MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. (65/61862)

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP-regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP-mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/ Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.  (+info)

Proadrenomedullin N-terminal 20 peptide hyperpolarizes the membrane by activating an inwardly rectifying K+ current in differentiated PC12 cells. (66/61862)

The mechanism of proadrenomedullin N-terminal 20 peptide (PAMP)-induced inhibition of catecholamine release from adrenergic nerve was investigated in nerve growth factor-treated PC12 cells that have differentiated characteristics somewhat similar to noradrenergic neurons. The effect of PAMP on the excitability of these cells was investigated with the use of perforated whole-cell clamp. PAMP hyperpolarized the membrane by increasing a K+ conductance in a dose-dependent manner. The current-voltage relationship (I-V) relationship of the PAMP-induced K+ conductance exhibited inward-going rectification. The activation was abolished by microinjecting GDPbetaS into the cells or pretreating the cells with pertussis toxin. These results indicate that a pertussis toxin-sensitive G protein is involved in the signal transduction. The PAMP-induced activation of the K+ conductance was attenuated by microinjecting antibody against the carboxyl terminus of Galphai3, but it was not influenced by microinjecting antibody against the common carboxyl termini of Galphai1 and Galphai2, which indicated that the G protein coupling the PAMP receptor to the inwardly rectifying K+ current is Galphai3. The PAMP-induced hyperpolarization may inhibit the catecholamine release from the neurons by attenuating the action potential frequency.  (+info)

Cbfa1 isoforms exert functional differences in osteoblast differentiation. (67/61862)

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. We investigated functional differences among three isoforms of Cbfa1: Type I (originally reported as Pebp2alphaA by Ogawa et al. (Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863), Type II (originally reported as til-1 by Stewart et al. (Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter, E., Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8646-8651), and Type III (originally reported as Osf2/Cbfa1 by Ducy et al. (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). A reverse transcriptase-polymerase chain reaction analysis demonstrated that these isoforms were expressed in adult mouse bones. The transient transfection of Type I or Type II Cbfa1 in a mouse fibroblastic cell line, C3H10T1/2, induced the expression of alkaline phosphatase (ALP) activity. This induction was synergistically enhanced by the co-introduction of Xenopus BMP-4 cDNA. In contrast, the transient transfection of Type III cDNA induced no ALP activity. In C3H10T1/2 cells stably transfected with each isoform of Cbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type II Cbfa1 but not in cells with Type III Cbfa1. Osteocalcin, osteopontin,and type I collagen gene expressions were induced or up-regulated in all of the cells stably transfected with each isoform of Cbfa1, and Type II transfected cells exhibited the highest expression level of osteocalcin gene. A luciferase reporter gene assay using a 6XOSE2-SV40 promoter (6 tandem binding elements for Cbfa1 ligated in front of the SV40 promoter sequence), a mouse osteocalcin promoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by each Cbfa1 isoform with or without its beta-subunit. These results suggest that all three of the Cbfa1 isoforms used in the present study are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation.  (+info)

CREB-binding [corrected] protein interacts with the homeodomain protein Cdx2 and enhances transcriptional activity. (68/61862)

Cdx2 encodes for a homeodomain protein that is expressed in intestinal epithelial cells. The Cdx2 protein triggers intestinal differentiation in cell lines and is necessary for maintenance of the intestinal phenotype in mice. CBP (cAMP response element-binding protein) is a transcriptional co-activator that interacts with many transcription factors and components of the basal transcriptional machinery. In this study, we demonstrate that CBP is markedly induced upon differentiation of the Caco-2 intestinal cell line and augments Cdx2-dependent transcriptional activity. Cdx2 interacts with the amino-terminal domain of CBP, and the two proteins coexist in vivo within the same nuclear protein complex. Moreover, expression of the CBP domain that interacts with Cdx2 acts as a dominant-negative inhibitor of transcriptional activation by Cdx2. These findings demonstrate a direct interaction between an intestinal homeodomain protein and CBP and suggest that CBP participates in the network of transcriptional proteins that direct intestinal differentiation.  (+info)

Anti-apoptotic role of telomerase in pheochromocytoma cells. (69/61862)

