Journal of Cell Science 111, 2043-2054 (1998) 2043 Printed in Great Britain © The Company of Biologists Limited 1998 JCS3760 Distinct subcellular localization of calcium binding S100 proteins in human smooth muscle cells and their relocation in response to rises in intracellular calcium Anna Mandinova1, Dan Atar2, Beat W. Schäfer3, Martin Spiess1, Ueli Aebi1 and Claus W. Heizmann3,* 1Maurice E. Müller-Institute, Biocentrum, University of Basel, 4056 Basel, Switzerland 2Division of Cardiology, Department of Internal Medicine, University Hospital, 8032 Zürich, Switzerland 3Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, 8032 Zürich, Switzerland *Author for correspondence (e-mail: [email protected]) Accepted 19 May; published on WWW 30 June 1998 SUMMARY Changes in cytosolic Ca2+ concentration control a wide associated with the sarcoplasmic reticulum and with actin range of cellular responses, and intracellular Ca2+-binding stress fibers. In contrast, S100A2 was located primarily in proteins are the key molecules to transduce Ca2+ signaling the cell nucleus. Using a sedimentation assay and via interactions with different types of target proteins. subsequent electron microscopy after negative staining, we Among these, S100 Ca2+-binding proteins, characterized by demonstrated that S100A1 directly interacts with a common structural motif, the EF-hand, have recently filamentous actin in a Ca2+-dependent manner. After attracted major interest due to their cell- and tissue-specific thapsigargin (1 µM) induced increase of the intracellular expression pattern and involvement in various pathological Ca2+ concentration, specific vesicular structures in the processes. The aim of our study was to identify the sarcoplasmic reticulum region of the cell were formed with subcellular localization of S100 proteins in vascular smooth high S100 protein content. In conclusion, we demonstrated muscle cell lines derived from human aorta and intestinal a distinct subcellular localization pattern of S100 proteins smooth muscles, and in primary cell cultures derived from and their interaction with actin filaments and the arterial smooth muscle tissue under normal conditions and sarcoplasmic reticulum in human smooth muscle cells. The after stimulation of the intracellular Ca2+ concentration. specific translocation of S100 proteins after intracellular Confocal laser scanning microscopy was used with a Ca2+ increase supports the hypothesis that S100 proteins specially designed colocalization software. Distinct exert several important functions in the regulation of Ca2+ intracellular localization of S100 proteins was observed: homeostasis in smooth muscle cells. S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found Key words: Smooth muscle cell, Calcium-binding S100 protein, predominantly in the cytosol where they were strongly Actin, Confocal microscopy, Immunohistochemistry INTRODUCTION F-helices of parvalbumin. The EF-hand domain consists of a helix-loop-helix motif that binds Ca2+ selectively and with high Calcium ions acting as second messengers transduce affinity (Kretsinger, 1980; Nakayama and Kretsinger, 1994). extracellular signals into a wide variety of intracellular Whereas calmodulin, the most prominent member of the EF- responses and thereby regulate many different biological hand protein family, is ubiquitously expressed and processes such as secretion, proliferation, differentiation, multifunctional (Cohen and Klee, 1988; James et al., 1995), transcription, apoptosis, and muscle contraction (for reviews most of the other EF-hand proteins are expressed in a tissue- see Parekh and Penner, 1997; Berridge, 1997). In recent years and cell-specific manner (Heizmann and Hunziker, 1991). This it has become clear that a number of human diseases, including is also true for the S100 proteins representing the largest cardiomyopathies, hypertension, neurodegenerative and subfamily of EF-hand Ca2+-binding proteins (Donato, 1991; neoplastic disorders are linked to altered Ca2+ handling Hilt and Kligman, 1991; Fano et al., 1995; Schäfer and mediated by Ca2+-binding proteins (Heizmann and Braun, Heizmann, 1996). 1992, 1995; van Eldik and Griffin, 1994; Richard et al., 1995; S100 proteins are acidic proteins of low molecular mass (10- Polans et al., 1996; Heizmann, 1996). 