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Aims: Cachexia is a severe consequence of cancer. Although cancer-induced heart atrophy leads to cardiac dysfunction and heart failure (HF), biomarkers for their diagnosis have not been identified. Neutrophil gelatinase-associated lipocalin (NGAL) is an aldosterone-responsive gene increased in HF. We studied NGAL and its association with aldosterone levels in a model of cancer cachexia-induced cardiomyopathy., Methods and Results: Rats were injected with Yoshida 10 8 AH-130 hepatoma cells to induce tumour. Cachectic rats were treated daily, for 16 days, with placebo or with 5 or 50 mg/kg/day of spironolactone. Cardiac function was analysed by echocardiography at baseline and at Day 11. Weight loss and atrophy of lean body and fat mass of cachectic rats were significantly attenuated by spironolactone. Cardiac dysfunction of tumour-bearing rats was improved by spironolactone. Plasma aldosterone was up-regulated from 337 ± 7 pg/mL in sham animals to 591 ± 31 pg/mL in the cachectic rats (P < 0.001 vs. sham). Treatment with 50 or 5 mg/kg/day of spironolactone reduced plasma aldosterone to 396 ± 22 and 391 ± 25 pg/mL (P < 0.01 vs. placebo). Plasma levels of NGAL were also increased in cachectic rats (1.462 ± 0.3603 μg/mL) than in controls (0.0936 ± 6 μg/mL, P < 0.001). Spironolactone treatment (50 mg/kg/day) significantly reduced cardiac mRNA and protein NGAL levels (P < 0.05 and P < 0.001 vs. placebo, respectively). NGAL mRNA and protein levels were overexpressed in cachectic animal hearts treated with placebo, compared with control (P < 0.05 and P < 0.01 vs. sham). Spironolactone treatment at 50 mg/kg/day reduced significantly cardiac NGAL (P < 0.05 and P < 0.001 vs. placebo)., Conclusions: Cancer cachexia induced increased levels of aldosterone and NGAL, contributing to worsening cardiac damage in cancer cachexia-induced cardiomyopathy. Spironolactone treatment may greatly attenuate cardiac dysfunction and lean mass atrophy associated with cancer cachexia., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Background: We sought to confirm that neutrophil gelatinase-associated lipocalin (NGAL) protects against apoptosis during endotoxemia., Methods: Endotoxemia was induced in rats with lipopolysaccharide (LPS; 3.5 mg/kg) and serum creatinine (SCr), urinary NGAL (uNGAL), renal histopathology confirmed acute kidney injury (AKI). Renal caspase 3 and NGAL were assayed with immunohistochemistry 6 h later. A HK-2 cell model was used in which NGAL and caspase 3 mRNA were evaluated by qRT-PCR within 6 h after LPS (50 μM) treatment, and correlations were studied. NGAL and caspase 3 mRNA expression were measured after delivering NGAL siRNA in HK-2 cells and apoptosis was measured with TUNEL and flow cytometry., Results: SCr and uNGAL were significantly increased after LPS treatment and renal morphology data indicated AKI and renal tubular epithelial cell apoptosis. Caspase 3 and NGAL were predominantly expressed in the tubular epithelial cells and there was a correlation between caspase 3 and NGAL protein (r = 0.663, p = 0.01). In vitro, there was a strong correlation between caspase 3 and NGAL mRNA in LPS-injured HK-2 cells within 24 h (r = 0.448, p < 0.05). Suppressing the NGAL gene in HK-2 cells increased caspase 3 mRNA 4.5-fold and apoptosis increased 1.5-fold after LPS treatment., Conclusions: NGAL is associated with caspase 3 in renal tubular cells with endotoxin-induced kidney injury, and may regulate its expression and inhibit apoptosis.

Diabetic nephropathy is a serious microvascular complication and one of the main causes of end-stage renal disease. L-Glutamine (LG) is naturally occurring amino acids with antidiabetic and antioxidant potential. The aim of present investigation was to evaluate the potential of LG against streptozotocin (STZ)-induced diabetic nephropathy (DN) in laboratory rats. DN was induced in male Wistar rats (200-220 g) by intraperitoneal administration of STZ (55 mg/kg). Animals were treated orally with either distilled water (10 mg/kg) or LG (250, 500, and 1000 mg/kg) or Sitagliptin (5 mg/kg). Various biochemical, molecular, and histological (hematoxylin-eosin and Masson's trichrome stain) parameters were assessed. Administration of LG (500 and 1000 mg/kg) significantly inhibited (p < .05) STZ-induced alterations in serum and urine biochemistry (urine creatinine, uric acid, albumin, and BUN). It also significantly increased creatinine clearance rate. STZ induced increase in renal oxidonitrosative stress was significantly decreased (p < .05) by LG (500 and 1000 mg/kg) treatment. Upregulated renal KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expression after STZ administration was significantly inhibited (p < .05) by LG (500 and 1000 mg/kg) treatment. Correlation analysis also revealed that antidiabetic potential of LG attenuates STZ-induced elevated renal KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expression. Histopathological alteration induced by STZ in renal tissue was ameliorated by LG treatment. In conclusion, results of present investigation suggest that treatment with LG ameliorated STZ-induced DN via the inhibition of oxidonitrosative stress as well as downregulation of KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expressions.

Introduction: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxified by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats., Materials and Methods: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were fixed in paraformaldehyde and cut in 20 micrometer slices by cryostat., Results: The expression of NGAL significantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene., Discussion: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.

Tumor initiating cancer stem-like cells (TICSCs) have recently become the object of intensive study. Human-Lipocalin-2 (hLCN2) acts as a biomarker for cancers. The aim of the present study was to explore new insights regarding the potential role of LCN2 in inducing epithelial to mesenchymal transition (EMT) by transfecting LCN2 into CD133+-A549-TICSCs and its cross-talk with the NF-κB signaling pathway in adenocarcinoma of the lung. Furthermore, EMT was confirmed by transcriptomic analysis, immunoblotting and immunocyto/histochemical analyses. Tumorigenesis and metastasis were confirmed by molecular therapeutics tracer 2DG infrared optical probe in BALB/cSIc-nude mice. It was observed that the CD133+-expressing-LCN2-A549 TICSCs population increased in adenocarcinoma of the lung compared to the normal lung tissue. The expressions of genes involved in stemness, adhesion, motility and drug efflux was higher in these cells than in their non-LCN2 expressing counterparts. The present study revealed that elevated expression of LCN2 significantly induced metastasis via EMT. Overexpression of LCN2 significantly increased stemness and tumor metastasis by modulating NF-κB cellular signaling. BRM270, a novel inhibitor of NF-κB plays a significant role in the EMT reversal. BRM270, a naturaceutical induces cell shrinkage, karyorrhexis and programmed cell death (PCD) which were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. Also, 2DG guided in vivo model revealed that BRRM270 significantly (P<0.0003) reduced tumor metastasis and increased percent survival in real-time with complete resection. An elaborate study on the novel concept with respect to linking of naturaceutics as selective and potential anticancer agent that eliminates the elevated LCN2 induced EMT and tumor dissemination through cooperation with the NF-κB signaling as the baseline data for the planning of new therapeutic strategies was conducted for the first time. Our results also illustrate a molecular mechanistic approach for 2DG-guided molecular imaging-based cancer therapy using BRM270 as a novel cancer therapeutic drug to enhance the effect of doxorubicin (Dox)-resistant LCN2 induced metastasis of solid tumors in nude mice.

Purpose: Neutrophil gelatinase associated lipocalin, also known as lipocalin-2, is a 25 kDa protein now considered the biochemical gold standard for the early diagnosis of acute kidney injury. Recently lipocalin-2 was suggested to have an important role in several human neoplasias. In this study we assess lipocalin-2 expression in 2 renal tumor types and analyze its association with clinicopathological parameters and prognosis., Materials and Methods: The study group included 189 patients who underwent surgery for renal lesions between 2003 and 2013. Of these patients 105 had clear cell renal cell carcinoma and 84 had papillary renal cell carcinoma. The association of lipocalin-2 immunoexpression and clinicopathological characteristics was evaluated. Cox proportional hazard regression was performed to estimate the prognostic significance of potential confounders in predicting overall and disease-free survival., Results: Lipocalin-2 expression in different histotypes of analyzed tumors was highly and significantly associated with papillary renal cell carcinoma (p <0.001). In papillary renal cell carcinoma high lipocalin-2 expression was associated with high Fuhrman grade (p <0.001), tumor size greater than 7 cm (p = 0.007), increased TNM stage (p <0.001) and lymph node metastasis (p = 0.009). On univariable and multivariable Cox survival analyses of papillary renal cell carcinoma, after a median followup of 49.1 months (range 7 to 136) lipocalin-2 expression was associated with reduced overall survival (HR 4.10, 95% CI 1.19-14.14, p = 0.026) and disease-free survival (HR 2.56, 95% CI 1.01-6.48, p = 0.047)., Conclusions: Lipocalin-2 over expression may be a prognostic factor in decreased overall and disease-free survival in papillary renal cell carcinoma. The association of lipocalin-2 over expression and poor prognosis with papillary renal cell carcinoma may have potential diagnostic and therapeutic utility., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

