Expression of Steroidogenic Enzymes and Transcription Factors in GIP-stimulated GIPR-expressing H295R Adrenocortical Cancer Cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 1-36-Adrenal Incidentaloma & Carcinoma
Clinical
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-10
Hiroko Fujii*1, Kosuke Uchida1, Mimi Tamamori-Adachi2, Yuji Tanaka1 and Tomoki Okazaki2
1National Defense Medical College, Tokorozawa Saitama, Japan, 2Teikyo Univ Schl of Med, Tokyo, Japan
The glucose-dependent insulinotrophic polypeptide (GIP) promotes postprandial insulin secretion. GIP is also known to stimulate glucocorticoid secretion in some exceptional morbidity such as food-dependent Cushing syndrome (CS). However, the pathogenesis underlying this disease entity is poorly understood. Here, we investigated whether GIP directly induces GIPR-mediated glucocorticoid secretion from the resected adrenal tumor of a food-dependent CS patient. In the primary cultured cells obtained from the patient’s adenoma, we found GIP stimulated cortisol secretion in a dose-dependent manner. The level of GIPR mRNA was remarkably increased, 75-fold compared with the control. Next, we transiently expressed flag-tagged human GIPR in H295R human adrenocortical cancer cells and stimulated the cells with GIP. Surprisingly, there are significant differences in level and pattern of GIPR expression between N-terminal and C-terminal flag-tagged GIPR. Western blot and immunofluorescence using anti-flag and -GIPR antibodies revealed that GIPR expression in N-terminal flag-tagged GIPR-expressing cells (flag-hGIPR-H295R) was much lower than that in C-terminal flag-tagged GIPR-expressing cells (hGIPR-flag-H295R). These results suggest that unoccupied N-terminus of GIPR might be required for some modifications for stable expression. Subsequent RT-qPCR experiments using hGIPR-flag-H295R showed that gene expression of NR5A1 [steroidogenic factor 1 (SF1)], an adrenal transcription factor known as a molecule downstream of ACTH receptor signaling pathway, was up-regulated. Further, mRNAs for steroidogenic enzymes including CYP11A1, HSD3b2 and CYP21 were significantly up-regulated compared with GIP-stimulated non-transfected H295R cells. Interestingly, we observed that phospho-ERK-positive cells were increased in GIP-stimulated hGIPR-flag-H295R. Our results indicate that GIP directly activates steroidogenesis with concomitant phosphorylation of ERK via GIPR in hGIPR-flag-H295R. Further studies for other important molecules of downstream signaling and regulation of glucocorticoid secretion are now in progress in our laboratory.

Nothing to Disclose: HF, KU, MT, YT, TO

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