Immunolocalization and Determination of Proteins Involved in the Signal Transduction Mechanism of p-ERK 1/2 in Canine Prostates with PIN
K.S. Oliveira; R. Laufer-Amorim; J.F. Pérez-Gutiérrez; J. Oliva-Hernandez; G.H. Toniollo
Dept. of Preventive Veterinary Medicine and Animal Reproduction, FCAV/UNESP, Jaboticabal, São Paulo, Brazil
The prostate is the only accessory gland of the genital tract in the canine specie, which can be affected by several diseases in mid-age and geriatric patients (Krawiec & Helfin 1992). Among the main causes of prostatic distress, prostatic intraepithelial neoplasia (PIN) is highlighted as one of the most common ones in dogs. PIN is considered as a preneoplastic lesion and morphological alterations such as disorganization of the basal cellular layer, increased proliferative capacity and microvascular density are factors that reinforce its pre-malignant characteristic (Waters & Bostwick 1997). Proliferation, differentiation and organization of the animal tissue cells are controlled by various mechanisms of intercellular communications. One of them involves the polypeptide chains, such as growth factors, which activate membrane receptors for the transduction of signs of proliferation and differentiation (Massagué 1987). This study aimed the immunolocalization the proteins CYR61, EGF, TGFα, EGFr and p-ERK1/2 in canine prostate owning PIN by means of immunohistochemistry (IHC) and to determinate their molecular weight by Western Blotting (WB).
Materials and Methods
In the present trial, 87 prostatic samples, taken from 29 dogs, were assessed. Each prostate was fractioned in three pieces based on three prostatic anatomical patterns: cranial (next to the bladder), medial (intermediary fraction), and caudal section (next to the penile urethra) of the gland. All sections were transected and stored in two ways: in buffered 10% formalin and frozen in -80°C. After histological evaluation, three prostatic fragments (3.44%) owning PIN and five (5.74) normal ones were identified. All the fragments (n = 8) were assessed by the IHC technique with anti-CYR61 (1/400), anti-EGF (1/20), anti-p-ERK1/2 (1/200) polyclonal antibodies and anti-TGF-α (1/20) and anti-EGFr (1/20) monoclonal antibodies. The secondary polyclonal and monoclonal bitionolate followed the concentration pattern indicated by the manufacturer. The histological samples treated with polyclonal and monoclonal antibodies were evaluated for the presence or absence of staining of stromal components under optic light microscopy: smooth muscular fibbers (SMF), fibroblasts (F), arterial or arteriolar smooth musculature (ASM), endothelial cells (EC) and mononuclear cell infiltration (MC); and acinar epithelial components: cytoplasm and nucleus of the secretory cells. In order to perform the WB, the frozen sections were lysed and 50μg of protein were added to polyacrylamide-SDS PAGE 10% gel in addition to the molecular weight markers, which were transferred to a membrane of PVDF in 110v voltage for one hour. The anti-CYR61 (1/3000), anti-EGF (1/250), anti-p-ERK1/2 (1/500) polyclonal and anti-TGF-α (1/200) e anti-EGFr (1/100) monoclonal antibodies were used overnight under 4°C. The concentrations employed for the secondary antibodies (Amersham Bioscience, NJ, USA) were 1/2000 for the polyclonal and 1/500 for the monoclonal ones with 30 minutes of incubation under room temperature. The membranes remained exposed to the revealing solution (Cat#34080, Pierce, MD, USA) for 3-10 minutes and to the radiographic film for 1030 minutes. The radiographic films were scanned (GS 800 Calibrated Densitometer, Bio-Rad labs, UK) and the molecular weights were calculated by the machine's specific software.
