Identification of a novel secretory leukocyte protease inhibitor‐binding protein involved in membrane phospholipid movement

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-␤1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-␤1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated. Secretory leukocyte protease inhibitor (SLPI),1 also known as anti-leukoprotease, human mucus proteinase inhibitor, and human seminal plasma inhibitor, is a 12-kDa member of the chelonianin class of serine protease inhibitors, which also includes SKALP/elafin (1, 2). SLPI is composed of two homologous cysteine-rich domains. The carboxyl-terminal domain manifests inhibitory activities against chymotrypsin, trypsin, granulocyte and pancreatic elastases, cathepsin G, and mast cell chymase (3, 4). SLPI is expressed primarily in secretory/glandular epithelial cells of a variety of human tissues including the bronchi, parotid glands, small and large intestines, skin, breast, pancreas, male and female genital tracts, and kidney (5-14). Its relative absence in serum, except in certain pathological conditions (15, 16), and its localization to extracellular matrix and subcellular sites not readily accessible to larger molecular weight protease inhibitors such as ␣ 1 -antitrypsin (17), suggests a primarily autocrine/paracrine mode of action. In addition to its anti-protease activity, SLPI has anti-inflammatory, anti-bacterial, anti-fungal, and anti-retroviral (human immunodeficiency virus) activities that appear to reside in its amino-terminal cysteine-rich domain (18 -23). Furthermore, SLPI has been shown to regulate intracellular enzyme synthesis, suppress matrix metalloproteinase production and activity, mediate normal wound healing, prevent scar formation, and augment fertility (24 -27). The higher uterine endometrial expression of SLPI during pregnancy ...

Section: Fig 8 Effects Of Tgf-␤1 On Ishikawa Cell Gene Expressionmentioning

confidence: 82%

Secretory Leukocyte Protease Inhibitor Mediates Proliferation of Human Endometrial Epithelial Cells by Positive and Negative Regulation of Growth-associated Genes

Zhang

Simmen

Michel

et al. 2002

“…The integrin ␤ 3 expression plasmid was a kind gift of Dr. Erkki Ruoslahti (Burnham Institute). pTOPO-U6, Dab2-2112, pCI-neo-hDAB2, pCI-neo-His 9, pGST-⌬B, p82, ⌬B, ⌬N, and p82-S24A (S24A) have been described previously (2,7,14,34).…”

Section: Methodsmentioning

confidence: 99%

Disabled-2 Is a Negative Regulator of Integrin αIIbβ3-mediated Fibrinogen Adhesion and Cell Signaling

Huang¹,

Cheng

Liao

et al. 2004

Self Cite

Disabled-2 (DAB2) is an adapter protein that is up-regulated during megakaryocytic differentiation of hematopoietic cells and is abundantly expressed in platelets. In this study, the role of DAB2 in integrin ␣ IIb ␤ 3 -mediated matrix protein fibrinogen adhesion and cell signaling was investigated. In K562 cells differentiating to the megakaryocytic lineage, down-regulation of DAB2 by DAB2 small interfering RNA augmented integrin ␣ IIb ␤ 3 activation and resulted in an increase in cell adhesion to fibrinogen. Ectopic expression of DAB2 reversed the DAB2 small interfering RNA effect or, by itself, decreased fibrinogen adhesion of K562 cells. Mutational analysis revealed that a DAB2 Ser 24 phosphorylation mutant (S24A) abrogated the inhibitory function of DAB2. The spatial and temporal association/interaction of DAB2 and platelet integrin ␣ IIb ␤ 3 (CD61) in both megakaryocytic cells and platelets led us to examine the effect of Ser 24 phosphorylation on the interaction between DAB2 and integrin ␤ 3 . Through cellular localization and co-immunoprecipitation analysis, we demonstrate for the first time that Ser 24 phosphorylation promotes membrane translocation of DAB2 and its subsequent interaction with integrin ␤ 3 , thereby defining a mechanism for DAB2 in regulating integrin ␣ IIb ␤ 3 activation and inside-out signaling. Consistent with the effect on fibrinogen adhesion, Ser 24 phosphorylation of DAB2 was also involved in the negative regulation of ␣ IIb ␤ 3 -induced T cell factor transcriptional activity. In contrast, the S24A mutant acted like wild-type DAB2 and inhibited both ␤-catenin-and plakoglobin-mediated T cell factor transactivation. Hence, DAB2 elicits distinct regulatory mechanisms in ␣ IIb ␤ 3 and ␤-catenin/plakoglobin signaling in a Ser 24 phosphorylation-dependent and -independent manner, respectively. These findings indicate Ser 24 phosphorylation as a molecular basis for DAB2 acting as a negative regulator in ␣ IIb ␤ 3 inside-out signaling and contribute to our understanding of DAB2 in megakaryocytic differentiation and platelet function.Disabled-2 (DAB2) is an adapter protein that has been implicated in growth factor signaling (1, 2), endocytosis (3-5), cell adhesive function (6, 7), and hematopoietic cell differentiation (8). Like other adapter proteins, DAB2 elicits its function through interaction with other cellular proteins. DAB2 interacts with Grb2, myosin VI, SMAD2/3, DIP1/2, Dvl-3, the integrin ␤ subunit, and c-Src through the N-terminal phosphotyrosine-binding (PTB) 1 domain and the C-terminal proline-rich region (1, 4, 5, 9 -13). These interactions have been shown to modulate cytoskeleton organization, transcriptional activity, and cell signaling of various receptor protein-tyrosine kinases. DAB2 thus plays in a pivotal role in the control of cellular homeostasis.In cells, protein phosphorylation of DAB2 modulates its functional activity during growth factor signaling, megakaryocytic differentiation, macrophage spreading, and cell cycle progression. To date, protein kinase C (PKC) and...

