Wenxian Nie

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.

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.

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.

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.

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 1x105 (n = 14), 1x106 (n = 9), or 5x106 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.

Sa1711 Primary Myofibroblasts Maintain Short-Term Viability Following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy

Backround: Myofibroblasts are a subpopulation of stromal cells that are believed to play an important regulatory role in inflammation and cancer in the GI tract. While the inflammatory cell signaling mechanisms that regulate myofibroblast function are being elucidated in vitro, few in vivo models exist to directly study the impact of myofibroblast signal modulation on the overlying epithelium. We report our early experience with a novel technique utilizing murine colonoscopy that may allow for the real-time in vivo study of myofibroblast-epithelial cell interactions in their natural tissue environment of the colon. Methods: Primary colonic myofibroblasts (MFB) were harvested from adult FVB mice and were grown in cell culture. MFB were then suspended in serum-free media (1x103 cells/50μL, 1x104 cells/50μL), mixed with Spot® ink, and were then re-implanted endoscopically in anesthetized immunecompetent syngeneic mice by submucosal injection approximately 4 cm from the anal verge by utilizing small animal colonoscopy (Karl Storz Coloview miniendoscopic system). Mice were put back in their housing and were fed a normal diet. A repeat colonoscopy was performed on post-procedure days 2 and 7. Mice were sacrificed on day 7 and histologic analysis was performed. Results: Submucosal injection of primary myofibroblasts is technically feasible, visualized endoscopically by the appearance of a transient mucosal wheal, and confirmed histologically. Follow up colonoscopy on post-procedure day 2 confirmed that the injection site can be reliably identified and the overlying mucosa appeared intact. Mice tolerate multiple endoscopic procedures and multiple submucosal injections without obvious sequelae. Colonoscopic findings on day 7 were notable for an intact-appearing mucosa with an associated bump at the inked site. Mice were sacrificed on day 7 and histologic analysis suggests that the injected myofibroblasts maintain viability, and do not illicit an inflammatory response or disrupt the normal histologic architecture of the colon wall. Conclusions: Submucosal injection of primary myofibroblasts using murine colonoscopy is technically feasible and well tolerated in mice. The injection site can be easily re-identified, serially evaluated, and directly compared to adjacent tissue under the same experimental conditions. Submucosally-injected primary myofibroblasts maintain short-term viability with minimal disruption to the normal colon architecture of an immune-competent, syngeneic mouse. Successful development of this technique may create a novel platform for the in vivo study of stromal-epithelial cell interactions that is applicable to any existing mouse model.

TNF-α Potentiates Lysophosphatidic Acid-Induced COX-2 Expression via the LPA1 Receptor, GIα and PKD in Human Colonic Myofibroblasts

on the contribution of M1R to barrier function. Aim: To determine the contribution of M1R and M3R to intestinal barrier function. Methods: Muscle-free sections of ilea from wild-type (WT), M1R-deficient (M1R) or M3R-deficient (M3R) mice were placed inmicrosnapwells to determine transepithelial electrical resistance (TEER) over 120 minutes. The mRNA expression of cytokines and mAchRs was assessed in full thickness sections of ilea using quantitative real-time PCR. Results: When compared to WT (31.6±4.5 Ω●cm2), TEER was lower in M1R (25.4±2.1 Ω●cm2) and M3R (19.9±2.0 Ω●cm2) mice indicating increased mucosal permeability. This was associated with upregulation of proinflammatory cytokines IFN-γ, IL-17a, and TNF-α (Table 1). Expression of M1R in M3R mice and of M3R in M1R mice was unchanged from WT indicating no compensatory upregulation of other epithelially expressed mAchRs in single mAchR-deficient mice. Conclusions: These data indicate that both M1R and M3R play a physiological role in maintaining ileal mucosal barrier function as deficiency of M1R or M3R is associated with impaired barrier function. Ach is a major enteric neurotransmitter. These data support a role for Ach release from nerves acting at mAchRs on epithelial cells, consistent with the reported role of enteric nerves in the maintenance of barrier function. Impaired barrier function is associated with increased expression of Th1/Th17 cytokines. This constitutively “leaky gut” is a feature of autoimmune diseases such as celiac disease and inflammatory bowel disease. Modulation of mAchR activity may be a therapeutic target in derangements of intestinal permeability in these and similar pathologies. Table 1: Gene expression of proinflammatory cytokines in WT, M1R, and M3R mice (fold change relative to WT).