Saulo J.A. Felizola

Adrenal CYP11B1/2 expression in primary aldosteronism: Immunohistochemical analysis using novel monoclonal antibodies

CYP11B1 and CYP11B2 play pivotal roles in adrenocorticosteroids synthesis. We performed semi-quantitative immunohistochemical analysis of these proteins in adrenals from patients with primary aldosteronism using novel monoclonal antibodies. Clusters of cortical cells positive for CYP11B2 were detected in the zona glomerulosa (ZG) of normal adrenal gland (NA), idiopathic hyperaldosteronism (IHA) and the adjacent adrenal of aldosterone-producing adenoma (APA). In APA, heterogenous immunolocalization of CYP11B2 and diffuse immunoreactivity of CYP11B1 were detected in tumor cells, respectively. The relative immunoreactivity of CYP11B2 in the ZG of adjacent adrenal of APA was significantly lower than that of NA, IHA and APA tumor cells, suggestive of suppressed aldosterone biosynthesis in these cells. These findings did indicate the regulatory mechanisms of aldosterone biosynthesis were different between normal/hyperplastic and neoplastic aldosterone-producing cells in human adrenals. CYP11B2 immunoreactivity in the ZG could also serve as a potential immunohistochemical marker differentiating morphologically hyperplastic ZG of IHA and APA adjacent adrenal.

Different Expression of 11β-Hydroxylase and Aldosterone Synthase Between Aldosterone-Producing Microadenomas and Macroadenomas

Aldosterone-producing adenoma is a major subtype of primary aldosteronism. The number of cases of these adenomas, which are below the detection limit of computed tomography but diagnosed by adrenal venous sampling, has recently been increasing. However, the pathophysiology of these adenomas, especially those manifesting clinically overt hyperaldosteronism despite their small size, remains unknown. Therefore, we examined the correlation between tumor size and the status of intratumoral steroidogenic enzymes involved in aldosterone biosynthesis using immunohistochemistry. Forty patients with surgically proven aldosterone-producing adenomas were retrospectively studied. Multidetector computed tomography, adrenal venous sampling, and laparoscopic adrenalectomy were performed in all of the patients studied. The tumor area at the maximum diameter of the sections was precisely measured by ImageJ software. The status of the steroidogenic enzymes was immunohistochemically analyzed, and the findings were evaluated according to the H-score system, based on both the number of immunopositive cells and relative immunointensity. Adrenal masses were not detected by computed tomography in 20 patients. Blood pressure, plasma aldosterone concentration, urinary aldosterone excretion, and the number of antihypertensive agents also decreased significantly after the surgery in these patients, as well as in the patients with adenomas detectable by computed tomography. Maximum tumor area obtained in the specimens was significantly correlated with preoperative plasma aldosterone concentration, urinary aldosterone excretion, and the H score of 11&bgr;-hydroxylase and was inversely correlated with the H score of aldosterone synthase. These results demonstrated that small adenomas could produce sufficient aldosterone to cause clinically overt primary aldosteronism because of the significantly higher aldosterone synthase expression per tumor area.

Cyclin D1 (CCND1) expression is involved in estrogen receptor beta (ERβ) in human prostate cancer.

Estrogen receptor beta (ERβ) has been demonstrated to be expressed in prostate carcinoma cells and estrogen signals through ERβ to act as a tumor suppressor in prostate cancer patients. ERβ is thought to regulate the cell cycle of prostate carcinoma cells by controlling the expression of cell cycle regulators including cyclin D1 (CCND1). This interaction is of particular interest as CCND1 has been implicated in the development of prostate cancer.

Voltage-gated calcium channels in the human adrenal and primary aldosteronism

Calcium channel blockers can efficiently be used in the treatment of primary aldosteronism (PA) related hypertension, but details on the localization of calcium channel (CC) in the human adrenal and its disorders, including PA, have remained unclear. Therefore, in this study we analyzed the known α subunits of L-, N- and T-type CCs in 74 adrenocortical aldosterone-producing adenomas (APA) and 16 cortisol-producing adenomas (CPA) using quantitative RT-PCR (qPCR). We also examined the status of L-(CaV1.2, CaV1.3), N-(CaV2.2) and T-(CaV3.2) CC subunits in five non-pathological adrenals (NA), five idiopathic hyperaldosteronism (IHA) cases, and 50 APA using immunohistochemistry. After qPCR evaluation, only CaV1.2, CaV1.3, CaV2.2, and CaV3.2 mRNA levels could be detected in APA and CPA. Among those, only CaV3.2 mRNA levels were significantly correlated with plasma aldosterone levels (P=0.0031), CYP11B2 expression levels (P<0.0001) and the presence of KCNJ5 mutations (P=0.0019) in APA. The immunolocalization of CCs in NA and IHA was detected in the zona glomerulosa (ZG), with a predominance of CaV3.2 in APA. These findings suggest that different types of CC can be involved in calcium-related aldosterone biosynthesis.

