The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation.

The mechanism and significance of synergistic induction of the expression of plasminogen activator inhibitor-1 by glucocorticoid and transforming growth factor beta in human ovarian cancer cells.

Plasminogen activator inhibitor-1 (PAI-1) plays a key role in tissue remodeling and tumor development by suppression of plasminogen activator function. Glucocorticoids (GCs) and transforming growth factor beta (TGF-ß) signal pathways cross-talk to regulate gene expression, but the mechanism is poorly understood. Here we investigated the mechanism and significance of co-regulation of PAI-1 by TGF-ß and dexamethasone (DEX), a synthetic glucocorticoid in ovarian cancer cells. We found that TGF-ß and DEX showed rapidly synergistic induction of PAI-1 expression, which contributed to the early pro-adhesion effects. The synergistic induction effect was accomplished by several signal pathways, including GC receptor (GR) pathway and TGF-ß-activated p38MAPK, ERK and Smad3 pathways. TGF-ß-activated p38MAPK and ERK pathways cross-talked with GR pathway to augment the expression of PAI-1 through enhancing DEX-induced GR phosphorylation at Ser211 in ovarian cancer cells. These findings reveal possible novel mechanisms by which TGF-ß pathways cooperatively cross-talk with GR pathway to regulate gene expression.

Up-regulation of stomatin expression by hypoxia and glucocorticoid stabilizes membrane-associated actin in alveolar epithelial cells.

Stomatin is an important lipid raft-associated protein which interacts with membrane proteins and plays a role in the membrane organization. However, it is unknown whether it is involved in the response to hypoxia and glucocorticoid (GC) in alveolar epithelial cells (AEC). In this study we found that hypoxia and dexamethasone (dex), a synthetic GC not only up-regulated the expression of stomatin alone, but also imposed additive effect on the expression of stomatin in A549 cells, primary AEC and lung of rats. Then we investigated whether hypoxia and dex transcriptionally up-regulated the expression of stomatin by reporter gene assay, and found that dex, but not hypoxia could increase the activity of a stomatin promoter-driven reporter gene. Further deletion and mutational studies demonstrated that a GC response element (GRE) within the promoter region mainly contributed to the induction of stomatin by dex. Moreover, we found that hypoxia exposure did not affect membrane-associated actin, but decreased actin in cytoplasm in A549 cells. Inhibiting stomatin expression by stomatin siRNA significantly decreased dense of peripheral actin ring in hypoxia or dex treated A549 cells. Taken all together, these data indicated that dex and/or hypoxia significantly up-regulated the expression of stomatin in vivo and in vitro, which could stabilize membrane-associated actin in AEC. We suppose that the up-regulation of stomatin by hypoxia and dex may enhance the barrier function of alveolar epithelia and mediate the adaptive role of GC to hypoxia.

BZS1, a B-box protein, promotes photomorphogenesis downstream of both brassinosteroid and light signaling pathways.

Photomorphogenesis is controlled by multiple signaling pathways, including the light and brassinosteroid (BR) pathways. BR signaling activates the BZR1 transcription factor, which is required for suppressing photomorphogenesis in the dark. We identified a suppressor of the BR hypersensitive mutant bzr1-1D and named it bzr1-1D suppressor1-Dominant (bzs1-D). The bzs1-D mutation was caused by overexpression of a B-box zinc finger protein BZS1, which is transcriptionally repressed by BZR1. Overexpression of BZS1 causes de-etiolation in the dark, short hypocotyls in the light, reduced sensitivity to BR treatment, and repression of many BR-activated genes. Knockdown of BZS1 by co-suppression partly suppressed the short hypocotyl phenotypes of BR-deficient or insensitive mutants. These results support that BZS1 is a negative regulator of BR response. BZS1 overexpressors are hypersensitive to different wavelengths of light and loss of function of BZS1 reduces plant sensitivity to light and partly suppresses the constitutive photomorphogenesis 1 (cop1) mutant in the dark, suggesting a positive role in light response. BZS1 protein accumulates at an increased level after light treatment of dark-grown BZS1-OX plants and in the cop1 mutants, and BZS1 interacts with COP1 in vitro, suggesting that light regulates BZS1 through COP1-mediated ubiquitination and proteasomal degradation. These results demonstrate that BZS1 mediates the crosstalk between BR and light pathways.

Induction of small G protein RhoB by non-genotoxic stress inhibits apoptosis and activates NF-κB.

It has been reported by us and other groups that the expression of small GTP binding protein RhoB can be induced by genotoxic stressors and glucocorticoid (GC), a stress hormone that plays a key role in stress response. Until now stress-induced genes that confer cytoprotection under stressed conditions are largely unknown. In this study, we investigated the effects and mechanism of non-genotoxic stressors, including scalding in vivo and heat stress in vitro on the expression of RhoB. We found for the first time that both scalding, which could induce typical neuroendocrine responses of acute stress and cellular heat stress significantly increased the expression of RhoB at mRNA and protein levels. Moreover, in vitro experiments in human lung epithelial cells (A549) showed that induction of RhoB by heat stress was in a glucocorticoid receptor (GR)-independent manner and through multiple pathways including stabilization of RhoB mRNA and activation of p38 MAPK. Further experiments demonstrated that up-regulation of RhoB significantly inhibited heat stress-induced apoptosis and elevated transcriptional activity of NF-κB, but did not affect the expression of Hsp70 in A549 cells. In conclusion, we showed for the first time that RhoB was up-regulated by scalding in vivo and heat stress in vitro and played an important cytoprotective role during heat stress-induced apoptotic cell death.

Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis.

Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here, we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR-signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light-signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways but also coregulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants that reveals a regulatory network that integrates hormonal and light-signaling pathways for plant growth regulation.

Up-regulation of RhoB by glucocorticoids and its effects on the cell proliferation and NF-kappaB transcriptional activity.

Although there is ample evidence that glucocorticoids (GCs) have an antiproliferative effect on many cell types, the molecular mechanism remains elusive. We reported in our previous study that Dex treatment led to cell growth arrest in a human ovarian cancer cell HO-8910. RhoB, as a member of Rho GTPases, have been implicated to be a negative regulator of cell proliferation. In this study, we provided novel evidence that Dex induced the expressions of small GTPase RhoB mRNA and protein, but not RhoA and RhoC mRNA in a dose- and time-dependent fashion via glucocorticoid receptor (GR). Over-expression of RhoB increased while inhibition of RhoB expression by RNA interference reversed Dex-induced growth arrest, indicating that RhoB signaling is involved in Dex-induced proliferation inhibition. We also presented the novel observation that over-expression or activation of RhoB signaling elevated the basal transcriptional activity of the transcription factor NF-kappaB in HO-8910 cells. Furthermore, elevating RhoB signaling enhanced the inhibitory effect of Dex on NF-kappaB activity, while attenuating RhoB signaling almost abrogated Dex suppression of NF-kappaB signaling, indicating that RhoB pathway is involved in the regulation of NF-kappaB activity and is essential for Dex transcriptional repression on NF-kappaB signaling in HO-8910 cells.

[Identification of a bacterium with activities of inhibiting growth of fungi].

HB02 is characterized as a new microbiological agent that has the potential to decrease the toxicity of mildewed wheat containing Deoxynivalenol (DON). To explore whether HB02 could inhibit the growth of fungi, a spore suspension of Aspergillus flavus or Gibberellazeae was incubated with or without HB02 in PYG medium at 28 degrees C for 15d. The mycelium was weighed after 3, 6, 9, 12, 15 d of incubation, respectively. The result showed that the growth of these two important fungi was significantly inhibited by co-cultivation with HB02. Besides, HB02 was confirmed as Lactobacillus curvatus by its microbiological characters and the 16s-23s rRNA sequence. In conclusion, HB02, identified as Lactobacillus curvatus, prevent the effects of mycotoxins in contaminated feed by inhibiting the growth of fungi. Other detoxification ways of HB02 remain further study.

Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays.

AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipHO2 cDNA array. METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR. RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5, ccl16, GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level. RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings. CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1 mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.