MAP kinase pathways and calcitonin influence CD44 alternate isoform expression in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Dysregulated expression and splicing of cell adhesion marker CD44 is found in many types of cancer. In prostate cancer (PC) specifically, the standard isoform (CD44s) has been found to be downregulated compared with benign tissue whereas predominant variant isoform CD44v7-10 is upregulated. Mitogen-activated protein kinase pathways and paracrine calcitonin are two common factors linked to dysregulated expression and splicing of CD44 in cancer. Calcitonin has been found to increase proliferation and invasion in PC acting through the protein kinase A pathway.</p> <p>Methods</p> <p>In androgen-independent PC with known high CD44v7-10 expression, CD44 total and CD44v7-10 RNA or protein were assessed in response to exogenous and endogenous calcitonin and to inhibitors of protein kinase A, MEK, JNK, or p38 kinase. Benign cells and calcitonin receptor-negative PC cells were also tested.</p> <p>Results</p> <p>MEK or p38 but not JNK reduced CD44 total RNA by 40%–65% in cancer and benign cells. Inhibition of protein kinase A reduced CD44 total and v7-10 protein expression. In calcitonin receptor-positive cells only, calcitonin increased CD44 variant RNA and protein by 3 h and persisting to 48 h, apparently dependent on an uninhibited p38 pathway. Cells with constitutive CT expression showed an increase in CD44v7-10 mRNA but a decrease in CD44 total RNA.</p> <p>Conclusion</p> <p>The MEK pathway increases CD44 RNA, while calcitonin, acting through the protein kinase A and p38 pathway, facilitates variant splicing. These findings could be used in the formulation of therapeutic methods for PC targeting CD44 alternate splicing.</p

Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo

Human coronavirus HKU1 infection of primary human type II alveolar epithelial cells: cytopathic effects and innate immune response.

Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease

HCoV-HKU1 Infection of Primary Human Alveolar Cells.

<p>Cells were inoculated with media alone or a 1∶10 dilution of the clinical isolate HKU1/DEN/2010/21 at 34^oC, maintained at the air-liquid interface, and fixed 96 hours post infection. Cell cultures were immunolabeled with polyclonal rabbit antibodies to purified HCoV-HKU1 spike protein and fluorescein labeled anti rabbit IgG (green). Nuclei were stained with DAPI (blue). Panels A and B shows infection of type I-like alveolar cells and alveolar macrophages, respectively. Panels C-D show mock and HCoV-HKU1 infection of alveolar type II cells, respectively. Neither the type 1-like cells nor alveolar macrophages were susceptible to infection. In contrast, alveolar type II cells supported infection with HCoV-HKU1.</p

Clinical and demographic characteristics of patients from Colorado HCoV-HKU1 isolates that were successfully propagated in primary human alveolar type II cells.

<p>ALL = acute lymphoblastic leukemia; CHD = congenital heart disease; DVT = deep venous thrombus; unk = unknown; URTI = upper respiratory tract infection.</p

Analysis of anti-viral gene expression in human alveolar epithelial cells in response to HCoV-HKU1 infection.

<p>Total mRNA from HCoV-HKU1 and control cells was analyzed at 72 hours post inoculation for immune response genes: CXCL10, IFNβ, IL29 (IFNλ), CCL5 (RANTES) and IL6. Gene expression values were normalized to expression of the cyclophilin B (CYB), a housekeeping gene. Figure (A) shows the average results from 5 human donors (p-values from Wilcoxon signed rank test, **p≤0.05) and (B) shows the response to HCoV-HKU1 infection by individual donor. Error bars represent the standard deviation between donors (A) or replicates (B).</p

Formation of large syncytia of primary human alveolar type II cells infected with HCoV-HKU1.

<p>Cells were inoculated with a 1∶10 dilution of the clinical isolate HKU1/DEN/2010/21 at 34^oC and fixed 120 hours post infection. Type II cell cultures were immunolabeled with polyclonal rabbit antibodies to purified HCoV-HKU1 spike protein and fluorescein labeled anti rabbit IgG (green). Nuclei were stained with DAPI (blue). Viral antigen is seen only within the cytoplasm of the cells. Efficient infection with cell to cell spread and formation of large, multinucleated giant cells is clearly evident.</p

Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.

<p>The cells were grown under air/liquid conditions as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070129#s2" target="_blank">methods</a> section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.</p

The Spike Glycoprotein of Murine Coronavirus MHV-JHM Mediates Receptor-Independent Infection and Spread in the Central Nervous Systems of Ceacam1a−/− Mice▿

The MHV-JHM strain of the murine coronavirus mouse hepatitis virus is much more neurovirulent than the MHV-A59 strain, although both strains use murine CEACAM1a (mCEACAM1a) as the receptor to infect murine cells. We previously showed that Ceacam1a−/− mice are completely resistant to MHV-A59 infection (E. Hemmila et al., J. Virol. 78:10156-10165, 2004). In vitro, MHV-JHM, but not MHV-A59, can spread from infected murine cells to cells that lack mCEACAM1a, a phenomenon called receptor-independent spread. To determine whether MHV-JHM could infect and spread in the brain independent of mCEACAM1a, we inoculated Ceacam1a−/− mice. Although Ceacam1a−/− mice were completely resistant to i.c. inoculation with 106 PFU of recombinant wild-type MHV-A59 (RA59) virus, these mice were killed by recombinant MHV-JHM (RJHM) and a chimeric virus containing the spike of MHV-JHM in the MHV-A59 genome (SJHM/RA59). Immunohistochemistry showed that RJHM and SJHM/RA59 infected all neural cell types and induced severe microgliosis in both Ceacam1a−/− and wild-type mice. For RJHM, the 50% lethal dose (LD50) is <101.3 in wild-type mice and 103.1 in Ceacam1a−/− mice. For SJHM/RA59, the LD50 is <101.3 in wild-type mice and 103.6 in Ceacam1a−/− mice. This study shows that infection and spread of MHV-JHM in the brain are dependent upon the viral spike glycoprotein. RJHM can initiate infection in the brains of Ceacam1a−/− mice, but expression of mCEACAM1a increases susceptibility to infection. The spread of infection in the brain is mCEACAM1a independent. Thus, the ability of the MHV-JHM spike to mediate mCEACAM1a-independent spread in the brain is likely an important factor in the severe neurovirulence of MHV-JHM in wild-type mice