Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth.

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell-cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear-encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

Quantitative RNA imaging in single live cells reveals age-dependent asymmetric inheritance.

Asymmetric inheritance of cellular content through cell division plays an important role in cell viability and fitness. The dynamics of RNA segregation are so far largely unaddressed. This is partly due to a lack of approaches to follow RNAs over multiple cellular divisions. Here, we establish an approach to quantify RNA dynamics in single cells across several generations in a microfluidics device by tagging RNAs with the diSpinach aptamer. Using S. cerevisiae as a model, we quantitatively characterize intracellular RNA transport from mothers into their buds. Our results suggest that, at cytokinesis, ENO2 diSpinach RNA is preferentially distributed to daughters. This asymmetric RNA segregation depends on the lifespan regulator Sir2 and decreases with increasing replicative age of mothers but does not result from increasing cell size during aging. Overall, our approach opens more opportunities to study RNA dynamics and inheritance in live budding yeast at the single-cell level.

Transcription coordinates histone amounts and genome content.

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.

Expression of colony-stimulating factor 1 and interleukin-34 in gingival tissue and gingival fibroblasts from periodontitis patients and controls.

BACKGROUND: Colony-stimulating factor 1 (CSF-1) and interleukin (IL)-34 are important for the functions of myeloid lineage cells and are involved in several chronic inflammatory conditions associated with tissue degeneration. The aim of this study is to evaluate the expression of CSF-1 and IL-34 in gingival tissue and gingival fibroblasts (GF) from patients with periodontitis and controls. METHODS: Gingival biopsies were obtained from 19 periodontitis patients and 15 controls. Expression of CSF-1 and IL-34 in gingival tissue was assessed by western blot and localization by immunohistochemistry. Expression of CSF1 and IL34 mRNA in GF was analyzed by real-time polymerase chain reaction and protein expression visualized by immunofluorescence stainings. CSF-1 and IL-34 secretion from GF was evaluated in response to tumor necrosis factor-alpha (TNF-α), IL-1ß, Escherichia coli lipopolysaccharide (Ec-LPS) and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) stimulation, using enzyme-linked immunosorbent assays. RESULTS: CSF-1 was increased in gingival tissue from periodontitis patients compared with controls (P < 0.05) whereas IL-34 expression was similar. In GF from a non-periodontitis donor, stimulation with either TNF-α, IL-1ß, Ec-LPS, or Pg-LPS, increased the secretion of CSF-1 (P < 0.05) and Ec-LPS stimulation increased IL-34 (P < 0.05). CSF-1 and IL-34 were expressed and secreted constitutively from GF, with comparable levels in GF from periodontitis patients and controls. Inflammatory stimuli increased the secretion of CSF-1 and IL-34 with comparable levels measured from GF from periodontitis patients and controls (P < 0.05). CONCLUSION: The expression of CSF-1 and IL-34 in gingival tissue and fibroblasts suggests involvement in myeloid cell functions during periodontal inflammation.

Gingival Tissue Inflammation Promotes Increased Matrix Metalloproteinase-12 Production by CD200Rlow Monocyte-Derived Cells in Periodontitis.

Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.

Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation.

IL-1ß is a potent player in cutaneous inflammation and central for the development of a Th17 micro-milieu in autoinflammatory diseases including psoriasis. Its production is controlled at the transcriptional level and by subsequent posttranslational processing via inflammatory caspases. In this study, we detected inflammatory caspase-5 active in epidermal keratinocytes and in psoriatic skin lesions. Further, interferon-γ and interleukin-17A synergistically induced caspase-5 expression in cultured keratinocytes, which was dependent on the antimicrobial peptide psoriasin (S100A7). However, diseases-relevant triggers for caspase-5 activity and IL-1ß production remain unknown. Recently, extranuclear DNA has been identified as danger-signals abundant in the psoriatic epidermis. Here, we could demonstrate that cytosolic double-stranded (ds) DNA transfected into keratinocytes triggered the activation of caspase-5 and the release of IL-1ß. Further, interleukin-17A promoted caspase-5 function via facilitation of the NLRP1-inflammasome. Anti-inflammatory vitamin D interfered with the IL-1ß release and suppressed caspase-5 in keratinocytes and in psoriatic skin lesions. Our data link the disease-intrinsic danger signals psoriasin (S100A7) and dsDNA for NLPR1-dependent caspase-5 activity in psoriasis providing potential therapeutic targets in Th17-mediated skin autoinflammation.

Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1.

Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1, RPTP-ζ), an additional IL-34 receptor, was recently identified. Here, we aimed to assess PTPRZ1 expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of PTPRZ1 was higher in non-IBD colon compared to ileum. PTPRZ1 expression was not altered with inflammation in IBD, however, correlated to IL34, CSF1, and CSF1R. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor α (TNF-α), IL-1ß, interferon γ (IFN-γ), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that PTPRZ1 was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions.

The antimicrobial peptides psoriasin (S100A7) and koebnerisin (S100A15) suppress extracellular matrix production and proliferation of human fibroblasts.

BACKGROUND/AIMS: Keloids result from aberrations in the normal wound healing cascade and can lead to pruritus, contractures and pain. The underlying mechanisms of excessive scarring are not yet understood, and most therapeutic strategies remain unsatisfactory. Psoriasin (S100A7) and koebnerisin (S100A15) are released by keratinocytes during physiological wound healing. We found S100 production is markedly decreased in keloid scar tissue. The disturbed epidermal S100 expression might contribute to keloid formation; thus, we studied their effect on dermal fibroblasts and extracellular matrix (ECM) production. METHODS: S100 peptides, ECM regulation and distribution were analysed in normal and keloid tissue by quantitative PCR (qPCR), immunoblotting and immunofluorescent staining. Isolated dermal fibroblasts were incubated with S100 proteins, and the regulation of ECM and transforming growth factor (TGF)-ß was determined using qPCR. Fibroblast proliferation and viability were determined by the 5-bromo-2'-deoxyuridine assay and crystal violet assay. RESULTS: Keloid tissue featured a pronounced expression of ECMs, such as collagen types 1 and 3, whereas the production of psoriasin and koebnerisin was markedly decreased in keloid-derived cells and keloid tissue. Both S100 proteins inhibited the expression of collagens, fibronectin-1, α-smooth-muscle actin and TGF-ß by fibroblasts. Further, they also suppressed fibroblast proliferation. CONCLUSION: Psoriasin and koebnerisin show antifibrotic effects and may lead to novel preventive and therapeutic strategies for fibroproliferative diseases.

Vitamin D analog calcipotriol suppresses the Th17 cytokine-induced proinflammatory S100 "alarmins" psoriasin (S100A7) and koebnerisin (S100A15) in psoriasis.

The antimicrobial peptides (AMP) psoriasin (S100A7) and koebnerisin (S100A15) are differently induced in psoriatic skin. They act synergistically as chemoattractants and "alarmins" to amplify inflammation in psoriasis. Th17 cytokines are key players in psoriasis pathogenesis and vitamin D analogs feature anti-psoriatic effects; both of these activities could be mediated through epidermal AMP regulation. We show that supernatants of cultured psoriatic T cells induce and release psoriasin and koebnerisin from keratinocytes and the Th17 cytokines IL-17A, tumor necrosis factor-α, and IL-22 differently regulate psoriasin and koebnerisin reflecting their distinct expression pattern in normal and psoriatic skin. IL-17A is the principal inducer of both S100 and their expression is further amplified by cooperating Th17 cytokines in the micromilieu of psoriatic skin. Increased extracellular psoriasin and koebnerisin also synergize as "alarmins" to prime epidermal keratinocytes for production of immunotropic cytokines that further amplify the inflammatory response. Treatment of psoriatic plaques with the vitamin D analog calcipotriol interferes with the S100-mediated positive feedback loop by suppressing the increased production of psoriasin and koebnerisin in psoriatic skin and their Th17-mediated regulation in epidermal keratinocytes. Thus, targeting the S100-amplification loop could be a beneficial anti-inflammatory approach in psoriasis and other inflammatory skin diseases.