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Role of CD44 in the organization of keratinocyte pericellular hyaluronan

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Abstract

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44−/− animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.

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Abbreviations

bHABC:

Biotinylated hyaluronan binding complex

ELSA:

Enzyme linked sorbent assay

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Acknowledgments

We are grateful to Ms. Eija Rahunen and Mr. Kari Kotikumpu for preparing the histological specimens, and Ms. Riikka Kärnä, Arja Venäläinen and Eija Kettunen for taking care of the cell cultures and performing the hyaluronan measurements. We thank the personnel of the Laboratory Animal Center for taking care of the animals, and Biomater Center for providing us the confocal and transmission electron microscopes. The work was supported by grants from Finnish Cancer Foundation (RT), Juselius Foundation (RT, MT), EVO Funds of the University Hospital of Kuopio (MT), funds from BioCenter and Cancer Center of University of Eastern Finland (RT, MT), and Academy of Finland (MT).

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Authors and Affiliations

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

    Sanna Pasonen-Seppänen, Juha M. T. Hyttinen, Kirsi Rilla, Tiina Jokela, Markku Tammi & Raija Tammi

  2. Duke University School of Medicine, Durham, NC, USA

    Paul W. Noble

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  1. Sanna Pasonen-Seppänen
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  2. Juha M. T. Hyttinen
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  4. Tiina Jokela
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  5. Paul W. Noble
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Correspondence to Sanna Pasonen-Seppänen.

Electronic supplementary material

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418_2011_883_MOESM1_ESM.pdf

Supplementary Figure 1. The effect of CD44 siRNA transfection on CD44 mRNA and protein expression. The mRNA level of CD44 was analyzed with quantitative RT-PCR in HaCaT cells (a). Cell lysates were collected 2 days after siRNA transfection and analyzed by Western blotting (b, c). The data represent means and ranges of two independent experiments. (PDF 301 kb)

418_2011_883_MOESM2_ESM.tif

Supplementary Figure 2. The transfection efficiency of HaCaT cells. HaCaT cells were co-transfected with fluorescent reporter (blue) and siRNAs to establish the transfection efficiency, and stained for CD44 (red) and hyaluronan (green). (TIFF 18450 kb)

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Pasonen-Seppänen, S., Hyttinen, J.M.T., Rilla, K. et al. Role of CD44 in the organization of keratinocyte pericellular hyaluronan. Histochem Cell Biol 137, 107–120 (2012). https://doi.org/10.1007/s00418-011-0883-2

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