To examine the significance of the Neutrophil gelatinase-associated lipocalin (NGAL) in diagnosing head and neck squamous cell carcinoma (HNSCC) and predicting regional metastasis. We first used GEO dataset to analyze the NGAL gene expression in HNSCC. Then, we summarized the characteristics of patients retrospectively selected in clinic. Expression of NGAL protein in human HNSCC tumor, lymph node and normal samples were analyzed using immunohistochemistry. Next, we further investigated the NGAL expression in a tissue microassay to analyze the relationship between NGAL protein expression and TNM stage. Finally, we tested the NGAL protein expression in head and neck cancer cell lines. Analysis of GEO dataset concluded that NGAL gene expression in HNSCC was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL gene expression between T-stage and N-stage (P>0.05). NGAL protein expression in tumor was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL protein expression between metastasis group and non-metastasis group (P>0.05). Expression of NGAL protein was not correlated with TNM stage of HNSCC. Aggressive HNSCC cell lines have lower NGAL protein expression. Our data demonstrated that the expression of NGAL protein was correlated with tumorigenesis of HNSCC, but not with regional metastasis. It may serve as a novel biomarker for prognostic evaluation of patients with HNSCC.

Background: Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood., Methods: LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126., Results: We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway., Conclusion: LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer., (© 2015 Wiley Periodicals, Inc.)

Purpose: The current study was done to determine the role of lipocalin-2 (LCN2) in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model., Methods: The EAON was induced by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG35-55) peptide in mice. The LCN2 expression was examined in the optic nerve after MOG peptide injection. Degree of demyelination, inflammatory infiltration, glial activation, and expression profile of inflammatory mediators in the optic nerve were compared between LCN2 knockout (KO) animals and wild-type littermates by histological analysis and real-time PCR following EAON induction. Plasma levels of LCN2 in patients with optic neuritis were measured by ELISA., Results: The expression of LCN2 was notably increased in the optic nerve after EAON induction. Expression of LCN2 was colocalized with reactive astrocytes. A significant reduction of demyelination, inflammatory infiltration, and gliosis was demonstrated in the optic nerve of LCN2 KO mice. The LCN2 KO mice also showed markedly reduced gene expression associated with the M1-polarized glia phenotype and toll-like receptor signaling in the optic nerve. The LCN2 levels in plasma were significantly higher in optic neuritis patients (71.6 ± 10.6 ng/mL) compared to healthy controls (37.4 ± 9.1 ng/mL, P = 0.0284)., Conclusions: In this study, we demonstrated a significant induction of LCN2 expression in astrocytes of the optic nerve following EAON induction. Our results imply that astrocyte-derived LCN2 may have a pivotal role in the development of demyelinating optic neuritis, and LCN2 can be a therapeutic target to alleviate immune and inflammatory damage in the optic nerve.

Prognosis in patients with lung cancer is poor. Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) are proteins involved in the invasion and metastases of cancer. The objective of this study is to determine if there is a relationship between tumor expression of NGAL and MMP-9 in lung adenocarcinoma patients with prognosis and overall survival. Retrospective analysis was made of patients with lung adenocarcinoma treated at Medica Sur Hospital between 2005 and 2013. Tumor tissue was analyzed for NGAL and MMP-9 expression by immunohistochemistry. We identified 41 patients. Mean overexpression in tumoral tissue of NGAL was 70 % and 30 % for MMP-9. Univariate analysis revealed that prognostic factors associated with overall survival (OS) were NGAL expression and stage at diagnosis. Median OS for NGAL expression < 70 % was 45.7 months (95 % CI; 15.2-76.2) and for patients with ≥ 70 % 4.6 months (95 % CI; 0.5-18.8; P < 0.0001), and for stage at diagnosis (stages I and II mean not reached), stage III mean OS 15.57 months (95 % CI; 9.8-21.2) and stage IV 9.6 months (95 % CI; 0.8-18.4. P = 0.002). No differences in OS were found for expression of MMP-9. Multivariate analysis revealed significance for OS in NGAL expression (HR 5.01 [95 % CI; 1.68-14.93] P = 0.004) and stage at diagnosis (HR 2.05 [95 % CI 1.30-3.22] P = 0.002). Tumoral tissue expression of NGAL ≥ 70 % confers a worse prognosis compared to those who did not. NGAL is an independent prognostic factor of stage at diagnosis.