12 kDa), containing two distinct EF-hands with significantly The largest family of Ca2+-binding proteins shares a different affinities for Ca2+. Both EF-hands are flanked by common structural motif, the EF-hand, named after the E- and hydrophobic regions at either terminal and are separated by a 2044 A. Mandinova and others central hinge region. The carboxy-terminal EF-hand contains were visualized by immunofluorescence using confocal laser- the canonical Ca2+-binding loop encompassing 12 amino acids, scanning microscopy. Our results indicate that S100 Ca2+- whereas the amino-terminal loop consisting of 14 amino acids binding proteins exhibit a distinct subcellular localization is specific for S100 proteins. To date, some 17 different pattern in human vascular smooth muscle cell lines and in proteins have been assigned to the S100 protein family that primary cell cultures derived from smooth muscle tissue. We display various degrees of amino acid sequence homology in provide evidence that the S100A1 protein specifically interacts the range of 25% to 65%. At least thirteen S100 genes were with filamentous actin (F-actin) in smooth muscle cells, thus found to be clustered on human chromosome 1q21 (Engelkamp possibly representing a new regulatory system together with et al., 1993; Wicki et al., 1996a,b), leading to the introduction calponin and caldesmon in modulating smooth muscle of a new S100 nomenclature (Schäfer et al., 1995). The contraction. In addition, they specifically translocate within localization of the S100 gene cluster on human chromosome these cells after an increase of the intracellular Ca2+ 1q21 is of special interest since a number of rearrangements concentration. These experiments provide new insights into the (deletions, translocations, duplications) have been found to function of S100 proteins through their interaction with occur in this chromosomal region in cancer cells (Gendler et different target proteins in the Ca2+-signal transduction cascade. al., 1990; Hoggard et al., 1995; Forus et al., 1995; Bartoli et al., 1996; Weterman et al., 1996). Alterations in S100 expression have been demonstrated in MATERIALS AND METHODS diseases such as Down’s syndrome (Allore et al., 1988), Alzheimer’s disease (van Eldik and Griffin, 1994), chronic Cell cultures inflammation (Rammes et al., 1997), and cardiomyopathies All experiments were performed with two stable smooth muscle cell (Remppis et al., 1996). lines derived from human aorta (HVSMC ATCC-13145 CRL-1999) S100 proteins are thought to exert their effect through Ca2+- and from human jejunum (HISM ATCC CRL-1692). Cells obtained regulated interactions with specific intracellular target proteins. from the American Type Culture collection (Maryland, USA) were Some intracellular S100 target proteins are myosin (Burgess et grown in Dulbecco’s modified Eagle’s medium (DMEM), al., 1984), tubulin (Donato et al., 1989), microtubule- supplemented with 1% L-glutamine, 10% fetal bovine serum, 100 units/µl penicillin and streptomycin (all from Gibco BRL). The cells associated tau-proteins (Baudier and Cole, 1988), glial were incubated in a humidified incubator (Heraeus, Switzerland) at fibrillary acidic protein (Bianchi et al., 1996), tropomyosin 5% CO2and 37°C. Cells in passages 3 through 5 were used for (Gimona et al., 1997), phosphoglutamase (Landar et al., 1996), analysis. twitchin kinase (Heierhost et al., 1996), cytosolic Primary cultures derived from human arterial smooth muscle tissue phospholipase A2 (Wu et al., 1997), Ca2+ release channel were a kind gift from Dr Z. Yang, University of Zurich, Switzerland. (ryanodine receptor) of the sarcoplasmic reticulum (SR) Briefly, cells were prepared from the left internal mammary artery of (Treves et al., 1997), and annexins (Garbuglia et al., 1996). patients undergoing aorto-coronary bypass surgery. The work was Here we demonstrate that smooth muscle cells represent a performed in accordance with the requirements of the institutional valuable model system to investigate the distinct subcellular review committee for the use of human material. Immediately upon localization and the functional role of S100 proteins since these tissue removal samples were taken to the laboratory and processed within 30 minutes. Following removal of the adventitia, tissues were cells co-express S100A1, S100A2, S100A4, and S100A6. minced into blocks of approximately 1 mm3. Tissue blocks were S100A1, found to be expressed in muscle tissues transferred to a sterile 25 cm2 culture flask covered with fresh DMEM (Engelkamp et al., 1992; Pedrocchi et al., 1993; Song and plus 10% fetal bovine serum, 1% L-glutamine, and 100 units/µl Zimmer, 1996; Remppis et al., 1996), was also shown to penicillin and streptomycin. Flasks were placed in a humidified activate invertebrate giant protein kinases involved in the incubator for 6 hours to allow time for the tissue blocks to attach to regulation of muscle function (Heierhorst et al., 1996), and the culture surface. After 6 hours, fresh DMEM containing L- modulates adenylate cyclase activity (Fano et al., 1989a) and glutamine, antibiotics, and FBS was added and flasks were returned the