MicroRNA miR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting Wnt2 signaling pathway

MicroRNA miR-199a-5p impairs tight junction formation leading to increased urothelial permeability in bladder pain syndrome. Now using transcriptome analysis in urothelial TEU-2 cells we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF and Wnt signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder and we altered its levels in bladder smooth muscle cells (SMC) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size and up-regulated miR-199a-5p targets, including Wnt2. Overexpression of Wnt2 protein or treating SMCs with recombinant Wnt2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of Wnt2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM alpha-actin and SM myosin heavy chain mRNA and protein levels. These changes, as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor (MRTF-A) downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of Wnt-dependent inhibitory Kruppel-like transcription factor 4 (KLF4) in miR-199a-5p overexpressing cells. In contrast, KLF4 was induced in antimiR-expressing cells following the activation of Wnt2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the Wnt2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, relevant for organ remodeling.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

Hypofibrinolysis in type 2 diabetes: the role of the inflammatory pathway and complement C3

AIMS/HYPOTHESIS Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes. METHODS Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay. RESULTS Clot lysis time correlated with C3 and PAI-1 plasma levels (r = 0.24, p < 0.001 and r = 0.22, p < 0.001, respectively). In a multivariable regression model involving age, sex, BMI, C3, PAI-1, CRP and fibrinogen, and using log-transformed data as appropriate, C3 was associated with clot lysis time (regression coefficient 0.227 [95% CI 0.161, 0.292], p < 0.001), as was PAI-1 (regression coefficient 0.033 [95% CI 0.020, 0.064], p < 0.05) but not fibrinogen (regression coefficient 0.003 [95% CI -0.046, 0.051], p = 0.92) or CRP (regression coefficient 0.024 [95% CI -0.008, 0.056], p = 0.14). No correlation was demonstrated between plasma levels of C3 and PAI-1 (r = -0.03, p = 0.44), consistent with previous observations that the two proteins affect different pathways in the fibrinolytic system. CONCLUSIONS/INTERPRETATION Similarly to PAI-1, C3 plasma levels are independently associated with fibrin clot lysis in individuals with type 2 diabetes. Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fibrinolysis potential in this population.

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5'UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export.

The role of glucosinolates and the jasmonic acid pathway in resistance of Arabidopsis thaliana against molluscan herbivores

Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant immunity to molluscs is limited. We found slugs to be natural herbivores of Arabidopsis thaliana and therefore investigated possible resistance mechanisms of this species against several molluscan herbivores. Treating wounded leaves with the mucus residue (‘slime trail’) of the Spanish slug Arion lusitanicus increased wound-induced jasmonate levels, suggesting the presence of defence elicitors in the mucus. Plants deficient in jasmonate biosynthesis and signalling suffered more damage by molluscan herbivores in the laboratory and in the field, demonstrating that JA-mediated defences protect A. thaliana against slugs and snails. Furthermore, experiments using A. thaliana mutants with altered levels of specific glucosinolate classes revealed the importance of aliphatic glucosinolates in defending leaves and reproductive structures against molluscs. The presence in mollusc faeces of known and novel metabolites arising from glutathione conjugation with glucosinolate hydrolysis products suggests that molluscan herbivores actively detoxify glucosinolates. Higher levels of aliphatic glucosinolates were found in plants during the night compared to the day, which correlated well with the nocturnal activity rhythms of slugs and snails. Our data highlight the function of well-known antiherbivore defence pathways in resistance against slugs and snails and suggest an important role for the diurnal regulation of defence metabolites against nocturnal molluscan herbivores.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.

IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions

The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

igments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 132 OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.

The sonic hedgehog signaling pathway and the development of pharyngeal arch Derivatives in Haplochromis piceatus, a Lake Victoria cichlid

Objectives Pharyngeal arches develop in the head and neck regions, and give rise to teeth, oral jaws, the hyoid bone, operculum, gills, and pharyngeal jaws in teleosts. In this study, the expression patterns of genes in the sonic hedgehog (shh), wnt, ectodysplasin A (eda), and bone morphogenetic protein (bmp) pathways were investigated in the pharyngeal arches of Haplochromis piceatus, one of the Lake Victoria cichlids. Furthermore, the role of the shh pathway in pharyngeal arch development in H. piceatus larvae was investigated. Methods The expression patterns of lymphocyte enhancer binding factor 1 (lef1), ectodysplasin A receptor (edar), shh, patched 1 (ptch1), bmp4, sp5 transcription factor (sp5), sclerostin domain containing 1a (sostdc1a), and dickkopf 1 (dkk1) were investigated in H. piceatus larvae by in situ hybridization. The role of the shh pathway was investigated through morphological phenotypic characterization after its inhibition. Results We found that lef1, edar, shh, ptch1, bmp4, dkk1, sostdc1a, and sp5 were expressed not only in the teeth, but also in the operculum and gill filaments of H piceatus larvae. After blocking the shh pathway using cyclopamine, we observed ectopic shh expression and the disappearance of ptch1 expression. After six weeks of cyclopamine treatment, an absence of teeth in the oral upper jaws and a poor outgrowth of premaxilla, operculum, and gill filaments in juvenile H. piceatus were observed. Conclusions These results suggest that the shh pathway is important for the development of pharyngeal arch derivatives such as teeth, premaxilla, operculum, and gill filaments in H. piceatus.

Association of lectin pathway proteins with intra-abdominal Candida infection in high-risk surgical intensive-care unit patients. A prospective cohort study within the fungal infection network of Switzerland

OBJECTIVES Human studies on the role of mannose-binding lectin (MBL) in patients with invasive candidiasis have yielded conflicting results. We investigated the influence of MBL and other lectin pathway proteins on Candida colonization and intra-abdominal candidiasis (IAC) in a cohort of high-risk patients. METHODS Prospective observational cohort study of 89 high-risk intensive-care unit (ICU) patients. Levels of lectin pathway proteins at study entry and six MBL2 single-nucleotide polymorphisms were analyzed by sandwich-type immunoassays and genotyping, respectively, and correlated with development of heavy Candida colonization (corrected colonization index (CCI) ≥0.4) and occurrence of IAC during a 4-week period. RESULTS Within 4 weeks after inclusion a CCI ≥0.4 and IAC was observed in 47% and 38% of patients respectively. Neither serum levels of MBL, ficolin-1, -2, -3, MASP-2 or collectin liver 1 nor MBL2 genotypes were associated with a CCI ≥0.4. Similarly, none of the analyzed proteins was found to be associated with IAC with the exception of lower MBL levels (HR 0.74, p = 0.02) at study entry. However, there was no association of MBL deficiency (<0.5 μg/ml), MBL2 haplo- or genotypes with IAC. CONCLUSION Lectin pathway protein levels and MBL2 genotype investigated in this study were not associated with heavy Candida colonization or IAC in a cohort of high-risk ICU patients.