Pollen germination, along with pollen tube growth, is an essential process for the reproduction of flowering plants. The germinating pollen with tip-growth characteristics provides an ideal model system for the study of cell growth and morphogenesis. As an essential step toward a detailed understanding of this important process, the objective of this study was to comprehensively analyze the transcriptome changes during pollen germination and pollen tube growth. Using Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 Genome Arrays, this study is, to our knowledge, the first to show the changes in the transcriptome from desiccated mature pollen grains to hydrated pollen grains and then to pollen tubes of Arabidopsis. The number of expressed genes, either for total expressed genes or for specifically expressed genes, increased significantly from desiccated mature pollen to hydrated pollen and again to growing pollen tubes, which is consistent with the finding that pollen germination and tube growth were significantly inhibited in vitro by a transcriptional inhibitor. The results of Gene Ontology analyses showed that expression of genes related to cell rescue, transcription, signal transduction, and cellular transport was significantly changed, especially for up-regulation, during pollen germination and tube growth. In particular, genes of the calmodulin/calmodulin-like protein, cation/hydrogen exchanger, and heat shock protein families showed the most significant changes during pollen germination and tube growth. These results demonstrate that the overall transcription of genes, both in the number of expressed genes and in the levels of transcription, was increased. Furthermore, the appearance of many novel transcripts during pollen germination as well as tube growth indicates that these newly expressed genes may function in this complex process.

The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. To probe these questions, we mutated specific lysines in the N-termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5. We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro. Moreover, in the absence of GCN5, cells accumulate in G 2 /M indicating that Gcn5p functions are important for normal cell-cycle progression. Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells. Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4. Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N-terminal tail, results in the death of gcn5 cells. Mutation of these same three sites to glutamine is not lethal. Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4-VP16, supporting an important role for histone acetylation in Gcn5p-mediated regulation of transcription. Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability.

Aldosterone is a major regulator of epithelial Na؉ absorption and acts in large part through induction of the epithelial Na ؉ channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaC␣ transcription through mediating histone H3 Lys-79 methylation associated with the ENaC␣ promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaC␣. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaC␣ promoter at most, but not all subregions examined, repression of endogenous ENaC␣ mRNA expression and acts synergistically with Dot1a to inhibit ENaC␣ promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaC␣ promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaC␣ promoter and represses ENaC␣ transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.The epithelial sodium channel (ENaC) 2 is a heteromultimeric protein composed of three partially homologous subunits (␣, ␤, and ␥) that is expressed in the apical membrane of salt-absorbing epithelia of kidney, colon, and lung where it constitutes the rate-limiting steps in active Na ϩ and fluid absorption. ENaC plays a major role in the regulation of salt homeostasis and blood pressure as evidenced by the fact that ENaC mutations are associated with genetic hypertensive and hypotensive diseases, such as Liddle's syndrome (1) and pseudohypoaldosteronism type 1 (2), and the fact that it is subject to tight and complex regulation by aldosterone. Aldosterone is a major regulator of epithelial Na ϩ absorption and acts in large part through ENaC induction in the renal collecting duct (3, 4). Aldosterone administration or hyperaldosteronism induced by a low-Na ϩ diet increases ENaC␣ gene transcription, without increasing ␤-or ␥-subunit expression (5-9), and without a separate effect on ENaC␣ mRNA turnover (10) in this segment. Although ENaC␣ synthesis is believed to be the rate-limiting step in Na ϩ channel formation in the collecting duct, only limited information exists regarding the specific mechanisms governing transcriptional regulation of this gene, in particular epigenetic mechanisms exerting such controls.Traditional models of aldosterone trans-activation of target genes, including ENaC␣, have emphasized interaction of the liganded mineralocorticoid receptor or g...

Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilator molecule that plays important roles in diverse biological effects in the kidney, vasculature, and many other tissues. Because of its high biological reactivity and diffusibility, multiple tiers of regulation, ranging from transcriptional to posttranslational controls, tightly control NO biosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed. Dimerization of the NOS isozymes, required for their activity, exhibits distinguishing features among these proteins and may serve as a regulated process and target for therapeutic intervention. An increasingly wide array of proteins, ranging from scaffolding proteins to membrane receptors, has been shown to function as NOS-binding partners. Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins. Several resident and recruited proteins of plasmalemmal caveolae, including caveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOS in the endothelium. Inducible NOS (iNOS) interacts with the inhibitory molecules kalirin and NOS-associated protein 110 kDa, as well as activator proteins, the Rac GTPases. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. The calpain and ubiquitin-proteasome pathways are the major proteolytic systems responsible for the regulated degradation of NOS isozymes. The experimental basis for these protein-protein interactions, their functional importance, and potential implication for renal and vascular physiology and pathophysiology is reviewed.

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes -including that encoding the epithelial Na + channel (ENaC), a key arbiter of Na + transport in the kidney and other epithelia -have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCα by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum-and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCα promoter and derepression of ENaCα transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCα and likely other aldosterone-induced genes. IntroductionThe renin-angiotensin-aldosterone system plays a major role in the control of blood pressure, extracellular fluid volume, and electrolyte balance, largely through the regulation of urinary Na + excretion. The aldosterone-sensitive distal nephron (ASDN), composed of the late distal convoluted tubule, connecting tubule, and cortical and medullary collecting ducts, is the final arbiter of renal Na + excretion. In the ASDN, transepithelial Na + absorption occurs by apical Na + entry via the epithelial Na + channel (ENaC) and basolateral Na + exit via the Na + ,K + -ATPase. ENaC, composed of 3 subunits (α, β, and γ), constitutes the rate-limiting step in this process, and changes in its activity and/or plasma membrane abundance constitute key regulatory steps. Aldosterone increases transepithelial Na + transport in the ASDN in large part through ENaCα induction in this region (1). Aldosterone increases ENaC function in 2 phases: an early phase involving upregulation of preexisting transport machinery and aldosterone-induced regulatory proteins, notably serum- and glucocorticoid-induced kinase-1 (Sgk1), which regulates the plasma membrane abundance of ENaC in part through phosphorylation of the ubiquitin ligase Nedd4-2 (2); and a delayed phase of aldosterone action involving de novo synthesis of ENaC, either from the liganded mineralocorticoid receptor directly binding hormone response elements in the ENaCα promoter to activate transcription (1) or through indirect

Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1beta- and lipopolysaccharide (LPS)+interferon (IFN)-gamma-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor kappaB (NF-kappaB) p65 and p50 complexed with STAT3 in the DNA-protein complex. The direct interaction of STAT3 and NF-kappaB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-kappaB p65 fusion protein and in vitro -translated STAT3alpha. Overexpression of STAT3 dramatically inhibited IL-1beta- or LPS+IFN-gamma-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-kappaB-dependent promoter devoid of STAT-binding elements without affecting NF-kappaB DNA-binding activity. Thus STAT3, via direct interactions with NF-kappaB p65, serves as a dominant-negative inhibitor of NF-kappaB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli.

Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)-and Ca 2+ -mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H 2 O 2 -, and Ca 2+ -induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H 2 O 2 compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca 2+ inhibition of inward K + currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity.