Applicability of Anti-Human Estrogen Receptor β Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor β Proteins

Abstract

Several lines of controversial evidence concerning estrogen receptor β (ERβ) remain to be solved because of the unavailability of specific antibodies against ERβ. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERβ. However, the specificity and cross-reactivity of PPZ0506 antibody against ERβ proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERβ proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERβ proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERβ expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERβ studies, and our results provide a fundamental basis for further examination of ERβ functions.

Keywords: ESR2; alternative promoter usage; alternative splicing; antibody validation.

Figure 1

Confirmation of specific immunoreactivity of PPZ0506 antibody against human, mouse, and rat estrogen receptor β (ERβ) proteins in immunoblotting analysis. (a) Immunoblot detection of human, mouse, and rat ERβ proteins in transfected HEK293 cells using anti-human ERβ monoclonal antibody (PPZ0506). (b) Immunoblot detection of FLAG (DYKDDDDK)-tagged ERα and ERβ proteins in transfected HEK293 cells using anti-DYKDDDDK monoclonal antibody (2H8). “h”, “m”, and “r” indicate human, mouse, and rat, respectively. Mock-transfected cells (mock) were used as negative controls. An equal amount of protein lysate was loaded in each lane (0.5 μg/lane). The representative images in panels (a) and (b) were obtained in parallel using the same samples. Similar results were obtained in three separate experiments (n = 3).

Figure 2

Confirmation of specific immunoreactivity of PPZ0506 antibody against human, mouse, and rat ERβ proteins in immunocytochemical analyses. (a) Immunocytochemical detection of human, mouse, and rat ERβ proteins in transfected COS-7 cells using anti-human ERβ monoclonal antibody (PPZ0506). (b) Immunocytochemical detection of FLAG-tagged ERα and ERβ proteins in transfected COS-7 cells using anti-DYKDDDDK monoclonal antibody (2H8). Transfected cells were treated with 10 nM E₂ (+) or 0.1% EtOH (–). “h”, “m”, and “r” indicate human, mouse, and rat, respectively. Mock-transfected cells (mock) were used as negative controls. Alexa Fluor 488 and 4′,6-diamino-2-phenylindole (DAPI) images were pseudocolored in green and red, respectively. Scale bar: 50 μm. Similar results were obtained in three separate experiments (n = 3).

Figure 2

Confirmation of specific immunoreactivity of PPZ0506 antibody against human, mouse, and rat ERβ proteins in immunocytochemical analyses. (a) Immunocytochemical detection of human, mouse, and rat ERβ proteins in transfected COS-7 cells using anti-human ERβ monoclonal antibody (PPZ0506). (b) Immunocytochemical detection of FLAG-tagged ERα and ERβ proteins in transfected COS-7 cells using anti-DYKDDDDK monoclonal antibody (2H8). Transfected cells were treated with 10 nM E₂ (+) or 0.1% EtOH (–). “h”, “m”, and “r” indicate human, mouse, and rat, respectively. Mock-transfected cells (mock) were used as negative controls. Alexa Fluor 488 and 4′,6-diamino-2-phenylindole (DAPI) images were pseudocolored in green and red, respectively. Scale bar: 50 μm. Similar results were obtained in three separate experiments (n = 3).

Figure 3

Immunohistochemical analysis of rat ERβ expression in rat tissues. Immunohistochemical signals against rat ERβ proteins were evaluated in the ovary (a), prostate (b), testis (c), AVPV (d), PVH (e), lung (f), anterior pituitary (g), uterus (h), and adrenal gland (i). Left panels (a–i), low magnification; middle panels (a₁–i₁, a₂–e₂), magnified images of the framed areas in the left panels; right panels (a_(-)–i_(-)), immunostaining without PPZ0506 antibody; the brain sections are thicker (16 μm) than the other sections (5 μm) and not counterstained with hematoxylin. The dotted lines in panels (i₁) and (i_(-)) indicate boundaries between the adrenal cortex and medulla. Scale bars: 100 μm in left panels; 50 μm in middle and right panels. Similar results were obtained in three separate experiments (n = 3).

Figure 3

Immunohistochemical analysis of rat ERβ expression in rat tissues. Immunohistochemical signals against rat ERβ proteins were evaluated in the ovary (a), prostate (b), testis (c), AVPV (d), PVH (e), lung (f), anterior pituitary (g), uterus (h), and adrenal gland (i). Left panels (a–i), low magnification; middle panels (a₁–i₁, a₂–e₂), magnified images of the framed areas in the left panels; right panels (a_(-)–i_(-)), immunostaining without PPZ0506 antibody; the brain sections are thicker (16 μm) than the other sections (5 μm) and not counterstained with hematoxylin. The dotted lines in panels (i₁) and (i_(-)) indicate boundaries between the adrenal cortex and medulla. Scale bars: 100 μm in left panels; 50 μm in middle and right panels. Similar results were obtained in three separate experiments (n = 3).

Figure 4

Genomic organization of the 5′-regions of human and rat ERβ genes. Structure of the 5′-regions of the (a) human and (b) rat ERβ gene, represented schematically. Human and rat ERβ genes are located at 14q23.2-23.3 on human chromosome 14 and at 6q24 on rat chromosome 6. Black, gray, and white boxes indicate coding exons, untranslated leader exons, and untranslated internal exons, respectively. Exon 0Y2 localizes to the 3′-end of human exon 0N. Exons 0X2, 0X4, and 0X7 have two alternative splice donor sites, and human exon 0N contains a splice acceptor site of exon 0Y2. Dotted lines in boxes and bent arrows represent alternative splice sites and transcriptional start sites, respectively. Nucleotide sequences of the leader and internal exons are shown in detail in Supplementary Figure S5. Human leader exons E1 and 0N are orthologous to rat leader exons E1/P2 and 0N/P1, respectively. Human leader exon 0K is not homologous to rat leader exon 0H. The image is not to scale.

Figure 5

Profiles of the alternative promoter usage of human and rat ERβ genes. (a) Expression patterns of human promoter-specific ERβ variants. (b) Expression patterns of rat promoter-specific ERβ variants. Total RNAs isolated from multiple organs were subjected to RT-PCR. The distribution and splice patterns of human E1, 0N, and 0K isoforms, and rat E1/P2 and 0N/P1 isoforms were analyzed. To assess the overall expression of the human and rat ERβ genes, the open reading frame (ORF) regions were amplified (ERβ ORF). As a comparison, the expression of the human and rat ERα genes was evaluated by amplifying the coding regions between human exons 2 and 3, and rat exons 6 and 8, respectively (ERα ORF). GAPDH was used as an internal control. From the upper to lower panels, the number of PCR cycles increased in increments of three cycles (indicated to the right of the respective panels). Human cDNAs require two more PCR cycles than rat cDNAs to detect target molecules due to the quality of their total RNAs.