Mastomys enterochromaffin-like (ECL) 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 +
EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor
PD-98059 (0.1-100 microM) 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 (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in
tumor ECL cell fractions (41.3 +/- 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 +
EGF stimulated normal ECL cell proliferation. CCN2 stimulated
tumor proliferation (EC50 approximately 0.01 ng/ml);
EGF significantly augmented (P < 0.01) CCN2-induced
tumor cell proliferation (EC50 = 20 pg/ml).
PD-98059 inhibited CCN2-induced proliferation (-12 +/- 3%, P < 0.05) and ERK1/2 phosphorylation (-34 +/- 5%, P < 0.05) in
tumor cells. In clinical samples, both CCN2 transcript and
protein were elevated in
gastrin-autonomous
carcinoids (P < 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.