Summary
Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high, confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein, (HLP) and growth factor-Livertine (GF-Livertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and, Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis and dome formation of A6 cells in altered gravity.
Similar content being viewed by others
References
Abaza, N. A.; Leighton, J.; Schultz, S. G. Effects of ouabain on the function and structure of a cell line (MDCK) derived from canine kidney. I. Light microscopic observations of monolayer growth. In Vitro Cell. Dev. Biol. 10A:172–183; 1974.
Aberger, F.; Schmidt, G.; Richter, K. The Xenopus homologue of hepatocyte growth factor-like protein is specially expressed in the presumptive neural plate during gastrulation. Mech. Dev. 54:23–27; 1996.
Amsler, K.; Cook, J. S. Development of Na+-dependent hexose transport in a cultured line of porcine kidney cells. Am. J. Physiol. 242:C94-C101; 1982.
Asashima, M.; Nakano, H.; Shimada, K. Mesoderm induction in early amphibians embryos by activin A. Roux's Arch. Dev. Biol. 198:330–335; 1990.
Bellusci, S.; Moens, G.; Guadino, G.; Comoglio, P. M.; Nakamura, T.; Thiery, J. P.; Jouanneau, J. Creation of an hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity. Oncogene 9:1091–1099; 1994.
Bivic, A. L.; Hirn, M.; Riggo, H. HT-29 cells are in vitro models for the generation of cell polarity in epithelia during embryonic differentiation. Proc. Natl. Acad. Sci. USA 85:136–140; 1988.
Bullock, T. H.; Bodznick, D. A.; Northcutt R. G. The phylogenetic distribution of electroreception: evidence for convergent evolution of a primitive vertebrate sense modality. Brain. Res. 287:25–46; 1983.
Cereijido, M.; Ehrenfeld, J.; Meza, I.; Martinez-Palomo, A. Structural and functional membrane polarity in cultured monolayers of MDCK cells. J. Membr. Biol. 52:147–159; 1980.
Cohen-Luria, R.; Rimon, G.; Moran, A. PGE2 inhibits Na-K-ATPase activity and ouabain binding in MDCK cells. Am. J. Physiol. 264 (1 Pt 2):F61-F65; 1993.
Fantini, J.; Abadie, B.; Tirard, A.; Remy, L.; Ripert, J. P.; El Battari, A.; Marvald, J. Spontaneous and induced dome formation by two clonal cell populations derived from a human adenocarcinoma cell line, HT-29. J. Cell. Sci., 123:89–100; 1985.
Frieda, C. P.; John, M. G.; Philip, S. R. The effect of ionophores and related agents on the induction of doming in a rat mammary epithelial cell line. J. Cell. Physiol. 123:89–100; 1985.
Furue, M.; Okamoto, T.; Hayashi, H.; Sato, J. D.; Asashima, M.; Saito, S. Effects of hepatocyte growth factor (HGF) and activin A on the morphogenesis of rat submandibular gland derived epithelial cells in serum free collagen gel culture. In Vitro Cell. Dev. Biol. 35A(3):131–135; 1999.
Garty, H.; Palmer, L. Epithelial sodium channels: function, structure and regulation. Physiol. Rev. 77:359–396; 1997.
Glynn, I. M.; Karlish, S. J. D. The sodium pump Annu. Rev. Physiol. 37:13–55; 1975.
Gomez-Lechon, M. J.; Guillen, I.; Ponsoda, X.; Fabra, R.; Trullenque, R.; Nakamura, T. Cell cycle progression proteins (cyclins), oncogene expression, and signal transduction during the proliferative response of human hepatocyte growth factor. Hepatology 23:1012–1019; 1996.
Grant, M. E.; Neufeld, T. K.; Cragoe, E. J., Jr.; Welling, L. W.; Grantham, J. J. Arginine vasopressin stimulates net fluid secretion in a polarized subculture of cyst-forming MDCK cells. J. Am. Soc. Nephrol. 2:219–227; 1991.
Hardman, P.; Spooner, B. S. Localization of extracellular matrix compounts in developing mouse salivary glands by confocal microscopy. Anat. Rec. 234(3):452–459; 1992.
Hiroe, T.; Kinoshita, T.; Yasuda, C.; Takaoki, M. Motion control of three-dimensional clinostat. Jpn. J. Biol. Sci. Space 12(3):314–315; 1998.
