Systemic evaluation of total Stat3 and Stat3 tyrosine phosphorylation in normal human tissues

Experimental and Molecular Pathology 80 (2006) 295 – 305 www.elsevier.com/locate/yexmp

Systemic evaluation of total Stat3 and Stat3 tyrosine phosphorylation in normal human tissues Chun-Liang Chen

a,1

, Fu-Chuan Hsieh

a,1

, Jiayuh Lin

a,b,⁎

a

b

Center for Childhood Cancer, Columbus Children’s Research Institute, Columbus, OH 43205, USA Department of Pediatrics, Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH 43205, USA Received 11 November 2005 Available online 19 January 2006

Abstract Stat3 plays important roles in many biological phenomena including cell survival, growth, proliferation, differentiation and cancer malignancies. As Stat3 emerges as a new therapeutic target for treatment of cancers in which the Stat3 is constitutively activated, the overall evaluation of basal expression of Stat3 and phosphorylated Stat3 at tyrosine residue 705 in human tissues would be very important and informative. We took a pilot study to examine the expression patterns of total Stat3 and phosphorylated Stat3 protein (p-Stat3) using immunohistochemistry in 47 different adult normal human tissues of 10 organ systems. Immunohistochemistry showed that total Stat3 protein was almost universally detected in all tissues except peripheral nerve. Interestingly, majorities of tissues showed to have moderate to high expression levels of total Stat3 protein. Several heart tissues displayed a unique perinuclear immunostaining for both Stat3 and p-Stat3, most likely in Golgi complexes. Based on the cell types, the p-Stat3 was also expressed in glandular, secretory, mucosal epithelial, circulatory endothelial, lymphoid, proliferative, and reabsorption-active cells. © 2005 Elsevier Inc. All rights reserved. Keywords: Stat3; Human tissues; Tissue microarray slides

Introduction The signal transducer and activator of transcription (Stat) protein family is a group of related proteins that mediate signaling from cytokines (e.g. IFN family and IL-6 family) and growth factors (e.g. EGF, VEGF, and PDGF) (Bowman et al., 2000; Duncan et al., 1997; Ernst and Jenkins, 2004; Kamimura et al., 2003; Kisseleva et al., 2002; Levy and Lee, 2002; Schindler and Darnell, 1995). Stat3, as a major member of Stat family consisting of Stat1, 2, 3, 4, 5α, 5β, and 6, shares high similarity in protein structures with other members and plays important roles in the acute phase response (Buettner et al., 2002; Chen et al., 1998; Darnell et al., 1994; Schaefer et al., 1995). The activation of Stat3 is based on the phosphorylation

⁎ Corresponding author. WA5020, Center for Childhood Cancer, 700 Children's Drive Columbus, OH 43205, USA. Fax: +1 614 355 2672. E-mail address: [email protected] (J. Lin). 1 These two authors contribute equally. 0014-4800/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.11.003

on the tyrosine 705 residue in the src homology-2 domain (SH2) by a wide range of cytokine-receptor-associated kinases, growth factor receptor tyrosine kinases, and nonreceptor tyrosine kinases originally defined as the signaling mechanism for IFN receptors (Levy and Lee, 2002). The activation assures the stable hetero- or homodimerization that leads to the translocation of Stat3 into the nucleus (Minami et al., 1996). Subsequently, the activated dimerized Stat3 binds to a palindromic IFN-γ-activated sequence (GAS) element and triggers the transcription of downstream-regulated genes in full scale as the serine residue 727 at the C terminus is also phosphorylated (Shen et al., 2004; Wen and Darnell, 1997). A growing body of evidence shows that Stat3 is widely expressed in different tissues and plays a role in a variety of biological functions: embryogenesis, postnatal development, and adult functionality (Ernst and Jenkins, 2004; Kamimura et al., 2003; Levy and Lee, 2002; Takeda and Akira, 2000, 2001). Besides the acute phase response, Stat3 is involved in cell survival, growth, proliferation, and differentiation (Levy and Darnell, 2002; Takeda et al., 1997). Polarized distribution of

