Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor

Abstract

Existing evidence indicates that, in addition to its neuroendocrine action, growth hormone-releasing hormone (GHRH) acts directly on several nonpituitary tissues, especially neoplasms, and stimulates cell proliferation. We have recently reported that a splice variant of the receptor (SV1) is expressed in various normal tissues and particularly in tumor tissues, producing mitogenic effects on GHRH binding. By using HEC-1A human endometrial carcinoma cells, which express endogenous SV1, we show that, in addition to its ability to mediate the mitogenic effects of GHRH, SV1 also possesses relatively high intrinsic, ligand-independent activity. By using an antisense RNA-based approach we found that SV1 ablation reduces the efficacy of colony formation and the rate of cell proliferation of HEC-1A cells in the absence of exogenous GHRH, and decreases their sensitivity to GHRH when the neurohormone is added to the culture media. This ligand-independent stimulation of cell proliferation appears to be a characteristic property of the truncated form of the receptor, because the expression of SV1 and not of the full-length GHRH receptor stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH. Evaluation of 21 specimens of human primary endometrial carcinoma for expression of SV1 by immunohistochemistry indicated that in contrast to the GHRH receptor, which is absent, SV1 is expressed in approximately 43% of the specimens. These findings indicate that SV1 can operate in a ligand-independent as well as a ligand-dependent manner. The overexpression of this form of GHRH receptor may be associated with carcinogenesis.

Fig. 1.

SV1 staining in HEC-1A endometrial carcinoma cells stably transfected with plasmids pcDNA3 (a) or pcDNA3-SV1(as) (b–d). Arrows in c and d indicate mitotic figures in Hec-1A cells, which show anti-SV1 immunoreactivity despite the presence of pcDNA3-SV1(as). Cells were counterstained with hematoxylin.

Fig. 2.

SV1 ablation by expression of pcDNA3-SV1(as) suppresses the rate of cell proliferation of HEC-1A endometrial carcinoma cells in both the absence and the presence of GHRH. Growth rate of Hec-1A cells transfected with plasmids pcDNA3 or pcDNA3-SV1(as), cultured in the absence of exogenously added GHRH in media containing 10% FBS (a), or in media containing 1% FBS with the addition of GHRH(1–29)NH₂ at the concentrations indicated (b). Average values of experiments performed in triplicate are shown. *, P < 0.05 vs. cells transfected with the vector pcDNA3-SV1(as).

Fig. 3.

Suppression of colony formation in HEC-1A endometrial carcinoma cells by pcDNA3-SV1(as). (a) Representative Petri dishes of cells transfected with pcDNA3 or pcDNA3-SV1(as). The experiment was repeated in triplicate and average values are shown in b. *, P < 0.05 vs. controls.

Fig. 4.

Effect of SV1 and GHRHR on the proliferation of 3T3 fibroblasts cultured in the absence or the presence of 0.1 and 1.0 μM hGHRH(1–29)NH₂. Graphs show absolute cell number (a) or percent cell number vs. corresponding controls (cultured in the absence of GHRH) (b). Both SV1 and GHRHR mediate the mitogenic effects of GHRH, but only SV1 stimulates cell proliferation in the absence of exogenously added ligand. Average values of experiments performed in triplicate are shown. *, P < 0.05 vs. controls (cells transfected with the same plasmid but cultured in the absence of GHRH); **, P < 0.05 vs. cells transfected with pcDNA3.

Fig. 5.

Anti-SV1 immunoreactivity in primary endometrial carcinoma of the endometrioid (a–c) or serous-papillary (d) type. Cytoplasmic staining is apparent in the epithelial cells of the glands, whereas stromal cells are negative. Arrows indicate areas of immunopositivity. Specimens were counterstained with hematoxylin. (×40.) This study was performed in the cytopathology laboratory of Aretaieion University Hospital in Athens.