mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD)

Abstract

Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.

Figure 1.

Phosphorylation of AKT/mTOR pathway proteins in adrenals of WT and AdKO mice. (A) Phosphorylated S6K1, rpS6, 4E-BP1 and AKT were detected in adrenal tissues from 12 to 14 month-old WT and AdKO mice. (B) Phosphorylation signal illustrated in A was quantified in five to six samples (values ± SEM). Values represent relative band density of the phosphorylated forms over total signal of the corresponding proteins, expressed as a percentage of the mean of WT. *P < 0.05. (C) Immunostaining for P-rpS6 in adrenal sections of vehicle-treated (Vhl) WT (a) and AdKO (b) compared with rapamycin-treated WT (c) and AdKO (d) mice. Dotted line delineates medullary (M) and cortical compartments, normal cortex (double headed arrow) and tumoral inner cortex (double dotted arrow) are indicated. Scale bars, 100 µm.

Figure 2.

Dexamethasone-induced apoptosis in adrenals of vehicle or rapamycin-treated WT and AdKO mice. (A) TUNEL staining of adrenal sections from dexamethasone/vehicle-treated WT and AdKO (Dex) compared with dexamethasone/rapamycin-treated WT and AdKO mice (Dex/Rapa). (B) The number of TUNEL-stained cells illustrated in A was quantified in four to seven individuals per condition (values ± SEM). Values represent the number of stained cells per adrenal section expressed as a percentage of the mean of WT. *P < 0.05. (C) Cleaved-caspase 3 staining in adrenal sections was also performed in four different treatment groups (vehicle, dexamethasone, rapamycin, dexamethasone/rapamycin) in both genotypes (WT and AdKO): the number of stained cells was quantified in four to eight individuals per condition (values ± SEM). Values represent the number of cleaved-caspase 3 stained cells per adrenal section expressed as a percentage of the mean of WT. *P < 0.05. Vhl, vehicle; Dex, dexamethasone; Rapa, Rapamycin. Scale bars, 100 µm.

Figure 3.

Phosphorylation of protein targets of PKA and AKT/mTOR signaling pathways in H295R human adrenocortical cells. (A) Phosphorylated mTOR, rpS6 and ATF1 were detected in cells treated for 15′ with various combinations of PKA activator 8-Br-cAMP, PKA inhibitor H89 and mTORC1 inhibitor rapamycin. (B) Phosphorylation signals of mTOR, rpS6 and ATF1 were quantified (values ± SEM). (C) Phosphorylation signals of mTOR, rpS6 and ATF1 in the presence of 0.1 mm of PKA activator 8-Br-cAMP were quantified after treatment by either PKA inhibitor H89 (5 µm) and/or mTORC1 inhibitor rapamycin (50 nm). (values ± SEM). Values in B and C represent relative band density over tubulin expressed as a percentage of the mean of the vehicle condition. Total mTOR, rpS6 and ATF1 protein signals were unaffected by these short-time treatments (not shown). n = 4; *P < 0.05. Vhl, vehicle; Rapa, rapamycin.

Figure 4.

Phosphorylation of protein targets of PKA and AKT/mTOR signalling pathways in murine ATC7 adrenocortical cells. (A) Phosphorylated mTOR, S6K1, rpS6 and CREB were detected in cells treated for 15′ with increasing concentrations of ACTH. (B) Phosphorylated AKT, mTOR, rpS6 and CREB were detected in cells treated for 2′ to 1 h with PKA hormonal activator ACTH or for 15′ with either the specific PKA activator 8-Br-cAMP or the PI3K/AKT/mTOR pathway hormonal activator insulin. (C) Phosphorylated mTOR, rpS6 and CREB were detected in cells treated in various combinations with ACTH, PKA inhibitor H89 and mTORC1 inhibitor rapamycin. (D) Corresponding phosphorylation signals of mTOR, rpS6 and CREB were quantified (values ± SEM). Values represent relative band density over tubulin expressed as a percentage of the mean of the control. Total mTOR, S6K1, rpS6 and CREB protein signals were unaffected by these short-time treatments (not shown). n = 4; *P < 0.05. Vhl, vehicle; 8Br, 8-Br-cAMP; Ins, insulin; Rapa, rapamycin.

Figure 5.

Apoptosis induction and BAD phosphorylation in murine ATC7 adrenocortical cells. (A) PARP and Caspase 3 cleavage and AKR1B7 levels were detected in ATC7 cells treated with PKA pathway activator forskolin (Fsk) and/or apoptosis inductor HA14-1 for 4 h. (B) PARP and Caspase 3 cleavage was quantified in response to HA14-1 in the presence or absence of forskolin (values ± SEM). Values represent relative band density over actin for cleaved-caspase 3 and relative cleaved protein over total protein for cleaved-PARP, expressed as a percentage of the mean of the control. n = 5; *P < 0.05. Vhl, vehicle. (C) BAD phosphorylation at Ser112 or Ser136 positions was detected in cells treated for 15′ with increasing concentrations of ACTH. (D) BAD phosphorylation at Ser112 or Ser136 positions was detected in cells treated for various amounts of time with the PKA pathway activator ACTH or for 15′ with either the specific PKA activator 8-Br-cAMP (8Br) or PI3K/AKT/mTOR pathway activator insulin (Ins). Total BAD and CREB protein signals were unaffected by these short-time treatments (not shown).

Figure 6.

Ser112 and Ser136 phosphorylation of BAD in adrenals from WT and AdKO mice. (A) P-rpS6, BAD, P-BAD Ser112 and P-BAD Ser136 were detected in adrenal tissues from ACTH-treated or from vehicle-treated WT mice (Vhl). (B) Results illustrated in A were quantified (values ± SEM). Values represent relative band density over actin expressed as a percentage of the mean of vehicle-treated mice for P-rpS6 and BAD, and relative band density over BAD expressed as a percentage of the mean of vehicle-treated mice for the two forms of P-BAD. n = 7. (C) BCL-XL, BAD, P-BAD Ser112 and P-BAD Ser136 were detected in adrenal tissues from 12 to 14 month-old WT and AdKO mice. (D) Results illustrated in C were quantified on five WT and six AdKO mice (values ± SEM). Values represent relative band density over actin expressed as a percentage of the mean of WT mice for BCL-XL and BAD, and relative band density over BAD expressed as a percentage of the mean of WT mice for the two forms of P-BAD. (E–G) P-BAD Ser112, P-BAD Ser136, P-rpS6 and P-AKT were detected in PPNAD human adrenal samples from patients carrying germline PRKAR1A-inactivating mutation (19) and counterstained with hematoxylin. *P < 0.05. Scale bars, 100 µm in magnification and 500 µm in entire adrenal.