Cell Type-specific Signaling Pathways Research Articles

Chrysanthemum Zawadskii Herbich var. latilobum (Maxim.) Kitamura water extract prevents BALB/c mice lung injury from particulate matter 10 toxicity

ABSTRACT Chronic exposure to airborne particulate matter (PM) causes respiratory damage in humans owing to oxidative stress and inflammation. Chrysanthemum zawadskii Herbich var. latilobum (Maxim.) Kitamura (CZL) has been used in traditional medicine to treat several inflammatory diseases; however, studies on inflammatory pulmonary diseases are scarce. This study investigated the protective effects of CZL extract against PM10-induced lung injury in BALB/c mice. Cell type specific signaling pathways were explored using A549 and RAW264.7 cell lines. CZL extract noticeably attenuated PM10-induced lung injury and inflammatory cell infiltration in a mouse model. Protein markers, such as p-AKT, p-ERK, and p-NF-κB for PM10 induced lung inflammation were effectively reduced in CZL extract-treated mice and cells. Furthermore, CZL extracts considerably reduced the generation of reactive oxygen species and nitric oxide in cells. Collectively, CZL extract effectively reduced PM10-induced lung injury by suppressing pulmonary inflammation, potentially due to its anti-inflammatory and antioxidant properties.

Ameliorative Effect of Citrus junos Tanaka Waste (By-Product) Water Extract on Particulate Matter 10-Induced Lung Damage.

Citrus junos Tanaka (CJ)-related products are well-accepted by consumers worldwide; thus, they generate huge amounts of waste (peel, pulp, and seed) through CJ processing. Although some CJ by-products (CJBs) are recycled, their use is limited owing to the limited understanding of their nutritional and economic value. The exposure to particulate matter (PM) increases the risk of respiratory diseases. In this study, we investigated the ameliorative effects of CJB extracts (100, 200 mg/kg/day, 7 days) on PM10-induced (10 mg/kg, intranasal, 6 h) lung damage in BALB/c mice. Cell type-specific signaling pathways are examined using the A549 (PM10, 200 μg/mL, 6 h) and RAW264.7 (LPS, 100 ng/mL, 6 h) cell lines. The CJB extracts significantly attenuated PM10-induced pulmonary damage and inflammatory cell infiltration in a mouse model. The essential protein markers in inflammatory signaling pathways, such as AKT, ERK, JNK, and NF-κB for PM10-induced phosphorylation, were dramatically reduced by CJB extract treatment in both the mouse and cell models. Furthermore, the CJB extracts reduced the production of reactive oxygen species and nitric oxide in a dose-dependent manner in the cells. Comprehensively, the CJB extracts were effective in reducing PM10-induced lung injuries by suppressing pulmonary inflammation, potentially due to their anti-inflammatory and antioxidant properties.

Energy and Dynamics of Caveolae Trafficking.

Caveolae are 70–100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study.

Proteome Analysis Reveals Distinct Mitochondrial Functions Linked to Interferon Response Patterns in Activated CD4+ and CD8+ T Cells.

While genetic traits and epigenetic modifications mainly encode cell type-specific effector functions, the eventual outcome is also prone to modulation by post-transcriptional regulation mechanisms. T cells are a powerful model for the investigation of such modulatory effects, as common precursor cells may differentiate either to helper CD4+ T cells or cytotoxic CD8+ cells, which elicit distinct functionalities upon TCR-stimulation. Human primary CD4+ and CD8+ T cells were purified from three individual donors and activated with anti-CD3/CD28 antibodies. Associated proteome alterations were analyzed by high-resolution mass spectrometry using a label-free shotgun approach. Metabolic activation was indicated by upregulation of enzymes related to glycolysis, NADH production, fatty acid synthesis, and uptake as well as amino acid and iron uptake. Besides various inflammatory effector molecules, the mitochondrial proteins CLUH, TFAM, and TOMM34 were found specifically induced in CD4+ T cells. Investigation of overrepresented conserved transcription binding sites by the oPOSSUM software suggested interferon type I inducer IRF1 to cause many of the observed proteome alterations in CD4+ T cells. RT qPCR demonstrated the specific induction of IRF1 in CD4+ T cells only. While the interferon regulatory factor IRF4 was found induced in both T cell subtypes at protein and mRNA level, IRF9 and the type I interferon-induced proteins IFIT1, IFIT3, and MX1 were only found induced in CD4+ T cells. As oxidative stress enhances mitochondrial DNA-dependent type I interferon responses, the present data suggested that mitochondrial activities regulate those cell type-specific signaling pathways. Indeed, we detected mitochondrial superoxide formation predominantly in CD4+ T cells via FACS analysis with MitoSOX™ and confirmed this observation by live cell imaging with confocal microscopy. As interferon signaling regulates important features such as resistance regarding immune checkpoint blockade therapy, the present data may identify potential new targets for the efficient control of highly relevant immune cell properties.

Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders.

Abstract B34: Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature.

Abstract Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Using a murine model of AML that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V), we have found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival (Sachs et al. in revisions). Similarly, recent work has shown that NRASG12V has bimodal, mutually exclusive effects in hematopoietic stem cells (HSCs) providing a proliferation advantage in some HSCs and a self-renewal advantage in others (Li et al. Nature 2013). These data show that mutant NRAS enforces different behaviors in similar cells and suggest that these discrepant effects may be mediated by different NRAS-activated pathways and gene expression pathways. In order to define the mechanisms by which NRASG12V directs self-renewal in AML, we investigated the NRASG12V-mediated transcriptional profile of self-renewing leukemic subgroups and single cells from our AML model. We sorted our primary murine AML into immunophenotypic subgroups and performed bulk cell RNA sequencing on these groups. The Mac-1High group displayed poor self-renewal capacity in colony forming assays and in vivo leukemia transplant assays. In contrast, the Mac-1Low/c-Kit+/Sca-1+ (MLowKS) subgroup was enriched for self-renewal capacity and displayed significantly increased colony forming ability and enhanced in vivo leukemia transplantability. Using principal component analysis and unsupervised hierarchical clustering, we found that each of our immunophenotypic subpopulations were transcriptionally distinct. Using this dataset, we identified the NRASG12V-directed leukemia self-renewal gene expression signature. Gene Set Enrichment Analysis (GSEA, et al. 2005) of this dataset showed that the MLowKS group preferentially expressed previously published transcriptional signatures of leukemia self-renewal. These data also show that NRASG12V enforces distinct gene expression signatures among leukemic subpopulations. Since others have shown that NRASG12V can enforce bimodal behaviors among cells within the same hematopoietic compartment (HSCs, Li et al. Nature 2013), we speculated that the NRASG12V–mediated transcriptional signature critical for leukemia self-renewal may be variable among individual cells. In order to identify that signature and to assess the cell-to-cell heterogeneity of NRASG12V-mediated transcription, we performed single cell RNA sequencing of unsorted and sorted leukemia cells from our murine AML model. In unsupervised hierarchical clustering algorithms, our single cells clustered into five distinct transcriptional signatures, demonstrating that individual leukemia cells are transcriptionally distinct and identifying the NRASG12V-mediated leukemia self renewal signature. Currently, we employ ultra-high parameter flow cytometry (mass cytometry, CyTOF) to identify the cell type specific signaling pathways associated with these discrepant transcriptional signatures. These data identify the NRASG12V-mediated self-renewal gene expression signature at the single cell level and will allow us to identify the specific signaling pathways that activate this signature. Citation Format: Zohar Sachs, Rebecca S. LaRue, Klara Noble, Susan K. Rathe, Aaron L. Sarver, Ngoc A. Ha, David A. Largaespada. Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B34.

Cell volume and monovalent ion transporters: their role in cell death machinery triggering and progression

Cell death is accompanied by the dissipation of electrochemical gradients of monovalent ions across the plasma membrane that, in turn, affects cell volume via modulation of intracellular osmolyte content. In numerous cell types, apoptotic and necrotic stimuli caused cell shrinkage and swelling, respectively. Thermodynamics predicts a cell type-specific rather than an ubiquitous impact of monovalent ion transporters on volume perturbations in dying cells, suggesting their diverse roles in the cell death machinery. Indeed, recent data showed that apoptotic collapse may occur in the absence of cell volume changes and even follow cell swelling rather than shrinkage. Moreover, side-by-side with cell volume adjustment, monovalent ion transporters contribute to cell death machinery engagement independently of volume regulation via cell type-specific signaling pathways. Thus, inhibition of Na(+)-K(+)-ATPase by cardiotonic steroids (CTS) rescues rat vascular smooth muscle cells from apoptosis via a novel Na(+)i-K(+)i-mediated, Ca(2+)i-independent mechanism of excitation-transcription coupling. In contrast, CTS kill renal epithelial cells independently of Na(+)-K(+)-ATPase inhibition and increased [Na(+)]i/[K(+)]i ratio. The molecular origin of [Na(+)]i/[K(+)]i sensors involved in the inhibition of apoptosis as well as upstream intermediates of Na(+)i/K(+)i-independent death signaling triggered by CTS remain unknown.

