Wenli Liu

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development.

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.

Olfactomedin 4 down-regulates innate immunity against Helicobacter pylori infection.

Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 KO mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with WT littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1α was increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF--κB pathway and has a negative feedback effect on NF-κB activation induced by H. pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization.

Reduced hGC-1 Protein Expression Is Associated with Malignant Progression of Colon Carcinoma

Purpose: hGC-1 (human granulocyte colony–stimulating factor–stimulated clone 1) is a gastrointestinal protein that is a member of the olfactomedin glycoprotein family. Its biological function remains poorly understood. Aberrant expression of hGC-1 in some human carcinomas has been recently reported. The purpose of this study was to examine hGC-1 expression in colon carcinoma and explore the relationship between hGC-1 expression and the clinicopathologic features of patients with colon cancer. Experimental Design: The expression of hGC-1 in colon adenocarcinoma tissues was examined by dot-blot analysis, in situ hybridization, and immunohistochemistry. The association of hGC-1 expression pattern with patient differentiation grade, tumor stage, metastasis, and survival were examined. To further investigate the involvement of hGC-1 in colon cancer progression, human colon carcinoma (HT-29) cells overexpressing hGC-1 were established and cell proliferation, adhesion, and migration were studied. Results: Compared with normal colon mucosa, the up-regulation of hGC-1 was more frequently detected in more differentiated colon cancers, whereas down-regulation or no expression was associated with poorly differentiated colon cancers. Interestingly, hGC-1 down-regulation was also found in late tumor-node-metastasis stage, metastasis, and in patients with shorter survival. The morphology and cortical actin distribution of HT-29 cells were altered by hGC-1 overexpression. However, this did not change cell proliferation, but decreased cell adhesion and migration. Conclusion: Our findings indicate that hGC-1 is involved in colon cancer adhesion and metastasis, and that hGC-1 may be a useful marker for tumor differentiation and progression of human colon carcinoma.

Expression of hGC-1 is correlated with differentiation of gastric carcinoma

Aims:  The human G‐CSF‐stimulated clone‐1 (hGC‐1) gene encodes a 510‐amino acid olfactomedin‐related glycoprotein whose exact in vivo localization and function still remain elusive. The aim of this study was to demonstrate hGC‐1 protein localization in the normal human gastrointestinal tract and to explore further a potential relationship between hGC‐1 expression and gastric carcinoma.

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4−/− mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4−/− mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4−/− mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4−/− neutrophils. The bacterial killing and clearance capabilities observed in OLFM4−/− mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.

Olfactomedin 4 suppresses prostate cancer cell growth and metastasis via negative interaction with cathepsin D and SDF-1

The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 is normally expressed in a limited number of tissues, including the prostate, but its biological functions in prostate are largely unknown. In this study, we found that OLFM4 messenger RNA was reduced or undetectable in prostate cancer tissues and prostate cancer cell lines. To study the effects of OLFM4 on prostate cancer progression, we transfected PC-3 prostate cancer cells with OLFM4 to establish OLFM4-expressing PC-3 cell clones. The OLFM4-expressing PC-3 cell clones were found to have decreased proliferation and invasiveness compared with vector-transfected control PC-3 cells in vitro. In addition, nude mice injected with OLFM4-expressing PC-3 cells demonstrated reduced tumor growth and bone invasion and metastasis compared with mice injected with vector-transfected control cells. Mechanistic studies revealed that OLFM4 may exhibit its anticancer effects through regulating cell autophagy by targeting cathepsin D, as OLFM4 reduced cathepsin D protein levels and enzymatic activity and attenuated cathepsin D-induced cancer cell proliferation. In addition, overexpression of OLFM4 abrogated stromal cell derived factor-1 (SDF-1)-induced PC-3 cell invasiveness in a Matrigel invasion assay, partially through blocking SDF-1-mediated AKT phosphorylation. Coimmunoprecipitation and immunofluorescence staining studies in OLFM4-expressing PC-3 cells demonstrated a direct interaction between OLFM4 and cathepsin D or SDF-1. Taken together, these results suggest that OLFM4 negatively interacts with cathepsin D and SDF-1 and inhibits prostate cancer growth and bone metastasis.

