CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth. 10.1111/(ISSN)1469-8137

WNT-Inflammasome Signaling Mediates NOD2-Induced Development of Acute Arthritis in Mice

In addition to its role in innate immunity, the intracellular pathogen sensor nucleotide-binding oligomerization domain 2 (NOD2) has been implicated in various inflammatory disorders, including the development of acute arthritis. However, the molecular mechanisms involved in the development of NOD2-responsive acute arthritis are not clear. In this study, we demonstrate that NOD2 signals to a cellular protein, Ly6/PLAUR domain-containing protein 6, in a receptor-interacting protein kinase 2-TGF-beta-activated kinase 1-independent manner to activate the WNT signaling cascade. Gain- or loss-of-function of the WNT signaling pathway in an in vivo experimental mouse arthritis model or in vitro systems established the role for WNT-responsive X-linked inhibitor of apoptosis during the development of acute arthritis. Importantly, WNT-stimulated X-linked inhibitor of apoptosis mediates the activation of inflammasomes. The subsequent caspase-1 activation and IL-1 beta secretion together contribute to the phenotypic character of the inflammatory condition of acute arthritis. Thus, identification of a role for WNT-mediated inflammasome activation during NOD2 stimulation serves as a paradigm to understand NOD2-associated inflammatory disorders and develop novel therapeutics.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is irn plicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream 1)38 MAPK in a time dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thiorcdoxin (Trxl), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase ji 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein ovariation netvvork analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mecliated TNF signaling through intrathecal administration of TNFneutralizing antibody prevented Trxl oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states. (C) 2015 Published by Elsevier Inc.

Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1-and prostaglandin E-2-induced regulatory T cell expansion

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are exploited by mycobacteria to subvert the protective host immune responses. The Treg expansion in the periphery requires signaling by professional antigen presenting cells and in particularly dendritic cells (DC). However, precise molecular mechanisms by which mycobacteria instruct Treg expansion via DCs are not established. Here we demonstrate that mycobacteria-responsive sonic hedgehog (SHH) signaling in human DCs leads to programmed death ligand-1 (PD-L1) expression and cyclooxygenase (COX)-2-catalyzed prostaglandin E-2 (PGE(2)) that orchestrate mycobacterial infection-induced expansion of Tregs. While SHH-responsive transcription factor GLI1 directly arbitrated COX-2 transcription, specific microRNAs, miR-324-5p and miR-338-5p, which target PD-L1 were downregulated by SHH signaling. Further, counter-regulatory roles of SHH and NOTCH1 signaling during mycobacterial-infection of human DCs was also evident. Together, our results establish that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in human DCs to expand Tregs that favour immune evasion of the pathogen.

Correct biological timing in Arabidopsis requires multiple light-signaling pathways.

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant's response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

Neurotransmitter signaling in the pathophysiology of microglia

Erratun publicado en Frontiers in Cellular Neuroscience 7 : (2013) // Article ID 107

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. Methods: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. Results: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. Conclusions: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.

Microfluidic effects of transporting signaling components in cell coculture chips

A theoretical model has been developed to investigate the microfluidic transport of the signaling chemicals in the cell coculture chips. Using an epidermal growth factor (EGF)-like growth factor as the sample chemical, the effects of velocities and channel geometry were studied for the continuous-flow microchannel bioreactors. It is found that different perfusion velocities must be applied in the parallel channels to facilitate the communication, i.e., transport of the signaling component, between the coculture channels. Such communication occurs in a unidirectional way because the signaling chemicals can only flow from the high velocity area to the low velocity area. Moreover, the effect of the transport of the signaling component between the coculture channels on the growth of the monolayer cells and the multicellular tumor spheroid (MTS) in the continuous-flow coculture environment were simulated using 3D models. The numerical results demonstrated that the concentration gradients will induce the heterogeneous growth of the cells and the MTSs, which should be taken into account in designing the continuous-flow perfusion bioreactor for the cell coculture research.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Negative regulators of a growth factor-mediated signaling pathway in the nematode Caenorhabditis elegans

Vulval differentiation in C. elegans is mediated by an Epidermal growth factor (EGF)- EGF receptor (EGFR) signaling pathway. I have cloned unc-101, a negative regulator of vulval differentiation of the nematode C. elegans. unc-101 encodes a homolog of AP47, the medium chain of the trans-Golgi clathrin-associated protein complex. This identity was confirmed by cloning and comparing sequence of a C. elegans homolog of AP50, the medium chain of the plasma membrane clathrin-associated protein complex. I provided the first genetic evidence that the trans-Golgi clathrin-coated vesicles are involved in regulation of an EGF signaling pathway. Most of the unc-101 alleles are deletions or nonsense mutations, suggesting that these alleles severely reduce the unc-101 activity. A hybrid gene that contains parts of unc-101 and mouse AP4 7 rescued at least two phenotypes of unc-101 mutations, the Unc and the suppression of vulvaless phenotype of let-23(sy1) mutation. Therefore, the functions of AP47 are conserved between nematodes and mammals.