All the studied prostatic sections (n = 8) presented positive staining for the proteins CYR61, EGF, TGF-α, EGFr and p-ERK1/2 and the stromal and acinar epithelial components also were positively stained in different intensity levels. The prostatic samples that were considered as normal presented greater SMF and ASM immunoreactivity to the protein CYR61 and the major acinar epithelial immunoreactivity was found in the nucleus. In the other hand, the fragments diagnosed PIN showed increased stromal immunoreactivity in the SMF and F. However, the reactivity was similar in the nucleus and cytoplasm in the epithelial component. The WB of this protein did not reveal any visible band diagnosed as normal. Nonetheless, concerning the samples diagnosed PIN, there was one visible band with 37.93 kDa of mean molecular weight. Concerning the growth factor EGF, the stromal immunoreactivity in the normal samples was detected in the SMF and ASM, and in the epithelial component, it was found in the nucleus. In the fragments with PIN, greater stromal immunoreactivity was found in the SMF and EC, and in the acinar epithelium, it was determined in the cytoplasm. WB test of this protein revealed two visible bands with normal diagnosis, which had 46.76 and 41.31 kDa of mean molecular weight. However, there was only one visible band revealing 42.25 kDa of mean molecular weight in the samples of prostates owning PIN. Regarding the growth factor TGF-α, there was no highlighted stromal component in the samples diagnosed normal and the cytoplasm was the structure of the acinar epithelium that presented greater reactivity. In the PIN samples, stromal immunoreactivity was given in SMF, F and MC, and in the epithelial component, it was found in the cytoplasm. In the cytoplasm of the samples diagnosed PIN and normal, there was a characteristic of immunoreaction which was classified as punctiform and perinuclear. WB test of this protein revealed a visible band diagnosed normal and mean molecular weight of 51.22. In the other hand, the PIN samples revealed three visible bands and their mean molecular weights were 191.79, 51.87 and 38.12 kDa. The membrane receptor EGFr presented similar result in the stromal component in the samples diagnosed normal and there was nuclear immunoreactivity in the epithelial component. In the PIN samples, there was immunoreactivity in the SMF, F, in the stroma and nucleus of the epithelial cells. WB test revealed a single visible band diagnosed normal presenting 48.08 kDa of mean molecular weight. However, there was no visible band in the PIN samples. The protein p-ERK1/2 presented stromal immunoreactivity in the SMF and F and nuclear, in the acinar epithelial cells in the samples diagnosed normal and PIN. WB test did not show any visible band in the fragments with no alterations. In contrast, there was a single visible band weighing 37.79 kDa of mean molecular weight in the PIN samples.
Discussion and Conclusions
The anti-CYR61, anti-EGF, anti-p-ERK1/2 polyclonal antibodies and anti-TGF-α e anti-EGFr monoclonal antibodies were effective in staining stromal and epithelial components in the diagnosis of the studied canine prostatic samples, even in different staining intensity between and inside each evaluated component. CYR61 protein is rapidly expressed in fibroblasts and endothelial cells in response to growth factors and cytokines (Kireeva et al. 1996) and was identified in smooth muscular cells both in cytoplasm and nucleus (Tamura et al. 2001). Studies involving placentas during the embryonic development (Kireeva et al. 1997) revealed immunoreactivity in cytoplasm and/or nucleus of the assessed cells. The anti-CYR61 can present three bands, in which there are two bands representing 25 and 37 kDa that are considered as modified forms of the protein, and another band of 45 kDa, which is its complete form. According to data from GenBank, the protein pro-CYR61 presents 41.64 kDa in the dog. Studies highlights that the components of the EGF family are extensively distributed in the organism. In dogs, the precursor of EGF was isolated in the urine (Kobayashi et al. 1985). Research verified the action of this growth factor in the ASM in dogs submitted to different anesthetic protocols, vasodilatation was obtained (Gan et al. 1987), which confirms that protein EGF is present systemically and actuates in SMF of dogs. In a study performed with different tissues of humans in ages varying from embryo to adult, the authors localized a nuclear staining in mammary and submandibular and sweat gland (Kasselberg et al. 1985). According to the GenBank, the molecular weight of the protein pro-EGF in the dog is 134.29 kDa. In a study, pro-EGF originates two reactive forms of EGF: one of them is a low molecular weight molecule (6 kDa) and the other one, low molecular weight (45kDa). It was