Differentiation (2000) 67:41–49 C Blackwell Wissenschafts-Verlag 2001 ORIGINAL ARTICLE Agnes Michalczyk ¡ Robert W . Brown ¡ John P. Collins M. Leigh Ackland Lactation affects expression of intermediate filaments in human breast epithelium Accepted in revised form: 24 August 2000 Abstract The human breast contains two epithelial lin- eages, luminal epithelial and myoepithelial. Specific pat- terns of expression of intermediate filaments have pre- viously been demonstrated in the resting breast. To de- termine how terminal differentiation and lactation influenced expression of intermediate filaments in breast epithelial cells, we used Western blot analysis to measure the levels of vimentin, a-smooth muscle actin, keratin 14, and keratin 18 in the resting and lactating breast. Confocal immunofluorescence was used to determine the subcellular site of localization of the intermediate filaments. Vimentin was localised to myoepithelial cells in both the resting and lactating gland. There was a four-fold increase in vimentin protein levels in lactating tissue relative to resting tissue, and this may be related to increased cellular activity of the myoepithelial cells which surround secretory alveoli. Alpha-smooth muscle actin and keratin 14 were detected in myoepithelial cells, and similar levels of expression were found in lactating and resting tissue. In the resting breast, keratin 18 and keratin 8 were detected in luminal epithelial cells in a filamentous form, whereas in lactating tissue it was pres- ent in a punctate form in luminal cells and also seen as granules in the lumen of alveoli. Our results indicate that A. Michalczyk ¡ M. Leigh Ackland ( ✉ ) Deakin University, Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Burwood Campus, Burwood, Victoria 3125, Australia e-mail: leigha/deakin.edu.au Tel: π 61 39251 7036 Fax: π 61 39251 7036 R. W. Brown Melbourne Pathology, Victoria 3066, Australia J. P. Collins Breast Unit, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia U.S. Copyright Clearance Center Code Statement: 0301–4681/2000/6701–41$ 15.00/0 intermediate filament expression patterns are altered in the lactating human breast, and this may reflect their role in the fully functional gland. Key words keratin 18, keratin 14, vimentin, human breast epithelial cells, lactation Introduction The human breast is unusual in that its fully differen- tiated state is not reached until adulthood when ter- minal differentiation and lactation occur in response to hormonal signals. At puberty and pregnancy, prolifer- ation and branching of epithelial tissue lead to the for- mation of a ductal network and terminal lobules [36]. During lactation, terminal lobules distend to form se- cretory alveoli into which milk is secreted [35]. Two major epithelial cell types can be distinguished in the breast, basal or myoepithelial cells and luminal cells. In the resting human breast, specific patterns of expression of intermediate filaments are found. Luminal epithelial cells express keratins 7, 8, 16, 18, and 19 and myo- epithelial cells express keratins 5 and 14 [28, 40, 32]. Myoepithelial cells are also identified by the presence of vimentin and a-smooth muscle actin [31]. Keratins comprise the intermediate filament cytoskel- eton of epithelial cells. Their function in providing struc- tural support is demonstrated in the skin blistering dis- order, epidermolysis bullosa, which is caused by muta- tions in keratins 14 and 5 [26, 6] and by keratin 14 null mice [23]. The role of non-epidermal keratins is not clear but expression of keratins is linked to developmental stage and differentiated state, suggesting a functional role in addition to structural [28, 25]. Intermediate filaments are not required for growth of simple epithelial cells [19, 43], and this may indicate a function in differ- entiation rather than structure [24]. In the developing