3β-hydroxysteroid dehydrogenase isoforms in human aldosterone-producing adenoma

It has become important to evaluate the possible involvement of 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) and 2 (HSD3B2) isoforms in aldosterone-producing adenoma (APA). In this study, we studied 67 and 100 APA cases using real-time quantitative PCR (qPCR) and immunohistochemistry, respectively. Results of qPCR analysis demonstrated that HSD3B2 mRNA was significantly more abundant than HSD3B1 mRNA (P < 0.0001), but only HSD3B1 mRNA significantly correlated with CYP11B2 (aldosterone synthase) mRNA (P <0.0001) and plasma aldosterone concentration (PAC) of the patients (P <0.0001). Results of immunohistochemistry subsequently revealed that HSD3B2 immunoreactivity was detected in the great majority of APA but a significant correlation was also detected between HSD3B1 and CYP11B2 (P <0.0001). In KCNJ5 mutated APA, CYP11B2 mRNA (P <0.0001) and HSD3B1 mRNA (P = 0.011) were significantly higher than those of wild type APA. These results suggest that HSD3B1 is involved in aldosterone production, despite its lower levels of expression compared with HSD3B2, and also possibly associated with KCNJ5 mutation in APA.

Glutamate receptors and the regulation of steroidogenesis in the human adrenal gland: The metabotropic pathway

BACKGROUND l-glutamate is a major excitatory neurotransmitter in the mammalian brain. Glutamate receptors have been reported in the rat adrenal cortex and in human aldosterone-producing adenomas (APA). However, details regarding the expression levels and functions of these receptors in human adrenocortical tissues remain unknown. METHODS The mRNA levels of glutamate receptors were evaluated by qPCR in: 12 normal adrenal cortex (NAC), 11 APA, and 12 cortisol-producing adenoma (CPA) tissues. Protein localization was evaluated by immunohistochemistry for 15 NAC, 5 idiopathic hyperaldosteronism cases, 15 APA and 15 CPA. H295R cells were treated with angiotensin-II or forskolin alone or combined with the GRM2/3 agonist LY354740. RESULTS The level of GRM3 mRNA was higher in APA than in CPA (P=0.0086) or NAC (P=0.0022). GRM1, IGLUR2, and IGLUR3 were also detected in adrenocortical tissues. When added to angiotensin-II/forskolin treatments, LY354740 decreased aldosterone and cortisol production in H295R cells. CONCLUSIONS GRM3 is considered to regulate steroidogenesis in adrenocortical tissues.

PCP4: a regulator of aldosterone synthesis in human adrenocortical tissues

Purkinje cell protein 4 (PCP4) is a calmodulin (CaM)-binding protein that accelerates calcium association and dissociation with CaM. It has been previously detected in aldosterone-producing adenomas (APA), but details on its expression and function in adrenocortical tissues have remained unknown. Therefore, we performed the immunohistochemical analysis of PCP4 in the following tissues: normal adrenal (NA; n=15), APA (n=15), cortisol-producing adenomas (n=15), and idiopathic hyperaldosteronism cases (IHA; n=5). APA samples (n=45) were also submitted to quantitative RT-PCR of PCP4, CYP11B1, and CYP11B2, as well as DNA sequencing for KCNJ5 mutations. Transient transfection analysis using PCP4 siRNA was also performed in H295R adrenocortical carcinoma cells, following ELISA analysis, and CYP11B2 luciferase assays were also performed after PCP4 vector transfection in order to study the regulation of PCP4 protein expression. In our findings, PCP4 immunoreactivity was predominantly detected in APA and in the zona glomerulosa of NA and IHA. In APA, the mRNA levels of PCP4 were significantly correlated with those of CYP11B2 (P<0.0001) and were significantly higher in cases with KCNJ5 mutation than WT (P=0.005). Following PCP4 vector transfection, CYP11B2 luciferase reporter activity was significantly higher than controls in the presence of angiotensin-II. Knockdown of PCP4 resulted in a significant decrease in CYP11B2 mRNA levels (P=0.012) and aldosterone production (P=0.011). Our results indicate that PCP4 is a regulator of aldosterone production in normal, hyperplastic, and neoplastic human adrenocortical cells.

