Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase‐2 (COX‐2) expression

Abstract

Tobacco use is the most important risk factor for the development of lung carcinoma. One characteristic shared by tobacco-related lung diseases is altered lung connective tissue content and composition. In particular, tobacco results in increased expression of fibronectin (FN), a matrix glycoprotein implicated in lung development, injury and repair and in tumor cell invasion. We hypothesized that excessive deposition of FN in lung might promote lung carcinoma cell proliferation. Consistent with this hypothesis, we found that FN stimulated human lung carcinoma cell proliferation and diminished apoptosis in vitro, and that this effect was mediated through the integrin ␣5␤1 and associated with upregulation of cyclooxygenase-2 (COX-2) mRNA and protein expression, and increased prostaglandin E 2 (PGE 2 ) biosynthesis. Key words: fibronectin; COX-2; human lung carcinoma cells; cAMP response element; C/EBP; NF-IL6Lung carcinoma is one of the most common malignant tumors in the world and is the leading cause of carcinoma death in men and women in the United States. 1 Despite recent advances in understanding the molecular biology of lung carcinoma and the introduction of multiple new chemotherapeutic agents for its treatment, its dismal 5-year survival rate (Ͻ 15%) has not changed substantially. 2 Tobacco use is the most important risk factor for the development of lung carcinoma. 3 Also, patients with emphysema, idiopathic pulmonary fibrosis and other lung diseases characterized by dramatic alterations in lung architecture are at increased risk of developing lung carcinoma. 3 One characteristic shared by tobacco-related and other chronic lung diseases is altered lung connective tissue content and composition. In particular, they are associated with increased expression and deposition in lung of fibronectin (FN), a 250 Kd heterodimeric extracellular matrix glycoprotein implicated in physiologic events during embryogenesis, angiogenesis, thrombosis and inflammation. 4,5 FN has also been implicated in carcinogenesis. Studies of solid human tumors have shown that among the early signs of malignant transformation is the fragmentation of pericellular FN that is concomitant with an increase in FN deposition in the peritumoral stroma. 6 FN has also been shown to be expressed in several carcinoma cell types. 7-9 Nanki et al. 10 showed that oncofetal FN is expressed in lung carcinoma cells, especially in non small cell lung carcinoma (NSCLC) cell lines. Jakowlew et al. 11 reported that treatment of NSCLC cells with TGF-␤1 resulted in a persistent increase in protein and mRNA expression for FN. The adhesion of lung carcinoma cells to FN enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic agents. 12 Despite the above, a link between FN and lung carcinoma cell growth has not been firmly established. Furthermore, the mechanisms by which FN exerts its effects on lung tumors remain unelucidated.Many of the biologic effects of FN are mediated via the integrin receptor ␣5␤1. 13,14 This hetero...

Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells

“…Cultures and Chemicals-The human non-small cell lung carcinoma cell lines H1838 and H2106 were obtained from American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 g of amphotericin (complete medium) (15). Afterward, cells were harvested and replated in serum-free medium on FN-coated culture plates for experiments described later.…”

“…To amplify the 590-bp MMP-9 and 200-bp GAPDH cDNA fragments, the sequences of PCR primers (Sigma Genosys, Woodlands, Texas) were as follows: for MMP-9 sense, 5Ј-CACTGTCCACCCCT-CAGAGC, and antisense, 5Ј-GCCACTTGTCGGCGATA-AGG; and for GAPDH sense, 5Ј-CCATGGAGAAGGCTG-GGG, and antisense, 5Ј-CAAAGTTGTCATGGATGACC, according to published data (15,17). The RT-PCR was carried out as previously described (15).…”

“…To amplify the 590-bp MMP-9 and 200-bp GAPDH cDNA fragments, the sequences of PCR primers (Sigma Genosys, Woodlands, Texas) were as follows: for MMP-9 sense, 5Ј-CACTGTCCACCCCT-CAGAGC, and antisense, 5Ј-GCCACTTGTCGGCGATA-AGG; and for GAPDH sense, 5Ј-CCATGGAGAAGGCTG-GGG, and antisense, 5Ј-CAAAGTTGTCATGGATGACC, according to published data (15,17). The RT-PCR was carried out as previously described (15). The samples were first denatured at 95°C for 30 s, followed by 32 PCR cycles, each with temperature variations as follows: 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The last cycle was followed by an additional extension incubation of 7 min at 72°C.…”

Integrin-mediated Adhesion and Soluble Ligand Binding Stabilize COX-2 Protein Levels in Endothelial Cells by Inducing Expression and Preventing Degradation

“…In view of our results, one should consider the possibility that COX-2 expression observed in these or other models may require the integration of signaling events that are originating from the ECM, which are transduced by integrins (7). Recent reports have implicated adhesion to purified ECM proteins such as vitronectin (71), fibronectin (72), collagen I (73), or a cell-derived matrix (74) in promoting COX-2 expression and prostaglandin production in various cell types. In one report, mAb LM609, an anti-␣ V ␤ 3 mAb that blocks ␣ V ␤ 3 -mediated endothelial cell adhesion and angiogenesis (75), or LIBS 6, an anti-␣ V ␤ 3 mAb specific for activated ␣ V ␤ 3 (76), but not other anti-␣ V or anti-␤ 3 mAbs were shown to stimulate prostaglandin production, induce endothelial cell proliferation, and inhibit in vitro angiogenesis in a COX-1-and COX-2-dependent and COX-independent manner (71).…”

Novel Role of Pin1 Induction in Type II Collagen-Mediated Rheumatoid Arthritis

“…4D). c-Jun/AP-1 phosphorylation and expression are controlled by MAPK (36), which also regulates COX-2 expression (37,38). To study whether MAPK pathways are activated in Pin1 cells, we measured the phosphorylated form of each MAPK.…”