Interleukin-1ß Induces mtDNA Release to Activate Innate Immune Signaling via cGAS-STING.

Interleukin-1 beta (IL-1ß) is a pleiotropic mediator of inflammation and is produced in response to a wide range of stimuli. During infection, IL-1ß production occurs in parallel with the onset of innate antimicrobial defenses, but the contribution of IL-1ß signaling to cell-intrinsic immunity is not defined. Here, we report that exogenous IL-1ß induces interferon regulatory factor 3 (IRF3) activation in human myeloid, fibroblast, and epithelial cells. IRF3 activation by IL-1ß is dependent upon the DNA-sensing pathway adaptor, stimulator of interferon genes (STING), through the recognition of cytosolic mtDNA by cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). IL-1ß treatment results in interferon (IFN) production and activation of IFN signaling to direct a potent innate immune response that restricts dengue virus infection. This study identifies a new function for IL-1ß in the onset or enhancement of cell-intrinsic immunity, with important implications for cGAS-STING in integrating inflammatory and microbial cues for host defense.

Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli.

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Intact and mutated Shigella diguanylate cyclases increase c-di-GMP.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

From Input to Output: The Lap/c-di-GMP Biofilm Regulatory Circuit.

Biofilms are the dominant bacterial lifestyle. The regulation of the formation and dispersal of bacterial biofilms has been the subject of study in many organisms. Over the last two decades, the mechanisms of Pseudomonas fluorescens biofilm formation and regulation have emerged as among the best understood of any bacterial biofilm system. Biofilm formation by P. fluorescens occurs through the localization of an adhesin, LapA, to the outer membrane via a variant of the classical type I secretion system. The decision between biofilm formation and dispersal is mediated by LapD, a c-di-GMP receptor, and LapG, a periplasmic protease, which together control whether LapA is retained or released from the cell surface. LapA localization is also controlled by a complex network of c-di-GMP-metabolizing enzymes. This review describes the current understanding of LapA-mediated biofilm formation by P. fluorescens and discusses several emerging models for the regulation and function of this adhesin.

The PDE-Opathies: Diverse Phenotypes Produced by a Functionally Related Multigene Family.

The phosphodiesterase (PDE)-opathies, an expanding set of disorders caused by germline mutations in cyclic nucleotide PDEs, present an intriguing paradox. The enzymes encoded by the PDE family all hydrolyze cAMP and/or cGMP, but mutations in different family members produce very divergent phenotypes. Three interacting factors have been shown recently to contribute to this phenotypic diversity: (i) the 21 genes encode over 80 different isoforms, using alternative mRNA splicing and related mechanisms; (ii) the various isoforms have different regulatory mechanisms, mediated by their unique amino-terminal regulatory domains; (iii) the isoforms differ widely in their pattern of tissue expression. These mechanisms explain why many PDE-opathies are gain-of-function mutations and how they exemplify uniqueness and redundancy within a multigene family.

A complete twelve-gene deletion null mutant reveals that cyclic di-GMP is a global regulator of phase-transition and host colonization in Erwinia amylovora.

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates biofilm formation and pathogenicity. To study the global regulatory effect of individual components of the c-di-GMP metabolic system, we deleted all 12 diguanylate cyclase (dgc) and phosphodiesterase (pde)-encoding genes in E. amylovora Ea1189 (Ea1189Δ12). Ea1189Δ12 was impaired in surface attachment due to a transcriptional dysregulation of the type IV pilus and the flagellar filament. A transcriptomic analysis of surface-exposed WT Ea1189 and Ea1189Δ12 cells indicated that genes involved in metabolism, appendage generation and global transcriptional/post-transcriptional regulation were differentially regulated in Ea1189Δ12. Biofilm formation was regulated by all 5 Dgcs, whereas type III secretion and disease development were differentially regulated by specific Dgcs. A comparative transcriptomic analysis of Ea1189Δ8 (lacks all five enzymatically active dgc and 3 pde genes) against Ea1189Δ8 expressing specific dgcs, revealed the presence of a dual modality of spatial and global regulatory frameworks in the c-di-GMP signaling network.

The c-di-GMP signalling component YfiR regulates multiple bacterial phenotypes and virulence in Pseudomonas plecoglossicida.

AIMS: Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3'-5') diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR-a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida. METHODS AND RESULTS: A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found. CONCLUSIONS: YfiR is the key regulator of virulence in P. plecoglossicida, which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model.

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity.

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR RpfR uniquely integrates two major signaling systems-quorum sensing and the motile-sessile switch mediated by cyclic-di-GMP-by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions.

Pseudomonas aeruginosa Strains from Both Clinical and Environmental Origins Readily Adopt a Stable Small-Colony-Variant Phenotype Resulting from Single Mutations in c-di-GMP Pathways.

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain. IMPORTANCE P. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because this bacterium is the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is considered related to the emergence of an alternative small-colony-variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various nonclinical environments and the involvement of c-di-GMP in SCV emergence from both clinical and environmental strains, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to adapt readily in all environments. Hindering this adaptation strategy could help control persistent infection by P. aeruginosa.

