Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing ␣21 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src ؊/؊ Fyn ؊/؊ fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an ␣21 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.
It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.Escherichia coli DNA topoisomerase I, a member of the type IA family of topoisomerases, specifically relaxes negatively supercoiled DNA (1, 2). This specificity is explained by the fact that this enzyme binds to the junction of single-stranded and double-stranded DNA regions. DNA opening, and hence the generation of single-stranded DNA regions, is promoted by negative but not positive supercoiling. Hot spots for relaxation by DNA topoisomerase I are provided during transcription elongation in the frame of the twin-domain model (3). Indeed, very high levels of negative supercoiling can be generated behind the moving RNA polymerase during transcription elongation (4, 5). An R-loop, in which the template strand is paired with the nascent RNA, leaving the nontemplate strand unpaired, also provides a hot spot for relaxation by this enzyme (6).The accumulated evidence over the last few years has allowed us to conclude that a major function of DNA topoisomerase I in E. coli is to inhibit R-loop formation during transcription elongation. Indeed, the growth problem of topA (encoding DNA topoisomerase I) null mutants was shown to be partially corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop (7). A correlation was also established between the level of DNA gyrase activity, the enzyme that introduces negative supercoiling within the chromosomal DNA, and the amount of RNase H required to stimulate the growth of topA null mutants (7) and to inhibit R-loop formation during transcription (8). The finding that several topA null mutants carry compensatory gyr mutations (in gyrA or gyrB) that reduce DNA gyrase activity and correct their growth defect (9, 10) was therefore explained by the supercoiling activity of DNA gyrase, which promotes R-loop formation (7). On the contrary, DNA topoisomerase I activity inhibits R-lo...
Cardiac arrest promotes activation of death-inducing molecules such as Bax and is associated with increased development of caspase-independent renal cell death during cold storage. Developing strategies, such as free radical scavenging, aimed at inhibiting cell death during cold storage, could prove useful for improving preservation of NHB kidneys.
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