Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A

Abstract

Objective: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD.

Design: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro.

Results: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation.

Conclusions: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.

Fig. 1. IGF axis/synaptophysin mRNA ratios in PPNAD

Expression of IGF-II, IGF-I, IGFBP-3 and IGFBP-2 were measured by Real-time quantitative RT-PCR. For each gene, the results are displayed according to PRKAR1A mutation status (positive,+; negative,−). Each white box depicts the message ratio of the IGF axis member to synaptophysin for an individual patient, expressed as fold difference from the ratio measured in the normal control RNA. Each box is the average of quadruplicate raw measures, each normalized to PGK. To facilitate visualization of any overlap between the patient groups, the normal control value of 1 is indicated by the black horizontal bar. Ratio values for each IGF axis member/synaptophysin significantly differed between patient groups, as shown.

Fig. 2. Representative IGFBP-2 immunohistochemistry in PPNAD

(A) PRKAR1A mutation-positive PPNAD section labeled with 1:50 rabbit anti-human IGFBP-2 antibody and visualized by the Envision Plus method using DAB color substrate and Harris’ haematoxylin counter-staining (original magnification X100). (B) Adjacent section to that in Panel A, simultaneously processed the same way except using rabbit anti-human β amyloid precursor protein antibody as negative control primary antibody (original magnification X100). (C) and (D) PRKAR1A mutation-negative PPNAD sections processed the same as (A) and (B), respectively (both original magnification X100).

Fig. 3. Protein kinase A inhibitors increase IGFBP-2 mRNA in NCI-H295R cells

Real-time quantitative RT-PCR of IGFBP-2 message, normalized to PGK, in NCI-H295R adrenocortical cells treated with PKA modulators for 6 hr. Each bar depicts the mean ± SD of duplicate values expressed as fold-difference in IGFBP-2 mRNA level relative to untreated NCI-H295R cells plated at equal densities. The modulators were added singly or in combination at concentrations of 5 μM forskolin, 50 nM H89 and 10 μg/ml PKI. Difference among all treatments was highly significant (P<0.001, ANOVA).

Fig. 4. Time course of mRNA changes in NCI-H295R cells treated with 50 nM H89

Real-time quantitative RT-PCR of IGFBP-2 (black diamonds), IGFBP-3 (gray triangles) and IGF-II (white circles) message levels was performed, normalized to PGK and expressed as fold change relative to control, untreated NCI-H295R cells plated at equal densities. Each point represents the mean ± SEM of 3 replications in each of two independent experiments.

Fig. 5. IGFBP-2 inhibition decreases NCI-H295R cell proliferation

NCI-H295R cells in culture were treated with anti-human IGFBP-2 antibody or anti-human CD8 antibody at1.2 μg/ml. After 72 hr, cellular proliferation was measured by colorimetric assay and compared to untreated NCI-H295R cells. Each bar represents the mean of triplicate wells.