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.

Objective: Over-expression of the aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated in many human cancers. Lipocalin 2 (LCN2) is reported to inhibit cervical cancer metastasis but little is known regarding its relationship with AKR1C3 in the development and progression of uterine cervical cancer. This study aimed to investigate the involvement of AKR1C3 and its relationship with LCN2 in cervical cancer., Methods: The roles of AKR1C3 and LCN2 were investigated using the lentivirus shRNA system in SiHa and Caski cervical cancer cells. LCN2 and matrix metalloproteinase-2 (MMP-2) promoters were constructed to demonstrate transcriptional regulation by shAKR1C3 and shLCN2, respectively. The influences of metastatic phenotypes were analyzed by wound healing, Boyden chamber, and immunofluorescence assays. The activity of MMP-2 was determined by zymography assay. The impacts of AKR1C3 and LCN2 on patient prognosis were evaluated using tissue microarrays by Cox regression and Kaplan-Meier models., Results: Silencing of the AKR1C3 gene increased the expression of LCN2 and decreased the migratory and invasive abilities and changed the cytoskeleton of cervical cancer cells. When AKR1C3 was over-expressed, it decreased LCN2 promoter activity and LCN2 expression and increased cell migration. The mRNA level and enzyme activity of MMP-2 increased in silenced LCN2 cells. Positive AKR1C3 and negative LCN2 were correlated with higher recurrence and poorer survival of cervical cancer patients., Conclusions: Silencing of AKR1C3 increases LCN2 expression and inhibits metastasis in cervical cancer. Both AKR1C3 and LCN2 serve as molecular targets for cancer therapy to improve the clinical outcome of cervical cancer patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

NGAL (neutrophil gelatinase-associated lipocalin) is a novel cancer-related protein involves multiple functions in many cancers and other diseases. We previously overexpressed NGAL to analyze its role in esophageal squamous cell carcinoma (ESCC). In this study, a protein-protein interaction (PPI) was constructed and the shortest paths from NGAL to transcription factors in the network were analyzed. We found 28 shortest paths from NGAL to RELA, most of them obeying the principle of extracellular to cytoplasm, then nucleus. These shortest paths were also prioritized according to their normalized intensity from the microarray by the order of interaction cascades. A systems approach was developed in this study by linking differentially expressed genes with publicly available PPI data, Gene Ontology and subcellular localizaton for the integrated analyses. These shortest paths from NGAL to DEG transcription factors or other transcription factors in the PPI network provide important clues for future experimental identification of new pathways.

Background: Lipocalin-2 is a multifaceted modulator in cancer progression. Its clinical significance is not clear in pancreatic cancer. The purpose of this study was to investigate whether lipocalin-2 is associated with good prognosis by reversing epithelial-to-mesenchymal transition (EMT) in pancreatic cancer., Methods: Lipocalin-2, E-cadherin, or vimentin expression was detected in 60 pancreatic adenocarcinoma specimens. Correlations between lipocalin-2 expression and EMT, the clinicopathologic characteristics, and prognosis were investigated. Whether pancreatic cancer cells' migration and invasion (some characteristics of EMT) were affected by lipocalin-2 was also explored., Results: High lipocalin-2 expression was significantly associated with a good prognosis in pancreatic cancer (p < 0.05). Overexpression of lipocalin-2 correlated with a lower extent of EMT (p < 0.05), increased E-cadherin expression (p < 0.05), decreased vimentin expression (p < 0.05), and reduced cancer cell migration and invasion in pancreatic cancer., Conclusions: Lipocalin-2 may be considered an epithelial inducer, which may reverse EMT and predict a good prognosis in pancreatic cancer.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers., (© 2013 by the Wound Healing Society.)

Unlabelled: Neutrophil gelatinase-associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase-associated lipocalin is expressed in a number of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation. For this reason, we hypothesized that levels of NGAL might be expressed at higher levels after blast injury compared with other ballistic injury., Purpose: The purpose of this study is to test the hypothesis that NGAL may be a marker of injury severity in blast injury., Materials: Twenty-three combat casualties (13 blast, 10 gunshot wounds) admitted to the multinational role 3 facility in Helmand province were studied. Serum NGAL was measured using a Biosite Triage point-of-care monitor at 5 time points after injury., Results: Neutrophil gelatinase-associated lipocalin rose in both groups of casualties and was significantly predictive of death or renal failure at intensive care unit admission, 12 and 24 hours after injury., Conclusions: Neutrophil gelatinase-associated lipocalin is not a specific marker of blast injury but is predictive of both renal failure and poor outcome., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival., Methods: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies., Results: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, β-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage., Conclusion: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.

