complexes revealed that the concentration of active CycE/CDK2-p that controls cell cycle arrest provides support for the above findings with CycE/CDK2-p undergoing the largest reduction (over 100%) under the combined CDK2&CKI conditions leading to the arrest of most of the damaged cells. Our study thus provides quantitative assessments for the previously published qualitative findings on senescence and highlights new avenues for bringing forward senescence bar.

Hiwi mediated tumorigenesis is associated with DNA hypermethylation.

Expression of Piwi proteins is confined to early development and stem cells during which they suppress transposon migration via DNA methylation to ensure genomic stability. Piwi's genomic protective function conflicts with reports that its human ortholog, Hiwi, is expressed in numerous cancers and prognosticates shorter survival. However, the role of Hiwi in tumorigenesis has not been examined. Here we demonstrate that (1) over-expressing Hiwi in sarcoma precursors inhibits their differentiation in vitro and generates sarcomas in vivo; (2) transgenic mice expressing Hiwi (mesodermally restricted) develop sarcomas; and (3) inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. Our data indicates that Hiwi is directly tumorigenic and Hiwi-expressing cancers may be addicted to Hiwi expression. We further show that Hiwi associated DNA methylation and cyclin-dependent kinase inhibitor (CDKI) silencing is reversible along with Hiwi-induced tumorigenesis, via DNA-methyltransferase inhibitors. Our studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.

Trichostatin A prevents neointimal hyperplasia via activation of Krüppel like factor 4.

The proliferation of vascular smooth muscle cells (VSMCs) is an integral part of the mechanism of vascular diseases such as restenosis. Post-translational modifications by histone deacetylase (HDAC) inhibitors play an important role in the regulation of gene expression by inducing cell cycle arrest. However, the role and mechanism of the HDAC inhibitor trichostatin A (TSA) on neointimal proliferation remain unknown. In this study, we investigated the effect and mechanism whereby TSA prevents the proliferation of VSMCs and neointimal hyperplasia induced by balloon injury in rat carotid artery. Local administration of TSA significantly prevented neointimal hyperplasia. TSA dramatically inhibited the proliferation and DNA synthesis of VSMCs in response to FBS or PDGF-BB. Overexpression of Krüppel like factor 4 (KLF4) blocked the cell proliferation and DNA synthesis, as determined by the MTT and [³H]thymidine incorporation assays, whereas knockdown of KLF4 resulted in an increase in VSMC proliferation. In VSMCs, TSA increased the mRNA level and protein expression of KLF4. Treatment with TSA or transfection of KLF4 increased the expression of both p21 and p27 and promoter activity. In addition, the anti-proliferative activity of TSA was recovered in KLF4-knockdown cells. These data demonstrate that TSA inhibits neointimal thickening and VSMC proliferation via activation of the KLF4/p21/p27 signaling pathway.

Risk loci for type 2 diabetes - quo vadis?

Reduced insulin sensitivity plays a role in the early pathogenesis of type 2 diabetes, and defects in insulin secretion by pancreatic beta-cells are instrumental in hyperglycemic progression. There is strong evidence that genetic factors play an important role in both of these components. Several of the single nucleotide polymorphisms (SNPs) of genes associated with an increased risk of type 2 diabetes are hypothesized to influence beta-cell function. The aim of the present study was to describe the function of the latter genes, to analyze the implications of the SNP positions within or near these genes, and to evaluate the suggested primary role of pancreatic beta-cells in the etiology of type 2 diabetes.

Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma.

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.

Multiple cyclin kinase inhibitors promote bile acid-induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling.

Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.