Sensory Perception in Bacterial Cyclic Diguanylate Signal Transduction.

Cyclic diguanylate (c-di-GMP) signal transduction systems provide bacteria with the ability to sense changing cell status or environmental conditions and then execute suitable physiological and social behaviors in response. In this review, we provide a comprehensive census of the stimuli and receptors that are linked to the modulation of intracellular c-di-GMP. Emerging evidence indicates that c-di-GMP networks sense light, surfaces, energy, redox potential, respiratory electron acceptors, temperature, and structurally diverse biotic and abiotic chemicals. Bioinformatic analysis of sensory domains in diguanylate cyclases and c-di-GMP-specific phosphodiesterases as well as the receptor complexes associated with them reveals that these functions are linked to a diverse repertoire of protein domain families. We describe the principles of stimulus perception learned from studying these modular sensory devices, illustrate how they are assembled in varied combinations with output domains, and summarize a system for classifying these sensor proteins based on their complexity. Biological information processing via c-di-GMP signal transduction not only is fundamental to bacterial survival in dynamic environments but also is being used to engineer gene expression circuitry and synthetic proteins with à la carte biochemical functionalities.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

INX-18 and INX-19 play distinct roles in electrical synapses that modulate aversive behavior in Caenorhabditis elegans.

In order to respond to changing environments and fluctuations in internal states, animals adjust their behavior through diverse neuromodulatory mechanisms. In this study we show that electrical synapses between the ASH primary quinine-detecting sensory neurons and the neighboring ASK neurons are required for modulating the aversive response to the bitter tastant quinine in C. elegans. Mutant worms that lack the electrical synapse proteins INX-18 and INX-19 become hypersensitive to dilute quinine. Cell-specific rescue experiments indicate that inx-18 operates in ASK while inx-19 is required in both ASK and ASH for proper quinine sensitivity. Imaging analyses find that INX-19 in ASK and ASH localizes to the same regions in the nerve ring, suggesting that both sides of ASK-ASH electrical synapses contain INX-19. While inx-18 and inx-19 mutant animals have a similar behavioral phenotype, several lines of evidence suggest the proteins encoded by these genes play different roles in modulating the aversive quinine response. First, INX-18 and INX-19 localize to different regions of the nerve ring, indicating that they are not present in the same synapses. Second, removing inx-18 disrupts the distribution of INX-19, while removing inx-19 does not alter INX-18 localization. Finally, by using a fluorescent cGMP reporter, we find that INX-18 and INX-19 have distinct roles in establishing cGMP levels in ASK and ASH. Together, these results demonstrate that electrical synapses containing INX-18 and INX-19 facilitate modulation of ASH nociceptive signaling. Our findings support the idea that a network of electrical synapses mediates cGMP exchange between neurons, enabling modulation of sensory responses and behavior.

Shigella flexneri Diguanylate Cyclases Regulate Virulence.

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. IMPORTANCE The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among Shigella species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

A Trigger Phosphodiesterase Modulates the Global c-di-GMP Pool, Motility, and Biofilm Formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

The cGMP system in normal and degenerating mouse neuroretina: New proteins with cGMP interaction potential identified by a proteomics approach.

The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over-activate specific cGMP-interacting proteins, like cGMP-dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP-interactors are present in the retina, which we, therefore, investigated in wild-type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP-interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and 10 potentially new retinal cGMP-interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration.

Bi-allelic Loss-of-Function Mutations in the NPR-C Receptor Result in Enhanced Growth and Connective Tissue Abnormalities.

The natriuretic peptide signaling pathway has been implicated in many cellular processes, including endochondral ossification and bone growth. More precisely, different mutations in the NPR-B receptor and the CNP ligand have been identified in individuals with either short or tall stature. In this study we show that the NPR-C receptor (encoded by NPR3) is also important for the regulation of linear bone growth. We report four individuals, originating from three different families, with a phenotype characterized by tall stature, long digits, and extra epiphyses in the hands and feet. In addition, aortic dilatation was observed in two of these families. In each affected individual, we identified a bi-allelic loss-of-function mutation in NPR3. The missense mutations (c.442T>C [p.Ser148Pro] and c.1088A>T [p.Asp363Val]) resulted in intracellular retention of the NPR-C receptor and absent localization on the plasma membrane, whereas the nonsense mutation (c.1524delC [p.Tyr508∗]) resulted in nonsense-mediated mRNA decay. Biochemical analysis of plasma from two affected and unrelated individuals revealed a reduced NTproNP/NP ratio for all ligands and also high cGMP levels. These data strongly suggest a reduced clearance of natriuretic peptides by the defective NPR-C receptor and consequently increased activity of the NPR-A/B receptors. In conclusion, this study demonstrates that loss-of-function mutations in NPR3 result in increased NPR-A/B signaling activity and cause a phenotype marked by enhanced bone growth and cardiovascular abnormalities.

cGMP-PKG dependent transcriptome in normal and degenerating retinas: Novel insights into the retinitis pigmentosa pathology.