Horic, S.; Higashihara, E.; Nutahara, K.; Mikami, Y.; Okubo, A.; Kano, M.; Kawabe, K. Mediation of renal cyst formation by hepatocyte growth factor. Lancet 344(8925):789–791; 1994.
Hoson, T.; Kamisaki, S.; Masuda, Y.; Yamashita, M.; Buchen, B. Evaluation of the three-dimensional clinostat as a simulator of weightlessness. Planta 203(Suppl.):S187-S197; 1997.
Ishibashi, K.; Sasaki, S.; Sakamoto, H.; Nakamura, Y.; Hata, T.; Nakamura, T.; Marumo, F. Hepatocyte growth factor is a paracrine factor for renal epithelial cells: stimulation of DNA synthesis, and Na+, K+-ATPase activity. Biochem. Biophys. Res. Commun. 182(2):960–965; 1992.
Kaneko, A.; Hayashi, N.; Tanaka, Y.; Horimoto, M.; Ito, T.; Sasaki, Y.; Fusamoto, H.; Kamada, T. Activation of Na+/H+ exchanger by hepatocyte growth factor in hepatocytes. Hepatology 22(2):629–636; 1995.
Khaoustov, V. I.; Darlington, G. I.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Yoffe, B. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell. Dev. Biol. 35A(9):501–509; 1999.
Klebe, R. J.; Grant, A.; Grant, G.; Ghosh, P. Cycli-AMP deficient MDCK cells form tubules. J. Cell. Biochem. 59:453–462; 1995.
Komatsu, N.; Yasuda, C.; Matsui, K.; Tsunewaki, H.; Takaoki, M. The development of 3D clinostat in Mitsubishi Heavy Industries. Jpn. J. Biol. Sci. Space 12(3):316–317; 1998.
Kumei, Y.; Nakajima, T.; Sato, A.; Kamata, N.; Fnomoto, S. Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity. J. Cell. Sci. 93(2):221–226; 1989.
Lamb, J. F.; Ogden, P.; Simmons, N. L. Autoradiographic localization of [3H]-ouabain bound to cultured epithelial cell monolayers of MDCK cells. Biochem. Biophys. Acta. 644:333–340; 1981.
Leighton, J.; Brada, Z.; Estes, L. W.; Justh, G. Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science 163:472–473; 1969.
Lever, J. E. Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK). Proc. Natl. Acad. Sci. USA 76:1323–1327; 1979.
Levrat, J. H.; Palevody, C.; Daumas, M.; Ratovo, G.; Hollande, E. Differentiation of the human pancreatic adenocarcinoma cell line (Capan-1) in culture and co-culture with fibroblasts dome formation. Int. J. Cancer. 42(4):615–621; 1988.
Louvard, D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc. Natl. Acad Sci. USA 77:4123–4136; 1980.
Matsumoto, K.; Nakamura, T. Roles of HGF as a pleiotropic factor in organ regeneration. EXS 65:225–249; 1993.
Michalopoulos, G.; Houck, K. A.; Dolan, M. L.; Luetteke, N. C. Control of hepatocyte replication by two serum factors. Cancer Res. 44:4414–4419; 1984.
Moberly, J. B.; Fanestil, D. D. A monoclonal antibody that recognizes a basolaternal membrane protein in A6 epithelial cells. J. Cell. Physiol. 135:63–70; 1988.
Molnar, G.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R. Skeletal muscle satellite cells cultured in simulated microgravity. In Vitro Cell. Dev. Biol. 33A(5):386–391; 1997.
Montesano, R.; Matsumoto, K.; Nakamura, T.; Orei, L. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67(5):901–908; 1991.
Morey-Holton, E. R.; Cone, C. M. Bone as a model system for organ/tissue response to microgrativy. In: Asashima, M.; Malacinski, G. M. ed. Fundamentals of space biology. Jpn. J. Sci. Soc. Press, Tokyo, 1990:113–122.
Nakamura, T.; Nawa, K.; Ichihara, A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem. Biophys. Res. Commun. 122:1450–1459; 1984.
Perkins, F.; Handler, J. Transport properties of toad kidney epithelia in culture. Am. J. Physiol. 241(12):C154-C159; 1981.
Pinto, M.; Robine, L. S.; Appay, M. D., et al. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell. 47:323–330; 1983.