supposed that 45 kDa form is an intermediary form between the pro-EGF and the 6 kDa EGF (Journe et al. 1997). In this context, the molecular weights for the EGF encountered in canine prostatic samples in the present study is probably molecular weight of the intermediary form of this protein. In canine specie, this growth factor was localized in the uterus in different phases of the estrous cycle (Tamada et al. 2005). TGF-α is involved in processes of normal growth and development of several tissues. Additionally, it plays an important role in wound repair, promotes angiogenesis and probably performs an immunologic function due to its presence in macrophages and eosinophils (Kumar et al. 1995), which explains the fact that greater immunoreactivity was found in the stromal component of the studied tissue samples. In humans, the prostatic epithelial cells are TGF-α producers (Taylor & Ramsdell 1993). Regarding the immunoreaction in the cytoplasm described as punctiform at the perinuclear zone, there was no data in the researched literature that could support the discussion. In an assessment where the secretion of TGF-α by alveolar macrophages was monitored in rabbits from zero to four months old, three isoforms of TGF-α were found, which presented 46; 30 and 14.3 kDa of molecular weight (Wagner et al. 1995). According to the GenBank, the dog pro-TGF-α presents 49.19kDa of molecular weight. The values encountered in the present study differ from those observed in the researched literature. A reasonable explanation for this fact could be the difference among the animal models and the degree of tissue alteration, because in other studies samples of tissue in development were taken. EGFr was identified in chain by Iglesias-Núñez et al. (2008) in normal uterus of bitches and in those owning the cystic endometrial hyperplasia complex (CEHC). In the reproductive tract of male dogs, EGFr was associated to the augmentation of fertility, due to its involvement in the proliferation of the Sertoli cells when it was stimulated by acetylcholine-muscarine receptors (Porto et al. 2008). The localization of this protein in the present assessment was in accordance to the findings of other studies for the EGF and TGF-α, as the EGFr is a common receptor for both growth factors and those structures of the stromal component also present considerable immunoreactivity to the EGF and TGFα. This growth factor was identified in the nucleus of healthy dog endometrial gland cells and those owning CEHC (Iglesias-Núñez et al. 2008). The nuclear localization of EGFr is related to the state of proliferation of the tissues and to the potential role of transcription or co-activation of mitosis (Lin et al. 2001). In studies involving the human placenta, two isoforms of the receptor EGFr of 170 kDa were identified, which had 60 and 110 kDa of molecular weight. The second one was related to the cellular surface, which may constitute an excellent indicator to the progression of diseases involved on the placental development (Reither & Maihle 2003). GenBank assures that dog pro-EGFr present 175,41 kDa of molecular weight. In the present study lower molecular weight when compared to those described in the researched literature. This fact suggests that more studies should be accomplished in order to enlighten this isoform in the dog prostate. The protein ERK1/2 is involved in several cellular events, such as cellular proliferation, integration of signs inside and among cells, apoptosis, genes transcription, cellular differentiation, neuronal plasticity, among other functions (Lenz et al. 2000). One of the main functions of the protein ERK1/2 is the activation by phosphorylation of effector proteins present in the nucleus of the cells that codify the received stimuli, promoting the specific action, i.e., genetic transcription, protein production, signalization to a new cellular cycle, tissue differentiation and development and beginning of the apoptosis process. According to data from the GenBank, the dog proteins p-ERK 1 and p-ERK 2 present 41,26 and 42,75 kDa of molecular weight, respectively. No data in the researched literature that could be compared to the values obtained in the present trial was found. The study of the proteins involved in the cellular differentiation and proliferation has been applied in various tissue alterations in the veterinary medicine. The comparison of the immunolocalization of those proteins in normal and altered tissue is crucial in order to understand their action, to establish routes of transduction of signs and to offer data to studies related to the pharmacology for blocking the overexpression and/or activation of those proteins, which may reduce the chances of neoformations of altered tissues.
Financing agencies: FAPESP, CAPES.
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