Retinitis Pigmentosa represents a group of genetic disorders that cause progressive vision loss via degeneration of photoreceptors, but there is in principle no treatment available. For any therapy development, a deeper comprehension of the disease-leading mechanism(s) at the molecular level is needed. Here we focused on the cGMP-PKG system, which has been suggested to be a driver in several models of the disease. To gain insights in its downstream signaling we manipulated the cGMP-PKG system with the aid of organotypic retinal explant cultures from either a mouse-based disease model, i.e. the rd1 mouse, or its healthy wild-type counterpart (wt), by adding different types of cGMP analogues to either inhibit or activate PKG in retinal explants from rd1 and wt, respectively. An RNA sequencing was then performed to study the cGMP-PKG dependent transcriptome. Expression changes of gene sets related to specific pathways or functions, that fulfilled criteria involving that the changes should match PKG activation and inhibition, were determined via bioinformatics. The analyses highlighted that several gene sets linked to oxidative phosphorylation and mitochondrial pathways were regulated by this enzyme system. Specifically, the expression of such pathway components was upregulated in the rd1 treated with PKG inhibitor and downregulated in the wt with PKG activator treatment, suggesting that cGMP-PKG act as a negative regulator in this context. Downregulation of energy production pathways may thus play an integral part in the mechanism behind the degeneration for at least several RP mutations.

Depletion of cyclic-GMP levels and inhibition of cGMP-dependent protein kinase activate p21Cip1 /p27Kip1 pathways and lead to renal fibrosis and dysfunction.

Cell-cycle regulatory proteins (p21Cip1 /p27Kip1 ) inhibit cyclin and cyclin-dependent kinase (CDK) complex that promotes fibrosis and hypertrophy. The present study examined the role of CDK blockers, p21Cip1 /p27Kip1 in the progression of renal fibrosis and dysfunction using Npr1 (encoding guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) gene-knockout (0-copy; Npr1-/- ), 2-copy (Npr1+/+ ), and 4-copy (Npr1++/++ ) mice treated with GC inhibitor, A71915 and cGMP-dependent protein kinase (cGK) inhibitor, (Rp-8-Br-cGMPS). A significant decrease in renal cGMP levels and cGK activity was observed in 0-copy mice and A71915- and Rp-treated 2-copy and 4-copy mice compared with controls. An increased phosphorylation of Erk1/2, p38, p21Cip1 , and p27Kip1 occurred in 0-copy and A71915-treated 2-copy and 4-copy mice, while Rp treatment caused minimal changes than controls. Pro-inflammatory (TNF-α, IL-6) and pro-fibrotic (TGF-ß1) cytokines were significantly increased in plasma and kidneys of 0-copy and A71915-treated 2-copy mice, but to lesser extent in 4-copy mice. Progressive renal pathologies, including fibrosis, mesangial matrix expansion, and tubular hypertrophy were observed in 0-copy and A71915-treated 2-copy and 4-copy mice, but minimally occurred in Rp-treated mice compared with controls. These results indicate that Npr1 has pivotal roles in inhibiting renal fibrosis and hypertrophy and exerts protective effects involving cGMP/cGK axis by repressing CDK blockers p21Cip1 and p27Kip1 .

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

Upregulation of lncRNA DARS-AS1 accelerates tumor malignancy in cervical cancer by activating cGMP-PKG pathway.

This paper investigates the function of lncRNA DARS-AS1 in cervical cancer (CC) as well as its in-depth mechanism. The differential expression of DARS-AS1 and ATP1B2 were analyzed based on The Cancer Genome Atlas and the Genotype-Tissue Expression databases, and the survival rate was measured using Kaplan-Meier survival analysis. Biological function experiments were performed to detect cell proliferation, invasion, and migration. Quantitative real-time polymerase chain reaction was carried out to detect the expression of DARS-AS1 and ATP1B2. Western blot analysis was utilized to assess the protein levels of ATP1B2 and cGMP-PKG pathway-related proteins. DARS-AS1 was expressed at high levels in CC tissues and cell lines, and high expression of DARS-AS1 indicated a lower survival rate. CCK-8 and colony formation assays revealed that the overexpression of DARS-AS1 promoted the proliferation of CC cells. Furthermore, bioinformatics analysis suggested that the cGMP-PKG pathway ranks as the first pathway enriched by the differential genes that correlated with DARS-AS1 (|r| > 0.4). ATP1B2, as a cGMP-PKG pathway-related gene, was significantly correlated with the overall survival of CC patients. We further confirmed that ATP1B2 was lowly expressed in CC and negatively correlated with the DARS-AS1 expression. Then, biological function experiments exhibited that the promotion of cell proliferation, invasion, and migration resulted due to the upregulation of DARS-AS1 could be canceled by ATP1B2 overexpression. Finally, Western blot revealed that upregulation of DARS-AS1 could activate the cGMP-PKG pathway, while overexpression of ATP1B2 reversed this activation. Our study revealed that DARS-AS1/ATP1B2 contributes to regulating the progression of CC at least partially by modulating the cGMP-PKG pathway.