Calcium-dependent protein kinase CPK9 negatively functions in stomatal abscisic acid signaling by regulating ion channel activity in Arabidopsis.

KEY MESSAGE: In this manuscript, we demonstrated the negative role of CPK9 in stomatal ABA signaling, and both CPK9 and CPK33 for accurate guard cell function was explored via cpk9/cpk33 double mutants' phenotype. Abscisic acid (ABA) can inhibit stomatal opening and promote stomatal closure by regulating ion channel activity in guard cell membranes. As an important second messenger, calcium (Ca2+) is essentially needed in ABA regulation of stomatal movement. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central Ca2+ signal transduction in plants. Here, we report the functional characterization of CPK9 in Arabidopsis stomatal ABA signaling. CPK9 had high expression in guard cells and the protein was subcellularly located in the cell membrane. A loss-of-function mutant cpk9 showed a much more sensitive phenotype to ABA regulation of stomatal movement and ion channel activity, while CPK9 overexpression lines had opposite phonotypes. These findings demonstrated the negative role of CPK9 in stomatal ABA signaling. As the closest homolog of CPK33, we also proved that stomatal movement of the cpk9/cpk33 double mutants was more sensitive to ABA than either single mutants. These results revealed the role of CPK9 in guard cells, and the need of both CPK9 and CPK33 for accurate guard cell function.

A Tonoplast-Associated Calcium-Signaling Module Dampens ABA Signaling during Stomatal Movement.

Stomatal movement, critical for photobiosynthesis, respiration, and stress responses, is regulated by many factors, among which abscisic acid (ABA) is critical. Early events of ABA signaling involve Ca2+ influx and an increase of cytoplasmic calcium ([Ca2+]cyt). Positive regulators of this process have been extensively studied, whereas negative regulators are obscure. ABA-induced stomatal closure involves K+ flux and vacuolar convolution. How these processes are connected with Ca2+ is not fully understood. We report that pat10-1, a null mutant of Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is hypersensitive to ABA-induced stomatal closure and vacuolar convolution. A similar phenotype was observed in cbl2;cbl3, the double mutant of CBL2 and CBL3, whose tonoplast association depends on PAT10. Functional loss of the PAT10-CBL2/CBL3 system resulted in enhanced Ca2+ influx and [Ca2+]cyt elevation. Promoting vacuolar K+ accumulation by overexpressing NHX2 suppressed ABA-hypersensitive stomatal closure and vacuolar convolution of the mutants, suggesting that PAT10-CBL2/CBL3 positively mediates vacuolar K+ accumulation. We have identified CBL-interacting protein kinases (CIPKs) that mediate CBL2/CBL3 signaling during ABA-induced stomatal movement. Functional loss of the PAT10-CBL2/3-CIPK9/17 system in guard cells enhanced drought tolerance. We propose that the tonoplast CBL-CIPK complexes form a signaling module that negatively regulates ABA signaling during stomatal movement.

Associations of dietary phytosterols with blood lipid profiles and prevalence of obesity in Chinese adults, a cross-sectional study.

BACKGROUND: It has been established in RCTs that high dose of phytosterols can significantly reduce blood cholesterol. However, it was uncertain whether low dose of phytosterols from daily diets was effective. In this study, we evaluated the associations between dietary phytosterols and body mass index (BMI), waist circumference (WC), blood glucose, serum lipid profiles and prevalence of overweight/obesity and abdominal obesity in healthy subjects. METHODS: Four hundred nine men and 503 women aged 18-60 years were included in this study. Dietary intakes of phytosterols were estimated using a validated food frequency questionnaire. Height, body weight, WC and blood pressure were measured, an oral glucose tolerance test was performed. Moreover, fasting serum triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDLc) and low density lipoprotein cholesterol (LDLc) were further determined. RESULTS: When comparing extreme quartiles of dietary phytosterols, significant differences of BMI, WC, systolic blood pressure (SBP), diastolic blood pressure (DBP), serum TC and LDLc were found. Dietary phytosterols presented a negative association with BMI, WC, SBP, DBP, serum TC and LDLc (with and without adjustment for energy). After adjustment for confounders, we found higher dietary phytosterols were linked with lower prevalence of overweight/obesity (OR highest vs. lowest quartile = 0.487; 95% CI 0.234, 0.918 for men; OR highest vs. lowest quartile = 0.277; 95% CI 0.124, 0.619 for women) and abdominal obesity (OR highest vs. lowest quartile = 0.344; 95% CI 0.144, 0.819 for men; OR highest vs. lowest quartile = 0.321; 95% CI 0.140, 0.571 for women). CONCLUSIONS: Higher dietary phytosterols were associated with lower BMI, WC, blood pressure, serum TC and LDLc and lower prevalence of overweight/obesity and abdominal obesity in Chinese adults.