Estrogen-related receptor α in normal adrenal cortex and adrenocortical tumors: Involvement in development and oncogenesis

AIMS The nuclear hormone receptor estrogen-related receptor α (ERRα) regulates the activation of mitochondrial genes in various human tissues, but its role in the adrenal gland and its disorders has not been defined. Therefore, we examined ERRα expression in both normal adrenal cortex (NAC) and adrenocortical tumor (ACT) in order to study the possible correlation of ERRα with adrenal development and tumor development. METHODS Human adrenal specimens (non-pathological fetal n=7; non-pathological post-birth n=40; aldosterone producing adenoma (APA) n=11; cortisol producing adenoma (CPA) n=11; adrenocortical carcinoma (ACC) n=8) were immunohistochemically examined in this study. NAC (n=13) and ACT (n=28) frozen tissue specimens were also available for studying ERRα mRNA levels. KEY FINDINGS In fetal NAC tissues, ERRα labeling index (LI) in fetal zone (FZ) was significantly higher that that in neocortex (NC), and the differences among age groups for overall mean LI was statistically significant when analyzed according to individual cortical layers. ERRα LI was also significantly higher in ACC than in other types of ACT. ERRα mRNA was detected in NAC and all types of ACT. SIGNIFICANCE Results of our present study suggest a possible role of ERRα in adrenal development and ACC.

Changes of tumor infiltrating lymphocyte subtypes before and after neoadjuvant endocrine therapy in estrogen receptor-positive breast cancer patients - an immunohistochemical study of cd8+ and foxp3+ using double immunostaining with correlation to the pathobiological response of the patients

Tumor-stromal interactions involve continuous crosstalk and interactions among different cell types and play pivotal roles in tumorigenesis, tumor development, disease progression, subsequent metastasis, and also tumor response to therapeutic agents. Tumor infiltrating lymphocytes (TILs) are important components of these tumor-stromal interactions. Specific TIL subtypes are known to be involved in the clinical course of individual patients. However, the status of TILs following endocrine therapy has not been studied in breast cancer patients. We evaluated the alterations of TIL subtypes in a cohort of East Asian patients with estrogen receptor-positive breast cancer during the course of neoadjuvant steroidal aromatase inhibitor (AI) therapy, using double immunohistochemical staining of CD8+ and T regulatory cells (Treg) or Foxp3+, yielding the CD8+/Treg ratio in individual patients. Changes in CD8+/Treg ratio before and after therapy were then correlated with pathobiological responses of individual patients based upon alterations of the Ki-67 labeling index (LI). A significant increase in the CD8+/Treg ratio was detected in responders (p=0.028) but not in non-responders, which may reflect the dynamic process in which the host immune response to tumor antigens changed in consequence of an interaction between tumor and stromal cells in its microenvironment following estrogen depletion caused by the AI. The CD8+/Treg ratio in breast cancer tissue can be a potential surrogate marker in surgical pathology specimens for predicting responses to neoadjuvant endocrine therapy, not only incorporating features of carcinoma cells as in Ki-67 LI but also those of adjacent stromal cells in the tumor microenvironment, especially in the early stage of treatment prior to any detectable clinical and/or histopathological changes.

Steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions

The sebaceous gland is a major site of steroid synthesis in human skin, but details of the status of steroidogenic enzymes and their regulation in human sebaceous glands under normal and pathological conditions have rarely been reported. Therefore, in this study, we examined the status of steroidogenic enzymes, sex steroid receptors and transcription factors in human sebaceous glands under normal and pathological conditions to explore their possible roles in in situ steroid production in human skin. Immunohistochemical analysis was performed in a total of 59 human skin specimens, including 22 normal human sebaceous glands, 12 with sebaceous nevus, 12 with sebaceous gland hyperplasia, 3 with sebaceoma and 10 with sebaceous carcinoma. Immortalised human SZ95 sebocytes were treated with forskolin or vehicle for 3h, 6h, 12h or 24h, and the mRNA levels of steroidogenic enzymes were evaluated at each time point using quantitative RT-PCR (qPCR). The results of immunohistochemistry demonstrated the immunoreactivity of 3β-HSD1, CYP11A1, StAR, 17β-HSD5, CYP17A1, 5α-red1, PRB, AR and NGFI-B in normal human sebaceous gland, with lower levels of expression in pathological sebaceous glands. The results of the in vitro study also indicated that the expression levels of 3β-HSD1, CYP11A1, StAR, 5α-red1 and NGFI-B were elevated by forskolin. 3β-HSD1 and other steroidogenic enzymes were expressed in sebaceous glands resulting in in situ androgen and progesterone synthesis and their functions.