CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense.

Pseudomonas aeruginosa is a key opportunistic pathogen causing disease in cystic fibrosis (CF) and other lung diseases such as chronic obstructive pulmonary disease (COPD). However, the pulmonary host defense mechanisms regulating anti-P. aeruginosa immunity remain incompletely understood. Here we demonstrate, by studying an airway P. aeruginosa infection model, in vivo bioluminescence imaging, neutrophil effector responses and human airway samples, that the chemokine receptor CXCR1 regulates pulmonary host defense against P. aeruginosa. Mechanistically, CXCR1 regulates anti-Pseudomonas neutrophil responses through modulation of reactive oxygen species and interference with Toll-like receptor 5 expression. These studies define CXCR1 as a novel, noncanonical chemokine receptor that regulates pulmonary anti-Pseudomonas host defense with broad implications for CF, COPD and other infectious lung diseases.

GMP-adapted overexpression of CXCR4 in human mesenchymal stem cells for cardiac repair.

BACKGROUND: Human mesenchymal stem cells (MSC) have been utilized for cardiac regeneration after myocardial damage. Their clinical effects are marginal and only a minority of administered cells could make their way into the myocardium. The chemokine receptor CXCR4 has been identified as crucial for migration and homing of stem cells. In this study we overexpressed CXCR4 on human MSC to improve cell trafficking and tissue repair. METHODS: Human MSC were isolated from the spongiosa of tibia and femur as well as from pelvic bone marrow. MSC were characterized by differentiation assays and FACS analysis. CXCR4 was overexpressed by mRNA-nucleofection. Intracellular signaling was analyzed to demonstrate functionality of CXCR4. The modified Boyden chamber, wounding assays and time lapse microscopy were utilized to investigate MSC migration. RESULTS: MSC did not express relevant amounts of CXCR4 spontaneously. CXCR4 could be overexpressed in 93% of MSC with a cell viability of 62%. Functionality of the overexpressed CXCR4 was demonstrated by a significant cytosolic Ca(2+) increase and activation of different MAP kinases followed by SDF-1α stimulation. In contrast no improvement of cell migration could be observed. There was a strong basal MSC chemokinesis independent from CXCR4 expression. CONCLUSIONS: CXCR4 could be effectively overexpressed in human MSC by mRNA-nucleofection. Despite functionality of CXCR4 MSC were characterized by a strong basal chemokinesis that could not be further enhanced by CXCR4 overexpression. As isolation, culture and nucleofection of pelvic bone marrow-derived MSC basically fulfill the GMP-requirements our approach seems suited for an in vivo application in patients.

Characterization of a Xenopus laevis CXC chemokine receptor 4: implications for hematopoietic cell development in the vertebrate embryo.

Previous reports have shown that the Gi-protein-coupled CXC chemokine receptor 4 is activated by stromal cell-derived factor 1 (SDF-1). The receptor is present in many cell types and regulates a variety of cellular functions, including chemotaxis, adhesion, hematopoiesis, and organogenesis. To examine the role of CXCR4 as a regulator of organogenesis in the vertebrate embryo, we have isolated a cDNA encoding the Xenopus laevis homologue of CXCR4 (xCXCR4). The encoded polypeptide was functionally reconstituted with recombinant Gi2 in baculovirus-infected insect cells. Although xCXCR4 shares only 42% of its extracellular residues with mammalian CXCR4, it is indistinguishable from human CXCR4 in terms of its activation by human SDF-1alpha and SDF-1beta. The fact that only 19 of these residues are specifically present in the extracellular portions of CXCR4 suggests that these residues may be involved in recognizing SDF-1 and/or mediating CXCR4 activation by SDF-1. Xenopus CXCR4 mRNA expression was up-regulated during early neurula stages and remained high during early organogenesis. Whole mount in situ hybridization analysis showed abundant expression of xCXCR4 mRNA in the nervous system, including forebrain, hindbrain, and sensory organs, and in neural crest cells. xCXCR4 mRNA was also detected in the dorsal lateral plate, the first site of definitive hematopoiesis in the amphibian embryo corresponding to aorta-gonad-mesonephros or para-aortic splanchnopleura in mammals. This observation suggests that SDF-1 and CXCR4 are involved in regulating the migratory behavior of hematopoietic stem cells colonizing the larval or fetal liver. The hematopoietic defects observed in mice lacking SDF-1 or CXCR4 may, at least in part, be explained by a disturbance of this migration.

Alternative splicing produces transcripts encoding four variants of mouse G-protein-coupled receptor kinase 6.

