RESUMO
Ethylene is a gas that has long been known to act as a plant hormone. We recently showed that a cyanobacterium, Synechocystis sp. PCC 6803 (Synechocystis) contains an ethylene receptor (SynEtr1) that regulates cell surface and extracellular components leading to altered phototaxis and biofilm formation. To determine whether other cyanobacteria respond to ethylene, we examined the effects of exogenous ethylene on phototaxis of the filamentous cyanobacterium, Geitlerinema sp. PCC 7105 (Geitlerinema). A search of the Geitlerinema genome suggests that two genes encode proteins that contain an ethylene binding domain and Geitlerinema cells have previously been shown to bind ethylene. We call these genes GeiEtr1 and GeiEtr2 and show that in air both are expressed. Treatment with ethylene decreases the abundance of GeiEtr1 transcripts. Treatment of Geitlerinema with 1000 nL L-1 ethylene affected the phototaxis response to white light as well as monochromatic red light, but not blue or green light. This is in contrast to Synechocystis where we previously found ethylene affected phototaxis to all three colors. We also demonstrate that application of ethylene down to 8 nL L-1 stimulates phototaxis of both cyanobacteria as well as biofilm formation of Synechocystis. We formerly demonstrated that the transcript levels of slr1214 and CsiR1 in Synechocystis are reduced by treatment with 1000 nL L-1 ethylene. Here we show that application of ethylene down to 1 nL L-1 causes a reduction in CsiR1 abundance. This is below the threshold for most ethylene responses documented in plants. By contrast, slr1214 is unaffected by this low level of ethylene and only shows a reduction in transcript abundance at the highest ethylene level used. Thus, cyanobacteria are very sensitive to ethylene. However, the dose-binding characteristics of ethylene binding to Geitlerinema and Synechocystis cells as well as to the ethylene binding domain of SynEtr1 heterologously expressed in yeast, are similar to what has been reported for plants and exogenously expressed ethylene receptors from plants. These data are consistent with a model where signal amplification is occurring at the level of the receptors.
RESUMO
Ethylene is well known as a plant hormone, but its role in bacteria is poorly studied. We recently showed that Synechocystis sp. Strain PCC 6803 has a functional receptor for ethylene, ethylene response 1 (Etr1), that is involved in various processes such as phototaxis in response to directional light and biofilm formation. Here, we use RNA sequencing to examine the changes in gene transcripts caused by ethylene under phototaxis conditions. Over 500 gene transcripts across many functional categories, of approximately 3700 protein-encoding genes, were altered by application of ethylene. In general, ethylene caused both up- and downregulation of genes within a functional category. However, the transcript levels of amino acid metabolism genes were mainly upregulated and cell envelope genes were mostly downregulated by ethylene. The changes in cell envelope genes correlate with our prior observation that ethylene affects cell surface properties to alter cell motility. Ethylene caused a twofold or more change in 62 transcripts with the largest category of upregulated genes annotated as transporters and the largest category of downregulated genes annotated as glycosyltransferases which sometimes are involved in changing the composition of sugars on the cell surface. Consistent with changes in cell envelope, glycosyltransferase, and transporter gene transcripts, application of ethylene altered the levels of specific sugar moieties on the surface of cells. Light signaling from Etr1 involves two proteins (Slr1213 and Slr1214) and a small, noncoding RNA, carbon stress-induced RNA1 (csiR1). Application of ethylene caused a rapid, but transient, decrease in the transcript levels of etr1, slr1213, and slr1214 and a rapid and prolonged decrease in csiR1 transcript. Deletion of Slr1214 caused a large increase in csiR1 transcript levels and ethylene lowered csiR1 transcript. These data combined with prior reports indicate that ethylene functions as a signal to affect a variety of processes altering the physiology of Synechocystis cells.
RESUMO
BACKGROUND: Distant prostate cancers are commonly hormone refractory and exhibit increased growth no longer inhibited by androgen deprivation therapy. Understanding all molecular mechanisms contributing to uncontrolled growth is important to obtain effective treatment strategies for hormone refractory prostate cancers (HRPC). The aryl hydrocarbon receptor (AhR) affects a number of biological processes including cell growth and differentiation. Several studies have revealed that exogenous AhR ligands inhibit cellular proliferation but recent evidence suggests AhR may possess intrinsic functions that promote cellular proliferation in the absence of exogenous ligands. METHODS/RESULTS: qRT-PCR and western blot analysis was used to determine AhR mRNA and protein expression in hormone sensitive LNCaP cells as well as hormone refractory DU145, PC3 and PC3M prostate cancer cell lines. LNCaP cells express AhR mRNA and protein at a much lower level than the hormone refractory cell models. Cellular fractionation and immunocytochemistry revealed nuclear localization of AhR in the established hormone refractory cell lines while LNCaP cells are devoid of nuclear AhR protein. qRT-PCR analysis used to assess basal CYP1B1 levels and a xenobiotic responsive element binding assay confirmed ligand independent transcriptional activity of AhR in DU145, PC3 and PC3M cells. Basal CYP1B1 levels were decreased by treatment with specific AhR inhibitor, CH223191. An in vitro growth assay revealed that CH223191 inhibited growth of DU145, PC3 and PC3M cells in an androgen depleted environment. Immunohistochemical staining of prostate cancer tissues revealed increased nuclear localization of AhR in grade 2 and grade 3 cancers compared to the well differentiated grade 1 cancers. CONCLUSIONS: Together, these results show that AhR is constitutively active in advanced prostate cancer cell lines that model hormone refractory prostate cancer. Chemical ablation of AhR signaling can reduce the growth of advanced prostate cancer cells, an effect not achieved with androgen receptor inhibitors or growth in androgen depleted media.