Mesenchymal stem cells attenuate the proinflammatory cytokine pattern in a guinea pig model of chronic cigarette smoke exposure.

AIMS: Cigarette smoke often induces pulmonary and systemic inflammation. In animal models, mesenchymal stem cells (MSC) tend to ameliorate these effects. We aimed to explore the local and systemic expression of cytokines in guinea pigs chronically exposed to cigarette smoke, and their modifications by MSC. MAIN METHODS: Concentrations of IL-1ß, IL-6, IL-8, IL-12, TNF-α, INF-É£, TSG-6, MMP-9, TIMP-1, and/or TIMP-2 in serum and bronchoalveolar lavage (BALF) from animals exposed to tobacco smoke (20 cigarettes/day, 5 days/week for 10 weeks) were determined, and mRNA expression of some of them was measured in lung tissue. Intratracheal instillation of allogeneic bone marrow MSC (5x106 cells in 1 ml) was done at week 2. KEY FINDINGS: After cigarette smoke, IL-6 and IFN-γ increased in serum and BALF, while IL-1ß and IL-12 decreased in serum, and TSG-6 and TIMP-2 increased in BALF. IL-1ß had a paradoxical increase in BALF. MSC had an almost null effect in unexposed animals. The intratracheal administration of MSC in guinea pigs exposed to cigarette smoke was associated with a statistically significant decrease of IL-12 and TSG-6 in serum, as well as a decrease of IL-1ß and IFN-γ and an increase in TIMP-1 in BALF. Concerning mRNA expression in lung tissue, cigarette smoke did not modify the relative amount of the studied transcripts, but even so, MSC decreased the IL-12 mRNA and increased the TIMP-1 mRNA. SIGNIFICANCE: A single intratracheal instillation of MSC reduces the pulmonary and systemic proinflammatory pattern induced by chronic exposure to cigarette smoke in guinea pigs. TRIAL REGISTRATION: Not applicable.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix-Loop-Helix Transcription Factors.

The basic helix-loop-helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell's fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

The Transcription Factor SCX is a Potential Serum Biomarker of Fibrotic Diseases.

Fibrosing diseases are causes of morbidity and mortality around the world, and they are characterized by excessive extracellular matrix (ECM) accumulation. The bHLH transcription factor scleraxis (SCX) regulates the synthesis of ECM proteins in heart fibrosis. SCX expression was evaluated in lung fibroblasts and tissue derived from fibrotic disease patients and healthy controls. We also measured SCX in sera from 57 healthy controls, and 56 Idiopathic Pulmonary Fibrosis (IPF), 40 Hypersensitivity Pneumonitis (HP), and 100 Systemic Sclerosis (SSc) patients. We report high SCX expression in fibroblasts and tissue from IPF patients versus controls. High SCX-serum levels were observed in IPF (0.663 ± 0.559 ng/mL, p < 0.01) and SSc (0.611 ± 0.296 ng/mL, p < 0.001), versus controls (0.351 ± 0.207 ng/mL) and HP (0.323 ± 0.323 ng/mL). Serum levels of the SCX heterodimerization partner, TCF3, did not associate with fibrotic illness. IPF patients with severely affected respiratory capacities and late-stage SSc patients presenting anti-topoisomerase I antibodies and interstitial lung disease showed the highest SCX-serum levels. SCX gain-of-function induced the expression of alpha-smooth muscle actin (α-SMA/ACTA2) in fibroblasts when co-overexpressed with TCF3. As late and severe stages of the fibrotic processes correlated with high circulating SCX, we postulate it as a candidate biomarker of fibrosis and a potential therapeutic target.

A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.

Altered expression pattern of immune response-related genes and isoforms in hypersensitivity pneumonitis lung fibroblasts.

