Cell-specific Manner Research Articles

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

IL-22 is rapidly induced by Pathogen Recognition Receptors Stimulation in Bone-Marrow-derived Dendritic Cells in the Absence of IL-23.

In vertebrates, microorganisms are recognized by pathogen recognition receptors (PRRs). Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of genes. Within these genes, the cytokines family plays a crucial function because of its role in adaptive immunity induction and in tissue-specific functional regulation, such as tissue repair and tissue homeostasis during steady state conditions. Within the myeloid compartment, dendritic cells (DCs) release a variety of inflammatory cytokines in response to microbes. In this study, we show that BMDCs release IL-22 directly upon PRRs activation without the need of IL-23 signaling as reported for other IL22-producing cells. Moreover, we demonstrate that cytokine IL-22 is rapidly released in a cell-specific manner as macrophages are not able to produce IL-22 through the same PRRs system. In addition, we characterize the intracellular signaling cascade required for IL-22 release in BMDCs. Myd88, MEK1/2, NFkb and AhR, but not p38, NFAT, and RORgt, were found to be involved in IL-22 regulation in DCs. Our study suggests that BMDCs possess a unique intracellular molecular plasticity which, once activated, directs different BMDCs functions in a cell-specific manner.

Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

Reconfiguring the AR-TIF2 Protein-Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen.

The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein-protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC.

Who watches the watchmen? Regulation of the expression and activity of sirtuins.

Sirtuins (SIRT1-7) are a family of nicotine adenine dinucleotide (NAD+)-dependent enzymes that catalyze post-translational modifications of proteins. Together, they regulate crucial cellular functions and are traditionally associated with aging and longevity. Dysregulation of sirtuins plays an important role in major diseases, including cancer and metabolic, cardiac, and neurodegerative diseases. They are extensively regulated in response to a wide range of stimuli, including nutritional and metabolic challenges, inflammatory signals or hypoxic and oxidative stress. Each sirtuin is regulated individually in a tissue- and cell-specific manner. The control of sirtuin expression involves all the major points of regulation, including transcriptional and post-translational mechanisms and microRNAs. Collectively, these mechanisms control the protein levels, localization, and enzymatic activity of sirtuins. In many cases, the regulators of sirtuin expression are also their substrates, which lead to formation of intricate regulatory networks and extensive feedback loops. In this review, we highlight the mechanisms mediating the physiologic and pathologic regulation of sirtuin expression and activity. We also discuss the consequences of this regulation on sirtuin function and cellular physiology.-Buler, M., Andersson, U., Hakkola, J. Who watches the watchmen? Regulation of the expression and activity of sirtuins.

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner.

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2ʹ-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state.

Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.

Comparison of photodynamic treatment produced cell damage between human breast cancer cell MCF-7 and its multidrug resistance cell

BackgroundMultidrug resistance (MDR) of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently the anti-tumor effects of Chlorin e6 (Ce6) mediated photodynamic therapy (Ce6-PDT) were reported in skin cancer and hepatoma in vitro. However, its therapeutic potential in killing human breast cancer especially those with MDR and the differences between MCF-7 and MCF-7/ADR after PDT treatment has not been fully investigated. MethodsMTT assay was used to measure cell survival rate of MCF-7 cells and MCF-7/ADR cells. Intracellular reactive oxygen species (ROS) generation was measured by monitoring the fluorescence intensity of dichlorofluorescein (DCF) by flow cytometry. Nuclear morphology changes and DNA damage in both MCF-7 and MCF-7/ADR after Ce6-PDT were analyzed by hochest33342 staining and comet assay. Western blot and monodansylcadaverine (MDC) staining were used to monitor autophagic response in MCF-7/ADR. ResultsCe6-PDT induced cell viability decrease, intracellular ROS generation, and DNA damage in concentration-dependent and cell-specific manner, and MCF-7 was more sensitive to Ce6-PDT than MCF-7/ADR cells at the same PDT condition. PDT treatment could trigger cell death via apoptosis in MCF-7 cells but autophagic cell death in MCF-7/ADR cells. ConclusionThese results suggested that MCF-7 was more sensitive to Ce6-PDT than MCF-7/ADR, and PDT treatment could trigger apoptotic response in MCF-7 cells, but stimulate autophagic response in MCF-7/ADR cells.