Dietary Sulfur-Containing Amino Acids Are Associated with Higher Prevalence of Overweight/Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study.

BACKGROUND/AIMS: Elevation of plasma sulfur-containing amino acids (SAAs) is generally associated with higher body mass index (BMI) and unfavorable lipid profiles. It is not known how dietary SAAs relate to these associations in humans. METHODS: A convenient tool named internet-based dietary questionnaire for Chinese (IDQC) was used to estimate dietary SAAs intake. A total of 936 participants were randomly recruited and asked to complete the IDQC. Furthermore, 90 subjects were randomly selected to perform a subgroup study. The associations between dietary SAAs and prevalence of obesity, lipid profiles, and status of insulin resistance (IR), inflammation and oxidative stress were assessed. RESULTS: Dietary total SAAs and cysteine of overweight/obese participants were significantly higher. Dietary total SAAs and cysteine were positively associated with BMI and waist circumference. Higher dietary total SAAs were associated with higher prevalence of overweight/obesity. Higher dietary total SAAs and cysteine also associated with higher serum triglyceride (total cholesterol), low density lipoprotein, fasting blood glucose, 2 h-postprandial glucose, and homeostasis model assessment of IR. In the subgroup study, positive associations between dietary SAAs and inflammation biomarkers were also observed. CONCLUSIONS: Dietary SAAs are associated with higher prevalence of overweight/obesity, unfavorable lipid profiles and status of IR, and inflammation.

Mitochondrial pyruvate carrier 1 mediates abscisic acid-regulated stomatal closure and the drought response by affecting cellular pyruvate content in Arabidopsis thaliana.

BACKGROUND: Stomata are micropores surrounded by pairs of guard cells, and their opening is finely controlled to balance water vapor loss as transpiration and CO2 absorption for photosynthesis. The regulatory signaling network for stomatal movement is complicated, and increasing numbers of new genes have been shown to be involved in this process. Our previous study indicated that a member of the plant putative mitochondrial pyruvate carrier (MPC) family, NRGA1, is a negative regulator of guard cell abscisic acid (ABA) signaling. In this study, we identified novel physiological roles of pyruvate and MPC1, another member of the MPC family, in the regulation of stomatal closure in Arabidopsis. RESULTS: Loss-of-function mutants of MPC1 (mpc1) were hypersensitive to ABA-induced stomatal closure and ABA-activated guard cell slow-type anion currents, and showed a reduced rate of water loss upon drought treatment compared with wild-type plants. In contrast, plants overexpressing MPC1 showed a hyposensitive ABA response and increased sensitivity to drought stress. In addition, mpc1 mutants accumulated more pyruvate after drought or ABA treatment. The increased pyruvate content also induced stomatal closure and activated the slow-type anion channels of guard cells, and this process was dependent on the function of RbohD/F NADPH oxidases and reactive oxygen species concentrations in guard cells. CONCLUSIONS: Our findings revealed the essential roles of MPC1 and pyruvate in stomatal movement and plant drought resistance.

THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis.

Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca(2+)-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling.

WRKY1 regulates stomatal movement in drought-stressed Arabidopsis thaliana.

