Flow-induced DNA synthesis requires signaling to a translational control pathway.

BACKGROUND: The mTOR translational control pathway that signals to the P70/P85 S6 kinase (pp70(S6k)) is essential for mitogenesis. We have previously shown that pp70(S6k) is activated by fluid flow. We hypothesized that oscillatory fluid flow in the absence of exogenous mitogens would induce endothelial cells to synthesize DNA via activation of the mTOR pathway. For comparison, we also studied the ERK1/2 transcriptional signaling pathway. METHODS: Confluent human umbilical vein endothelial cells (HUVECs) were exposed to oscillatory flow (12 dyn/cm(2) peak shear stress; 3.3 Hz) or kept static in serum-deprived culture medium. Rapamycin or PD98059 was used to inhibit pp70(S6k) or ERK1/2 activation, respectively. RESULTS: Oscillatory flow activated both the pp70(S6k) and ERK1/2 signaling pathways. Rapamycin blocked activation of pp70(S6k) but not ERK1/2, while PD98059 blocked ERK1/2 but not pp70(S6k). DNA synthesis, as measured by [3H]thymidine uptake, increased by approximately twofold (P < 0.01) in HUVEC cultures exposed to oscillatory flow compared with those kept static. Rapamycin completely abolished the flow-induced increase in DNA synthesis while PD98059 did not. Oscillatory flow upregulated expression of cyclin-dependent kinases 1 and 4 mRNA in a temporal pattern consistent with cell cycle entry; rapamycin also inhibited these changes. CONCLUSIONS: Oscillatory flow activates both the ERK 1/2 and pp70(S6k) signaling pathways in HUVECs and induces DNA synthesis in the absence of other exogenous mitogens. Complete blockade of [3H]thymidine uptake by the mTOR pathway inhibitor rapamycin indicates that separate and distinct signaling to a translational control pathway is necessary to mediate flow-induced DNA synthesis by endothelial cells. Oscillatory flow-induced endothelial proliferation may contribute to atherogenesis.

Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila.

Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells.

Cellular phenotype is determined not only by genetic transcription but also by subsequent translation of mRNA into protein. Extracellular signals trigger intracellular pathways that distinctly activate translation. The 70/85-kDa S6 kinase (pp70(S6k)) is a central enzyme in the signal-dependent control of translation, but its regulation in endothelial cells is largely unknown. Here we show that fluid flow (in the absence of an exogenous mitogen) as well as humoral agonists activate endothelial pp70(S6k). Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), and wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked flow-induced pp70(S6k) activation; FK-506, a rapamycin analog with minimal mTOR inhibitory activity, and PD-98059, an inhibitor of the flow-sensitive mitogen-activated protein kinase pathway, had no effect. Synthesis of Bcl-3, a protein whose translation is controlled by an mTOR-dependent pathway, was induced by flow and inhibited by rapamycin and wortmannin. Transcriptional blockade did not abolish the flow-induced upregulation of Bcl-3. Fluid forces may therefore modify endothelial phenotype by specifically regulating translation of certain mRNA transcripts into protein.

Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex.

Regulation of N-methyl-D-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor-associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

Extensive zygotic control of the anteroposterior axis in the wasp Nasonia vitripennis.

Insect axis formation is best understood in Drosophila melanogaster, where rapid anteroposterior patterning of zygotic determinants is directed by maternal gene products. The earliest zygotic control is by gap genes, which determine regions of several contiguous segments and are largely conserved in insects. We have asked genetically whether early zygotic patterning genes control similar anteroposterior domains in the parasitoid wasp Nasonia vitripennis as in Drosophila. Nasonia is advantageous for identifying and studying recessive zygotic lethal mutations because unfertilized eggs develop as males while fertilized eggs develop as females. Here we describe recessive zygotic mutations identifying three Nasonia genes: head only mutant embryos have posterior defects, resembling loss of both maternal and zygotic Drosophila caudal function; headless mutant embryos have anterior and posterior gap defects, resembling loss of both maternal and zygotic Drosophila hunchback function; squiggy mutant embryos develop only four full trunk segments, a phenotype more severe than those caused by lack of Drosophila maternal or zygotic terminal gene functions. These results indicate greater dependence on the zygotic genome to control early patterning in Nasonia than in the fly.