Sialylation of Glycosylphosphatidylinositol (GPI) Anchors of Mammalian Prions Is Regulated in a Host-, Tissue-, and Cell-specific Manner

Prions or PrP(Sc) are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrP(C) PrP(C) is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrP(C) gives rise to glycosylated and GPI-anchored PrP(Sc) The question of the sialylation status of GPIs within PrP(Sc) has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrP(Sc), with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrP(C) with sialylo-GPIs could be recruited into PrP(Sc), whereas PrP(C) with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrP(Sc) generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrP(C) with both sialo- and asialo-GPIs is a common feature of PrP(Sc) The mixtures of sialo- and asialo-GPIs were observed in PrP(Sc) universally regardless of prion strain as well as host, tissue, or type of cells that produced PrP(Sc) Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrP(C) with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrP(Sc) is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis.

Binding of transition metals to S100 proteins.

The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.

Apoptotic properties of Citrus sudachi Hort, ex Shirai (Rutaceae) extract on human A549 and HepG2 cancer cells

Purpose: To investigate whether Citrus sudachi harvested at two stages of maturity can induce toxicity in a cell-specific manner and to determine the possible mechanisms of Citrus sudachi-induced cytotoxic responses in two types of cancer cells (human lung adenocarcinoma A549 and hepatocellular carcinoma HepG2 cells) and two normal cell lines (lung 16HBE140- and liver CHANG cells).Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and annexin V/propidium iodidle assay were used to test the antiproliferative activity and apoptosis of methanol extract of Citrus sudachi, respectively. Griess reaction and reverse transcriptase-polymerase chain reaction (RT-PCR) were carried out to evaluate nitric oxide (NO•) production and the mRNA levels of inhibitors of apoptosis (IAP).Results: Citrus sudachi exerted cytotoxicity in a time-dependent manner in cancer cells which increased with increase in maturity but did not affect normal cells. Citrus sudachi was found to induce accumulation of cells in the sub-G1 cell cycle phase, fragmentation of DNA and cell death with characteristics of apoptosis, in both types of cancer cells. Moreover, Citrus sudachi upregulated cellular NO• produced by activation of nitric oxide synthase (NOS), while it suppressed the levels of IAP mRNA in both types of cancer cells.Conclusion: The results obtained suggest that Citrus sudachi induces apoptosis in A549 and HepG2 cells, which may be mediated by NO•. There is need for further studies on the role of Citrus sudachi in cancer treatment.Keywords: Apoptosis, Citrus sudachi, Human lung and liver cancer cells, Inhibitors of apoptosis, Nitric oxide

Contradicting interplay between insulin-like growth factor-1 and miR-486-5p in primary NK cells and hepatoma cell lines with a contemporary inhibitory impact on HCC tumor progression

In this study, an impaired natural killer (NK) cell cytolytic activity in 135 hepatocellular carcinoma (HCC) patients parallel to a reduced expression level of insulin-like growth factor (IGF)-1 in NK cells of HCC patients has been revealed. Ectopic expression of miR-486-5p, a direct upstream regulator of IGF-1, restored the endogenous level of IGF-1 in NK cells of HCC patients, thus augmenting its cytolytic activity against Huh7 cells in an opposite manner to the IGF-1 siRNAs. Unorthodoxly, over-expression of miR-486-5p in target hepatocytes resulted in the repression of IGF-1, suppression of Huh7 cells proliferation and viability in a similar pattern to the IGF-1 siRNAs. Therefore, this study highlights a potential role of IGF-1 in modulating cytolytic potential of NK cells of HCC patients. miR-486-5p acts in a cell-specific manner, differentially modulating IGF-1 expression in NK cells and their target hepatocytes with a contemporary inhibitory impact on HCC progression.

