Human SLURP-1 Research Articles

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.

Recombinant SLURP-1 Inhibits Growth and Migration of U251 MG Glioma by Cell Cycle Arrest and Modulation of MAPK and AKT Signaling Pathways

A recombinant analog of the human SLURP-1 protein, rSLURP-1, effectively inhibits the growth of carcinomas by interaction with the α7-type nicotinic acetylcholine receptor. Recently, rSLURP-1 inhibition of glioma growth in vitro was shown by the authors; however, the action of rSLURP-1 was not studied. Here, we showed that rSLURP-1 selectively inhibits the growth of U251 MG glioma cells, but not of normal astrocytes, and controls glioma cell migration. In addition, rSLURP-1 induces cell cycle arrest in the G2/M phase in U251 MG glioma cells, but does not result in apoptosis. Incubation of U251 MG cells with rSLURP-1 causes inhibition of phosphorylation of ERK, p38 MAPK, and AKT kinases, whose activation contributes to the progression of gliomas. At the same time, rSLURP-1 does not affect the activity of JNK kinase. Thus, rSLURP-1 is an endogenous protein promising for the development of drugs based on it for the treatment of not only carcinomas but also gliomas.

Recombinant SLURP-1 Inhibits Growth and Migration of U251 MG Glioma by Cell Cycle Arrest and Modulation of MAPK and AKT Signaling Pathways

A recombinant analog of the human SLURP-1 protein (rSLURP-1) effectively inhibits the growth of carcinomas by interaction with the α7-type nicotinic acetylcholine receptor. Recently, rSLURP-1 inhibition of gliomas growth in vitro was shown by the authors, although, the mechanism of rSLURP-1 action was not studied. Here, we showed that rSLURP-1 selectively inhibits the growth of U251 MG glioma cells but not of normal astrocytes, and controls glioma cell migration. In addition, rSLURP-1 induces cell cycle arrest in the G2/M phase in U251 MG glioma cells, but does not result in apoptosis. Incubation of U251 MG cells with rSLURP-1 causes inhibition of phosphorylation of ERK, p38 MAPK, and AKT kinases, the activation of which contributes to the progression of gliomas. At the same time, rSLURP-1 does not affect the activity of JNK kinase. Thus, rSLURP-1 is an endogenous protein promising for the development of drugs based on it for the treatment of not only carcinomas, but also gliomas.

From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs.

The proteins of the Ly6 family have a three-finger folding as snake venom α-neurotoxins, targeting nicotinic acetylcholine receptors (nAChRs), and some of them, like mammalian secreted Ly6/uPAR protein (SLURP1) and membrane-attached Ly-6/neurotoxin (Lynx1), also interact with distinct nAChR subtypes. We believed that synthetic fragments of these endogenous proteins might open new ways for drug design because nAChRs are well-known targets for developing analgesics and drugs against neurodegenerative diseases. Since interaction with nAChRs was earlier shown for synthetic fragments of the α-neurotoxin central loop II, we synthesized a 15-membered fragment of human Lynx1, its form with two Cys residues added at the N- and C-termini and forming a disulfide, as well as similar forms of human SLURP1, SLURP2, and of Drosophila sleepless protein (SSS). The IC50 values measured in competition with radioiodinated α-bungarotoxin for binding to the membrane-bound Torpedo californica nAChR were 4.9 and 7.4 µM for Lynx1 and SSS fragments, but over 300 µM for SLURP1 or SLURP2 fragments. The affinity of these compounds for the α7 nAChR in the rat pituitary tumor-derived cell line GH4C1 was different: 13.1 and 147 µM for SSS and Lynx1 fragments, respectively. In competition for the ligand-binding domain of the α9 nAChR subunit, SSS and Lynx1 fragments had IC50 values of about 40 µM, which correlates with the value found for the latter with the rat α9α10 nAChR expressed in the Xenopus oocytes. Thus, the activity of these synthetic peptides against muscle-type and α9α10 nAChRs indicates that they may be useful in design of novel myorelaxants and analgesics.

Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors

Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [125I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

Corneal Expression of SLURP-1 by Age, Sex, Genetic Strain, and Ocular Surface Health.

Although secreted Ly6/urokinase-type plasminogen activator receptor-related protein-1 (Slurp1) transcript is highly abundant in the mouse cornea, corresponding protein expression remains uncharacterized. Also, SLURP1 was undetected in previous tear proteomics studies, resulting in ambiguity about its baseline levels. Here, we examine mouse corneal Slurp1 expression in different sexes, age groups, strains, and health conditions, and quantify SLURP1 in human tears from healthy or inflamed ocular surfaces. Expression of Slurp1 in embryonic day-13 (E13), E16, postnatal day-1 (PN1), PN10, PN20, and PN70 Balb/C, FVBN, C57Bl/6, and DBA/2J mouse corneas, Klf4Δ/ΔCE corneas with corneal epithelial-specific ablation of Klf4, migrating cells in wild-type corneal epithelial wound edge, and in corneas exposed to pathogen-associated molecular patterns (PAMPs) poly(I:C), zymosan-A, or Pam3Csk4 was examined by QPCR, immunoblots, and immunofluorescent staining. Human SLURP1 levels were quantified by ELISA in tears from 34 men and women aged 18 to 80 years. Expression of Slurp1, comparable in different strains and sexes, was low in E13, E16, PN1, and PN10 mouse corneas, and increased rapidly after eyelid opening in a Klf4-dependent manner. We found Slurp1 was downregulated in corneas exposed to PAMPs, and in migrating cells at the wound edge. Human SLURP1 expression, comparable in different sexes and age groups, was significantly decreased in tears from inflamed ocular surfaces (0.34%) than those from healthy individuals (0.77%). These data describe the influence of age, sex, genetic background, and ocular surface health on mouse corneal expression of Slurp1, establish the baseline for human tear SLURP1 expression, and identify SLURP1 as a useful diagnostic and/or therapeutic target for inflammatory ocular surface disorders.

Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells

Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13C-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.

SLURP1 mutation-impaired T-cell activation in a family with mal de Meleda

Mal de Meleda (MDM) is palmoplantar erythrokeratoderma with an autosomal recessive inheritance and is caused by a mutation in the gene encoding SLURP-1 (lymphocyte antigen 6/urokinase-type plasminogen activator receptor related protein-1). SLURP-1 is an allosteric agonist to the nicotinic acetylcholine receptor (nAchR) and it regulates epidermal homeostasis. In addition, murine studies have shown that nAchR signalling is important for the regulation of T-cell function. Among the family members, patients with the homozygous SLURP1 (previously known as ARS component B) mutation are prone to melanoma and viral infection, which might link to defective T-cell function as well as a derangement of epidermal homeostasis. To investigate the association of the SLURP1 gene mutation with T-cell activation in a Taiwanese family with MDM. To test that SLURP-1 is essential for T-cell activation. Human peripheral blood mononuclear cells (PBMCs) were isolated from a Taiwanese MDM family bearing the G to A substitution in nucleotide 256 in the SLURP1 gene, corresponding to a glycine to arginine substitution at amino acid 86 (G86R) in the SLURP-1 protein. PBMCs from homozygotes and wild-type controls were stimulated with anti-CD3/anti-CD28 antibodies and the level of T-cell activation was determined by the stimulation index. PBMCs with the heterozygous and homozygous SLURP-1 G86R mutation had defective T-cell activation. This was restored by the addition of 0·5 μg mL(-1) recombinant human SLURP-1 protein. Patients with MDM with the homozygous SLURP-1 G86R mutation may have an impaired T-cell activation. The presence of wild-type SLURP-1 is essential for normal T-cell activation.

Immune system expression of SLURP-1 and SLURP-2, two endogenous nicotinic acetylcholine receptor ligands

A novel transduction pathway via which apoptosis of keratinocytes is regulated through nicotinic acetylcholine (ACh) receptors (nAChRs) has emerged in studies of secreted mammalian Ly6/urokinase plasminogen-type activator receptor-related protein-1 and-2 (SLURP-1 and SLURP-2, respectively). SLURP-1 reportedly binds to α7 nAChRs and enhances the amplitude of macroscopic currents induced by ACh, leading to facilitation of apoptosis, whereas SLURP-2 binds to α3 nAChRs and prevents apoptosis. These observations prompted us to test whether SLURPs are expressed in immune cells and are involved in the regulation of immune function. We initially used reverse transcription-polymerase chain reaction analysis to characterize the expression profiles of SLURP mRNAs in several murine tissues and organs. Although SLURP-1 mRNA was not expressed in the pancreas, all other tissues and organs tested, including spleen and thymus, expressed both SLURP-1 and SLURP-2 mRNAs. Expression of both mRNAs also was detected in T and B cells, bone marrow-derived dendritic cells (DCs) and macrophages. Moreover, as in keratinocytes, stimulation of MOLT-3 human leukemic T cells with recombinant human SLURP-1 evoked intracellular Ca2+ signaling. These results suggest that both SLURP-1 and SLURP-2 are expressed in various immune cells and organs, and that not only ACh but also SLURPs may be involved in regulating lymphocyte function via nAChR-mediated pathways.

Biological Effects of SLURP-1 on Human Keratinocytes

A novel paradigm of keratinocyte (KC) regulation via nicotinic acetylcholine receptors (nAChR) has been discovered in studies of SLURP (secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein)-1 in Mal de Meleda. We cloned human SLURP-1 and produced recombinant protein and the monoclonal antibody 336H12-1A3 that visualized native SLURP-1. SLURP-1 ligated the conventional ligand-binding site of KC nAChR, showing a higher affinity to the [(3)H]nicotine-, compared with the [(3)H]epibatidine-sensitive nAChR. SLURP-1 significantly (p<0.05) increased the activities of caspases 3 and 8, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling-positive cells. The pro-apoptotic activity of SLURP-1 exceeded that of tumor necrosis factor-alpha, suggesting the involvement of separate pathways. In a series of real-time PCR and in-cell western experiments, SLURP-1 significantly (p<0.05) upregulated expression of transglutaminase type I cytokeratin 10, p21, and caspase-3. In the presence of the agonist carbachol, the effects of SLURP-1 on gene expression were augmented, which is in keeping with the notion that SLURP-1 acts as an allosteric agonist at the KC nAChR. Thus, the changes in the cell state induced by SLURP-1 could result from nAChR-mediated effects on the KC gene expression. These results suggest that the biological role of SLURP-1 in the epidermis is to provide fine tuning of the physiologic regulation of KC functions through the cholinergic pathways.