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Stat3 is present in early mammalian embryogenesis and becomes more widely distributed in multiple tissues with variant intensity in postnatal and adult stages (Antczak and Van Blerkom, 1997; Miyoshi et al., 2001; Zhong et al., 1994). Moreover, many tissues express Stat3 with variant intensity (Miyoshi et al., 2001; Zhong et al., 1994). Activation of Stat3 signaling pathway in normal tissues is transient and under a scrutinizing orchestrated regulatory control as revealed by IL6-gp130 paradigm (Ernst and Jenkins, 2004; Kamimura et al., 2003). With counter balance SHP2/ERK signal cascade, Stat3 signal pathway dictates expression of target genes including those involved in apoptosis, cell cycle regulation, and induction of growth arrest such as Bcl-xL, cyclin D1, p21WAF1/CIP1 , and c-myc (Levy and Lee, 2002; Real et al., 2002). The timely confinement of Stat3 activation assures that the execution of Stat3 function would not lead to pathological outcomes. A growing number of human malignancies and tumorigenesis are associated with high levels of activation of signal transducers and activators of transcription (Stats), very frequently Stat3 and Stat5 (Bowman et al., 2000; Garcia and Jove, 1998; Garcia et al., 1997). In a variety of human cancers, constitutive activation of Stat3 is sufficient to induce cell tumorigenesis (Bromberg et al., 1998, 1999; Buettner et al., 2002). Stat3 is also involved in the initiation and promotion of skin cancer (Chan et al., 2004; Pedranzini et al., 2004) and in angiogenesis of human gastric cancer (Gong et al., 2005). Since targeted interference to Stat3 pathway induces cancer cell death and restricts tumor growth (Ling and Arlinghaus, 2005; Proietti et al., 2005; Song et al., 2005), constitutive Stat3 signal cascade has become an important target for cancer therapy (Chan et al., 2004; Clevenger, 2004; Konnikova et al., 2003; Lee et al., 2004; Leong et al., 2003; Niu et al., 1999; Selander et al., 2004; Song et al., 2005). Phosphorylated Stat3 immunohistochemistry on tissue microarray with clinical cancer tissue section samples provides valuable prognosis for prostate and breast cancers (Buettner et al., 2002; Dolled-Filhart et al., 2003). Stat3 is activated both during apoptotic involution of mammary glands and during the highly proliferative phase of early pregnancy. Subsequent conditional knockout studies in mice have shown that Stat3 is essential in mammary gland epithelial cell apoptosis and involution. In human, Stat3 is activated in several mammary epithelial cells and breast carcinoma cell lines. There is evidence of increased Stat3 binding in the nuclei of breast cancer tumors compared with normal breast tissue or benign lesions. An immunohistochemical study of 364 cases of invasive malignant breast cancer tumors shows that Stat3 is expressed only in the cytoplasm of nontumor regions but was expressed in both the cytoplasm and nuclei of malignant regions of the specimens (Dolled-Filhart et al., 2003). It is found that not Stat3 but p-Stat3 subcellular localization expression is tightly correlated with survival. In prostate tissue, the activated form of Stat3 is localized predominantly to the nuclei of malignant glands. This activated Stat3 form may be a better probe for function than total Stat3 (both p-Stat3 and non-p-Stat3). Tissue microarray technology is a highly

efficient and economical way to evaluate hundreds of tumors. As Stat3 signal pathway emerges as an important player in the cancer malignancies and target for their treatment, there appears a great need for a large scale of screening on different cancer types in variant tissues using Stat3 immunohistochemistry combined with tissue microarray technology. Basal expression profiles of Stat3 and p-Stat3 in human tissues would be very important for comparison. Meanwhile, the systemic histological survey on the Stat3 expression pattern would also shed light on the Stat3 functions in human as a whole and bridge to the molecular and genetic evidence predominantly based on animal models. Here, we systematically evaluated the expression and subcellular localization of both Stat3 and p-Stat3 by immunohistochemistry on normal human adult tissue microarrays with 47 tissues of 10 organ systems. Materials and methods Immunohistochemistry Forty-seven human tissues used in this experiment represented all of the major organ systems of human body and cover exhaustively the majority of tissues and cell types. Normal human tissue microarray slides were obtained from two individual sources: the Biochain Institute, Inc. (Hayward, CA) and Imgenex Corporation (San Diego, CA). The list of tissues was shown in Table 1 including digestive system (8 tissues), nervous system (8), endocrine system (4), immune system (4), circulatory system (9), reproductive (8), respiratory (1), urinary (2), integumentary system (1), and connective system (2). There were 1– 4 individual specimens from each tissue subject to either total Stat3 or P-Stat3 antibody (Cell Singling Technology, Inc., Beverly, MA) immunohistochemical staining, respectively. These tissue microarray slides were baked at 60°C according to the manufacturer's instruction. Then, the slides were deparaffinized with xylene rinses three times, transferred through two changes of 100% ethanol, and then rehydrated with graded ethanol. Endogenous peroxidase activity was quenched by a 10-min incubation in 3% hydrogen peroxide. Antigen retrieval was carried out by boiling the slides in a pressure cooker filled with 10 mM sodium citrate (pH 6.0) or 1 mM EDTA (pH 8.0). After antigen retrieval, the slides were briefly rinsed with 0.1% Tween/1× TBS (TBST) two times and then washed three times for 10 min each at room temperature. To prevent nonspecific immunobinding, the tissue specimens were incubated in 5% normal goat serum in 0.1% TBST for 1 h. Primary antibody (1:30 dilution of rabbit anti-p-Stat3 (Tyr705) antibody or 1:100 dilution of rabbit anti-Stat3 antibody in 0.1% TBST with 0.3% normal serum) was applied for 1 h at room temperature. After a series of TBST rinses as described above, bound antibody was subsequently detected using a VETASTATIN ABC kit from VECTOR Laboratories, Inc. (Burlingame, CA). The immunoreactivity was then visualized with a 5–30 min incubation of 3-amino-9-ethylcarbazole (AEC) high sensitivity substrate chromogen from DakoCytomation (Carpinteria, CA). Finally, the slides were counterstained with hematoxylin and mounted with CRYSTAL/ MOUNT (Biomeda Corp., Foster City, CA) for preservation.

