Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development

Abstract

cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 ϩ EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1-100 M) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (ϩ2.36-fold, P Ͻ 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (Ͼ98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 Ϯ 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 ϩ EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC 50 ϳ0.01 ng/ml); EGF significantly augmented (P Ͻ 0.01) CCN2-induced tumor cell proliferation (EC50 ϭ 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (Ϫ12 Ϯ 3%, P Ͻ 0.05) and ERK1/2 phosphorylation (Ϫ34 Ϯ 5%, P Ͻ 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids (P Ͻ 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.connective tissue growth factor; epithelial growth factor AGENTS involved in regulating cell proliferation during the development of carcinoids and neuroendocrine (NE) tumors remain an elusive issue. Little information in this area is available since few models and NE cell lines exist to evaluate the problem. The rodent Mastomys develops gastric carcinoids both spontaneously (12-18 mo) and very rapidly (8 -12 wk) when acid suppression and hypergastrinemia are engendered (26, 34). In the stomach, the enterochromaffin-like (ECL) cell, which regulates acid secretion under the effect of both gastrin and PACAP (31, 40), transforms into a neoplastic phenotype when exposed to hypergastrinemia (34). Gastrin itself has no proliferative effect on ECL cells once neoplastic transformation has occurred and the ECL cells become gastrin autonomous (34). Nevertheless, proliferation continues even if the gastrin stimulus is withdrawn, indicating that an alternative proliferativ...

KRJ-I and BON Cell Lines: Defining an Appropriate Enterochromaffin Cell Neuroendocrine Tumor Model

“…In contrast, TGFβ stimulated KRJ-I proliferation, consistent with our previous observations of an altered-TGFβ-mediated regulatory pathway in SI-NETs [65, 66]. Further differences in growth regulation were noted by the observation that CTGF, a proliferative and profibrotic factor synthesized by GI-NETs [67, 70], stimulated KRJ-I but not BON cell proliferation. Fibrosis, identified in 40–60% of SI-NETs [1], is not a usual feature of pancreatic NETs.…”

Connective tissue growth factor expression in endocrine tumors is associated with high stromal expression of α-smooth muscle actin

“…CTGF has been shown to stimulate proliferation of cells isolated from a ECL cell tumor but not of ECL cells isolated from normal mucosa (35). Ileal carcinoids display generally low proliferation index (36), and CTGF expression was not associated with tumor proliferation index in this study.…”

Gastric NeuroendocrineTumours

“…[19]. In the Mastomys murine model, rodents have a polymorphism in the CCK-2 receptor gene, leading to constitutive activation and are, therefore, predisposed to the development of gastric NETs [20], with 50-80% developing tumours within 2 years [21]. This process is accelerated in the presence of H. pyloriinduced hypergastrinaemia [22].…”

“…Microarray technology has been used to identify altered gene expression signatures in type III compared with type I and II gastric NETs [20]. Of 270 genes differentially altered, CgA [30], MAGE-D2 (adhesin), MTA1 (histone deacetlyase regulator) [31] and CCN2 (growth factor) [20] can differentiate between type III and I tumours.…”