Role Of Fn Research Articles

Impaired incorporation of fibronectin into the extracellular matrix during aging exacerbates the senescent state of dermal cells

Fibronectin (Fn) is a ubiquitous extracellular matrix (ECM) glycoprotein that acts as an ECM scaffold organizer and is essential in many biological functions, including tissue repair, differentiation or cancer dissemination. Evidence suggests that the amount of Fn changes during aging. However, how these changes influence the aging process remains unclear. This study aims to understand Fn influence on cell aging. First, we assess the relative level of Fn abundance in both different biopsies of skin donors and replicative senescence cellular model. In skin biopsies, we observed that Fn level decreases with aging in the reticular dermis, while its expression remains relatively stable in the papillary dermis, likely to sustain the dermis-epidermis junction. During replicative senescence, in BJ skin fibroblasts, while intracellular Fn increases, we found that secretion and Fn fibrils formation are less effective. Reduced Fn fibrils leads to disorganization of the ECM. This could be explained by the expression of different Fn isoforms observed in the secretome of senescent cells. Surprisingly, the knockdown of Fn delays the onset of senescence while cultivating cells onto a Fn-coated support promotes it. Taken together, these new insights on the role of Fn during aging may emerge new therapeutic strategies on aged-related diseases.

Adhesion of human pathogenic bacteria to endothelial cells is facilitated by fibronectin interaction

Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection.

Abstract A05: Fusobacterium nucleatum and clinicopathologic features of colorectal carcinoma: Results from the ColoCare Cohort

Abstract Background: Fusobacterium nucleatum (Fn) plays an active role in normal host physiology and has been associated with colorectal carcinogenesis. To date, the role of Fn in newly diagnosed colorectal cancer patients is unclear. This is the first study to evaluate associations between Fn abundance and clinicopathologic characteristics among neoadjuvant chemotherapy naïve colorectal cancer patients. Methods: We used presurgery collected fecal stool samples from n=101 prospectively followed, newly diagnosed colorectal cancer patients from the ColoCare Cohort. Quantitative real-time polymerase chain reaction was used to amplify/detect Fn DNA in neoadjuvant chemotherapy-naïve patients, not using antibiotics for at least 1 month. Patients were classified into Fn-high (n=50) or Fn-low (n=51) groups based on median Fn Cycle thresholding (Ct) values (low≤16.72; high>16.72) in fecal DNA. Multivariable regression was utilized to analyze associations between Fn and clinicopathologic characteristics such as tumor stage, tumor site, and tumor grade. Results: We did not observe statistically significant differences in clinicopathologic or demographic characteristics by Fn abundance groups. Compared to the Fn-low patient group, Fn-high patients were 69% more likely to be diagnosed with stage III colorectal cancer (odds ratio [OR]=1.69, 95% confidence interval [CI] 1.56-15.93, P=0.0067), while there was no difference for stage I or II. Conclusion: Human gut microbial composition, including Fusobacterium nucleatum abundance, may be linked to colorectal cancer progression. Citation Format: Patrick Mallea, Christy A. Warby, W. Zac Stephens, Biljana Gigic, Kate Buhrke, Tengda Lin, Juergen Boehm, Sheetal Hardikar, Elisa K. Santori, Petra Schrotz-King, Martin Schneider, Alexis Ulrich, Eric A. Swanson, Adetunji T. Toriola, June Round, Cornelia M. Ulrich, Andreana N. Holowatyj, Jennifer Ose. Fusobacterium nucleatum and clinicopathologic features of colorectal carcinoma: Results from the ColoCare Cohort [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A05.

Abstract A01: Fusobacterium nucleatum and clinicopathologic features of colorectal carcinoma: Results from the ColoCare Study