Evaluation of immunohistochemical staining The intensity of immunohistochemical staining was scored by eye under a microscope. The intensity of total Stat3 staining on each sample was scored, whereas p-Stat3 staining in nuclei and cytoplasm was determined separately on each specimen for nuclear p-Stat3 is more closely correlated with Stat3 activation according to the current Stat3 activation paradigm. The staining intensity was graded relatively based on the following scales: 0, 1, 2, and 3 from no staining to strong staining. An averaged score was reached as the final score for each tissue with multiple samples. According to the final immunostaining scores, the normal human tissues were classified into four groups: negative group (score 0–0.49), weak staining group (score 0.5–1), moderate staining

C.-L. Chen et al. / Experimental and Molecular Pathology 80 (2006) 295–305 Table 1 Expression of total Stat3 and p-Stat3 in 47 normal human tissues Tissue

Total p-Stat3 p-Stat3-positive cells Stat3 Cytoplasm Nucleus

Digestive system Appendix vermiformis Colon

+

+

++

Lymphoid follicle

++

+

+

Esophagus

+

+

+

Salivary gland Small intestine Stomach

+ + ++

+ + +

+ + +

Pancreas Gall bladder

++ +

+ +

+ +

Colonal epithelium, follicular and scattered lymphoid Squamous epithelium submucosal lymphocytes Salivary lobules Mucosal epithelium Columnar mucus-secreting cells Acinar cells Mucosal epithelium

+

+

+

Neurons

+ +

+ +

− −

Neurons Neurons

+

+

−

Neurons

+ −

− −

− −

+ +

− −

− −

Nervous system Temporal cortex Cerebellum Corpus callosum Medulla oblongana Mesencephalon Peripheral nerve Pons Spinal cord

+

+

Pituitary gland Parathyroid Thyroid

++ ++ ++

+ + −

+ + +

Immune system Lymph node Thymus

++ +

+ +

+ +

Spleen Tonsil

+ +

+ +

+ +

Lymphocytes Epithelium, lymphocytes, and capillary Lymphoid cells Lymphoid cells

+ + + + +

− − + + +

− − − − −

Myocardiocytes Myocardiocytes Myocardiocytes

+ +

+ +

− −

Myocardiocytes Myocardiocytes

+

+

−

Reproductive system Placenta +

+

+

Cervix Ovarian stroma Ovary

− + −

+ + +

+ + +

Table 1 (continued ) Tissue

Total p-Stat3 p-Stat3-positive cells Stat3 Cytoplasm Nucleus

Uterus

+

+

+

Breast Prostate Testis

+ + ++

+ − +

+ + +

+

+

Infiltrated macrophages, respiratory epithelium

Arteries, veins, and lymphoid cells in outer adventitial coat and lamina propria Proximal/distal convoluted tubular cells and glomerular capillary

Respiratory system Lung +

Luminal and glandular epithelium Mammary glandular cells Glandular cells Developing spermatogonia and spermatid

Urinary system Urinary bladder

+

+

+

Kidney

+

+

+

Integumentary system Skin +

+

+

Epidermal cells

Connective system Skeletal muscle + Fat +

+ +

+ +

Myocytes Adipocytes

group (scores 1.1–2), and strong staining group (scores 2.1–3). The four groups were designated as “−”, “+”, “++”, and “+++” as in Table 1.

