Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog

TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.     

A growth factor-repressible gene associated with protein kinase C- mediated inhibition of adipocyte differentiation

The conversion of determined adipoblasts to fully differentiated adipocytes requires appropriate environmental conditions. A strict dependence on cell confluence and a facilitation by glucocorticoid hormones have previously been described. We have found that agents that are capable of activating protein kinase C, such as basic fibroblast growth factor and phorbol esters, inhibit the differentiation of the adipogenic cell line TA1 without stimulating cell growth. Here we describe the sequence and characterization of a cDNA (clone 5) that detects an RNA, the expression of which is enhanced by glucocorticoids and increasing cell density. In contrast, activators of protein kinase C including basic fibroblast growth factor, phorbol esters, and synthetic diacylglycerols inhibit clone 5 gene expression. It appears that clone 5 expression is closely linked to environmental and hormonal factors that promote the differentiation of adipogenic cells.     

Pyruvate Kinase M2 Expression,but Not Pyruvate Kinase Activity,Is Up-Regulated in a Grade-Specific Manner in Human Glioma

Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose de novo or progressed from lower grade tumors, showed a 3–5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth in vitro and in vivo. These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.     

Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men

Serine/threonine kinase 33 (STK33/Stk33) is a recently discovered gene whose inferred amino acid sequence translation displays characters typical for a calcium/calmodulin dependent kinase (CAMK). In this study we analysed the STK33/Stk33 RNA and protein distribution and the localization of the protein. The STK33/Stk33 expression pattern resembles those of some related members of the CAMK group. STK33/Stk33 displays a nonubiquitous and, in most tissues, low level of expression. It is highly expressed in testis, particularly in cells from the spermatogenic epithelia. Moreover, significant expression is detected in lung epithelia, alveolar macrophages, horizontal cells in the retina and in embryonic organs such as heart, brain and spinal cord. A possible role of STK33/Stk33 in spermatogenesis and organ ontogenesis is discussed.     

MicroRNAs in breast cancer: Roles,functions, and mechanism of actions

Breast cancer is one of the most lethal malignancies in women in the world. Various factors are involved in the development and promotion of the malignancy; most of them involve changes in the expression of certain genes, such as microRNAs (miRNAs). MiRNAs can regulate signaling pathways negatively or positively, thereby affecting tumorigenesis and various aspects of cancer progression, particularly breast cancer. Besides, accumulating data demonstrated that miRNAs are a novel tool for prognosis and diagnosis of breast cancer patients. Herein, we will review the roles of these RNA molecules in several important signaling pathways, such as transforming growth factor, Wnt, Notch, nuclear factor-κ B, phosphoinositide-3-kinase/Akt, and extracellular-signal-regulated kinase/mitogen activated protein kinase signaling pathways in breast cancer.     

Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways

Matrix metalloproteinases (MMP) play a pivotal role in the pathogenesis of cardiovascular diseases. Their expressions are altered in response to a variety of stimuli, including growth factors, inflammatory markers, and cytokines. In this study, we demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces a dose- and time-dependent increase in MMP-2 expression in rat vascular smooth muscle cells (VSMC). Treatment with either the Rho-associated protein kinase (ROCK) inhibitor Y-27632 or suppression of ROCK-1/2 by small interfering RNA technology significantly reduced the MMP-2 expression, thus suggesting that ROCK regulates such expression. Similar results were observed when VSMC were pretreated with either U0126 or SB203580, which are selective inhibitors of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, respectively, thus suggesting that these kinases are important for the induction of MMP-2 expression by PDGF-BB. In conclusion, these results described a novel mechanism in atherosclerosis through PDGF-BB signaling in VSMC, in which MMP-2 expression is induced via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase phosphorylation, as well as ROCK.     

Differential enhancement of neural and photoreceptor cell differentiation of cultured pineal cells by FGF-1, IGF-1, and EGF

There are several common features between the pineal organ and the lateral eye in their developmental and evolutionary aspects. The avian pineal is a photoendocrine organ that originates from the diencephalon roof and represents a transitional type between the photosensory organ of lower vertebrates and the endocrine gland of mammals. Previous cell culture studies have shown that embryonic avian pineal cells retain a wide spectrum of differentiative capacities, although little is known about the mechanisms involved in their fate determination. In the present study, we investigated the effects of various cell growth factors on the differentiation of photoreceptor and neural cell types using pineal cell cultures from quail embryos. The results show that IGF-1 promotes differentiation of rhodopsin-immunoreactive cells, but had no effect on neural cell differentiation. Simultaneous administration of EGF and IGF-1 further enhanced differentiation of rhodopsin-immunoreactive cells, although the mechanism of the synergistic effect is unknown. FGF-1 did not stimulate proliferation of neural progenitor cells, but intensively promoted and maintained expression of a neural cell phenotype. FGF-1 appeared to lead to the conversion from an epithelial (endocrinal) to a neuronal type. It also enhanced phenotypic expression of retinal ganglion cell markers but rather suppressed expression of an amacrine cell marker. These results indicate that growth factors are important regulatory cues for pineal cell differentiation and suggest that they play roles in determining the fate of the pineal organ and the eye. It can be speculated that the differences in environmental cues between the retina and pineal may result in the transition of the pineal primordium from a potentially ocular (retinal) organ to a photoendocrine organ.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Organ-specific expression of Arabidopsis genome during development

The development of complex eukaryotic organisms can be viewed as the selective expression of distinct fractions of the genome in different organs or tissue types in response to developmental and environmental cues. Here, we generated a genome expression atlas of 18 organ or tissue types representing the life cycle of Arabidopsis (Arabidopsis thaliana). We showed that each organ or tissue type had a defining genome expression pattern and that the degree to which organs share expression profiles is highly correlated with the biological relationship of organ types. Further, distinct fractions of the genome exhibited expression changes in response to environmental light among the three seedling organs, despite the fact that they share the same photo-perception and transduction systems. A significant fraction of the genes in the Arabidopsis genome is organized into chromatin domains exhibiting coregulated expression patterns in response to developmental or environmental signals. The knowledge of organ-specific expression patterns and their response to the changing environment provides a foundation for dissecting the molecular processes underlying development.     

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA-dependent protein kinase PKR

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.     

RAM pathway contributes to Rpb4 dependent pseudohyphal differentiation in Saccharomyces cerevisiae

Rpb4, a subunit of RNA Polymerase II plays an important role in various stress responses in budding yeast, Saccharomyces cerevisiae. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathway. In the present study, we show that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4Delta/rpb4Delta cells exhibit pseudohyphae even in the absence of functional MAP kinase and cAMP-PKA pathways. Genome-wide expression profiling showed that in the absence of Rpb4 several genes controlling mother daughter cell separation are down regulated. Our genetic studies also provide evidence for involvement of RNA Pol II subunit Rpb4 in the expression of genes downstream of the RAM pathway. Finally, we show that this effect on expression of RAM pathway may at least be partially responsible for the pseudohyphal phenotype of rpb4Delta/rpb4Delta cells.     

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.