Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E 2 that depended on cyclooxygenase-2 activity, and PGE 2 stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE 2 to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE 2 , and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.

Stimulated endothelial cells (EC) assume an activated phenotype with pro-inflammatory and prothrombotic features, requiring new gene and protein expression. New protein synthesis in activated EC is largely regulated by transcriptional events controlled by a variety of transcription factors. However, post-transcriptional control of gene expression also influences phenotype and allows the cell to alter protein expression in a faster and more direct way than is typically possible with transcriptional mechanisms. We sought to demonstrate that post-transcriptional control of gene expression occurs during EC activation. Using thrombin-activated EC and a high-throughput, microarray-based approach, we identified a number of gene products that may be regulated through post-transcriptional mechanisms, including the AP-1 transcription factor JunB. Using polysome profiling, cytoplasts and other standard cell biologic techniques, JunB is shown to be regulated at a post-transcriptional level during EC activation. In activated EC, the AP-1 transcription factor JunB, is regulated on a post-transcriptional level. Signal-dependent control of translation may regulate transcription factor expression and therefore, subsequent transcriptional events in stimulated EC.

Objective Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRES). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (EC) is unknown. Methods and Results We performed microarray analysis of polyribosomal mRNA from EC to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eIF4G cleavage, and decreased protein synthesis. 87.4% of mRNAs were dissociated from polysomes in virus-infected EC. 12% of mRNAs remained associated with polysomes and 0.6% were enriched ≥2-fold in polysome fractions from infected EC. Quantitative RT-PCR confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of PDCD8 and JunB mRNA resulted in increased protein expression in PV-infected EC. The presence of IRES in the 5’UTR of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. Conclusions We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected EC successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.

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