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HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

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Abstract

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.

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Abbreviations

bHABC:

Biotinylated hyaluronan binding complex

ELSA:

Enzyme-linked sorbent assay

fHABC:

Fluorescent hyaluronan binding complex

FBS:

Fetal bovine serum

GFP:

Green fluorescent protein

HA:

Hyaluronan

HAS:

Hyaluronan synthase

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscopy

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Acknowledgments

Special thanks are due to Laura Halttunen, Heli Huttunen, Eija Kettunen and Eija Rahunen for expert technical assistance. We also want to thank Satu Myllymäki for valuable help in mitotic spindle orientation analysis. This work was supported by the Academy of Finland, grants #131771 (S P–S.), #114330&#135560 (A.M.) and #107173 (M.T.) and by Sigrid Juselius Foundation (A.M., R.T. and M.T.) and the special funds (Kärkihanke) allocated by the University of Eastern Finland to the Cancer Center of Eastern Finland.

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Authors and Affiliations

  1. Department of Anatomy, School of Medicine, Institute of Biomedicine, University of Eastern Finland, P.O.B. 1627, 70211, Kuopio, Finland

    Kirsi Rilla, Sanna Pasonen-Seppänen, Riikka Kärnä, Hannu M. Karjalainen, Kari Törrönen, Ville Koistinen, Markku I. Tammi & Raija H. Tammi

  2. Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu, Oulu Center for Cell–Matrix Research, Oulu, Finland

    Terhi Teräväinen & Aki Manninen

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  1. Kirsi Rilla
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  2. Sanna Pasonen-Seppänen
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Rilla, K., Pasonen-Seppänen, S., Kärnä, R. et al. HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium. Histochem Cell Biol 137, 153–164 (2012). https://doi.org/10.1007/s00418-011-0896-x

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