Pollack, L. R.; Tate, E. H.; Cook, J. S. Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain. J. Cell. Physiol. 106(1):85–97; 1981.
Polulyakh, Y. A. Phospholipid and fatty acid composition of the pea root cell plasma membrane under clinostating. Dokl. Akad. Nauk. UKR. SSR 10:67–69; 1988.
Rahmani, M.; Nadori, F.; Durand-Schneider, A. M.; Lardeux, B.; Bernuau, D. Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes. J. Hepatol. 30(5):916–925; 1999.
Rodriguez-Boulan, E.; Nelson, W. J. Morphogenesis of the polarized epithelial cell phenotype. Science 245:718–725; 1989.
Romand, R.; Romand, M. R. Qualitative and quantitative observations of spiral ganglion development in the rat. Hear. Res. 18(2):111–20; 1985.
Rose, M. I.; Brown, D. C.; Pellis, N. R.; Crisera, C. A.; Colen, K. L.; Longaker, M. T.; Gittes, G. K. Effects of microgravity on the embrhonic pancreas. In Vitro Cell. Dev. Biol. 35A(10):560–563; 1999.
Schatz, A.; Reitstetter, R.; Linke-Hommes, A.; Briegleb, W.; Slenzka, K.; Rahmann, H. Gravity effects on membrane processes. Adv. Space Res. 14(8):35–43; 1994.
Stern, C. D.; Ireland, G. W.; Herrick, S. E.; Gherardi, E.; Gray, J.; Perryman, M.; Stoker, M. Epithelial scatter factor and development of the chick embryonic axis. Development 110:1271–1284; 1990.
Stocker, M.; Gherardi, E.; Perryman, M.; Gray, J. Scatter factor is a fibroblast-derived modulator. Nature 327:239–242; 1987.
Streit, A.; Stern, C. D.; Thery, C.; Ireland, G. W.; Aparico, S.; Sharpe, M. J.; Gherardi, E. A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation. Development 121:813–824; 1995.
Susan, C. K. Dome formation by a human colonic adenocarcinoma cell line (HCA-7). Cancer Res. 45:3790–3795; 1985.
Takahasi, H. Gravimorphogenesis: gravity-regulated formation of the peg in cucumber seedlings. Planta 203:S164-S169; 1997.
Thomas, S. R.; Schultz, S. G.; Lever, J. E. Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation: dissociation from effects on transepithelial resistance and cyclic AMP levels. J. Cell. Physiol. 113:427–432; 1982.
Uochi, T.; Takahashi, S.; Ninomiya, H.; Fukui, A.; Asashima, M. The Na+, K+-ATPase a subunit requires gastrulation in the Xenopus embryo. Dev. Growth Differ. 39:571–580; 1997.
Vallet, V.; Chraibi, A.; Gaeggeler, H. P.; Horisberger, J. D.; Rossier, B. C. An epithelial serine protease activates the amiloride sensitive sodium channel. Nature 389(6651):607–610; 1997.
Verrey, F.; Kairouz, P.; Schaerer, E.; Fuentes, P.; Greering, K.; Rossier, B. C.; Kraehenbuhl, J. P. Primary sequence of Xenopus laevis Na+, K+-ATPase and its localization in A6 kidney cells. Am. J. Physiol. 256(25):F1034-F1043; 1989.
Wang, A. Z.; Ojakian, G. K.; Nelson, W. J. Steps in the morphogenesis of a polarized epithelium 1. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts. J. Cell. Sci. 95:137–151; 1990.
Watari, H.; Ogiso, Y.; Abe, K., et al. Dome formation induced by v-H-ras oncogene in a human choriocarcinoma cell line. Placenta 17:443–449; 1996.
Weidner, K. M.; Behrens, J.; Vanderkerckhove, J.; Birchmeier, W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol. 111:2097–2108; 1990.
Yoneyama, Y.; Lever, J. E. Induction of microvillar hydrolase activities by cell density and exogenous differentiation inducers in an established kidney epithelial cell line (LLC-PK). J. Cell. Physiol. 121:64–73; 1984.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ichigu, J., Asashima, M. Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity. In Vitro Cell.Dev.Biol.-Animal 37, 31–44 (2001). https://doi.org/10.1290/1071-2690(2001)037<0031:DFATMB>2.0.CO;2
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1290/1071-2690(2001)037<0031:DFATMB>2.0.CO;2