A key response of plants to moisture stress is stomatal closure, a process mediated by the phytohormone abscisic acid (ABA). Closure is affected by changes in the turgor of the stomatal guard cell. The transcription factor WRKY1 is a part of the regulatory machinery underlying stomatal movements, and through this, in the plant's response to drought stress. The loss-of-function T-DNA insertion mutant wrky1 was particularly sensitive to ABA, with respect to both ion channel regulation and stomatal movements, and less sensitive to drought than the wild type. Complementation of the wrky1 mutant resulted in the recovery of the wild type phenotype. The WRKY1 product localized to the nucleus, and was shown able to bind to the W-box domain in the promoters of MYB2, ABCG40, DREB1A and ABI5, and thereby to control their transcription in response to drought stress or ABA treatment. WRKY1 is thought to act as a negative regulator in guard cell ABA signalling.

Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions.

IL-6/STAT3/TFF3 signaling regulates human biliary epithelial cell migration and wound healing in vitro.

Interleukin-6 (IL-6), through activation of the signal transducer and activator of transcription 3 (STAT3) and trefoil factor family 3 (TFF3), has been implicated in the promotion of mouse biliary epithelial cell (BEC) proliferation and migration. However, it is still unclear whether the IL-6/STAT3/TFF3 signaling had similar effects on human BECs. Here, we showed that exposure of human BECs to recombinant IL-6 resulted in STAT3 phosphorylation and increased the expression of TFF3 at both mRNA and protein levels. Moreover, inhibition of STAT3 using RNA interference significantly abrogated IL-6-induced TFF3 expression. In an in-vitro wound healing model, IL-6 facilitated human BEC migration. This promotion of cell migration by IL-6 was blocked when STAT3 was knocked down. Interestingly, the addition of exogenous TFF3 could rescue the cell migration defects caused by STAT3 silencing. In conclusion, our data indicate that STAT3 plays a critical role in IL-6-induced TFF3 expression in human BECs and the IL-6/STAT3/TFF3 signaling is involved in human BEC migration and wound healing.

Reconstitution of secreted frizzled-related protein 1 suppresses tumor growth and lung metastasis in an orthotopic model of hepatocellular carcinoma.

BACKGROUND: Secreted Frizzled-related protein 1 (sFRP1) is frequently silenced in many types of cancer, including hepatocellular carcinoma (HCC), leading to aberrant activation of Wnt signaling and thereby facilitating tumor development. In this study, we aimed to investigate whether restoration of sFRP1 affected HCC growth and metastasis. METHODS: We generated stable cell lines overexpressing sFRP1 in MHCC97-H cells, which naturally do not express detectable sFRP1 messenger RNA (mRNA) and have high metastatic properties. The effects of sFRP1 reexpression on tumor growth and metastasis were assessed in vitro and in vivo. It was also tested whether ß-catenin signaling mediated the function of sFRP1 in tumor progression. RESULTS: Overexpression of sFRP1 substantially diminished the proliferation and invasion potentials of MHCC97-H cells. Furthermore, sFRP1 expression significantly inhibited MHCC97-H xenograft growth and metastasis in vivo, which was accompanied by decreased angiogenesis and increased tumor cell apoptosis. Moreover, sFRP1 overexpression caused less expression of ß-catenin and its downstream effector genes cyclin D1 and matrix metalloproteinase (MMP)-2. CONCLUSION: Together these findings demonstrate that sFRP1 reconstitution suppresses tumor growth, angiogenesis, and metastasis in MHCC97-H xenografts, which may be associated with inactivation of ß-catenin signaling, thus providing a possible therapeutic strategy against HCC.

Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study.

Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC). Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p < 0.05) and obese (p < 0.01) participants of both sexes. Dietary histidine was inversely associated with body mass index (BMI), waist circumference (WC) and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), 2-h postprandial glucose (2 h-PG), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), C-reactive protein (CRP), malonaldehyde (MDA) and vaspin and higher glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults.