Abstract Background: Fusobacterium nucleatum (Fn), an oral commensal involved in a wide spectrum of infections, has recently been implicated in colorectal cancer (CRC) etiology. However, the role of Fn in treatment-naïve CRC patients remains unclear. Therefore, we assessed whether Fn abundance is associated with clinicopathologic characteristics among treatment-naïve CRC patients enrolled in the prospective ColoCare Study. Methods: Quantitative real-time PCR was used to amplify and detect Fn DNA in fecal samples collected prior to surgery from 105 patients. We utilized multivariable regression analysis to investigate associations between Fn abundance and sex, age at surgery, BMI, tumor stage, tumor grade, tumor site, infection with H. pylori, microsatellite instability, alcohol consumption, and smoking history by adjusting for sex, age at surgery, cohort, and BMI. Results: Compared to patients with undetectable or low abundance of Fn, patients with higher abundance of Fn were more likely to be diagnosed with rectal cancer than colon cancer (Odds Ratio [OR] = 3.01, 95% CI 1.06-8.57 P=0.04). Categorizing the colon into right-sided (proximal) and left-sided (distal) showed that patients with a high abundance of Fn were also more likely to be diagnosed with rectal cancer compared to right-sided colon cancer (OR=5.32, 95% CI 1.23-22.98 P=0.03), thus suggesting an increasing risk of cancer diagnosis along the bowel towards the rectum. Conclusion: Our study sheds light on the association of high abundance of Fn in fecal biospecimen with colorectal carcinogenesis, which may support future preventive or diagnostic measures. Citation Format: Yannick Eisele, Patrick M. Mallea, Biljana Gigic, W. Zac Stephens, Christy A. Warby, Kate Buhrke, Tengda Lin, Petra Schrotz-King, Sheetal Hardikar, Lyen C. Huang, T. Bartley Pickron, Courtney Scaife, Torsten Koelsch, Anita R. Peoples, Maria A. Pletneva, Mary Bronner, Martin Schneider, Alexis B. Ulrich, Eric A. Swanson, Adetunji T. Toriola, Hans Hauner, June Round, Cornelia M. Ulrich, Andreana N. Holowatyj, Jennifer Ose. Fusobacterium nucleatum and clinicopathologic features of colorectal carcinoma: Results from the ColoCare Study [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A01.

Abstract 2840: Fusobacterium nucleatum stimulates colorectal cancer progression by activating Annexin A1 in cancerous cells

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death in the U.S., affecting one in 20 individuals. Numerous studies have implicated Fusobacterium nucleatum (Fn), a Gram-negative oral commensal, in CRC; however, mechanistic insight on the role of Fn in this debilitating disease is scarce. Previously, we have reported that Fn stimulates CRC growth through its unique adhesin FadA, which binds to E-cadherin and modulates β-catenin signaling. In the present study, we tested the specificity of Fn-induced cell growth. Fn did not promote the growth of lung, prostate, breast and bladder cancer cells, indicating specific component(s) on CRC cells are required for stimulation. To identify such component(s), we utilized a CRC progression model consisting of a series of cell lines sequentially derived from a human colonic adenoma. Fn specifically stimulates growth of the cancerous cells without affecting the non-cancerous cells. The growth stimulation is mediated by Annexin A1 (ANXA1), a member of the Annexin family of Ca2+-dependent phospholipid-binding proteins. ANXA1 is specifically expressed in the proliferating cancerous cells, but not in non-cancerous or non-proliferating cells. Analysis of a database of 466 colon cancer patients reveals that increased level of ANXA1 is associated with cancer reoccurrence independent of cancer stage, grade, sex and age. Knocking down of ANXA1 in CRC cells inhibits cell proliferation due to down-regulation of Cyclin D1. An E-cadherin mediated positive feedback loop between FadA and ANXA1 is identified in the cancerous cells. Fn preferentially binds to ANXA1-expressing cancerous cells, which in turns stimulates ANXA1 expression. FadA, E-cadherin and Annexin A1 form a complex in CRC cells leading to activation of β-catenin signaling. The correlation between FadA and ANXA1 was investigated in vivo using the APCmin/+ mice. Mice gavaged with wild-type Fn produced significantly increased number of tumors in the colon, compared to those gavaged with sterile PBS, E. coli DH5α, or the fadA-deletion mutant US1. In both mice and humans, increased ANXA1mRNA levels were detected in the tumors compared to the matching normal colonic tissues, and a positive correlation between the fadA gene levels and ANXA1 mRNA levels was identified in the colorectal tumors. Immunofluorescent staining of paired normal and carcinoma tissues from CRC patients confirmed this finding and revealed co-localization of FadA and Annexin A1 in the carcinomas. We have thus elucidated a novel mechanism by which Fn promotes CRC, i.e. through stimulating Annexin A1, a novel Wnt/β-catenin modulator, in the cancerous cells. Given the non-cancerous cells are not affected by Fn, we propose a “two-hit” model in which the first hit would be the somatic mutation(s) causing normal cells to become cancerous, and Fn serves as the second hit to exacerbate cancer progression. Citation Format: Mara R. Rubinstein, Jung Eun Baik, Stephen M. Lagana, William J. Raab, Dabashis Sahoo, Piero Dalerba, Timothy C. Wang, Yiping W. Han. Fusobacterium nucleatum stimulates colorectal cancer progression by activating Annexin A1 in cancerous cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2840.