Endocrine system Adrenal gland ++

Circulatory system Pericardium Mitral valve Tricusp valve Atrium Interventricular septum Ventricle Unspecified heart tissue Vein

297

Glandular cells in fasculata and reticularis Glandular cells Glandular cells Glandular cells and lymphoid

Results Expression of total Stat3 in normal human tissues Expression levels of total Stat3 and p-Stat3 in normal human tissues were evaluated based on immunohistochemistry. The results were summarized in Table 1 and described in details as below. Total Stat3 immunoreactivity was detectable in almost all of the tissues screened, except for one tissue, periphery nerve (Table 1). Stomach, colon, pancreas, adrenal gland, parathyroid, thyroid, pituitary gland, lymph node, and testis showed the highest intensity of immunostaining (scores 2.1–3, +++, see details in Materials and methods) and were consisted of 19% of 47 tissues (Table 2). The immunostaining of the rest of tissues

Table 2 Numbers and portions of tissues that display variant expression levels of total Stat3 and p-Stat3 among 47 normal human tissues Cytotrophoblastic cells and capillary Endocervical glands Stromal cells Follicular cells and stromal cells

Stat3 status and location Total Stat3 p-Stat3 (cytoplasm) p-Stat3 (nucleus) a

Expression level Negative, − a

1 (2% ) 10 (21%) 16 (34%)

Percentage of total 47 tissues.

Weak, +

Moderate, ++

Strong, ++

17 (36%) 32 (68%) 13 (28%)

20 (43%) 5 (11%) 17 (36%)

9 (19%) 0 (0%) 1 (2%)

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appeared either weak (0.5–0.99, +) (36% of total tissues) or moderate (1–1.99, ++) (43% of total tissues). This indicated that total Stat3 was widely expressed in normal human tissues (98%), and the majority of tissues (62%) displayed moderate to strong expression level. Expression of p-Stat3 in normal human tissues Expression of p-Stat3 is shown to be more correlatively informative of Stat3 activation, particularly nuclear p-Stat3 (Dolled-Filhart et al., 2003). In order to understand the phosphorylation status of Stat3, different sets of normal human tissues equivalent to those used in Stat3 immunostaining were subject to p-Stat3 immunohistochemistry. PStat3 immunostaining for those tissues was summarized in Table 1 with separate scorings of the cytoplasmic and nuclear portions for two reasons. The first reason was that the presence of p-Stat3 in cytoplasmic and nuclear subcellular compartments was not homogenous throughout all the tissues. Secondly, p-Stat3 was favorably localized to nucleus rather than cytoplasm. Temporal cortex, lymph node, ovarian stroma, testis, kidney, and skeletal muscle showed moderate staining (++) in cytoplasm and were composed of only 11% of total tissues (Table 2). The rest of tissues (89%, 42/47) appeared with either weak (68%) or negative (21%) expression of p-Stat3 in cytoplasmic compartment. For nuclear p-Stat3 immunoreactivity, only appendix vermiformis (2% of total tissues) had strong scoring (+++) (Tables 1 and 2). Lymph node, cervix, uterus, kidney, small intestine, gall bladder, pituitary gland, lymph node, ovarian stroma, ovary, breast, testis, urinary bladder, think skin, and skeletal muscle had moderate expression (++) of p-Stat3 present in nuclei and represented 36% of total tissues (Table 2). Among tissues from both nervous and circulatory systems, no detectable nuclear p-Stat3 immunostaining was found, except for temporal cortex. Tissues that exhibited weak (+) or negative (−) p-Stat3 immunoreactivity in nuclei were 28% and 34% of total tissues in our screen (Table 2). This indicated that very minimum of Stat3 activation was required in those tissues in normal conditions. Six tissues (13%) did not show any p-Stat3 immunoreactivity in either cytoplasm or nuclei. They were mesencephalon, peripheral nerve, pons, spinal cord, pericardium, and mitral valve (Table 1), although most of them had at least weak expression of total Stat3. Altogether, low and negative pStat3 immunostaining levels implicated minimum or undetectable activation of Stat3 in tissues of the two organ systems. In each p-Stat3-positive tissue, some certain cell types exhibited p-Stat3 immunoreactivity, whereas other cell types were negative for immunostaining. These p-Stat3-positive cell types were glandular epithelial cells, secretory cell, mucosal epithelial cells, lymphoids, proliferative cells, reabsorptionactive cells, and circulatory endothelium. The phosphorylation status of Stat3 might be associated with the functions of these cell types.

