*2.2. Regulation of CRY1, CRY2, HSD3B1, and HSD3B2 Expression in HAC15 Cells*

To investigate the potential roles of *CRY1* and *CRY2* genes in adrenal cell function and aldosterone production, we used HAC15 adrenocortical cells as an in vitro model. *CRY1* and *CRY2* genes were transcribed in HAC15 cells to a level comparable to that of a pooled set of APA samples, while *HSD3B2* was 35-fold (25–61, *p* < 0.001) more expressed than *HSD3B1*.

**Figure 2.** (**A**–**C**) Representative immunohistochemistry staining for *CRY1* in APAs. (**D**–**F**) Representative immunohistochemistry staining for *CRY2* in APAs. Magnifications in (**B**,**C**), and in (**E**,**F**) correspond to the boxed sections in (**A**,**D**), respectively.

**Figure 3.** Representative immunohistochemistry staining for *HSD3B1* (**A**,**B**) and *HSD3B2* (**C**,**D**) in APAs, according to the cellular composition.

HAC15 cells, which were previously reported to express the AngII type 1 receptor [16], were stimulated with AngII (±1 μM irbesartan) or forskolin for 6, 12, and 24 h, and were then harvested for RNA extraction and gene-expression studies.

As expected, treatment with AngII (100 nM) resulted in a significant increase in *CYP11B2* expression at 12 h (68 ± 20-fold over basal, *p* < 0.001).

Treatment with AngII significantly increased the expression of *CRY1* mRNA within 6 h (1.7 ± 0.25-fold, *p* < 0.001). Following a peak in expression, the levels of *CRY1* mRNA returned to basal levels after 12 h of AngII treatment (Figure 4A). With respect to *CRY2* expression, stimulation with AngII resulted in a significant downregulation (0.6 ± 0.1-fold, *p* < 0.001) at 12 h (Figure 4B), followed by a return to basal levels at 24 h.

Treatment with forskolin, which mimics adrenocorticotropin (ACTH)-mediated elevation of intracellular cyclic adenosine monophosphate (cAMP), resulted in a downregulation of *CRY1* at 6, 12, and 24 h, and a downregulation of *CRY2* and at 12 and 24 h (Figure 4A,B).

Additionally, AngII and forskolin treatment positively regulated the transcription of both *HSD3B1* and *HSD3B2*. Following a 6-h stimulation with AngII, we observed that *HSD3B1* was 3.2 ± 2.4-fold (*p* = 0.035) more expressed when compared with basal conditions, while the maximum upregulation of *HSD3B2* was observed at 12 h (3.7 ± 0.4-fold, *p* = 0.002) (Figure 4C,D). Similarly, forskolin treatment induced a significant upregulation of both *HSD3B1*, with a peak at 6 h, and *HSD3B2*, with a peak at 12 h, (2.1 ± 1.2-fold and 5.1 ± 2.1-fold, *p* = 0.03 and *p* = 0.001, respectively).

Consistently, after 6 h of AngII stimulation, we detected a 1.5 ± 0.2-fold upregulation of *PER1*, that acts as a negative regulator of the core clock together with *CRY*, followed by a 42% reduction at 12 h, when compared with basal levels (Figure 4E).

Pre-treatment with irbersartan (1 μM) reverted the effects of AngII on *PER1*, *CRY1*, and *CRY2* expression (Figure 4F–H), indicating that the observed effects on gene expression were mediated by the activation of the AngII type 1 receptor.