Abstract C85: ck2 inhibitors in luminal and basal-like breast cancer cells.

Abstract Protein kinase casein kinase II (CK2) is a heterotetrameric serine/threonine kinase involved in proliferation, apoptosis and embryonic development. It is expressed at elevated levels in proliferating tissue, human cancer cell lines and virtually all forms of solid cancers. Recently it has been shown that CK2 inhibition induces apoptosis in cell models of acquired anti-estrogen resistance. These findings suggest that there may be tumor phenotype specific effects of CK2 in breast cancer and consequently phenotype specific sensitivity to CK2 inhibition. As such we sought to determine if luminal and basal cell types display differential sensitivity to selective CK2 inhibitors. Utilizing crystal violet and alamar blue assays, it was found that CK2 inhibition decreased viability of both luminal and basal breast cancer cell lines, with basal-like ER(−) MDA MB 231 cells showing notable sensitivity. Hypertrophic morphologic changes consistent with cellular senescence were noted upon inhibition of CK2. Additionally, flow cytometric analysis revealed an increase in mean vimentin expression in basal but not luminal breast cancer cells following CK2 inhibition. These studies suggest that CK2 utilizes multiple molecular mechanisms to promote tumorigenesis, and that CK2 inhibition may disrupt cell-type specific signaling pathways to induce breast cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C85.

PI3Kγ Mediates Kaposi's Sarcoma-Associated Herpesvirus vGPCR-Induced Sarcomagenesis

Angioproliferative tumors induced by the Kaposi's sarcoma-associated herpesvirus (KSHV) have been successfully treated with rapamycin, which provided direct evidence of the clinical activity of mTOR inhibitors in human malignancies. However, prolonged mTOR inhibition may raise concerns in immunocompromised patients, including AIDS-Kaposi's sarcoma (KS). Here, we explored whether KSHV oncogenes deploy cell type-specific signaling pathways activating mTOR, which could be exploited to halt KS development while minimizing immune suppressive effects. We found that PI3Kγ, a PI3K isoform exhibiting restricted tissue distribution, is strictly required for signaling from the KSHV-encoded vGPCR oncogene to Akt/mTOR. Indeed, by using an endothelial-specific gene delivery system modeling KS development, we provide genetic and pharmacological evidence that PI3Kγ may represent a suitable molecular target for therapeutic intervention in KS.

IFN-γInduces Apoptosis in HL-60 Cells Through Decreased Bcl-2 and Increased Bak Expression

Interferons (IFNs) are pleiotropic cytokines responsible for inducing innate and adaptive immunities against a wide range of viruses and other microbial pathogens. In addition, IFNs also exert antitumor activities due to their antiproliferative, immunomodulatory, proapoptotic functions. In the last decades, the successful clinical application of IFNs for treatment of cancer, particularly leukemia, has improved the quality and longevity of life for many patients. The induction of tumor cell apoptosis by IFNs is believed to contribute, at least in part, to the beneficial effects. IFN subtypes, such as IFN-alpha, IFN-beta, and IFN-gamma, induce apoptosis through cell type-specific signaling pathways, and several putative IFN-stimulated genes (ISGs) with proapoptotic functions have been identified. Here, we analyzed the ability of IFN-alpha, IFN-beta, or IFN-gamma to induce apoptosis in several malignant hematologic cell lines. We found that treatment with IFN-gamma, but not IFN-alpha, or IFN-beta, specifically induces HL-60 leukemia cells to undergo apoptosis. Roughly 30% of HL-60 cells treated for 48 h with IFN-gamma, but not IFN-gamma, or IFN-beta, underwent apoptosis as monitored by annexin V labeling to determine changes in phosphatidylserine (PS) asymmetry and TUNEL assay to detect DNA fragmentation. Consistent with these results, treatment with IFN-gamma, but not IFN-alpha or IFN-beta, induced the release of cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), a well-characterized caspase-3 substrate. Further investigation into the potential mechanism responsible for mitochondrial disruption revealed that treatment with IFN-gamma caused decreased levels of Bcl-2 and increased levels of Bak. This study thus provides the basis for additional research to uncover the molecular mechanism by which IFN-gamma regulates the expression of Bcl-2 family members in various cell types.