Identifying survival-associated ceRNA clusters in cholangiocarcinoma.

Competing endogenous RNAs (ceRNAs) represent a novel layer regulations of long non-coding RNAs (lncRNAs) and genes that play important roles in cancer pathogenesis by binding microRNAs (miRNAs). However, the competition mechanism of ceRNAs in cholangiocarcinoma (CHOL) is not fully understood. In this study, we constructed a dysregulated ceRNA competitive network (CCEN) to globally characterize the competing difference between CHOL and normal tissues. Then, we integrated affinity propagation and Kaplan­Meier (K-M) methods to identify functional clusters associated with survival. A total of 7 key ceRNA clusters were identified. Further functional annotation analyses found that Cluster23 and Cluster32 involved cell based functions, and the loss of ceRNA competitive relations in clusters may contribute to CHOL, by disturbing important biological processes, such as 'Pathway in cancer', MAPK and Neurotrophin signaling pathway. This study provides further insights into understanding the competitive mechanism of ceRNAs in CHOL.

Enhanced expression of suppresser of cytokine signaling 3 inhibits the IL-6-induced epithelial-to-mesenchymal transition and cholangiocarcinoma cell metastasis.

It was recently demonstrated that interleukin-6 (IL-6) induces the epithelial-to-mesenchymal transition (EMT) in cholangiocarcinoma (CCA), but the underlying molecular mechanism remains to be explored. In this study, we studied the role of suppresser of cytokine signaling 3 (SOCS3), a negative feedback regulator of IL-6/STAT3, in the IL-6-induced EMT in CCA. Treatment with IL-6 induced the EMT by decreasing the E-cadherin expression and increasing the expression of N-cadherin and vimentin. Using wound healing and invasion assays, we found that IL-6 promoted cell motility. Further, a stably transfected cell line overexpressing SOCS3 was constructed. Enhanced SOCS3 expression decreased IL-6-induced cell invasion and EMT in parallel with downregulating the IL-6/STAT3 pathway. In contrast, SOCS3 silencing using siRNA exhibited no effect on the cell invasive ability and EMT. Finally, an in vivo study indicated that the enhancement of SOCS3 expression decreased metastasis compared with the control, and this effect was achieved by the repression of p-STAT3, N-cadherin and vimentin, and the induction of E-cadherin assessed by Western blot analysis. Our results suggest that enhanced expression of SOCS3 can antagonize IL-6-induced EMT and cell metastasis by abrogating the IL-6/STAT3 pathway. These data establish that SOCS3 plays a role in the EMT in CCA and may provide novel therapeutic strategies for CCA.

NRGA1, a putative mitochondrial pyruvate carrier, mediates ABA regulation of guard cell ion channels and drought stress responses in Arabidopsis.

Abscisic acid (ABA) regulates ion channel activity and stomatal movements in response to drought and other stresses. Here, we show that the Arabidopsis thaliana gene NRGA1 is a putative mitochondrial pyruvate carrier which negatively regulates ABA-induced guard cell signaling. NRGA1 transcript was abundant in the A. thaliana leaf and particularly in the guard cells, and its product was directed to the mitochondria. The heterologous co-expression of NRGA1 and AtMPC1 in yeast complemented a loss-of-function mitochondrial pyruvate carrier (MPC) mutant. The nrga1 loss-of-function mutant was very sensitive to the presence of ABA in the context of stomatal movements, and exhibited a heightened tolerance to drought stress. Disruption of NRGA1 gene resulted in increased ABA inhibition of inward K(+) currents and ABA activation of slow anion currents in guard cells. The nrga1/NRGA1 functional complementation lines restored the mutant's phenotypes. Furthermore, transgenic lines of constitutively overexpressing NRGA1 showed opposite stomatal responses, reduced drought tolerance, and ABA sensitivity of guard cell inward K(+) channel inhibition and anion channel activation. Our findings highlight a putative role for the mitochondrial pyruvate carrier in guard cell ABA signaling in response to drought.