A family of protein kinases, termed G-protein-coupled receptor kinases (GRK1-6), is known to phosphorylate agonist-occupied G-protein-coupled receptors. We have identified mRNAs encoding four distinct mouse GRK6 isoforms (mGRK6), designated mGRK6-A through mGRK6-D. Mouse GRK6-B and mGRK6-C diverge from the known human GRK6 (577 residues) at residue 560 and are 13 residues longer and 16 residues shorter, respectively, than human GRK6, while mGRK6-A very likely represents the mouse equivalent of human GRK6. Mouse GRK6-D is identical to the other mGRK6 variants in the amino-terminal region, but comprises only 59 of the 263 amino acids of the putative catalytical domain. As mGRK6-D retains the region involved in interacting with activated receptors, but most likely lacks catalytic activity, this variant might represent a naturally occurring inhibitor of other GRKs. Analysis of the genomic organization of mGRK6 gene revealed that the four mRNAs are generated by alternative RNA splicing from a single approximately 14. 5-kb gene, made up of at least 17 exons and located on mouse chromosome 13. Similar to human GRK6, mGRK6-A contains three cysteine residues within its carboxyl-terminal region known to serve as substrates for palmitoylation. Mouse GRK6-B lacks these palmitoylation sites, but carries a basic carboxyl-terminus containing consensus sequences for phosphorylation by protein kinases C and cAMP/cGMP-dependent protein kinases. Mouse GRK6-C displays none of these motifs. Thus, mGRK6-A, mGRK6-B, and mGRK6-C are predicted to differ in terms of their regulation by carboxyl-terminal posttranslational modification. Analysis of mRNA expression revealed that the four mGRK6 mRNAs are differentially expressed in mouse tissues, suggesting that the four mGRK6 isoforms are involved in regulating tissue- or cell type-specific functions in vivo.

Genomic organization and expression of the CXCR4 gene in mouse and man: absence of a splice variant corresponding to mouse CXCR4-B in human tissues.

The human CXC chemokine receptor CXCR4 is activated by stromal cell-derived factor 1. The receptor is present in many cell types and regulates a variety of cellular functions, including chemotaxis, adhesion, hematopoiesis, and organogenesis. Human CXCR4 also serves as a cofactor for cell entry by certain strains of HIV-1 and HIV-2. In the mouse, alternative RNA splicing produces two transcripts encoding two CXCR4 isoforms, mCXCR4-A and mCXCR4-B, differing by the presence of two amino acids in the amino terminal portion of the longer protein, mCXCR4-B. Only one CXCR4 transcript, encoding the human counterpart of mCXCR4-A, is known in man. The involvement of the aminoterminal-most portion of CXCR4 in both ligand and HIV envelope protein recognition led us to determine whether a CXCR4 variant corresponding to mCXCR4-B is present in human tissues. To this end, the genomic organization and expression of the human CXCR4 gene was examined. Both the human and the mouse CXCR4 gene consist of two exons separated by an approximately 2.1 kbp intron between codons five and six and carry splice donor sites at the 5' end of their introns. These similarities notwithstanding, single nucleotide primer extension, reverse transcriptase PCR amplification, and sequencing of CXCR4 cDNA clones show that a splice variant of CXCR4 corresponding to mCXCR4-B is absent in man.

Two murine homologues of the human chemokine receptor CXCR4 mediating stromal cell-derived factor 1alpha activation of Gi2 are differentially expressed in vivo.

Previous results have shown that pertussis toxin-sensitive Gi proteins are likely to be involved in regulating the emigration of mature thymocytes from the thymus. In this study, a low stringency polymerase chain reaction (PCR) approach was used to identify Gi protein-coupled cell surface receptors expressed in mouse thymocytes. Among the ten G protein-coupled receptor cDNA isolated, the most prevalent cDNA encoded a polypeptide highly homologous to the human leukocyte-expressed seven-transmembrane-domain receptor LESTR, also referred to as HIV entry cofactor, fusin, or CXCR4. Isolation of full-length cDNA revealed that alternative RNA splicing produces transcripts encoding two isoforms of the murine LESTR, differing by the presence of two amino acids in the N-terminal portion of the longer protein. Functional reconstitution of recombinant murine LESTR with recombinant heterotrimeric G proteins in baculovirus-infected insect cells showed that both receptor variants mediate stromal cell-derived factor 1alpha activation of the pertussis toxin-sensitive G protein Gi2. Receptor subtype-specific reverse transcriptase-PCR analysis revealed differential expression of the two receptor mRNA in lymphoid tissues and brain, indicating that distinct functions are mediated by the two receptor isoforms in these tissues. The presence of LESTR mRNA in very early thymocytes as well as in immature (CD4+ CD8+) thymocytes suggests that both CD4 and LESTR are co-expressed and render developing human thymocytes susceptible for HIV entry, which may affect generation of both CD4+ CD8- and CD4- CD8+ mature lineages.

Isolation, characterization and sequence of a cDNA encoding gamma-tubulin protein from the fern Anemia phyllitidis L. Sw.

A complete cDNA encoding gamma-tubulin protein from Anemia phyllitidis is presented. The deduced amino acid sequence shows an average similarity of 81-86% to known gamma-tubulin genes from insects, mammals and fungi. Northern blots indicate the existence of transcripts of ca. 1.9 kb which are expressed during gametogenesis of A. phyllitidis. The gene described is the first cloned and entirely sequenced gamma-tubulin gene in any plant.

PCR-dependent amplification and sequence characterization of partial cDNAs encoding myosin-like proteins in Anemia phyllitidis (L.) Sw. and Arabidopsis thaliana (L.) Heynh.

Partial cDNAs encoding for myosin-like proteins from Anemia phyllitidis and Arabidopsis thaliana have been isolated using PCR technology. The deduced amino acid sequences show an average similarity up to 62% with known myosin heavy chain genes. From northern blot analysis we were able to estimate that transcripts of ca. 6.1 kb size are expressed in A. phyllitidis.