Regulation of Flowering Time and Other Developmental Plasticities by 3' Splicing Factor-Mediated Alternative Splicing in Arabidopsis thaliana.

Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3' splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3' splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in Arabidopsis thaliana.

Cannabidiol Enhances Cabozantinib-Induced Apoptotic Cell Death via Phosphorylation of p53 Regulated by ER Stress in Hepatocellular Carcinoma.

Cannabidiol (CBD), a primary constituent in hemp and cannabis, exerts broad pharmacological effects against various diseases, including cancer. Additionally, cabozantinib, a potent multi-kinase inhibitor, has been approved for treating patients with advanced hepatocellular carcinoma (HCC). Recently, there has been an increase in research on combination therapy using cabozantinib to improve efficacy and safety when treating patients. Here, we investigated the effect of a combination treatment of cabozantinib and CBD on HCC cells. CBD treatment enhanced the sensitivity of HCC cells to cabozantinib-mediated anti-cancer activity by increasing cytotoxicity and apoptosis. Phospho-kinase array analysis demonstrated that the apoptotic effect of the combination treatment was mainly related to p53 phosphorylation regulated by endoplasmic reticulum (ER) stress when compared to other kinases. The inhibition of p53 expression and ER stress suppressed the apoptotic effect of the combination treatment, revealing no changes in the expression of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-8, or cleaved caspase-9. Notably, the effect of the combination treatment was not associated with cannabinoid receptor 1 (CNR1) and the CNR2 signaling pathways. Our findings suggest that the combination therapy of cabozantinib and CBD provides therapeutic efficacy against HCC.

Two Arabidopsis Splicing Factors, U2AF65a and U2AF65b, Differentially Control Flowering Time by Modulating the Expression or Alternative Splicing of a Subset of FLC Upstream Regulators.

We investigated the transcriptomic changes in the shoot apices during floral transition in Arabidopsis mutants of two closely related splicing factors: AtU2AF65a (atu2af65a) and AtU2AF65b (atu2af65b). The atu2af65a mutants exhibited delayed flowering, while the atu2af65b mutants showed accelerated flowering. The underlying gene regulatory mechanism of these phenotypes was unclear. We performed RNA-seq analysis using shoot apices instead of whole seedlings and found that the atu2af65a mutants had more differentially expressed genes than the atu2af65b mutants when they were compared to wild type. The only flowering time gene that was significantly up- or down-regulated by more than two-fold in the mutants were FLOWERING LOCUS C (FLC), a major floral repressor. We also examined the expression and alternative splicing (AS) patterns of several FLC upstream regulators, such as COOLAIR, EDM2, FRIGIDA, and PP2A-b'ɤ, and found that those of COOLAIR, EDM2, and PP2A-b'ɤ were altered in the mutants. Furthermore, we demonstrated that AtU2AF65a and AtU2AF65b genes partially influenced FLC expression by analyzing these mutants in the flc-3 mutant background. Our findings indicate that AtU2AF65a and AtU2AF65b splicing factors modulate FLC expression by affecting the expression or AS patterns of a subset of FLC upstream regulators in the shoot apex, leading to different flowering phenotypes.

The splicing factor 1-FLOWERING LOCUS M module spatially regulates temperature-dependent flowering by modulating FLOWERING LOCUS T and LEAFY expression.