unc-101 mutations can cause a greater than wild-type vulval differentiation in combination with certain mutations in sli-1, another negative regulator of the vulval induction pathway. A mutation in a new gene, rok-1, causes no defect by itself, but causes a greater than wild-type vulval differentiation in the presence of a sli-1 mutation. The unc-101; rok-1; sli-1 triple mutants display a greater extent of vulval differentiation than any double mutant combinations of unc-101, rok-1 and sli-1. Therefore, rok-1 locus defines another negative regulator of the vulval induction pathway.

I analyzed a second gene encoding an AP47 homolog in C. elegans. This gene, CEAP47, encodes a protein 72% identical to both unc-101 and mammalian AP47. A hybrid gene containing parts of unc-101 and CEAP47 sequences can rescue phenotypes of unc-101 mutants, indicating that UNC- 101 and CEAP47 proteins can be redundant if expressed in the same set of cells.

Wnt and FGF signaling in C. elegans vulval cell lineage polarity

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The C. elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Herein I discuss the interaction of Wnt and FGF signaling in controlling vulval cell lineage polarity with emphasis on the posterior-most cell that forms the vulva, P7.p.

The mirror symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. Opposing Wnt signals control the division patterns of the VPCs by controlling the localization of SYS-1/ β-catenin toward the direction of the Wnt gradient. Multiple Wnt signals, expressed at the axis of symmetry, promote the wild-type, anterior-facing, P7.p orientation, whereas Wnts EGL-20 and CWN-1 from the tail and posterior body wall muscle, respectively, promote the daughter cells of P7.p to face the posterior. EGL-20 acts through a member of the LDL receptor superfamily, LRP-2, along with Ror/CAM-1 and Van Gogh/VANG-1. All three transmembrane proteins control orientation through the localization of the SYS-1.

The Fibroblast Growth Factor (FGF) pathway acts in concert with LIN-17/Frizzled to regulate the localization of SYS-1. The source of the FGF ligand is the 1° VPC, P6.p, which controls the polarity of the neighboring 2° VPC, P7.p, by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt, cwn-1, is expressed in the posterior body wall muscle of the worm as well as the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the Wnt gradient. The FGF pathway leads to the regulation of cwn-1 transcripts in the SMs. These results illustrate the first evidence of the interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity as well as highlight the promiscuous nature of Wnt signaling within C. elegans.

Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

Experimental and theoretical studies of Notch signaling-mediated spatial pattern

Notch signaling acts in many diverse developmental spatial patterning processes. To better understand why this particular pathway is employed where it is and how downstream feedbacks interact with the signaling system to drive patterning, we have pursued three aims: (i) to quantitatively measure the Notch system's signal input/output (I/O) relationship in cell culture, (ii) to use the quantitative I/O relationship to computationally predict patterning outcomes of downstream feedbacks, and (iii) to reconstitute a Notch-mediated lateral induction feedback (in which Notch signaling upregulates the expression of Delta) in cell culture. The quantitative Notch I/O relationship revealed that in addition to the trans-activation between Notch and Delta on neighboring cells there is also a strong, mutual cis-inactivation between Notch and Delta on the same cell. This feature tends to amplify small differences between cells. Incorporating our improved understanding of the signaling system into simulations of different types of downstream feedbacks and boundary conditions lent us several insights into their function. The Notch system converts a shallow gradient of Delta expression into a sharp band of Notch signaling without any sort of feedback at all, in a system motivated by the Drosophila wing vein. It also improves the robustness of lateral inhibition patterning, where signal downregulates ligand expression, by removing the requirement for explicit cooperativity in the feedback and permitting an exceptionally simple mechanism for the pattern. When coupled to a downstream lateral induction feedback, the Notch system supports the propagation of a signaling front across a tissue to convert a large area from one state to another with only a local source of initial stimulation. It is also capable of converting a slowly-varying gradient in parameters into a sharp delineation between high- and low-ligand populations of cells, a pattern reminiscent of smooth muscle specification around artery walls. Finally, by implementing a version of the lateral induction feedback architecture modified with the addition of an autoregulatory positive feedback loop, we were able to generate cells that produce enough cis ligand when stimulated by trans ligand to themselves transmit signal to neighboring cells, which is the hallmark of lateral induction.