Tumor invasion is a critical step in the spread of cancer. S2R (sigma-2 receptor)/Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b(5)-related drug-binding orphan receptor essential for tumor formation and invasion. Secretory proteins drive these processes, so we screened for S2R(Pgrmc1)-dependent secreted proteins using antibody arrays. S2R(Pgrmc1) markedly regulated the expression of NGAL/LCN2 (neutrophil gelatinase-associated lipocalin/lipocalin 2), a secreted glycoprotein that binds to MMP-9 (matrix metalloproteinase 9) and protects it from degradation. S2R(Pgrmc1) knock-down blocked NGAL/LCN2 expression at the protein and RNA levels and decreased MMP9 activity. NGAL expression was required for MMP-9 activity and tumor formation. S2R(Pgrmc1) associates with EGFR and increases EGFR levels at the plasma membrane, and the EGFR inhibitors erlotinib and AG1478, as well as Akt and ERK inhibitors, suppressed the NGAL/LCN2 RNA and protein levels. NGAL is transcriptionally regulated by NFκB, and S2R(Pgrmc1) knock-down decreased the NFκB subunit p65/RelA acetylation, phosphorylation, and activation. In S2R(Pgrmc1) knock-down cells, p65 acetylation was reversed by inhibitors of histone deacetylase 1, and the inhibitors partially restored NGAL levels. Our results are consistent with a model in which S2R(Pgrmc1) increases NGAL/LCN2 levels by activating NFκB via EGFR.

Neutrophil gelatinase-associated lipocalin (NGAL) is a protein which participates in iron trafficking and which is involved in cancerogenesis and cancer progression. Since its over-expression has been documented in thyroid malignancies in comparison to thyroid normal gland, in the present study, we aimed to determine whether the evaluation of NGAL immunoexpression may be of help in the differential diagnosis of follicular-patterned thyroid lesions. Our additional aim was to test the possible interference of endogenous biotin on the immunohistochemical findings. Thus, all the immunohistochemical procedures, carried out with labeled streptavidin biotin method, were doubly performed, with or without the preliminary inhibition of endogenous biotin. No NGAL staining was found in the normal thyroid gland nor in the nodular colloid goiters or in Hashimoto's thyroiditis. NGAL expression appeared to be significantly more frequent in the malignant tumors in comparison to benign ones (P < 0.000001). Even more, NGAL expression appeared to be specific (specificity 93%) for carcinoma and represented a sensitive method (sensitivity 84%), with high negative (88%) and positive (94%) predictive values, as well as high diagnostic accuracy (88%), in the identification of follicular-patterned thyroid malignant tumors. The specificity, positive predictive value and diagnostic accuracy lowered when biotin was not preliminary inhibited, due to the presence of false positives among benign Hürthle cell tumors. In conclusion, the immunohistochemical detection of NGAL may be helpful in the differential diagnosis between malignant and benign follicular-patterned lesions of the thyroid. The use of biotin free system or the preliminary biotin inhibition is warranted for the detection of NGAL in thyroid samples, especially when dealing with Hürthle cell tumors.

Background: Patients with pre-existing impaired renal function are prone to develop acute contrast media induced nephropathy (CIN). Neutrophil gelatinase-associated lipocalin (NGAL), a new biomarker predictive for acute kidney injury (AKI), has been shown to be useful for earlier diagnosis of CIN; however, urinary NGAL values may be markedly increased in chronic renal failure at baseline. Results from those studies suggested that urinary NGAL values may not be helpful for the clinician. An intravenous volume load is a widely accepted prophylactic measure and possibly a reasonable intervention to prevent deterioration of renal function. The aim of our study is to evaluate NGAL as an early predictor of CIN and to investigate the clinical benefit of early post-procedural i.v. hydration., Methods/design: The study will follow a prospective, open-label, randomized controlled design. Patients requiring intra-arterial contrast media (CM) application will be included and receive standardized, weight-based, intravenous hydration before investigation. Subjects with markedly increased urinary NGAL values after CM application will be randomized into one of two study groups. Group A will receive 3-4 ml/kg BW/h 0.9% saline intravenously for 6 hours. Group B will undergo only standard treatment consisting of unrestricted oral fluid intake. The primary outcome measure will be CIN defined by an increase greater than 25% of baseline serum creatinine. Secondary outcomes will include urinary NGAL values, cystatin C values, contrast media associated changes in cardiac parameters such as NT-pro-BNP/troponin T, changes in urinary cytology, need for renal replacement treatment, length of stay in hospital and death.We assume that 20% of the included patients will show a definite rise in urinary NGAL. Prospective statistical power calculations indicate that the study will have 80% statistical power to detect a clinically significant decrease of CIN of 40% in the treatment arm if 1200 patients are recruited into the study., Discussion: A volume expansion strategy showing a benefit from earlier intervention for patients with markedly elevated urinary NGAL values, indicating a CIN, might arise from data from this study., Trial Registration: ClinicalTrials.gov NCT01292317.

