Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

A TRPC1/TRPC3-mediated increase in store-operated calcium entry is required for differentiation of H19-7 hippocampal neuronal cells

Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba2+ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigargin-stimulated Ba2+ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.     

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C     

Effect of incremental exercise on airway and systemic inflammation in patients with COPD

Airway and systemic inflammation are features of chronic obstructive pulmonary disease (COPD), and there is growing interest in clarifying the inflammatory processes. Strenuous exercise induces an intensified systemic inflammatory response in patients with COPD, but no study has investigated the airway inflammatory and anti-inflammatory responses to exercise. Twenty steroid-na?ve, ex-smokers with diagnosed COPD (forced expired volume in 1 s = 66 ± 12%) underwent baseline collection of venous blood and induced sputum followed by an incremental exercise test to symptom limitation 48 h later. Additional venous blood samples were collected following exercise at 0, 2, and 24 h, while induced sputum was collected 2 and 24 h after exercise. Sputum and blood samples were analyzed for differential cell count, CD4(+) and CD8(+) T lymphocytes (serum only), interleukin (IL)-6, IL-8, IL-10, chemokine (C-C motif) ligand 5 (CCL5), and high sensitivity C-reactive protein (serum only). There was an increase in the number of sputum eosinophils (cells/gram, P = 0.012) and a reduction in sputum IL-6 (P = 0.01) 24 h postexercise. Sputum IL-8 and CCL5 were also persistently decreased after exercise (P = 0.0098 and P = 0.0012, respectively), but sputum IL-10 did not change. There was a decrease in serum eosinophils 2 h after exercise (P = 0.0014) and a reduction in serum CCL5 immediately following and 2 h postexercise (P < 0.0001). Both serum eosinophils and CCL5 returned to baseline levels within 24 h. An acute bout of exercise resulted in a significant increase in the number of sputum eosinophils, which may be mediated by serum CCL5. However, there was also a reduction in sputum proinflammatory cytokines, suggesting some anti-inflammatory effect of exercise in the lungs of steroid-na?ve patients with COPD.     

Sorcery's the curse: modernity, envy and the flow of sociality in a Melanesian society

This article examines the widely reported phenomenon of the efflorescence of magical beliefs and practices, often explained as a response by some societies to their incorporation into the cash and market economy. Rather than accepting this view that the phenomenon is a generalizable response to modernity, I seek to understand it in its own terms as a singular response to the introduction of new forms of wealth into a specific cultural framework. Taking the example of a particular society, the Lelet of Papua New Guinea, I show how the expansion of forms of wealth has led to a loss of control over the processes by which sociality was previously regulated. This discussion proceeds through consideration of Lelet management of valuables and desire, and the importance of visuality in this.     

p53 Down Regulates PDGF-Induced Formation of Circular Dorsal Ruffles in Rat Aortic Smooth Muscle Cells

The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.     

Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.     

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.