p-Stat3 expression in glandular epithelium, secretory, and mucosal cells Glandular mucosal epithelium, secretory, and mucosal cells contained low or moderate p-Stat3 expression as observed in many tissues. These tissues included adrenal glands, stomach, pancreas, pituitary gland, thyroid, cervix, breast, lung, small intestine, gall bladder, mandibular salivary gland, and adipocytes (Table 1 and Fig. 1). Cytoplasmic and nuclear immunoreactivity to p-Stat3 was present in adrenal glandular cells in zona fasculata and reticularis, mucus-secreting cells of stomach, pancreatic acinar cells, pituitary secretory cells, thyroid glandular epithelium, endocervical gland, mammary glandular epithelial cells of breast, respiratory epithelium of lung, mandibular salivary gland cells, and adipocytes. In addition, mucosal epithelial cells of small intestine, colon, and gall bladder were p-Stat3-positive. p-Stat3 expression in lymphatic tissues Activation of Stat3 has been reported to be a key player of development of maturation of B and T cells (Minami et al., 1996; Takeda and Akira, 2000, 2001; Takeda et al., 1997, 1998, 1999). Normal human tissues containing lymphatic tissues showed p-Stat3 expression (Table 1 and Figs. 2A–H). Apparently, Stat3 was activated in aggregated cells in lymphatic follicles or germination centers residing in esophagus, small intestine, appendix, colon, tonsil, thymus, lymph nodes, and spleen. P-Stat3-immunoreactive cells in the lymphatic tissues showed strong p-Stat3 immunostaining in nuclei, whereas the cytoplasm displayed either moderate or weak staining. Some of outstanding p-Stat3-positive cells were randomly scattered throughout the tissues and they might be infiltrated surveillance lymphocytes. p-Stat3 expression in proliferative or reabsorption-active cells Proliferative or reabsorption-active cells also showed immunoreactivity to p-Stat3. Majority of cells in seminiferous tubules of testis, follicles of ovary, epidermis, and kidney's distal and proximal convoluted tubules (DCT and PCT) were either highly engaged in proliferation or execution of the sodium ions reabsorption from urine (Fig. 3). Proliferationactive cells in testes, ovaries, and epidermis were expressing p-Stat3 intensively (Figs. 3A–D). Differential expressions of p-Stat3 were discovered in the developing sperm in seminiferous tubules of testes and tightly associated with spermagogenesis and spermiogenesis. Spermatid during maturation displayed intense expression of p-Stat3 (+++), and primary spermatocytes undergoing proliferation showed moderate expression of p-Stat3 (Figs. 3A and B), while mature spermatogonia were negative for the immunostaining. In ovary, follicular cells and stromal theca folliculi were moderately labeled in nuclei (Fig. 3C). The epidermis of skin also exhibited moderate immunostaining of p-Stat3 (Fig. 3D). Most of epidermal epithelial cells were moderately labeled by anti-p-Stat3 antibody in cytoplasm

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Fig. 1. p-Stat3 is expressed in glandular and mucosal epithelium and secretory cells in human tissues. (A) Adrenal gland. (B) Stomach. (C) Pancreas. (D) Pituitary gland. (E) Thyroid. (F) Cervix. (G) Breast. (H) Lung. (I) Small intestine. (J) Gall bladder. (K) Mandibular salivary gland. (L) Adipocytes. ADC: adipocyte, TF: thyroidal follicle, GP: gastric pit, IM: infiltrated macrophage, ME: mucosal epithelial cell, MSC: mucus secreting cell, RE: respiratory epithelial cell. Arrows indicate some glandular cells with expression of nuclear p-Stat3. Magnification: A–I = 400×.

and particularly strong in few nuclei. Particularly, few cells with high content of p-Stat3 in nuclei might be keratinocytes with high mitotic potential. In kidney, the nuclei of distal convoluted tubule (DCT) cells and glomerular cells expressed p-Stat3 much higher than proximal convoluted tubules (PCT) cells (Fig. 3E). DCT cells were responsible for the reabsorption of sodium coupled with secretion of hydrogen and potassium ions into DCT. PCT cells expressed moderate level of p-Stat3, and they contribute to 75% of ions and water reabsorption from urine.

p-Stat3 ubiquitously expressed in endothelium of circulatory system The endothelium of circulatory system was also with ubiquitously strong expression of p-Stat3 in all tissues they were located in. Capillaries and veins of circulatory system were penetrating and distributed with variant extent in all the tissues that we observed. In addition, the endothelium of heart, the main organ of circulatory system, was not exceptional. Several representative tissues were shown (Figs. 3F–I). Most endothelial

Fig. 2. p-Stat3 is expressed in lymphatic tissues. (A) Appendix. (B) Colon. (C) Esophagus. (D) Tonsil. (E) Thymus. (F) Lymph node. (G) Spleen. (H) Small intestine. LF: lymphatic follicle, GC: germination center. Magnification: A–H = 400×.