The regulation of OLFM4 expression in myeloid precursor cells relies on NF-κB transcription factor

The human olfactomedin 4 gene (OLFM4, also known as hGC‐1, GW112) is thought to be a useful marker for early myeloid development. To understand the molecular mechanisms underlying granulocyte colony‐stimulating factor (G‐CSF)‐stimulated OLFM4 expression, we characterized the promoter region of OLFM4. The 35‐bp region (−101 to −66) of the proximal promoter regulated reporter gene expression, and mutation of the nuclear factor (NF)‐κB binding site within the promoter abolished the binding of the transcription factor and the ability to regulate OLFM4 expression. G‐CSF increased reactive oxygen species (ROS) production in human CD34+ cells, which was abrogated by inhibition of phosphatidylinositol 3‐kinase (PI3K) or NADPH oxidase. Phosphorylation of ERK1/2 mitogen‐activated protein kinase (MAPK) induced by G‐CSF inhibited by the antioxidant N‐acetyl‐L‐cysteine (NAC), ERK1/2 inhibitor PD98059, or U0126. However, phosphorylation of signal transducer and activator of transcription (STAT)3 was only partially inhibited by NAC, but not by PD98059 or U0126. Inhibition of the ERK pathway remarkably decreased OLFM4 expression and this inhibition required NF‐κB transcription factor. Inhibition of ROS or the ERK pathway remarkably decreased G‐CSF‐induced OLFM4 expression. Our results suggest that G‐CSF‐induced expression of OLFM4 is regulated by the transcription factor NF‐κB, and that this induction is mediated by the ERK1/2 MAPK signaling pathway through PI3K‐driven ROS production.

Olfm4 deletion enhances defense against Staphylococcus aureus in chronic granulomatous disease

Chronic granulomatous disease (CGD) patients have recurrent life-threatening bacterial and fungal infections. Olfactomedin 4 (OLFM4) is a neutrophil granule protein that negatively regulates host defense against bacterial infection. The goal of this study was to evaluate the impact of Olfm4 deletion on host defense against Staphylococcus aureus and Aspergillus fumigatus in a murine X-linked gp91phox-deficiency CGD model. We found that intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox and Olfm4 double-deficient mice compared with CGD mice. The activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) and serum levels of IL-1β, IL-6, IL-12p40, CXCL2, G-CSF, and GM-CSF in Olfm4-deficient as well as gp91phox and Olfm4 double-deficient mice were significantly higher than those in WT and CGD mice after challenge with S. aureus. We did not observe enhanced defense against A. fumigatus in Olfm4-deficient mice using a lung infection model. These results show that Olfm4 deletion can successfully enhance immune defense against S. aureus, but not A. fumigatus, in CGD mice. These data suggest that OLFM4 may be an important target in CGD patients for the augmentation of host defense against bacterial infection.

Hydroxyurea exerts bi-modal dose-dependent effects on erythropoiesis in human cultured erythroid cells via distinct pathways

Summary. Hydroxyurea (HU) has been shown to increase the proportion of fetal haemoglobin (HbF) in most sickle cell patients. A low‐dosage regimen increased total haemoglobin (Hb) levels in some thalassaemia intermedia patients by preferentially increasing β‐globin biosynthesis. To further characterize these apparent dose‐dependent effects of HU, we examined erythroid cells exposed to HU (5–100 µmol/l) in two‐phase liquid culture. Low doses (from 5 to 25 µmol/l) increased Hb levels by up to 2·7‐fold, and a high dose (100 µmol/l) increased Hb levels when added at d 3–6 of phase II, with no significant changes in response to HU during the late stage of phase II culture (≥ 9 d). HU exposure during d 0–3 of phase II culture increased the number of erythroid colonies to a maximum of fivefold at 5 µmol/l HU. GATA‐1 mRNA was downregulated at a high dose and GATA‐2 was dose dependently upregulated over a lower dosage range. Treatment with 100 µmol/l HU dramatically upregulated the death receptor DR‐5, caspase 3, as determined by cDNA microarray analysis. In contrast, 10 µmol/l HU modestly upregulated mRNA levels of the early growth response gene. Our results suggest that HU exerts concentration‐dependent effects on HbF production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.

Thrombopoietin has a differentiative effect on late-stage human erythropoiesis.

To further explore the mechanism of the effect of thrombopoietin (TPO) on erythropoiesis, we used a two‐phase culture system to investigate the effect of TPO on late‐stage human erythroid lineage differentiation. In serum‐free suspension and semisolid cultures of human peripheral blood derived erythroid progenitors, TPO alone did not produce benzidine‐positive cells. However, in serum‐containing culture, TPO alone stimulated erythroid cell proliferation and differentiation, demonstrated by erythroid colony formation, production of benzidine‐positive cells and haemoglobin (Hb) synthesis. Monoclonal anti‐human erythropoietin antibody and anti‐human erythropoietin receptor antibody completely abrogated the erythroid differentiative ability of TPO in the serum‐containing systems. This implied that binding of EPO and EPO‐R was essential for erythropoiesis and the resultant signal transduction may be augmented by the signals emanating from TPO–c‐Mpl interaction. Experiment of withdrawal of TPO further demonstrated the involvement of TPO in late‐stage erythropoiesis. RT‐PCR results showed that there was EPO‐R but not c‐Mpl expression on developing erythroblasts induced by TPO in serum‐containing system. Our results establish that TPO affects not only the proliferation of erythroid progenitors but also the differentiation of erythroid progenitors to mature erythroid cells.