Melissa G. Hunter

MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines

Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.

Integrating the MicroRNome into the Study of Lung Disease

Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.

SHIP2 Is Recruited to the Cell Membrane upon Macrophage Colony-Stimulating Factor (M-CSF) Stimulation and Regulates M-CSF-Induced Signaling

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-κB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.

Phospholipase A2 activation regulates cytotoxicity of methylmercury in vascular endothelial cells.

Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A2 (PLA2) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [3H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA2 activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA2 in a dose-dependent (5 to 15 μM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiolprotective) agents, calcium chelators, antioxidants, and PLA2-specific inhibitors attenuated the MMC-induced PLA2 activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA2 in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA2 inhibitors. This study established that PLA2 activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA2 signaling in the methylmercury-induced vascular endothelial dysfunctions.

Intermittent hypoxia suppresses adiponectin secretion by adipocytes.

Obstructive sleep apnea (OSA), characterized by cyclic intermittent hypoxia (IH) during sleep, is an independent risk factor for cardiovascular disease. Adiponectin (APN), an adipocytokine secreted exclusively by adipocytes, possesses antiatherogenic properties. Low levels of APN, particularly the high-molecular-weight (HMW) form, are associated with an increased risk of cardiovascular disease. Here, we hypothesized that IH would result in the dysregulation of APN expression and secretion. 3T3-L1 adipocytes were exposed to IH at 12 cycles/h for 6 h/d to simulate the IH condition similar to that encountered in OSA. Control adipocytes were exposed to 21% O(2) under identical conditions. After 48 h of incubation, IH caused a decrease in the secretion of total and HMW APN in spite of a significant upregulation of APN mRNA expression by adipocytes. This study suggested a novel mechanism of how the cyclic hypoxemia in OSA predisposes OSA patients to cardiovascular disease through the dysregulation of secretion of APN by adipocytes. Further studies are needed to determine the exact molecular mechanism how IH reduces the release of APN by adipocytes.

Activation of Estrogen Receptor-α Reduces Aortic Smooth Muscle Differentiation

Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-&agr; (ER&agr;) by agonists or by growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ER&agr; expression on SMC differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower spontaneous expression of ER&agr; and higher levels of the differentiation markers calponin and smooth muscle &agr;-actin than SMC from women. When SMC containing low expression of ER&agr; were transduced with a lentivirus containing ER&agr;, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ER&agr; expression by small interfering RNA (siRNA) in cells expressing high levels of ER&agr; enhanced the expression of differentiation markers. ER&agr; expression and activation reduced the phosphorylation of Smad2, a signaling molecule important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ER&agr; activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability.

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.

Alveolar macrophages lack CCR2 expression and do not migrate to CCL2

BackgroundThe recruitment of mononuclear cells has important implications for tissue inflammation. Previous studies demonstrated enhanced CCR1 and CCR5 expression and decreased CCR2 expression during in vitro monocyte to macrophage differentiation. To date, no study examined the in vivo differences in chemokine receptor expression between human peripheral blood monocytes and alveolar macrophages.MethodsWe examined the expression of these receptors in human peripheral blood monocytes and alveolar macrophages using microarray analysis, reverse-transcriptase PCR, flow cytometry and migration analyses.ResultsIn contrast to peripheral blood monocytes, alveolar macrophages did not express the CCL2 receptor, CCR2, and did not migrate toward CCL2. In contrast, monocytes and freshly isolated resident alveolar macrophages both migrated towards CCL3. However, up to 6-fold more monocytes migrated toward equivalent concentrations of CCL3 than did alveolar macrophages from the same donor. While peripheral blood monocytes expressed the CCL3 receptor, CCR1, alveolar macrophages expressed the alternate CCL3 receptor, CCR5. The addition of anti-CCR5 blocking antibodies completely abrogated CCL3-induced migration in alveolar macrophages, but did not affect the migration of peripheral blood monocytes.ConclusionThese data support the specificity of CCL2 to selectively drive monocyte, but not alveolar macrophage recruitment to the lung and CCR5 as the primary macrophage receptor for CCL3.

LRG-Accelerated Differentiation Defines Unique G-CSFR Signaling Pathways Downstream of PU.1 and C/EBPε That Modulate Neutrophil Activation

Expression of leucine‐rich α2 glycoprotein (LRG), a member of the leucine‐rich repeat family of proteins, was recently shown to be up‐regulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBPε that regulate the expression of multiple myeloid‐specific genes also bind to the LRG promoter. We also demonstrate that LRG localizes to the same cytoplasmic compartment as myeloperoxidase and that G‐CSF treatment of the 32Dcl3 myeloid cell line induces nuclear translocation of LRG. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated, G‐CSF‐mediated neutrophil differentiation and induction of CD11b expression. In contrast, constitutive expression of LRG in 32Dwt18 cells, expressing a chimeric erythropoietin (Epo)/G‐CSFR consisting of the EpoR extracellular domain fused to the G‐CSFR transmembrane and cytoplasmic domains, failed to induce accelerated neutrophil differentiation and CD11b expression in response to Epo stimulation. LRG‐mediated accelerated differentiation and CD11b expression were found to correlate with an increased level of phospho‐Stat3 but not with PU.1 or p27kip1 levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBPε. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via nonredundant G‐CSFR signals.

Role of the proteasome in modulating native G-CSFR expression.

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.