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Fig. 3. p-Stat3 is expressed in nuclei of proliferative cells, reabsorption-active cells, and endothelium of circulatory system. (A) Testis. (B) Shows the higher power magnification of panel A. (C) Ovary. (D) Skin. (E) Kidney. (F) Urinary bladder. (G) Uterus. (H) Pericardium. (I) Mitral valve. BD: bile duct, DCT: distal convoluted tubule, E: epidermis, F: follicle; O: oocyte, PCT: promixal convoluted tubule, PV: portal vein, ST: seminiferous tubule, S1: primary spermatocyte, S2: spermatid, S3: spermatozoa, SC: Sertoli cell. Arrows specify endothelium. Magnifications: A, C–I = 400×; B = 1000×.

cells had the characteristic flattened nuclei with strong expression of p-Stat3. Their cytoplasm showed only weak or moderate expression of p-Stat3. This indicated that nuclear translocation of p-Stat3 in these cells was favored over the transport in the reverse direction. The strong expression of pStat3 in nuclei of endothelial cells might be induced unavoidably by the blood-containing upstream signals since they were exposed to the blood flowing through. Unique cytoplasmic localization of p-Stat3 of endomyocardiocytes A group of tissues in heart provided another interesting cytoplasmic distribution of p-Stat3 and Stat3 that was not found in other human tissues. This unique expression pattern of pStat3 and Stat3 occurred in the myocardiocytes of atrium, tricusp valve, interventricular septum, and ventricle (Figs. 4A– I). The perinuclear cytosolic pattern appeared to be in the Golgi complex. In pericardium, vein, and artery, however, for unknown reason, the similar pattern of immunostaining appeared for Stat3 but not p-Stat3 (Figs. 4J and K). This indicated that phosphorylation of Stat3 might not be required for retention of this molecule in the specific organelle. It seemed unlikely that the uncommon immunostaining for heart was caused by nonspecific immunostaining because two

independent monoclonal antibodies (anti p-Stat3 and anti-total Stat antibody) cross-reacted with the same antigen. Discussions There are accumulating studies showing Stat3 and p-Stat3 expressions in mice and other models. Their counterpart expression patterns in normal human tissues are largely unknown. We have examined a series of 47 normal human tissues on tissue microarray slides. The tissues were subject to immunostaining using two different monoclonal antibodies specific to Stat3 and p-Stat3. These tissues covered the major systems of human and displayed representative Stat3 and pStat3 expression profiles in normal condition. Expression of total Stat3 in human tissues Stat3 was widely expressed in most tissues we screened. Most of human tissues (98%) turned out to be positive for total Stat3 immunostaining except peripheral nerve tissue. This indicates that at least minimal level of Stat3 is present or required in normal tissues. The prevalence of Stat3 seems to be consistent with previous studies in animal models. Northern and Western blots have shown that Stat3 expression is in multiple adult mouse organs, such as heart, kidney, thymus, brain,

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Fig. 4. Perinulcear localization of total Stat3 and p-Stat3 in heart and vein tissues. (A, B, C) Atrium. (D, E, F) Ventricle. (G, H, I) Tricuspid valve. (J, K) Vein. Panels C, F, and I show the higher power magnifications of panels B, E, and H, respectively. Arrows indicate the locations of perinuclear p-Stat3 staining. Magnifications: A, B, D, E, G, H, J, K = 400×; C, F, I = 1000×.

