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Vol. 17, Issue 7, 3291-3303, July 2006

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Rho Kinase, Myosin-II, and p42/44 MAPK Control Extracellular Matrix-mediated Apical Bile Canalicular Lumen Morphogenesis in HepG2 Cells

Hilde Herrema^*, Dominika Czajkowska^*, Delphine Théard^*, Johanna M. van der Wouden^*, Dharamdajal Kalicharan, Behnam Zolghadr^*, Dick Hoekstra^*, and Sven C.D. van IJzendoorn^*

Sections of *Membrane Cell Biology and Electron Microscopy, Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands

Submitted January 24, 2006; Revised April 18, 2006; Accepted April 27, 2006
Monitoring Editor: Keith Mostov

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Sven C.D. van IJzendoorn (s.c.d.van. ijzendoorn{at}med.umcg.nl)

Abbreviations used: BC, bile canalicula(i); BDM, 2,3-butanedione; BSA, bovine serum albumin; C6-NBD, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid; ECM, extracellular matrix; ETA, eicosatetranoic acid; FCS, fetal calf serum; HBSS, Hank's balanced salt solution; MLC2, myosin light chain 2; NDGA, nordihydrouaiaretic acid; R123, rhodamine 123; ROCK, rho kinase; SM, sphingomyelin; TEM, transmission electron microscopy

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