ORF13-05 - Functional Adrenal Cortex Growth Hormone-Releasing Hormone Receptor (GHRHR) Expressed in Vitro and in Vivo

Introduction: Growth hormone–releasing hormone (GHRH) is classically recognized for its role in stimulating pituitary GH production. However, accumulating evidence indicates that GHRH and its receptor (GHRHR) are also expressed in peripheral tissues where they exert direct growth-regulatory and tissue-specific functions. However, expression and function of adrenal GHRHR is unclear.

Methods: Murine adrenal and pituitary glands and human HAC-15 adrenal cell-line and normal and tumoral human adrenals were used for this study. GHRHR, StAR, CYP11A1, pERK and pPKA antibodies were used in Western blots. Specific GHRHR, CYP11B1, and Ki67 were used for IHC. Cell proliferation was assessed by WST-1 Cell Proliferation Assay. DNA damage markers and P21 were analyzed by Flow Cytometry.

Results: GHRHR expression was detected in both murine and human adrenal glands, as well as in HAC-15 cells as demonstrated by both Western blot and IHC. GHRHR was abundantly expressed in the adrenal cortex but not in the medulla, and co-localized with CYP11B1 expression.
In HAC-15 cells acute 5-minute treatment with the long-acting GHRH analog CJC-1295 induced RAFbSer445 (p = 0.0063) and ERKThr202/Tyr204 (p = < 0.0001) phosphorylation, and reduced PKATHR197 (p = 0.0188) phosphorylation as compared to controls. Although extended 24-hour treatment with CJC-1295 resulted in marked reduction of GHRHR expression (p = 0.0091), cell proliferation increased over 24 (p = < 0.0001) to 72 hours (p = < 0.0001) along with increased p21 (p= < 0.0001) as well as pATM (p= 0.0001) and γH2AX (p=0.0002) levels.
In vivo studies showed that sustained 2-month treatment of adult C57BL/6 male mice for with CJC-1296 (10ng/kg) resulted in decreased phosphorylated ERK1/2 Thr202/185, Tyr204/187 (42kd, 44kd p=0.0068, 0.0072 respectively), StAR (p = 0.0032), EPAC1 (p = 0.0012), and RAS (p = 0.0007), with a trend toward reduced CYP11A1 expression. Notably, the number of Ki67-positive cells increased compared with vehicle-treated controls (p = 0.0002).
Analysis of surgically resected human adrenal tumor sections demonstrated phenotype–dependent GHRHR expression levels, with adrenal adenomas causing Cushing syndrome showing highest GHHR expression levels (30–52 %; n = 3), adrenal cortical carcinoma (9% and 13%; n = 2) and aldosteronomas (10% and 17%; n = 2) exhibiting low-moderate heterogeneous staining patterns as compared to 60% expression in normal adrenal cortex. Pheochromocytomas expressed lowest GHRHR expression (8% n =1), with both GHRHR-positive and-negative areas.

Conclusion: We show that functional GHRHR is expressed in both human and murine adrenal cortex and is associated with increased proliferation and modulation of the DNA damage pathway. The results indicate that the role of GHRHR in adrenal physiology and pathology warrants further investigation.