CIP2A mediates fibronectin-induced bladder cancer cell proliferation by stabilizing \u03b2-catenin

BackgroundFibronectin (FN) is associated with tumorigenesis and progression in bladder cancer, however, the underlying mechanisms causing this remain largely unknown. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A) has been shown to play important regulatory roles in cancer proliferation. Here, we investigated whether FN regulates CIP2A expression to promote bladder cancer cell proliferation.MethodsThe correlations of stromal FN with CIP2A and proliferating cell nuclear antigen (PCNA) expression were analyzed in a cohort bladder cancer patients. The roles of FN and CIP2A in regulating bladder cancer cell proliferation were evaluated in cell and animal models. Cycloheximide treatment was used to determine the effects of CIP2A on β-catenin stabilization. The CIP2A-β-catenin interaction was confirmed by immunofluorescence staining and co-immunoprcipitation.ResultsIn this study, we found that stromal FN expression correlated positively with the levels of CIP2A and PCNA in bladder cancer tissues. Meanwhile, in human bladder cancer cell lines (T24 and J82), exogenous FN significantly promoted cell proliferation, however, CIP2A depletion inhibited this process. Furthermore, the interaction between CIP2A and β-catenin enhanced the stabilization of β-catenin, which was involved in FN-induced cell proliferation. In vivo, CIP2A depletion repressed FN-accelerated subcutaneous xenograft growth rates.ConclusionsThese data reveal that CIP2A is a crucial mediator of FN-induced bladder cancer cell proliferation via enhancing the stabilization of β-catenin. Promisingly, FN and CIP2A could serve as potential therapeutic targets for bladder cancer treatment.

Platelet adhesion to polystyrene-based surfaces preadsorbed with plasmas selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor.

Four plasma proteins have been shown to be able to mediate platelet adhesion to synthetic materials when they are adsorbed as purified proteins: fibrinogen (Fg), fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWF). Among them, Fg is thought to play a leading role in mediating platelet adhesion to plasma-preadsorbed biomaterials, but this has been established for only three types of materials so far in our laboratory. Furthermore, the role of Fn, Vn, and vWF in mediating platelet adhesion to plasma-preadsorbed surfaces is still unclear. The aim of the current study was to assess the importance of Fg, Fn, Vn, and vWF in mediating platelet adhesion to a series of polystyrene-based surfaces. The strategy applied in the present investigation was to compare platelet adhesion to surfaces preadsorbed with normal plasma, plasma selectively depleted in Fn or Vn or both Fn and Vn, plasma from donors who were genetically deficient in vWF, and serum. Few platelets adhered to the surfaces preadsorbed with serum, whereas depletion of Fn, Vn, or vWF from plasma did not decrease platelet adhesion significantly. Replenishment of exogenous Fg to serum before protein adsorption restored platelet adhesion to the surfaces, suggesting that Fg was the major plasma protein that mediated platelet adhesion. Also, we found that a surface density of adsorbed Fg far below the amount that usually adsorbs to synthetic surfaces was sufficient to support full-scale platelet adhesion.

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells.

Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) which recognize and bind FN. The interaction of bacteria with FN is believed to contribute significantly to the virulence of a number of microorganisms, including staphylococci and streptococci. Several FN-binding MSCRAMMs of staphylococci and streptococci exhibit a similar structural organization and mechanism of ligand recognition. The ligand-binding domain consists of tandem repeats of a approximately 45 amino acid long unit which bind to the 29-kDa N-terminal region of FN. The binding mechanism is unusual in that the repeat units are unstructured and appear to undergo a conformational change upon ligand binding. Apart from supporting bacterial adherence, FN is also involved in bacterial entry into non-phagocytic mammalian cells. A sandwich model has been proposed in which FN forms a molecular bridge between MSCRAMMs on the bacterial surface and integrins on the host cell. However, the precise mechanism of bacterial invasion and the roles of FN and integrins in this process have yet to be fully elucidated.

Analysis of fibronectin on human sperm.

The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = -0.68), motility (r = -0.70), and morphology (r = -0.63). This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm is primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Fibronectin and circulating immune complexes in Behçet's disease

Being a high-molecular-weight adhesive glycoprotein, fibronectin (Fn) is suggested to be a component of immune complexes and may participate in the clearance of immune complexes. In Behçet's disease (BD), a multisystem disorder of unknown etiology, immune complexes have been shown to be deposited in affected tissue during disease activity, suggesting an immune mechanism. This study investigates the relationship between Fn and circulating immune complexes (CIC) and evaluates the changes in the levels of Fn and CIC along with disease activity. In 63 patients (31 active, 32 inactive) with BD, plasma Fn and serum CIC, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and the third and fourth components of the complement system (C3, C4) were studied. The mean ESR, CRP, C3 and C4 levels of active BD patients were found to be significantly higher than those of the inactive BD patient group. Although the mean Fn and CIC levels of BD patients were not significantly different from those of the healthy control group, Fn and CIC values of active BD patients were significantly lower than in the inactive group. Moreover, no significant correlation was observed among Fn levels and ESR, CRP, C3, C4 and CIC levels in BD patients. The result of this study suggest that the variation in Fn concentration is independent of the acute-phase response. The lack of relationship between the CIC and Fn concentrations indicates that IC deposition in the vessel wall is independent of the CIC levels. In order to determine the exact roles of Fn and IC, further studies in tissue specimens are required.

Biomaterial-associated infection with Candida albicans in mice.

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (M phi). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).

Role of Fibronectin in Immune Glomerulonephritis

There is already a considerable amount of evidence suggesting that fibronectin (Fn) plays an important role in the pathogenic process in some forms of glomerulonephritis (GN). It has been postulated that Fn may participate in the progression or regression of glomerular diseases. The Fn is presented in the kidney as a normal component of the mesangium, and it is increased in the expanded mesangium in various forms of GN. This paper reports our efforts to investigate the role of Fn in plasma and kidney in patients with GN. Using monoclonal antibodies against human Fn in the ELISA and immunohistoperoxidase techniques to evaluate Fn, we investigated its quantity in connection with clinical state and morphological findings. We studied 93 patients with GN and 26 renal biopsies. The patients with active forms of mesangial proliferative, membranoproliferative and membranous GN showed increased plasma Fn, and the highest levels were in patients with nephrotic syndrome. Increased tissue Fn correlated with mesangial expansion and with IgG and C3 deposits. We speculate on possible mechanisms of the involvement of Fn in human chronic GN.

Fibronectin binding and neutrophil aggregation in burn injury.

Fibronectin (Fn) plays an important role in the adhesive function of many cells including neutrophils (PMN). We examined the hypothesis that activated PMN develop binding sites for fibronectin which allows for the aggregation of contiguous PMN. Because PMN adhesive function is altered in acute burn injury, we also investigated the role of Fn in the aggregation of PMN from subjects with acute thermal injury. The chemotactic peptide, n-formylmethionyl leucyl phenylalanine, induced rapid binding of radioiodinated plasma Fn to PMN. Significant binding of Fn was detected as early as 30 sec poststimulus and maximal binding occurred at 5 min. Fn binding was only partially reversible and nonsaturable. The chemotactic peptide induced aggregation and binding of Fn to PMN with similar kinetics, concentration dependence, temperature, and cation requirements. In burn patients, PMN demonstrated a significant decrease in chemotactic peptide-induced aggregation which was associated with decreased binding of Fn. Alterations in the binding of Fn to PMN may be responsible, in part, for diminished aggregation responses of PMN in the early stages of thermal injury.

Thrombin increases expression of fibronectin antigen on the platelet surface.

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.