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been suggested to behave like a negative prognostic marker in stage I colorectal carcinoma. In the aim of clarifying whether its association with adverse outcome may descend from NGAL's ability to regulate matrix metallo-proteinase-9 (MMP-9), we analyzed the correlation, prognostic value, and association with neo-angiogenesis of NGAL and MMP-9 immunohistochemical expression in a series of stage I colorectal carcinomas. A variable NGAL immunoexpression was demonstrated in 17 of the 48 analyzed cases with a significantly higher frequency of positive cases among patients showing disease progression. NGAL expression was also positively correlated with VEGF expression detected in the same cases. MMP-9 immunostaining was present in the cytoplasm of the neoplastic cells in 30 cases; no significant correlations were evidenced with NGAL expression, as well as with the various clinico-pathological parameters or with progression of the colorectal carcinomas. By contrast, NGAL expression was confirmed as a significant independent negative prognostic marker related to a shorter disease-free survival in stage I colorectal carcinoma. Our preliminary results suggest that the association of NGAL with poor outcome might be independent from MMP-9 regulation, thus highlighting its prognostic value in this neoplasia. If our findings are confirmed in further analyses, NGAL assessment might be used in order to select those patients with a higher progression risk and to submit them to adjuvant therapies useful to prevent adverse outcome., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background: Obesity is widely recognised as an important risk factor for colorectal cancer (CC)., Aim: The study aimed to evaluate the effect of CC on circulating concentrations and gene expression levels of inflammatory and angiogenesis-related factors in human visceral adipose tissue (VAT)., Methods: VAT biopsies were obtained from 18 healthy individuals and 11 patients with CC. Real-time polymerase chain reactions were performed to quantify gene expression levels and zymographic analyses were used to determine the activity of matrix metalloproteinases (MMPs)., Results: Patients with CC exhibited increased mRNA expression levels of lipocalin-2 (P=.014), osteopontin (P=.027), tumor necrosis factor-α (TNF-α) (P=.016) and chitinase-3 like-1 (P=.006) compared to control subjects in VAT. Gene expression levels of hypoxia-inducible factor-1 α, vascular endothelial growth factor and MMP-2 were significantly higher (P<.05) in VAT of patients with CC. The expression of insulin-like growth factor I, insulin growth factor binding protein 3 and MMP-9 followed the same trend, although no significant differences were reached. The enzymatic activity of MMP-9 was increased (P<.001) in patients with CC. Furthermore, individuals with CC showed increased (P<.05) circulating concentrations of the inflammatory markers interleukin-6, tumour necrosis factor α and hepatocyte growth factor, whereas levels of the anti-inflammatory adipokine adiponectin were decreased (P<.01)., Conclusion: These findings represent the first observation that mRNA levels of the novel inflammatory factors lipocalin-2, chitinase-3 like-1 and osteopontin are increased in human VAT of subjects with CC. This observation together with the up-regulation of angiogenic factors suggests that adipokines secreted by VAT may be involved in the development of colon cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by an irreversible cognitive decline and neuronal loss associated with neurofibrillary tangles and senile plaques. Mild cognitive impairment (MCI) is a prodromal stage of AD and is associated with memory loss and a high risk of developing AD. Lipocalin 2 (LCN2) is an acute phase protein. Our previous studies have shown that exposure to inflammatory stimuli resulted in elevated LCN2 levels in brain microglia and astrocytes implicating LCN2 in brain inflammation. Therefore, we hypothesize that there may be a significant change in the plasma LCN2 levels in patients with MCI and AD when compared to healthy control subjects., Methods: Forty-one patients with MCI, 62 patients with AD and 38 healthy elderly control subjects were recruited for this study. They were given a comprehensive battery of neuropsychological tests including a mini-mental status examination (MMSE) and clinical dementia rating (CDR). A variety of clinical information was collected from the semi-structured questionnaire administered. The LCN2 levels were measured using a specific enzyme-linked immunosorbent assay in the plasma, which had been collected early in the morning after overnight fasting., Results: The LCN2 levels were significantly higher in MCI patients compared to the healthy control subjects and AD patients [control vs. MCI (p=0.005); MCI vs. AD (p=0.009)]. There was a significant negative correlation between the LCN2 levels and CDR scores (r=-0.245, p=0.014), and there was a positive correlation between the LCN2 levels and MMSE scores (r=0.317, p=0.001) among all of the MCI and AD patients., Conclusion: MCI represents a prodromal stage of AD, and inflammation occurs as one of the earliest pathological events in AD. Thus, increased plasma LCN2 levels during MCI could be helpful in predicting the progression from MCI to AD., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production., Methods: We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways., Results: Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation., Conclusions: We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation. We demonstrated previously that the gene encoding NGAL (LCN2) is strongly up-regulated by interleukin (IL)-1beta in an NF-kappaB-dependent manner but not by tumor necrosis factor (TNF)-alpha, another potent activator of NF-kappaB. This is due to an IL-1beta-specific synthesis of the NF-kappaB-binding co-factor IkappaB-zeta, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-alpha in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4(+) T helper cells, T(H)-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-beta or C/EBP-delta for NGAL induction by IL-17 and TNF-alpha as neither small interfering RNAs against the two C/EBP mRNAs nor mutation of the C/EBP sites in the LCN2 promoter abolished IL-17- and TNF-alpha-induced up-regulation of NGAL. NGAL induction is governed solely by NF-kappaB and its co-factor IkappaB-zeta. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with small interfering RNA against IkappaB-zeta and a total lack of activation of an LCN2 promoter construct with a mutated NF-kappaB site. As IL-17 stimulation stabilizes the IkappaB-zeta transcript, we propose a model where TNF-alpha induces activation and binding of NF-kappaB to the promoters of both NFKBIZ and LCN2 genes but induce only transcription of IkappaB-zeta. Co-stimulation with IL-17 leads to accumulation of IkappaB-zeta mRNA and IkappaB-zeta protein, which can bind to NF-kappaB on the LCN2 promoter and thus induce NGAL expression.

ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers, including breast carcinomas. NGAL can inhibit erythroid cell production, leading to anemia. Anemia usually occurs in cancer patients and negatively affects quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we show that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor-kappaB (NF-kappaB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by a NF-kappaB inhibitor and ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-kappaB activation is involved in cancer-related anemia, and the ErbB2, NF-kappaB, and NGAL pathways may serve as potential therapeutic targets for cancer-related anemia.

We recently identified lipocalin2 (LCN2) as being upregulated in ovarian cancer cell lines. The purpose of this study was to validate LCN2 upregulation in ovarian cancers and to investigate its potential as a serum biomarker. We assayed LCN2 expression in ovarian cancers using real-time PCR and IHC. To evaluate the potential of LCN2 as a biomarker, we measured serum LCN2 levels in 54 ovarian cancers, 15 borderline and 53 benign ovarian tumors, and 90 healthy controls. SYBR green PCR and IHC showed LCN2 overexpression in ovarian cancers. LCN2 immunoreactivity was significantly associated with tumor differentiation (p=0.009), as well-differentiated tumors showed the highest LCN2 expression. Serum LCN2 level in ovarian cancer was significantly higher than in the other study groups (p<0.001), and in accordance with IHC results, it also correlated with tumor differentiation, with well-differentiated tumors having the highest value. The sensitivity and specificity of LCN2 in detecting ovarian cancer was 72.2% and 50.4%, respectively. By Cox univariate analysis, LCN2 positivity was an independent prognostic factor for overall survival (hazard ratio = 1.47, p=0.012). In conclusion, LCN2 expressions are upregulated and related to tumor differentiation in ovarian cancers and should be included in future research assessing potential biomarkers for ovarian cancer.

Neutrophil gelatinase-associated lipocalin (NGAL) is a small, secreted glycoprotein with proposed functions in cell proliferation, survival and morphogenesis. NGAL is expressed in a variety of tumor types including breast carcinomas, but it is not known whether NGAL contributes directly to breast cancer progression. This study examines the relationship between NGAL expression in breast carcinomas and established clinical prognostic markers as well as clinical outcome. Using immunohistochemistry in tissue microarrays containing well characterized tumor samples from 207 breast cancer patients, NGAL was detected in 68 breast carcinomas in a cytoplasmic location. NGAL expression correlated strongly with negative steroid receptor status, HER-2/neu overexpression, poor histologic grade, the presence of lymph node metastases and a high Ki-67 proliferation index. In univariate survival analysis, NGAL expression was associated with decreased disease-specific survival and decreased disease-free survival in the entire cohort. In multivariate analysis, NGAL remained an independent prognostic marker for disease-free survival. In a subset of patients with estrogen receptor positive tumors, NGAL was significantly associated with decreased disease-free survival. The results show that NGAL expression is a predictor of poor prognosis in primary human breast cancer and suggest that NGAL detection may provide information for risk assessment and identify a subset of patients requiring more aggressive adjuvant therapy.