pancreas, muscle, and spleen (Gallmeier et al., 2005; Miyoshi et al., 2001; Philp et al., 1996; Zhong et al., 1994). As immunohistochemical staining reveals, Stat3 protein is expressed in mouse brain, mammary glandular epithelium, respiratory epithelium, endometrial glandular epithelium, pineal gland, ovaries, and testes (Chapman et al., 1999; Cheng et al., 2001; Hokuto et al., 2004; Murphy et al., 2005; Planas et al., 1997; Takamiya et al., 2002). Stat3 is found in the external granule cell layer and within the molecular layer in developing brain. From postnatal 15 days to adult, the internal granule cell layer and Purkinje cells are also stained for Stat3 in mouse brain (Planas et al., 1997). Stat3 is also activated during the mammary gland development (Philp et al., 1996). For more details of expression pattern, the expression of Stat3 is found in the cytoplasm of oocytes from primordial, primary, and secondary

follicles in the adult mouse ovary and in the developing acrosome in the adult testis. In embryonic and neonatal gonads, Stat3 is expressed in oogonia and oocytes in the ovary as well as in gonocytes and prespermatogonia in the testes (Murphy et al., 2005). These tissues in our screen were positive for Stat3 immunoreactivity and shared similar expression patterns as seen in rat and mouse models. Interestingly, majority (62%, 29/47) of human tissues showed from moderate (++) to strong (+++) levels of Stat3 expression. Particularly, colon, pancreas, stomach, adrenal gland, parathyroid, thyroid, lymph node, and testis possessed strong expression (+++) of Stat3. Expression of STATs proteins was shown to be regulated by the same signals that activated Stat3, such as IFN-β/γ (Schindler and Darnell, 1995). Lately, some evidence argued that high level of unphosphorylated Stat3

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could act as a transcription factor for expressions of certain genes (Yang et al., 2005). It is widely accepted that Stat3 is not activated until upon upstream signal stimuli the protein was phosphorylated, dimerized, and translocated into the nucleus where it subsequently triggered downstream gene transcription via binding to GAS elements (Levy and Lee, 2002; Schindler and Darnell, 1995). However, it would be interesting to investigate whether high levels of unphosphorylated Stat3 in these human tissues have any biological functions. Expression of p-Stat3 in human tissues The expression of p-Stat3 was widely distributed in human tissues because 87% of the 47 tissues were detected to have either cytoplasmic or nuclear p-Stat3 expression. The widespread of p-Stat3 expression pattern was supported by the ubiquitous presence of latent Stat3 in most of tissues on one hand. On the other hand, animal model showed that the upstream cytokines and growth factors and their receptors were widely available in most of tissues (Ernst and Jenkins, 2004; Kamimura et al., 2003). Expression of p-Stat3 was relatively low in the nervous system and circulatory systems. Fifteen of 16 tissues (94%) from these two systems, except temporal cortex, were p-Stat3negative in nuclear compartments. Furthermore, 6 of the 16 tissues (38%) did not have p-Stat3 expression at all in both cytoplasm and nucleus. The tissues from these two organ systems were from 16 different donor sources of ages 22–88 (data not shown), so undetectable level of p-Stat3 might not be age-specific. Both postnatal neuronal survival and normal heart functions were shown to be dependent on the activation of Stat3 (Hilfiker-Kleiner et al., 2004; Jacoby et al., 2003; Schweizer et al., 2002). In addition, Stat3 deficiency phenotype became more apparent as the knockout mouse ages grew, so age seemed to be correlated to the increased Stat3 dependency. Undetectable nuclear p-Stat3 seemed likely to suggest least requirement of pStat3 for maintenance in the two organ systems rather than complete inactivation of Stat3. Nuclear p-Stat3 in certain cell types and its possible biological functions Immunohistochemistry for p-Stat3 indicated that nuclear pStat3 expression was not present in all cells but restricted to three major groups of cell types in human tissues (Table 1 and Figs. 1–4). The three types of cells were (1) lymphoid and assessory cells in immune, digestive, and respiratory systems, (2) glandular, mucosal, and secretory epithelium in digestive system, endocrine and reproductive systems, and endothelium of circulatory system (heart, veins, and capillaries), (3) proliferative and reabsorption-active cells in productive, urinary, and integumentary systems. It is still not clear whether the p-Stat3 immunostaining reflected the activation of Stat3 in these cell types and needs further verification using Northern blot and gel shift assay. Moreover, the expression patterns of the upstream signal components, such as gp130, leukemia inhibitory factor receptor (LIFR), and others in human tissues would