Telomerase is a protein-RNA enzyme complex that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes and thereby prevents their shortening. Reduced telomerase activity is associated with cell differentiation and accelerated cellular senescence, whereas increased telomerase activity is associated with cell transformation and immortalization. Because many types of cancer have been associated with reduced apoptosis, whereas cell differentiation and senescence have been associated with increased apoptosis, we tested the hypothesis that telomerase activity is mechanistically involved in the regulation of apoptosis. Levels of telomerase activity in cultured pheochromocytoma cells decreased prior to cell death in cells undergoing apoptosis. Treatment of cells with the oligodeoxynucleotide TTAGGG or with 3,3'-diethyloxadicarbocyanine, agents that inhibit telomerase activity in a concentration-dependent manner, significantly enhanced mitochondrial dysfunction and apoptosis induced by staurosporine, Fe2+ (an oxidative insult), and amyloid beta-peptide (a cytotoxic peptide linked to neuronal apoptosis in Alzheimer's disease). Overexpression of Bcl-2 and the caspase inhibitor zVAD-fmk protected cells against apoptosis in the presence of telomerase inhibitors, suggesting a site of action of telomerase prior to caspase activation and mitochondrial dysfunction. Telomerase activity decreased in cells during the process of nerve growth factor-induced differentiation, and such differentiated cells exhibited increased sensitivity to apoptosis. Our data establish a role for telomerase in suppressing apoptotic signaling cascades and suggest a mechanism whereby telomerase may suppress cellular senescence and promote tumor formation.  (+info)

Morphological variations in transplanted tumors developed by inoculation of spontaneous mesothelioma cell lines derived from F344 rats. (70/61862)

Morphological and immunohistochemical features of the abdominal mesotheliomas that were developed by inoculation of 3 cell lines (MeET-4, -5 and -6) established from spontaneous abdominal mesotheliomas in male F344 rats. Although the original tumors of three cell lines showed signs of epithelioid growth with a predominantly simple papillary pattern, transplanted tumors revealed a variety of morphologic features including epithelioid with glandular structures, sarcomatous, and a mixture of these components. All tumor cells of transplanted tumors were positive for alpha-smooth muscle actin (ASMA) but almost negative for desmin as were epithelioid cells of the original tumors, and the cell lines were positive for desmin but not for ASMA. These results suggested that mesothelioma in the F344 rat had the potential for wide spectrum differentiation under in vitro conditions. The microenvironmental factors obtained in vivo can modify their potential ability and their morphological aspects. These factors may be related to tumor cell reexpression of ASMA of tumor cells that were masked under in vitro culture conditions.  (+info)

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity. (71/61862)

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.  (+info)

A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development? (72/61862)

A well-described population of cardiac neural crest (NC) cells migrates toward the arterial pole of the embryonic heart and differentiates into various cell types, including smooth muscle cells of the pharyngeal arch arteries (but not the coronary arteries), cardiac ganglionic cells, and mesenchymal cells of the aortopulmonary septum. Using a replication-incompetent retrovirus containing the reporter gene LacZ, administered to the migratory neural crest of chicken embryos, we demonstrated another population of cardiac neural crest cells that employs the venous pole as entrance to the heart. On the basis of our present data we cannot exclude the possibility that precursors of these cells might not only originate from the dorsal part of the posterior rhombencephalon, but also from the ventral part. These NC cells migrate to locations surrounding the prospective conduction system as well as to the atrioventricular (AV) cushions. Concerning the prospective conduction system, the tagged neural crest cells can be found in regions where the atrioventricular node area, the retroaortic root bundle, the bundle of His, the left and right bundle branches, and the right atrioventricular ring bundle are positioned. The last area connects the posteriorly located AV node area with the retroaortic root bundle, which receives its neural crest cells through the arterial pole in concert with the cells giving rise to the aortopulmonary septum. The NC cells most probably do not form the conduction system proper, as they enter an apoptotic pathway as determined by concomitant TUNEL detection. It is possible that the NC cells in the heart become anoikic and, as a consequence, fail to differentiate further and merely die. However, because of the perfect timing of the arrival of crest cells, their apoptosis, and a change in electrophysiological behavior of the heart, we postulate that neural crest cells play a role in the last phase of differentiation of the cardiac conduction system. Alternatively, the separation of the central conduction system from the surrounding working myocardium is mediated by apoptotic neural crest cells. As for the presence of NC cells in both the outflow tract and the AV cushions, followed by apoptosis, a function is assigned in the muscularization of both areas, resulting in proper septation of the outflow tract and of the AV region. Failure of normal neural crest development may not only play a role in cardiac outflow tract anomalies but also in inflow tract abnormalities, such as atrioventricular septal defects.  (+info)