TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR.

BACKGROUND: Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α. METHODS: 18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10 mm cell culture dishes and were used from passages 10-14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 µM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors. RESULTS: Exposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR. CONCLUSION: Synergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.

Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-α and bradykinin in human colonic myofibroblasts.

Stromal myofibroblasts regulate extracellular matrix components through the secretion of matrix metalloproteinases such as MMP-3. Both myofibroblasts and MMP-3 have been implicated in colonic inflammation and cancer but the regulatory signaling mechanism(s) are unknown. Exposure of the human colonic myofibroblast cell line 18Co to TNF-α and bradykinin induced synergistic MMP-3 mRNA and protein expression, which were blocked by the preferential PKC inhibitors GF109203X and Go6983 and by the MEK inhibitor U0126. Transfection with siRNA targeting PKD1, a known downstream target of both bradykinin and PKC, completely inhibited MMP-3 mRNA and protein expression. Our results imply that TNF-α and bradykinin amplify MMP-3 expression at a transcriptional level through a signaling cascade involving PKC, PKD1, and MEK. PKD1 plays a critical role in the expression of MMP-3 in human colonic myofibroblasts, and may contribute to the pathophysiology underlying colitis-associated cancer.

Bacteriophage T4 nanoparticle capsid surface SOC and HOC bipartite display with enhanced classical swine fever virus immunogenicity: a powerful immunological approach.

The phage T4 HOC, SOC bipartite display system is attractive for the expression of cDNA and display of peptides or proteins at high copy numbers on the phage capsid surface. Until recently, using T4 phage vector to display large foreign molecular immunogens resulted only from either an SOC or HOC single site. In this report, the main advantages of the phage T4 system over other display technologies are substantiated by using the phage T4 SOC, HOC dual site display vector T4-Zh(-) to express: (1) on the SOC site, the classical swine fever virus (CSFV) major antigenic determinant cluster mE2 (123 amino acid, aa) through gene fusion to the SOC gene C-terminus of T4 genome, and (2) on the HOC site, full-length CSFV primary antigen E2 (371 aa) through a co-transformed plasmid, hence leading to a simultaneous display of both proteins on the T4 capsid surface. The immunogenicities of these constructs were measured by ID-ELISA, dot-ELISA, Western blotting, and immunogenic response in mice including humoral and cellular immunity tests. The immunological efficiencies both in vitro and in mice of phage T4 with both single site and dual site displays, as well as conventional Escherichia coli plasmid expression, were evaluated. The animal immune response data showed that the antibody titers elicited by the T4 phage-CSFV recombinants were significantly higher than those obtained by E. coli plasmid expression, and the unpurified double site display T4 phage particles were around two times higher than either single site display or plasmid expression while being at lower phage concentrations than the single site phages. The immunogens were effective in the absence of eukaryotic protein modifications. Therefore, the phage T4 dual site display emerges as a powerful method with an enhanced immune response in animals for research and development of immunological products.

Primary Myofibroblasts Maintain Short-Term Viability following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x10(5) (n = 14), 1x10(6) (n = 9), or 5x10(6) cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

Isolation of primary myofibroblasts from mouse and human colon tissue.

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

White tea extract induces apoptosis in non-small cell lung cancer cells: the role of peroxisome proliferator-activated receptor-{gamma} and 15-lipoxygenases.

Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non-small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-gamma with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-gamma activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-gamma activation and upregulation of PPAR-gamma mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non-small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-gamma and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer.

Recruitment strategies for a lung cancer chemoprevention trial involving ex-smokers.

The ability to recruit qualified subjects who are willing to adhere to the study protocol in clinical trials is an essential component of translational research. Such tasks can be particularly challenging for chemoprevention studies when the targeted study population is healthy, at risk individuals who do not have signs or symptoms of the disease, and the study participation involves complex scheduling and invasive procedures such as bronchoscopy. In this report, we describe the recruitment process and evaluated the effectiveness of various recruitment strategies utilized in our National Cancer Institute sponsored lung cancer chemoprevention study with celecoxib. Heavy ex-smokers were recruited into the study through various methods such as radio advertisements, print media, mass mailings, flyers, internet postings and others. The number of inquiries, on-site screenees and randomization generated by each method determined the efficacy of that recruitment strategy. We prescreened 4470 individuals, invited 323 people for on-site screening and randomized 137 subjects. Radio advertisements (ads) generated the most inquiries (71.1%), followed by internet posting (11.8%), print media (6.0%), posted and racked flyers (4.4%), mass mailings (2.7%) and other strategies such as referrals from friends or family members or health care providers (2.3%). Radio ads, although costly, yielded the most subjects for on-site screening and randomization. Moreover, among the various types of radio stations, news radio stations were by far the most successful. Our results suggest that advertising on news radio is a highly effective recruitment method for successful accrual of ex-smokers into lung cancer chemoprevention trials.

Transcriptional regulation of murine Slc22a1 (Oct1) by peroxisome proliferator agonist receptor-alpha and -gamma.

The transport and metabolism of organic cationic endobiotics, nutrients, and drugs are essential hepatic functions. Slc22A1 is the basolateral liver transporter mediating the uptake of organic cations; however, little is known about the regulation of this transport protein. Peroxisome proliferator agonist receptor (PPAR)-alpha and -gamma agonists are commonly used agents that regulate many hepatocellular transport functions. Thus the purpose of this study was to examine the effects of PPAR agonists on the hepatic regulation and function of Slc22a1. Mice and H35 cells were administered PPAR-alpha and -gamma agonists, and the effect on Slc22a1 gene expression was measured. We subsequently cloned the Slc22a1 promoter and employed chimeric constructs to assay Slc22a1 gene transcription. The effects of PPAR-agonist treatment on organic cation uptake was also assayed. Slc22a1 expression was increased by PPAR-alpha and -gamma agonist treatment in both murine livers and H35 cells. Gene expression in H35 cells was further increased following transfection with expression vectors of PPAR transcription factors and PPAR agonist treatment. We cloned the promoter region of Slc22a1 and identified a PPAR-response element, and transfections with chimeric Slc22a1; promoter-reporter gene constructs demonstrate that the increased gene expression was transcriptionally regulated. Functional assays confirmed that cells treated with PPAR agonists displayed significant increases in organic cation uptake. PPAR-alpha and -gamma agonists transcriptionally increase Slc22a1 gene expression, and the increased Slc22a1 expression results in enhanced cellular organic cation uptake. These studies may have implication for the uptake of organic cationic drugs and for lipid metabolism.