Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia

Abstract

The intracellular serine protease inhibitors (serpins) are an important family of proteins that protect cells form proteinase-mediated injury. Understanding the tissue and cellular expression pattern of this protein family can provide important insights into their physiologic roles. For example, high expression in epithelial tissues, such as lung, may suggest a biologic function in cellular defense, secretion, or selective absorption. Although the expression pattern of many of the intracellular serpins has been well described, one member of this class, SERPINB12, has not been carefully examined. We generated a mouse monoclonal antibody directed against human SERPINB12 and delineated its specificity and tissue and cell type distribution pattern through immunoblotting and immunohistochemistry, respectively. This monoclonal antibody was human specific and did not cross-react with other human intracellular serpins or mouse Serpinb12. SERPINB12 was found in nearly all the tissues investigated. In addition, this serpin was found in multiple cell types within individual tissues but primarily the epithelium. These data suggest that SERPINB12, like some other intracellular serpins, may play a vital role in barrier function by providing protection of epithelial cells.

Keywords: epithelium; human; immunohistochemistry; protease; serpin; tissue array.

Figure 1.

Specificity of H3-1B monoclonal antibody against SERPINB12. (A) Immunoblot of H3-1B against 6×His-SERPINB2 (B2), GST-tagged recombinant proteins: GST-SERPINB3 (B3), -B4 (B4), -B13 (B13), -B12 (B12), and GST-Serpinb12 (mB12). Note that only SERPINB12 is detected. (B) Parallel immunoblot of monoclonal anti-GST antibody (1:1000 dilution) with the same recombinant serpins (B2, B3, B4, B12, B13, and mB12) showing detection of all GST-tagged proteins (B3, B4, B12, B13, and mB12). (C) Parallel immunoblot of monoclonal anti-His antibody (1 in 100 dilution) with the same recombinant serpins (B2, B3, B4, B12, B13, and mB12) showing detection of only 6×His SERPINB2. (D) Parallel Coomassie SDS-PAGE to show approximately equal amounts of proteins loaded. MW markers for all immunoblots and SDS-PAGE are in KDa.

Figure 2.

Specificity of the H3-1B MAb by immunohistochemistry. Immunohistochemical peroxidase staining of tissue array sections of prostate (A–C), fallopian tubes (D–F), and thyroid (G–I) with nonspecific mouse IgG1 (A, D, G), H3-1B blocked with recombinant GSTSERPINB12 (B, E, H), and H3-1B (C, F, I). HRP secondary antibody was detected using the DAB peroxidase detection kit. Positive staining is only seen in the H3-1B tissues. Scale bar = 100 µm. Images were captured on an Olympus BH2 microscope mounted with a Jenoptik ProgRes C5 camera. Images were acquired using ProgRes CapturePro 2.7 software.

Figure 3.

Respiratory tract. SERPINB12 staining in the lung and tracheobronchial tree showed that the distal alveolated lung parenchyma had little staining of alveolar lining cells (A) but did highlight alveolar macrophages (inset) and some endothelium of interstitial blood vessels. The bronchial (B) and tracheal (C) epithelium in general demonstrated weak intensity and variable distribution of cytoplasmic staining. Upon closer inspection, some bronchial epithelial cells appeared to demonstrate nuclear staining; however, this may be related to the overlapping nature of the pseudostratified epithelium (inset). Scale bar = 100 µm. Images were captured on an Olympus BH2 microscope mounted with a Jenoptik ProgRes C5 camera. Images were acquired using ProgRes CapturePro 2.7 software.

Figure 4.

Gastrointestinal tract. Immunohistochemistry using H3-1B shows staining in all epithelial compartments within the major segments of the gastrointestinal tract. The squamous epithelium of the esophagus (A) and glandular epithelium of the stomach (B), small intestine (C), colon (D), and rectum (E) exhibit diffuse, variably intense cytoplasmic staining (insets). Some stromal elements also exhibit staining, including lamina propria stromal cells and macrophages. Scale bar = 100 µm. Images were captured on an Olympus BH2 microscope mounted with a Jenoptik ProgRes C5 camera. Images were acquired using ProgRes CapturePro 2.7 software.

Figure 5.

SERPINB12 immunohistochemistry of heart (A), liver (B), skin epidermis (C), and kidney (D) shows diffuse, variably intense granular cytoplasmic staining in the cells of all four tissue types. Insets highlight the cytoplasmic staining. Cardiomyocytes (A) and hepatocytes (B) demonstrated the most intense staining. Scale bar = 100 µm. Images were captured on an Olympus BH2 microscope mounted with a Jenoptik ProgRes C5 camera. Images were acquired using ProgRes CapturePro 2.7 software.

Figure 6.

Summary of immunohistochemistry with anti-SERPINB12 monoclonal antibody (H3-1B); ×20 magnification with ×40 magnification insets. (A) Adrenal gland, (B) bladder, (C) bone marrow, (D) breast, (E) bronchus, (F) cerebellum, (G) cervix, (H) colon, (I) cortex, (J) esophagus, (K) fallopian tube, (L) heart, (M) kidney, (N) liver, (O) lung, (P) trachea/bronchus, (Q) muscle-skeletal, (R) ovary, (S) pituitary, (T) placenta, (U) prostate, (V) rectum, (W) small intestine, (X) skin, (Y) spleen, (Z) stomach, (AA) testis, (AB) thymus, (AC) thyroid, (AD) tonsils, (AE) trachea, and (AF) uterus. ×20 scale bars 100 µm and all ×40 scale bars 30 µm. Images were captured on an Olympus BH2 microscope mounted with a Jenoptik ProgRes C5 camera. Images were acquired using ProgRes CapturePro 2.7 software.