help answer the question. However, these p-Stat3 cell types have been reported to utilize Stat3 signaling for their maintenance and normal function in animal models. The previous studies implicated potential functions of Stat3 for those cell types in human tissues. Nuclear p-Stat3 was an important indicator for the activation of Stat3 signal pathway (Kisseleva et al., 2002; Schindler and Darnell, 1995). The activation of Stat3 is responsible to the upstream signals: such as IL-6 cytokines, IL-11, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1, other cytokines and hormones, leukemia inhibition factor (LIF), and IFN-β/γ through the receptor-associated kinase to phosphorylate the tyrosine residue and allow the Stat3 protein to form homodimers or heterodimers. In vitro Stat3 studies suggested that activation of Stat3 was involved in cellular function of myeloid cells, adipocytes, acinar cells, Sertoli cell, proximal tubular epithelial cells, colon epithelial cells, and central nervous system cells (Aggarwal et al., 2001; Chen et al., 2003; De Miguel et al., 1996; Minami et al., 1996; Nagalakshmi et al., 2004; Rajan et al., 1996; Stephens et al., 1996; Zhong et al., 1994). IL-6 and IL-2 synergistically induce homo- and heterodimerized STAT1 alpha and STAT3 in both NK and T cells (Yu et al., 1998). Stat3 was expressed in differentiated adipocytes and indicated that it might be required for maintenance of the cell function (Stephens et al., 1996). Stat3 was activated through IL-6, IFN-γ, and LIF in developing Sertoli cells and gonocytes (De Miguel et al., 1996; Jenab and Morris, 1996, 1998). IL-22 induced the JAK-STAT pathway in epithelial cells of the colon (Nagalakshmi et al., 2004). Acinar cells are responsive of IFN and IL-6 inflammatory signal through the Stat3 pathway (Gallmeier et al., 2005; Vona-Davis et al.). Renin–agiotensin system (RAS) is tightly associated with the function of promixal tubular cells and regulates reabsorption and production of TGFβ-1 through the activation of Stat3 (Chen et al., 2003). CNTF and LIF activated Stat3 pathway in CNS cells (Rajan et al., 1996). The expression pattern and activation of Stat3 in different cell types and tissues strongly support a notion that Stat3 is one of the major prevailing signaling pathways. However, Stat3 function was only possibly dissected through phenotype analysis of Stat3 loss-of-function mouse models (Takeda and Akira, 2001). Stat3 deficiency causes early embryonic lethality in mouse (Takeda et al., 1997). Stat3 is activated at the onset of uterine receptivity and embryo implantation (Cheng et al., 2001) and during early postimplantation development (Duncan et al., 1997). Data also suggest that Stat3 sustains postnatal survival and growth (Shen et al., 2004). Embryonic lethality induced by Stat3 deficiency raised a technical challenge to dissect the contributions of Stat3 in late developmental and adult stages. However, Stat3 signaling functionality in adult tissues has been intensely investigated in conditional loss-offunction mouse model using Cre-LoxP system (Takeda and Akira, 2001). Cell- or tissue-specific targeted disruption of Stat3 shows that Stat3 is required for normal function and activities in myeloid (Jenkins et al., 2002; Minami et al., 1996), thymus (Sano et al., 2001; Zhao et al., 2004), respiratory epithelium (Hokuto et al., 2004), heart (Hilfiker-Kleiner et al., 2004;

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Jacoby et al., 2003), mammary gland (Chapman et al., 1999; Humphreys et al., 2002), brain, and skin (Sano et al., 1999). Lack of Stat3 signaling pathway leads to defects in thymic hypoplasia architecture, chronic enterocolitis, delayed mammary gland involution, increased heart interstitial fibrosis and failure, elevated lung inflammatory response to hyperoxia, accumulation of immature hematopoietic progenitor cells, overreaction of macrophage and neutrophil, retarded skin healing, insulin resistance, and abnormal expression profile of gluconeogenic genes. Unique cyctoplasmic localization of Stat3 and p-Stat3 The uncommon distribution of Stat3 and p-Stat3 in cytoplasm of endomyocardiocytes raises some questions. The mechanism for differential immunoreactivity to Stat3 and pStat3 between nuclear and cytoplasmic compartments is not clear. The transport of p-Stat3 between nuclear and cytoplasm has been well studied (Kisseleva et al., 2002). Undetectable nuclear p-Stat3 in heart tissues could be due to the favored direction of transport of p-Stat3 from nuclei to cytoplasm. This may be temporary since mouse model shows that Stat3 is required for normal heart function (Hilfiker-Kleiner et al., 2004; Jacoby et al., 2003). Perinuclear distribution of Stat3 was previously observed in acrosome of mouse mature spermatogonia (Murphy et al., 2005). Similar pattern was not found in human counterpart but in several human heart tissues. Stat3 has been shown to interact with several molecules in the cytoplasm (Guo et al., 2002; Ndubuisi et al., 1999). The perinuclear pattern indicates the localization of Stat3 in Golgi complex and might provide new insight into Stat3 function in cytoplasm.

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