Hypersensitivity pneumonitis (HP) is an immune-mediated inflammatory interstitial lung disease that may evolve to pulmonary fibrosis, a progressive disorder with a poor prognosis characterized by fibroblast activation and extracellular matrix accumulation. In HP lung fibroblasts, the gene expression of proteins involved in the interaction with the immune response, their isoforms, and how they influence their phenotype have yet to be elucidated. We analyzed the expression and splicing variants of 16 target genes involved in the interaction between HP fibroblasts and immune signaling and evaluated possible correlations with clinical data. The comparison of HP and control fibroblasts revealed distinct gene expression patterns. HP lung fibroblasts displayed an increased expression of IFI27 and PDFGRA and a downregulation of IL17RC and TGFBR3. IFI27 immunoreactive protein was markedly increased in HP lung tissues and normal fibroblasts treated with TGF-ß. Furthermore, IFI27 overexpression in normal fibroblasts increased α-SMA and decreased cell number over time. The isoform analysis showed similar expression patterns for most genes, except for the AGER receptor with increased soluble variants relative to full-length AGER in HP fibroblasts. These findings indicate important differences in the expression of genes related to the immune response by HP fibroblasts, highlighting their unique characteristics and providing further insight into a possible profibrotic role of IFI27 in the disease.

Mir-155-5p targets TP53INP1 to promote proliferative phenotype in hypersensitivity pneumonitis lung fibroblasts.

Background: Hypersensitivity pneumonitis (HP) is an inflammatory disorder affecting lung parenchyma and often evolves into fibrosis (fHP). The altered regulation of genes involved in the pathogenesis of the disease is not well comprehended, while the role of microRNAs in lung fibroblasts remains unexplored. Methods: We used integrated bulk RNA-Seq and enrichment pathway bioinformatic analyses to identify differentially expressed (DE)-miRNAs and genes (DEGs) associated with HP lungs. In vitro, we evaluated the expression and potential role of miR-155-5p in the phenotype of fHP lung fibroblasts. Loss and gain assays were used to demonstrate the impact of miR-155-5p on fibroblast functions. In addition, mir-155-5p and its target TP53INP1 were analyzed after treatment with TGF-ß, IL-4, and IL-17A. Results: We found around 50 DEGs shared by several databases that differentiate HP from control and IPF lungs, constituting a unique HP lung transcriptional signature. Additionally, we reveal 18 DE-miRNAs that may regulate these DEGs. Among the candidates likely associated with HP pathogenesis was miR-155-5p. Our findings indicate that increased miR-155-5p in fHP fibroblasts coincides with reduced TP53INP1 expression, high proliferative capacity, and a lack of senescence markers compared to IPF fibroblasts. Induced overexpression of miR-155-5p in normal fibroblasts remarkably increases the proliferation rate and decreases TP53INP1 expression. Conversely, miR-155-5p inhibition reduces proliferation and increases senescence markers. TGF-ß, IL-4, and IL-17A stimulated miR-155-5p overexpression in HP lung fibroblasts. Conclusion: Our findings suggest a distinctive signature of 53 DEGs in HP, including CLDN18, EEF2, CXCL9, PLA2G2D, and ZNF683, as potential targets for future studies. Likewise, 18 miRNAs, including miR-155-5p, could be helpful to establish differences between these two pathologies. The overexpression of miR-155-5p and downregulation of TP53INP1 in fHP lung fibroblasts may be involved in his proliferative and profibrotic phenotype. These findings may help differentiate and characterize their pathogenic features and understand their role in the disease.

Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the ß-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Short-Term Exposure to Wood Smoke Increases the Expression of Pro-Inflammatory Cytokines, Gelatinases, and TIMPs in Guinea Pigs.

Exposure to air pollutants in wildfire smoke and indoor pollution causes lung diseases. Short-term exposure to wood smoke (WS) is partially known to alter the expression of human matrix metalloproteinases (MMPs), inflammatory cytokines, and tissue inhibitors of metalloproteinases (TIMPs). Accordingly, we investigated the effect of exposing guinea pigs to WS for two and four three-hour periods on different days. The daily content of particles reported by indoor pollution was produced by 60 g of pinewood. We analyzed the cell profile and collagen content in bronchoalveolar lavages (BAL). The mRNA expression of pro-inflammatory cytokines, MMPs, and TIMPs was studied in lung tissue. Cytokines and gelatinolytic activity were analyzed in BAL and serum. The results showed that total cells, macrophages, neutrophils, and collagen increased in BAL, whereas neutrophils and lymphocytes decreased. TGF-ß1, TNF-α, IFN-γ, IL-1ß, IL-6, IL-8, MMP-2, MMP-9, TIMP-1, and TIMP-2 were upregulated in lungs, downregulating IL-12. TNF-α, IFN-γ, TGF-ß1, IL-1ß, IL-6, and IL-8 were increased in BAL and serum, decreasing IL-12. Gelatinase activity was increased in serum. Thus, guinea pigs exposed to short-term domestic doses of WS overexpressed pro-inflammatory cytokines, MMPs, and TIMPs. These results are similar to ECM remodeling and pulmonary and systemic inflammation reported in humans.