Expression and distribution of forkhead activin signal transducer 2 (FAST2) during follicle development in mouse ovaries and pre-implantation embryos

Xenopus forkhead activin signal transducer 1 (xFAST 1) was first characterized in Xenopus as the transcriptional partner for Smad proteins. FAST2, which is the xFAST 1 homologues in mouse, is expressed during early developmental stages of the organism. However, the function of FAST2 in mouse ovaries and pre-implantation embryos is unclear. Therefore, we investigated its expression during these processes. In postnatal mice, FAST2 was expressed in oocytes and thecal cells from postnatal day (PD) 1 to PD 21. In gonadotropin-induced immature mice, FAST2 was expressed in oocytes, thecal cells and newly formed corpora lutea (CLs), but was expressed at a lower level in degenerated CLs. Similar results were observed upon western blot analyses. In meloxicam-treated immature mice, ovulation was inhibited and FAST2 was expressed in thecal cells, luteinized granulosa cells and entrapped oocytes. Immunofluorescence results showed that FAST2 was expressed in the cytoplasm and nucleus but not the nucleolus from the zygote to 8-cell embryo stage, after which it was localized to the cytoplasm of the morulae and inner cell mass of the blastocysts. Taken together, these observations suggest that FAST2 is expressed in a cell-specific manner during ovarian follicle development, ovulation, luteinization and early embryonic development, and that FAST2 might play important roles in these physiological processes.

LSm14A Plays a Critical Role in Antiviral Immune Responses by Regulating MITA Level in a Cell-Specific Manner.

Viral infection triggers induction of antiviral cytokines and effectors, which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice, in this study, we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs, which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses.

Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean–rhizobia symbiosis.

Analysis of the structure, evolution, and expression of CD24, an important regulator of cell fate

BackgroundCD24 is a small, glycophosphatidylinositol-anchored cell surface receptor, expressed in a variety of cells types and tissues. CD24 gene and protein expression is highly dynamic in response to cellular differentiation and stimulation in a cell-specific manner. Furthermore, CD24 interacts with a diverse collection of ligands, including cell adhesion molecules such as P-selectin, and the immune-associated siglec family of transmembrane proteins. While much is known regarding the biological roles of CD24 in regulating cell survival, death and differentiation, little is known about the evolution and organization of CD24 across species or the relationship between CD24 expression and its known ligands. ResultsWe analyzed the organization and evolution of the CD24 gene from 56 mammalian, avian and reptilian species. We further examined the mRNA expression of CD24 and its known ligands in Mus musculus in immune cells, immunologically privileged tissues, developing brain, and developing and regenerating liver. CD24 arose prior to the reptilian–avian divergence and is conserved across many mammalian species, although we were unable to identify CD24 in marsupials or monotremes. The CD24 genomic structure is diverse between and within species, with varying numbers of exons, introns, and the presence of untranslated regions. Of note, we found no obvious criteria distinguishing CD24 genes from those annotated as CD24-like. The expression of CD24 is similarly complex, with immune cells showing dynamic changes in mRNA levels during development, while immunologically privileged and developing tissues show a high, static expression level that decreases in mature tissues. Furthermore, the expression of CD24 correlated with some but not all of its known ligands in a tissues-specific manner, suggesting that novel ligands have yet to be identified and that cell-specific ligand expression can influence CD24 function. ConclusionsWe find that CD24 arose prior to the divergence of reptiles, birds and mammals. Furthermore, the most highly conserved areas of the protein are the amino acids which can be glycosylated. We also find that CD24 expression is highly tissue-specific and in many cases, not well conserved with known CD24 ligands, suggesting yet-unknown CD24–ligand interactions. Together, these data are a valuable resource for furthering studies in CD24 biology.

Cell-specific synaptic plasticity induced by network oscillations.

Gamma rhythms are known to contribute to the process of memory encoding. However, little is known about the underlying mechanisms at the molecular, cellular and network levels. Using local field potential recording in awake behaving mice and concomitant field potential and whole-cell recordings in slice preparations we found that gamma rhythms lead to activity-dependent modification of hippocampal networks, including alterations in sharp wave-ripple complexes. Network plasticity, expressed as long-lasting increases in sharp wave-associated synaptic currents, exhibits enhanced excitatory synaptic strength in pyramidal cells that is induced postsynaptically and depends on metabotropic glutamate receptor-5 activation. In sharp contrast, alteration of inhibitory synaptic strength is independent of postsynaptic activation and less pronounced. Further, we found a cell type-specific, directionally biased synaptic plasticity of two major types of GABAergic cells, parvalbumin- and cholecystokinin-expressing interneurons. Thus, we propose that gamma frequency oscillations represent a network state that introduces long-lasting synaptic plasticity in a cell-specific manner.

Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation.

Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues.