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. The hGC-1 gene encodes a 510-amino acid glycoprotein whose exact in vivo localization and function still remains elusive. A recent study showed that hGC-1 mRNA expression is upregulated in gastric caner. To further explore a potential relationship between hGC-1 and gastric carcinoma, we investigated the expression pattern of hGC-1 protein in 173 cases of gastric carcinoma. Using immunohistochemistry, we demonstrated that hGC-1 is expressed in the esophagus, stomach, small intestine and colon. We demonstrated a striking correlation between hGC-1 expression and the histological type and differentiation grades of gastric carcinoma. Enhanced hGC-1 expression is more frequently seen in intestinal- type adenocarcinoma, while loss of expression tends to occur in the diffuse-type. hGC-1 is highly expressed in well or moderately differentiated cancer tissues and remarkably reduced or lost in poorly differentiated or undifferentiated tissues. These investigations define for the first time the expression pattern of hGC-1 in the normal human gastrointestinal tract and provide a novel and sensitive marker for the differentiation of gastric carcinoma. On further analysis, we find high level expression of the mouse-analogue (mGC-1) in pro-B and pre-B cells and low-level expression in mature B and T-cells that co-localizes in this region of the small intestine. When the myeloid progenitor 32D cell line was exposed to G-CSF for 7 days, mGC-1 expression was induced. Taken together, these results suggest that mGC-1 play an important role in granulocytic differentiation, and quite likely in mucousal immunity. To gain further insight into the potential pathways involved in GC-1 effects we performed further analysis in the 293 cell line. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abundantly expressed in tissues which has secretion capability. Based on the data that hGC-1 is a glycoprotein and associated with cell adhesion, experiments have been performed to screen some lectins and cell adhesion proteins which might interact with hGC-1. Agarose bound lectin pull down assay shows hGC-1 strongly bound RCAI, weakly with ConA and WGA and no binding was detected with DSA, PNA and SNA. The purified hGC-1 enhances NIH3T3 and 293T/17 cell spread and attachment. hGC-1 enriched cell culture supernatants of 293T/17 cells transfected with hGC-1 expression vector also enhance cell spread and cell attachment. Co-immunoprecipitation demonstrated a association of hGC-1 with cadherin in 293 cells transiently transfected with hGC-1 cDNA. The interaction of hGC-1 with cadherin depends on the C-terminal olfactomedin domain, but does not require the five well conserved cysteine residues in hGC-1. However, cysteine residue at 83, 85, 246 and 437 is essential for hGC-1 secretion and cysteine 226 is critical for hGC-1 multimer formation. The potential association of hGC-1 with cadherin might explain the involvement of hGC-1 with cell adhesion. E-cadherin in particular, serves as a widely acting suppressor of invasion and growth of epithelial cancers, and its functional elimination represents a key step in the acquisition of the invasive phenotype for many tumors. Further analysis of the interaction of hGC-1 and the cadherin family of adhesion proteins may help clarify the role of this putative tumor suppessor gene. Finally, to further explore the hGC-1 gene function, we successfully established two mouse models: hGC-1 null mice and hGC-1 transgenic micse. The hGC-1 gene expression knock-out was confirmed by RT-PCR and Western blot in hGC-1 null mouse. The hGC-1 over-expression in transgenic mouse was confirmed by Western blot with tag (V5) antibody. At 12 weeks, these mice do not show any obvious (abnormal) phenotype. No abnormality was observed in HE histology examination of major internal organs. Blood profile and some biochemistry parameter tests were within the normal range. We are preparing to induce chronic colonitis by Dextran sulfide (DSS) in hGC-1+/+ and hGC-1-/- mice to determine the potential role of hGC-1 in intestinal innate immunity.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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Medicinal Chemistry B Study Section (MCHB)
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U.S. National Inst Diabetes/Digst/Kidney
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Liu, W; Zhu, J; Cao, L et al. (2007) Expression of hGC-1 is correlated with differentiation of gastric carcinoma. Histopathology 51:157-65
Liu, Wenli; Chen, Ling; Zhu, Jianchong et al. (2006) The glycoprotein hGC-1 binds to cadherin and lectins. Exp Cell Res 312:1785-97
Zhuang, Zhengping; Huang, Steve; Kowalak, Jeff A et al. (2006) From tissue phenotype to proteotype: sensitive protein identification in microdissected tumor tissue. Int J Oncol 28:103-10
Zhang, Jiachang; Liu, Wen Li; Tang, Delia C et al. (2002) Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development. Gene 283:83-93