Comparative analyses among the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S ribosomal RNA genes.

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.

Chromatin Regulation by the NuA4 Acetyltransferase Complex Is Mediated by Essential Interactions Between Enhancer of Polycomb (Epl1) and Esa1.

Enzymes that modify and remodel chromatin act in broadly conserved macromolecular complexes. One key modification is the dynamic acetylation of histones and other chromatin proteins by opposing activities of acetyltransferase and deacetylase complexes. Among acetyltransferases, the NuA4 complex containing Tip60 or its Saccharomyces cerevisiae ortholog Esa1 is of particular significance because of its roles in crucial genomic processes including DNA damage repair and transcription. The catalytic subunit Esa1 is essential, as are five noncatalytic NuA4 subunits. We found that of the noncatalytic subunits, deletion of Enhancer of polycomb (Epl1), but not the others, can be bypassed by loss of a major deacetylase complex, a property shared by Esa1 Noncatalytic complex subunits can be critical for complex assembly, stability, genomic targeting, substrate specificity, and regulation. Understanding the essential role of Epl1 has been previously limited, a limitation now overcome by the discovery of its bypass suppression. Here, we present a comprehensive in vivo study of Epl1 using the powerful tool of suppression combined with transcriptional and mutational analyses. Our results highlight functional parallels between Epl1 and Esa1 and further illustrate that the structural role of Epl1 is important for promotion of Esa1 activity. This conclusion is strengthened by our dissection of Epl1 domains required in vivo for interaction with specific NuA4 subunits, histone acetylation, and chromatin targeting. These results provide new insights for the conserved, essential nature of Epl1 and its homologs, such as EPC1/2 in humans, which is frequently altered in cancers.

Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation.

The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington's disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

The Set3 Complex Antagonizes the MYST Acetyltransferase Esa1 in the DNA Damage Response.

Acetylation is a dynamic posttranslational modification that contributes to chromatin-regulated processes, including DNA replication, repair, recombination, and gene expression. Acetylation is controlled by complexes containing opposing lysine and histone acetyltransferase (KAT and HAT) and deacetylase (KDAC and HDAC) activities. The essential MYST family Esa1 KAT acetylates core histones and many nonhistone substrates. Phenotypes of esa1 mutants include transcriptional silencing and activation defects, impaired growth at high temperatures, and sensitivity to DNA damage. The KDAC Rpd3 was previously identified as an activity opposing Esa1, as its deletion suppresses growth and silencing defects of esa1 mutants. However, loss of Rpd3 does not suppress esa1 DNA damage sensitivity. In this work, we identified Hos2 as a KDAC counteracting ESA1 in the damage response. Deletion of HOS2 resulted in changes of esa1's transcriptional response upon damage. Further, loss of HOS2 or components of the Set3 complex (Set3C) in which it acts specifically suppressed damage sensitivity and restored esa1 histone H4 acetylation. This rescue was mediated via loss of either Set3C integrity or of its binding to dimethylated histone H3K4. Our results thus add new insight into the interactions of an essential MYST acetyltransferase with diverse deacetylases to respond specifically to environmental and physiological challenges.

Bypassing the requirement for an essential MYST acetyltransferase.

Histone acetylation is a key regulatory feature for chromatin that is established by opposing enzymatic activities of lysine acetyltransferases (KATs/HATs) and deacetylases (KDACs/HDACs). Esa1, like its human homolog Tip60, is an essential MYST family enzyme that acetylates histones H4 and H2A and other nonhistone substrates. Here we report that the essential requirement for ESA1 in Saccharomyces cerevisiae can be bypassed upon loss of Sds3, a noncatalytic subunit of the Rpd3L deacetylase complex. By studying the esa1∆ sds3∆ strain, we conclude that the essential function of Esa1 is in promoting the cellular balance of acetylation. We demonstrate this by fine-tuning acetylation through modulation of HDACs and the histone tails themselves. Functional interactions between Esa1 and HDACs of class I, class II, and the Sirtuin family define specific roles of these opposing activities in cellular viability, fitness, and response to stress. The fact that both increased and decreased expression of the ESA1 homolog TIP60 has cancer associations in humans underscores just how important the balance of its activity is likely to be for human well-being.

Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.