Investigation of apoptotic activity in the lumen formation of salivary gland ducts: comparative analysis between animal and human based models

Author(s)

Tathyane Harumi Nakajima Teshima

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2016-03-22

Examining board members

Silvia Vanessa Lourenço; Cláudia Malheiros Coutinho Camillo; Victor Elias Arana Chavez; Regina Schultz

• Advisor: Silvia Vanessa Lourenço

Abstract

Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions. (AU)

• FAPESP's process: 11/18865-2 - Investigation of apoptotic activity in the lumen formation of salivary gland ducts: comparative analysis between animal and human based models
• Grantee: Tathyane Harumi Nakajima Teshima
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)