Constitutive collagenase‐1 synthesis through MAPK pathways is mediated, in part, by endogenous IL‐1α during fibrotic repair in corneal stroma

Collagenase-1 is a protease expressed by active fibroblasts that is involved in remodeling of the extracellular matrix (ECM). In this study, we characterize the intracellular signaling mechanism of collagenase-1 production by IL-1alpha in subcultured normal fibroblasts (NF) from uninjured normal corneas, compared to that in repair wound fibroblasts (WF). In NF, collagenase-1 was induced specifically after the exogenous addition of IL-1alpha via activation of ERK and p38MAPK. Collagenase-1 expression was strongly suppressed upon treatment with either a MEK or p38MAPK inhibitor. In contrast, repair WF constitutively synthesized both IL-1alpha and collagenase-1. Combined treatment with both mitogen-activated protein kinase (MAPK) inhibitors dramatically reduced collagenase-1 synthesis, while individual MEK1 or p38 inhibitors weakly modulated the collagenase-1 level. The results indicate that both pathways are crucial in the regulation of collagenase-1 synthesis. Furthermore, an IL-1alpha receptor antagonist (IL-1ra) could not abolish constitutive collagenase-1 synthesis, even at high doses, suggesting that other cytokines/factors are additionally involved in this process. We propose that induction of collagenase-1 by IL-1alpha in both WF and NF depends on a unique combination of cell type-specific signaling pathways.

The Lipoxin Receptor ALX: Potent Ligand-Specific and Stereoselective Actions in Vivo

Lipoxins (LXs) and aspirin-triggered LX (ATL) are trihydroxytetraene-containing eicosanoids generated from arachidonic acid that are distinct in structure, formation, and function from the many other proinflammatory lipid-derived mediators. These endogenous eicosanoids have now emerged as founding members of the first class of lipid/chemical mediators involved in the resolution of the inflammatory response. Lipoxin A(4) (LXA(4)), ATL, and their metabolic stable analogs elicit cellular responses and regulate leukocyte trafficking in vivo by activating the specific receptor, ALX. ALX was the first receptor cloned and identified as a G protein-coupled receptor (GPCR) for lipoxygenase-derived eicosanoids with demonstrated cell type-specific signaling pathways. ALX at the level of DNA has sequence homology to the N-formylpeptide receptor and as an orphan GPCR was initially referred to as the N-formylpeptide receptor-like 1. Although LXA(4) is the endogenous potent ligand for ALX activation, a number of peptides can also activate this receptor to stimulate calcium mobilization and chemotaxis in vitro. In contrast with LXA(4), the counterparts of many of these peptides in vivo remain to be established. The purpose of this review is to highlight the molecular characterization of the ALX receptor and provide an overview of the ALX-LXA(4) axis responsible for anti-inflammatory and proresolving signals in vivo. The information in this review provides further support for the initial nomenclature proposition for this GPCR as ALX.

Transforming Growth Factor β Activation of c-Abl Is Independent of Receptor Internalization and Regulated by Phosphatidylinositol 3-Kinase and PAK2 in Mesenchymal Cultures

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.

Anti-inflammatory circuitry: Lipoxin, aspirin-triggered lipoxins and their receptor ALX

Endogenous chemical mediators or autacoids play key roles in controlling inflammation and its programmed resolution. Among them, it is known that lipoxins (LX) and aspirin-triggered LX (ATL) evoke bioactions in a range of physiologic and pathophysiologic processes and serve as endogenous lipid/chemical mediators that stop neutrophilic infiltration and initiate resolution. LXA 4, ATL and their metabolic stable analogs elicit cellular responses and regulate PMN in vivo via interacting with their specific receptor, namely ALX. ALX is the first cloned and identified lipoxygenase-derived eicosanoid receptor with cell type-specific signaling pathways. Also, ALX could regulate PMN by interacting with each class of ligands (lipid vs. peptide) within specific phases of an inflammatory response. Together LX, ATL and ALX may provide new opportunities to design “resolution-targeted” therapies with high degree of precision in controlling inflammation. In this chapter, we give an overview and update of the current actions for LX and ATL, the identification of ALX and their novel anti-inflammatory and pro-resolving signals.

Comparative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on Aldosterone Secretion in Normal Bovine and Human Tumorous Adrenal Cells*

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.