KEY MESSAGE: The AtSF1-FLM module spatially controls temperature-dependent flowering by negatively regulating the expression of FT and LFY in the leaf and shoot apex, respectively. Alternative splicing mediated by various splicing factors is important for the regulation of plant growth and development. Our recent reports have shown that a temperature-dependent interaction between Arabidopsis thaliana splicing factor 1 (AtSF1) and FLOWERING LOCUS M (FLM) pre-mRNA introns controls the differential production of FLM-ß transcripts at different temperatures, eventually resulting in temperature-responsive flowering. However, the molecular and genetic interactions between the AtSF1-FLM module and floral activator genes remain unknown. Here, we aimed to identify the interactions among AtSF1, FLM, FLOWERING LOCUS T (FT), and LEAFY (LFY) by performing molecular and genetic analyses. FT and TWIN SISTER OF FT (TSF) expression in atsf1-2 mutants significantly increased in the morning and middle of the night at 16 and 23 °C, respectively, under long-day conditions. In addition, ft mutation suppressed the early flowering of atsf1-2 and atsf1-2 flm-3 mutants and masked the temperature response of atsf1-2 flm-3 mutants, suggesting that FT is a downstream target gene of the AtSF1-FLM module. LFY expression significantly increased in the diurnal samples of atsf1-2 mutants and in the shoot apex regions of atsf1-2 ft-10 mutants at different temperatures. The chromatin immunoprecipitation (ChIP) assay revealed that FLM directly binds to the genomic regions of LFY but not of APETALA1 (AP1). Moreover, lfy mutation suppressed the early flowering of flm-3 mutants, suggesting that LFY is another target of the AtSF1-FLM module. Our results reveal that the AtSF1-FLM module spatially modulates temperature-dependent flowering by regulating FT and LFY expressions.

Role of Arabidopsis Splicing factor SF1 in Temperature-Responsive Alternative Splicing of FLM pre-mRNA.

Small changes in temperature affect plant ecological and physiological factors that impact agricultural production. Hence, understanding how temperature affects flowering is crucial for decreasing the effects of climate change on crop yields. Recent reports have shown that FLM-ß, the major spliced isoform of FLOWERING LOCUS M (FLM)-a flowering time gene, contributes to temperature-responsive flowering in Arabidopsis thaliana. However, the molecular mechanism linking pre-mRNA processing and temperature-responsive flowering is not well understood. Genetic and molecular analyses identified the role of an Arabidopsis splicing factor SF1 homolog, AtSF1, in regulating temperature-responsive flowering. The loss-of-function AtSF1 mutant shows temperature insensitivity at different temperatures and very low levels of FLM-ß transcript, but a significantly increased transcript level of the alternative splicing (AS) isoform, FLM-δ. An RNA immunoprecipitation (RIP) assay revealed that AtSF1 is responsible for ambient temperature-dependent AS of FLM pre-mRNA, resulting in the temperature-dependent production of functional FLM-ß transcripts. Moreover, alterations in other splicing factors such as ABA HYPERSENSITIVE1/CBP80 (ABH1/CBP80) and STABILIZED1 (STA1) did not impact the FLM-ß/FLM-δ ratio at different temperatures. Taken together, our data suggest that a temperature-dependent interaction between AtSF1 and FLM pre-mRNA controls flowering time in response to temperature fluctuations.

Arabidopsis U2AF65 Regulates Flowering Time and the Growth of Pollen Tubes.

During pre-mRNA splicing, U2 small nuclear ribonucleoprotein auxiliary factor 65 (U2AF65) interacts with U2AF35 and splicing factor 1 (SF1), allowing for the recognition of the 3'-splice site by the ternary complex. The functional characterization of U2AF65 homologs has not been performed in Arabidopsis thaliana yet. Here, we show that normal plant development, including floral transition, and male gametophyte development, requires two Arabidopsis U2AF65 isoforms (AtU2AF65a and AtU2AF65b). Loss-of-function mutants of these two isoforms displayed opposite flowering phenotypes: atu2af65a mutants showed late flowering, whereas atu2af65b mutants were characterized by slightly early flowering, as compared to that in the wild-type (Col-0) plants. These abnormal flowering phenotypes were well-correlated with the expression patterns of the flowering time genes such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). However, the two atu2af65 mutants did not display any morphological abnormalities or alterations in abiotic stress tests. Double mutation of the AtU2AF65a and AtU2AF65b genes resulted in non-viable seeds due to defective male gametophyte. In vitro pollen germination test revealed that mutations in both AtU2AF65a and AtU2AF65b genes significantly impaired pollen tube growth. Collectively, our findings suggest that two protein isoforms of AtU2AF65 are differentially involved in regulating flowering time and display a redundant role in pollen tube growth.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms.

KEY MESSAGE: The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes.

KEY MESSAGE: The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-ß transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

RASSF1A Directly Antagonizes RhoA Activity through the Assembly of a Smurf1-Mediated Destruction Complex to Suppress Tumorigenesis.

RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256-277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial-mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847-59. ©2016 AACR.

Functional conservation of rice OsNF-YB/YC and Arabidopsis AtNF-YB/YC proteins in the regulation of flowering time.

KEY MESSAGE: Rice Os NF - YB and Os NF - YC complement the late flowering phenotype of Arabidopsis nf - yb double and nf - yc triple mutants, respectively. In addition, OsNF-YB and OsNF-YC interact with AtNF-YC and AtNF-YB, respectively. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors play important roles in plant development and abiotic stress. In Arabidopsis thaliana, two NF-YB (AtNF-YB2 and AtNF-YB3) and five NF-YC (AtNF-YC1, AtNF-YC2, AtNF-YC3, AtNF-YC4, and AtNF-YC9) genes regulate photoperiodic flowering by interacting with other AtNF-Y subunit proteins. Three rice NF-YB (OsNF-YB8, OsNF-YB10, and OsNF-YB11) and five rice OsNF-YC (OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6, and OsNF-YC7) genes are clustered with two AtNF-YB and five AtNF-YC genes, respectively. To investigate the functional conservation of these NF-YB and NF-YC genes in rice and Arabidopsis, we analyzed the flowering phenotypes of transgenic plants overexpressing the respective OsNF-YB and OsNF-YC genes in Arabidopsis mutants. Overexpression of OsNF-YB8/10/11 and OsNF-YC2 complemented the late flowering phenotype of Arabidopsis nf-yb2 nf-yb3 and nf-yc3 nf-yc4 nf-yc9 mutants, respectively. The rescued phenotype of 35S::OsNF-YC2 nf-yc3 nf-yc4 nf-yc9 plants was attributed to the upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). In vitro and in planta protein-protein analyses revealed that OsNF-YB8/10/11 and OsNF-YC1/2/4/6/7 interact with AtNF-YC3/4/9 and AtNF-YB2/3, respectively. Our data indicate that some OsNF-YB and OsNF-YC genes are functional equivalents of AtNF-YB2/3 and AtNF-YC3/4/9 genes, respectively, and suggest functional conservation of Arabidopsis and rice NF-Y genes in the control of flowering time.

OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice.

MAIN CONCLUSION: OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

Scoparone interferes with STAT3-induced proliferation of vascular smooth muscle cells.

Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin D1, phosphorylated Rb and survivin in the VSMCs. Cell adhesion markers, such as MCP-1 and ICAM-1, were significantly reduced by scoparone. Interestingly, this compound attenuated the increase in cyclin D promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.

A homolog of splicing factor SF1 is essential for development and is involved in the alternative splicing of pre-mRNA in Arabidopsis thaliana.

During initial spliceosome assembly, SF1 binds to intron branch points and interacts with U2 snRNP auxiliary factor 65 (U2AF65). Here, we present evidence indicating that AtSF1, the Arabidopsis SF1 homolog, interacts with AtU2AF65a and AtU2AF65b, the Arabidopsis U2AF65 homologs. A mutant allele of AtSF1 (At5g51300) that contains a T-DNA insertion conferred pleiotropic developmental defects, including early flowering and abnormal sensitivity to abscisic acid. An AtSF1 promoter-driven GUS reporter assay showed that AtSF1 promoter activity was temporally and spatially altered, and that full AtSF1 promoter activity required a significant proportion of the coding region. DNA chip analyses showed that only a small proportion of the transcriptome was altered by more than twofold in either direction in the AtSF1 mutant. Expression of the mRNAs of many heat shock proteins was more than fourfold higher in the mutant strain; these mRNAs were among those whose expression was increased most in the mutant strain. An RT-PCR assay revealed an altered alternative splicing pattern for heat shock transcription factor HsfA2 (At2g26150) in the mutant; this altered splicing is probably responsible for the increased expression of the target genes induced by HsfA2. Altered alternative splicing patterns were also detected for the transcripts of other genes in the mutant strain. These results suggest that AtSF1 has functional similarities to its yeast and metazoan counterparts.

Scoparone exerts anti-tumor activity against DU145 prostate cancer cells via inhibition of STAT3 activity.

Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3) contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2) or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C) was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.

The sequence variation responsible for the functional difference between the CONSTANS protein, and the CONSTANS-like (COL) 1 and COL2 proteins, resides mostly in the region encoded by their first exons.

Although the protein CONSTANS (CO) and its close relatives CONSTANS-like (COL) 1 and COL2 exhibit high amino acid sequence similarities, only the CO protein regulates floral induction in Arabidopsis. To investigate the structural basis for the functional differences between CO, COL1, and COL2 in flowering, we performed domain-swapping between CO, COL1, and COL2, and site-directed mutagenesis on the first exon of CO. The results suggest that the lack of flowering promotion activity by COL1 and COL2 is mainly attributed to the differences between CO and the COL1 and COL2 proteins in the amino acid sequence encoded by their first exons.

Identification of marneral synthase, which is critical for growth and development in Arabidopsis.

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3-oxidosqualene (OS), which is cyclized by 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.

Genome-scale screening and molecular characterization of membrane-bound transcription factors in Arabidopsis and rice.

Controlled proteolytic activation of membrane-bound transcription factors (MTFs) is recently emerging as a versatile way of rapid transcriptional responses to environmental changes in plants. Here, we report genome-scale identification of putative MTFs in the Arabidopsis and rice genomes. The Arabidopsis and rice genomes have at least 85 and 45 MTFs, respectively, in virtually all major transcription factor families. Of particular interest is the NAC MTFs (designated NTLs): there are at least 18 NTLs in Arabidopsis and 5 NTL members (OsNTLs) in rice. While the full-size OsNTL forms are associated with the membranes, truncated forms lacking the transmembrane domains are detected exclusively in the nucleus. Furthermore, transcript levels of the OsNTL genes were elevated after treatments with abiotic stresses, supporting their roles in plant stress responses. We propose that membrane-mediated transcriptional control is a critical component of gene regulatory network that serves as an adaptive strategy under unfavorable growth conditions.

Novel bifunctional nucleases, OmBBD and AtBBD1, are involved in abscisic acid-mediated callose deposition in Arabidopsis.

Screening of the expressed sequence tag library of the wild rice species Oryza minuta revealed an unknown gene that was rapidly and strongly induced in response to attack by a rice fungal pathogen (Magnaporthe oryzae) and an insect (Nilaparvata lugens) and by wounding, abscisic acid (ABA), and methyl jasmonate treatments. Its recombinant protein was identified as a bifunctional nuclease with both RNase and DNase activities in vitro. This gene was designated OmBBD (for O. minuta bifunctional nuclease in basal defense response). Overexpression of OmBBD in an Arabidopsis (Arabidopsis thaliana) model system caused the constitutive expression of the PDF1.2, ABA1, and AtSAC1 genes, which are involved in priming ABA-mediated callose deposition. This activation of defense responses led to an increased resistance against Botrytis cinerea. atbbd1, the knockout mutant of the Arabidopsis ortholog AtBBD1, was susceptible to attack by B. cinerea and had deficient callose deposition. Overexpression of either OmBBD or AtBBD1 in atbbd1 plants complemented the susceptible phenotype of atbbd1 against B. cinerea as well as the deficiency of callose deposition. We suggest that OmBBD and AtBBD1 have a novel regulatory role in ABA-mediated callose deposition.

Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells.

OBJECTIVE: Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action. METHODS AND RESULTS: Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-alpha-stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21(cip1/waf1) but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-alpha-induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-alpha. CONCLUSIONS: These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.