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium-induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis.

Objective: Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an approximately 125-130-kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF., Methods: MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase-associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay., Results: Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL-MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL-MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9-mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05)., Conclusion: Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.

Neutrophil gelatinase-associated lipocalin (NGAL) is expressed and secreted by immune cells, hepatocytes, and renal tubular cells in various pathologic states. NGAL exerts bacteriostatic effects, which are explained by its ability to capture and deplete siderophores, small iron-binding molecules that are synthesized by certain bacteria as a means of iron acquisition. Consistently, NGAL deficiency in genetically modified mice leads to an increased growth of bacteria. However, growing evidence suggests effects of the protein beyond fighting microorganisms. NGAL acts as a growth and differentiation factor in multiple cell types, including developing and mature renal epithelia, and some of this activity is enhanced in the presence of siderophore:iron complexes. This has led to the hypothesis that eukaryotes might synthesize siderophore-like molecules that bind NGAL. Accordingly, NGAL-mediated iron shuttling between the extracellular and intracellular spaces may explain some of the biologic activities of the protein. Interest in NGAL has been sparked by the observation that NGAL is massively upregulated after renal tubular injury and may participate in limiting kidney damage. This review summarizes the current knowledge about the dual effects of NGAL as a siderophore:iron-binding protein and as a growth factor and examines the role of these effects in renal injury.

Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, is a 25-kDa lipocalin initially purified from neutrophil granules. It is thought to play a role in regulating cellular growth since its expression is highly upregulated in a variety of proliferative cells such as cancer cells. However, experimental evidence showing a clear causal relationship between NGAL expression and the proliferation of tumor cells is lacking. Here, we found NGAL expression in highly and poorly metastatic colon cancer cell lines of the same genetic origin correlated inversely with the metastatic potential of these cells, which suggests NGAL participates in the metastatic process. To explore the role NGAL plays in tumor growth and metastasis, the KM12SM human colon cancer cell line, which is highly metastatic while showing decreased NGAL expression, was genetically manipulated to overexpress NGAL. The effects of this on tumor growth and liver metastasis were then analyzed using experimental animal models established by injecting BALB/c nude mice with tumor cells subcutaneously or intrasplenically. Ectopic expression of NGAL in the colon cancer cells had little effect on the growth and viability of the tumor cells both in vitro and in vivo. However, NGAL expression not only suppressed the ability of the colon carcinoma cells to invade Matrigel in vitro, it also substantially inhibited liver metastasis in an experimental animal model. Collectively, these results indicate that NGAL may be a candidate metastasis suppressor in colon cancer cells., (Copyright (c) 2005 Wiley-Liss, Inc.)

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding protein that exerts a bacteriostatic effect by sequestering iron. Strong induction of NGAL synthesis has been observed in inflamed epithelium of the lungs and colon. Expression of NGAL is up-regulated in the lung epithelial cell line A549 by IL-1beta, but not by TNF-alpha, despite an induction of NF-kappaB binding to the NGAL promoter by both cytokines. In this study, we present evidence that the IL-1beta specificity is caused by a requirement of the NGAL promoter for the NF-kappaB-binding cofactor IkappaB-zeta for transcriptional activation. Up-regulation of NGAL expression in A549 cells following IL-1beta stimulation was dependent on de novo protein synthesis and was greatly diminished by a small interfering against IkappaB-zeta mRNA. Cotransfection of A549 cells with a plasmid expressing IkappaB-zeta made TNF-alpha capable of inducing NGAL transcription, indicating that IkappaB-zeta induction is the only factor discriminating between IL-1beta and TNF-alpha in their ability to induce NGAL expression. Coexpression of the cofactor Bcl-3, which is closely related to IkappaB-zeta, did not enable TNF-alpha to induce NGAL transcription. A functional NF-kappaB site of the NGAL promoter was required for IkappaB-zeta to exert its effect. The human beta defensin 2 gene also required IkappaB-zeta for its IL-1beta-specific induction in A549 cells. Our findings indicate that a common regulatory mechanism has evolved to control expression of a subset of antimicrobial proteins expressed in epithelial cells.

Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for alpha-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.