Cloning of BRAK, a novel divergent CXC chemokine preferentially expressed in normal versus malignant cells

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.     

Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.     

2P1, a novel male mouse cDNA specifically expressed during meiosis

We screened a mouse germinal cell expression library with a probe derived from Sob1, a human testis-specific cDNA, and identified 2P1, a new mouse cDNA. A database search revealed that 2P1 was 91% identical to ORF1 of E3-3, a rat gene probably involved in the regulation of alternative splicing. Sequencing showed that 2P1 has a destabilization motif in its 3'-untranslated region. Northern blotting showed strong gene expression in the testis and weak expression in the epididymis, with no signal detected in other tissues. RT-PCR analysis confirmed testis and epididymis expression. In situ hybridization revealed that 2P1 mRNA was absent in spermatogonia but expressed in spermatocytes. This last result was confirmed by RT-PCR of FACS isolated primary spermatocytes (pachytene stage). Using RT-PCR, purified spermatids were also shown to express 2P1.     

CXC chemokine receptor 4 expressed in T cells plays an important role in the development of collagen-induced arthritis

Introduction  

Chemokines and their receptors are potential therapeutic targets in rheumatoid arthritis (RA). Among these, several studies suggested the involvement of CXC chemokine 4 (CXCR4) and its ligand CXC ligand 12 (SDF-1) in RA pathogenesis. However, the role of these molecules in T-cell function is not known completely because of embryonic lethality of Cxcr4- and Cxcl12- deficient mice. In this report, we generated T cell-specific Cxcr4 -deficient mice and showed that the CXCR4 in T cells is important for the development of collagen-induced arthritis (CIA).     

Biochemical and biological characterization of neutrophil chemotactic protein, a novel rabbit CXC chemokine from alveolar macrophages

The role of interleukin-8 (IL-8) and related CXC chemokines has been demonstrated in many human diseases. However, more profound studies, e.g., by blocking the effect of these inflammatory mediators, request animal models and hence the identification of all human counterparts for commonly used laboratory animals. In this study, we describe the identification of a novel neutrophil chemotactic protein (NCP) of the rabbit. Intact and NH(2)-terminally truncated NCP forms and IL-8 were isolated from LPS-stimulated rabbit alveolar macrophages and purified to homogeneity by a four-step purification procedure. Determination of the complete primary structure of NCP by mass spectrometry and NH(2)-terminal sequencing of natural protein revealed high structural homology with human epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2), two related ELR(+)CXC chemokines. Intact NCP(1-76) was found to be 10-fold less potent than truncated NCP(7, 8-76) at inducing neutrophil chemotaxis. NCP(7,8-76) was equally potent as intact rabbit IL-8 at chemoattracting human neutrophils and at inducing calcium fluxes in rabbit neutrophils, 1 ng/mL being the minimal effective concentration. However, like IL-8, NCP failed to induce monocyte or eosinophil migration at 300-fold higher concentrations. IL-8 desensitized the calcium increase induced by NCP and vice versa. Finally, intradermal injection of NCP induced a dose-dependent and significant infiltration of neutrophils in mice skin. It can be concluded that NCP is a novel rabbit CXC chemokine that is, like IL-8, implicated in animal models used to study various human disorders in which neutrophils play an important role.     

The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs,targets lymphocytes including activated B cells,and supports antibody responses to a bacterial pathogen in vivo

Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.     

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.     

FDC-SP,a novel secreted protein expressed by follicular dendritic cells

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-alpha-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.     

Ankrd7, a novel gene specifically expressed in Sertoli cells and its potential roles in Sertoli cell maturation

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immuno-histochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Tat1, a novel sulfate transporter specifically expressed in human male germ cells and potentially linked to rhogtpase signaling

RhoGTPases (Rho, Rac, and Cdc42) are known to regulate multiple functions, including cell motility, adhesion, and proliferation; however, the signaling pathways underlying these pleiotropic effects are far from fully understood. We have recently described a new RhoGAP (GTPase activating protein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We report here the isolation, through two-hybrid cloning, of a new partner of MgcRacGAP, very specifically expressed in the male germ line and showing structural features of anion transporters. This large protein (970 amino acids and a predicted size of 109 kDa), we provisionally designated Tat1 (for testis anion transporter 1), is closely related to a sulfate permease family comprising three proteins in human (DRA, Pendrin, and DTD); it is predicted to be an integral membrane protein with 14 transmembrane helices and intracytoplasmic NH(2) and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAP genes are coexpressed in male germ cells at the spermatocyte stage. On testis sections, Tat1 protein can be immunodetected in spermatocytes and spermatids associated with plasma membrane. Two-hybrid and in vitro binding assays demonstrate that MgcRacGAP stably interacts through its NH(2)-terminal domain with the Tat1 COOH-terminal region. Expression of Tat1 protein in COS7 cells generates a 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and chloride-sensitive sulfate transport. Therefore we conclude that Tat1 is a novel sulfate transporter specifically expressed in spermatocytes and spermatids and interacts with MgcRacGAP in these cells. These observations raise the possibility of a new regulatory pathway linking sulfate transport to Rho signaling in male germ cells.     

Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage.

A novel cDNA clone designated as HS1, which show an expression pattern limited to human hematopoietic cells, was isolated. About 2kb mRNA of the clone was accumulated in all the mature and immature lymphoid and myeloid cell lines tested, and two of three erythroblastoid cell lines, but not in any cell lines of non-hematopoietic tissues. The same mRNA was also detected in normal lymphoid and myeloid tissues and peripheral blood lymphocytes, granulocytes and macrophages, but again not in non-hematopoietic tissues. Nucleotide sequence of the HS1 predicts a protein of 486 amino acids (Mr 53,931). N-terminal half of the protein retains unique repeating motifs, each of which shows a significant homology with the helix-turn-helix DNA-binding motif of several proteins reported previously. C-terminal half of the protein retains a region conserved between non-receptor tyrosine kinases (src family), phospholipase C(PLC)-148 and the crk oncogene product. A unique feature of HS1 suggests that the protein may be involved in signal transduction and regulation of gene expression.     

Identification and characterization of TSAP, a novel gene specifically expressed in testis during spermatogenesis

Through in silico screens, we have identified many previously uncharacterized genes that display similar expression patterns as the mouse Dazl gene, a germ line-specific marker. Here, we report the identification and characterization of one of these novel genes. TSAP gene encodes a protein with 350 amino acids and contains five ankyrin repeats and a PEST sequence motif. Furthermore, we have generated an anti-TSAP antibody and have used three different approaches (RT-PCR, in situ hybridization, and immunohistochemistry) to investigate the expression profiles of TSAP mRNAs and proteins. TSAP is specifically expressed in testis, but not in other tissues such as ovary. Within the testis, TSAP is detected 10 days after birth and is mainly expressed in spermatocytes (ST) and later stage of germ cells, but not in spermatogonia (SG) or sertoli cells. Therefore, TSAP protein likely plays a role in spermatogenesis.     

Munster, a novel paired-class homeobox gene specifically expressed in the Drosophila larval eye.

Munster (Mu) is a homeobox-containing gene of the Paired-class which is specifically expressed in the developing Bolwig organs, the Drosophila larval eyes. This expression is first detected during early germ band retraction stage (stage 12 from 7 h 20 at 25 degrees C) and persists until the end of embryogenesis. Mu homeodomain is most similar to that of Aristaless and D-Goosecoid. Strikingly, the Munster gene maps within 6 kb of D-goosecoid, in the same genomic region as aristaless, suggesting that these genes are part of a homeobox gene cluster.     

A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.     

Molecular cloning and characterization of a novel rat CXC chemokine, rPBP, the homologue of human and mouse PBP

We have previously cloned the mouse platelet basic protein (mPBP), a homologue of human PBP, from mouse thymic stromal cells. Using EST alignment and RT-PCR, the rat homologue of human and mouse PBP was cloned from lung and named as rPBP. The complete open reading frame and part of the 3'- and 5'-non-coding regions were obtained through rapid amplification of cDNA ends. The rPBP cDNA encodes a protein of 111 amino acids containing a signal peptide of 37 amino acids at the N-terminus, with the mature protein of 74 amino acids. The rPBP is a new member of ELR+CXC chemokines. The mature protein of rPBP shares 69% and 45% homology with mouse and human PBP, respectively. In situ hybridization assay revealed rPBP to be predominantly localized in the pulmonary vascular endothelial cells. The eukaryotic expression vector pCDNA3-rPBP was constructed and transiently transfected into COS-7 cells. In the in vitro chemotaxis assay, the polymorphonuclear leukocytes (PMNs) were chemoattracted to the supernatants from transfected COS-7 cells in a dose-dependent manner. The implication of rPBP found in rat lung is that this chemokine may have the function to recruit PMNs to fight against pulmonary infection.     

Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2alphabeta, hence is designated as MIP-2gamma. Mouse MIP-2gamma was identified by electrocloning and is highly homologous to human MIP-2gamma. Northern blotting revealed that MIP-2gamma was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2gamma was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2gamma expression by monocyte-derived DC. Recombinant MIP-2gamma from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2gamma is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2gamma does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.