“…In addition, another heparinbinding serine protease inhibitor, the secretory leukocyte protease inhibitor (SLP), is able to inhibit HIV-1. It has been suggested that SLP is an entry inhibitor but blocks a receptor distinct from chemokine receptors and CD4 (37)(38)(39)(40)(41)(42). Furthermore, another serine protease inhibitor, ␣ 1 -antitrypsin, also has been described as an inhibitor of HIV-1.…”

Section: Cd8 ϩ T-cells Of Seropositive Individuals Modify Serum Atiiimentioning

confidence: 99%

Purification of a Modified Form of Bovine Antithrombin III as an HIV-1 CD8+ T-cell Antiviral Factor

Geiben-Lynn

Brown

Walker

et al. 2002

CD8 ؉ T-cells secrete soluble factor(s) capable of inhibiting both R5-and X4-tropic strains of human immunodeficiency virus type 1 (HIV-1). CCR5 chemokine ligands, released from activated CD8 ؉ T-cells, contribute to the antiviral activity of these cells. These CC-chemokines, however, do not account for all CD8 ؉ T-cell antiviral factor(s) (CAF) released from these cells, particularly because the elusive CAF can inhibit the replication of X4 HIV-1 strains that use CXCR4 and not CCR5 as a coreceptor. Here we demonstrate that activated CD8 ؉ T-cells of HIV-1-seropositive individuals modify serum bovine antithrombin III into an HIV-1 inhibitory factor capable of suppressing the replication of X4 HIV-1. These data indicate that antithrombin III may play a role in the progression of HIV-1 disease.Soluble inhibitory factors produced by CD8 ϩ T-cells have been shown to inhibit HIV-1 1 replication and may play a critical role in vivo in antiviral host defense (1). These inhibitory factors include CC-chemokines (2-4), which bind to the CCR5 coreceptor and inhibit R5 viral entry into cells (1) (5-7), as well as less well characterized soluble factor(s) produced by CD8 ϩ T-cells and termed CD8 ϩ T-cell antiviral factor(s) (CAF), which are capable of inhibiting both R5 and X4 .Recently, we demonstrated that there are two factors produced by activated CD8 ϩ T-cells capable of inhibiting the X4 strain HIV-1 IIIB (16). These factors are distinctive in size and the ability to bind heparin. One of these factors bound heparin at physiological salt concentration, eluted at 350 mM NaCl, and was retained by a 50-kDa cut-off Centricon filter. The other factor did not bind heparin at physiological salt concentration and was filtered through a 50-kDa cut-off Centricon filter. The HIV-1 inhibitory activity of these factors was higher with bulk CD8 ϩ T-cells of seropositive individuals and HIV-1-specific cytotoxic T-lymphocytes (CTL) compared with bulk CD8 ϩ Tcells of HIV-1-seronegative individuals (16). In the present study we identified the heparin binding inhibitory activity as a CD8 ϩ T-cell modified form of antithrombin, which is produced in higher amounts by HIV-1-specific CTL and bulk CD8 ϩ Tcells of seropositive individuals than by bulk CD8 ϩ T-cells of seronegative individuals. In this study for the first time we demonstrate that CD8 ϩ T-cells can activate a serum protein to become inhibitory for HIV, a possibility that has not been addressed previously. EXPERIMENTAL PROCEDURESHIV-specific CTL Clones and Bulk CD8 ϩ T-Cells-Polyclonal CD8 ϩ cells that were 90 -99% CD3 ϩ -and CD8 ϩ -positive were generated by fluorescence-activated cell sorting from the six seronegative and six HIV-1-seropositive long-term nonprogressors by positive selection with anti-CD8 antibody-coated immunomagnetic beads (PerSeptive Biosystems, Framingham, MA) as described (16). HIV-1-specific CTL clones were used as described (16). Bulk CD8 ϩ cell lines from seropositive and seronegative persons were established by incubating purified CD8 ϩ cells (2 ϫ 10 6 )...