“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

The Hybrid Type Polyketide Synthase SteelyA Is Required for cAMP Signalling in Early Dictyostelium Development

Background

In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development.

Principal Findings

Our stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation.

Conclusion

Our data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.     

cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin

Background

Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown.

Methods and Findings

We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX) and forskolin enhances adipogenesis, the gene expression of PPARγ2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-κB Ligand to Osteoprotegerin (RANKL/OPG) gene expression – the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish.

Conclusions

Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.     

Complete mitochondrial genomes of the human follicle mites Demodex brevis and D. folliculorum: novel gene arrangement,truncated tRNA genes,and ancient divergence between species

Background

Follicle mites of the genus Demodex are found on a wide diversity of mammals, including humans; surprisingly little is known, however, about the evolution of this association. Additional sequence information promises to facilitate studies of Demodex variation within and between host species. Here we report the complete mitochondrial genome sequences of two species of Demodex known to live on humans—Demodex brevis and D. folliculorum—which are the first such genomes available for any member of the genus. We analyzed these sequences to gain insight into the evolution of mitochondrial genomes within the Acariformes. We also used relaxed molecular clock analyses, based on alignments of mitochondrial proteins, to estimate the time of divergence between these two species.

Results

Both Demodex genomes shared a novel gene order that differs substantially from the ancestral chelicerate pattern, with transfer RNA (tRNA) genes apparently having moved much more often than other genes. Mitochondrial tRNA genes of both species were unusually short, with most of them unable to encode tRNAs that could fold into the canonical cloverleaf structure; indeed, several examples lacked both D- and T-arms. Finally, the high level of sequence divergence observed between these species suggests that these two lineages last shared a common ancestor no more recently than about 87 mya.

Conclusions

Among Acariformes, rearrangements involving tRNA genes tend to occur much more often than those involving other genes. The truncated tRNA genes observed in both Demodex species would seem to require the evolution of extensive tRNA editing capabilities and/or coevolved interacting factors. The molecular machinery necessary for these unusual tRNAs to function might provide an avenue for developing treatments of skin disorders caused by Demodex. The deep divergence time estimated between these two species sets a lower bound on the time that Demodex have been coevolving with their mammalian hosts, and supports the hypothesis that there was an early split within the genus Demodex into species that dwell in different skin microhabitats.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1124) contains supplementary material, which is available to authorized users.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

Background

Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.

Methodology/Principal Findings

We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem ^−/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.

Conclusion/Significance

Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.     

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

Background

Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection.

Objective

This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.

Study Design

Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined.

Results

Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4⁺CD25⁺Foxp3⁺ and CD4⁺Foxp3⁺ T cells compared with patients with HFMD or BE. The expression frequency of CD4⁻CD8⁻ was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4⁺Foxp3⁺ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.

Conclusions

These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.     

Hybrid proline-rich proteins: novel players in plant cell elongation?

Background and Aims

Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process.

Methods

To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants.

Key Results

In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta.

Conclusions

Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion.