Expression Patterns of Connexins in Merkel Cell Carcinoma and Adjacent Epidermis

Nikolas K. Haass, Pia Houdek, Johanna M. Brandner and Ingrid Moll

Department of Dermatology and Venerology, University Hospital Hamburg­Eppendorf, Hamburg, Germany

Summary

Alterations in cell-cell communication play an important role in carcinogene­sis. The specificity of communication is controlled by the type of connexins (Cx) forming gap junctions (GJ). In this work, we show an induction of Cx30 and Cx26 in normal interfollicular epidermis adjacent to Merkel cell carcinoma. This observation and previous inves­tigations of gap junctional communication in other malignant skin tumours give a hint that the change of the composition of gap junctions in the epidermis adjacent to malignant skin tumours may play a role in their metastasization.

Introduction

Gap junctions (GJ) play an important role in the communication of cells. They form channels between adjacent cells allowing the diffusion of molecules with a molecular weight of up to 1000 Da. These include ions, metabolites and second messenger molecules (Loewenstein 1981; Spray 1994). GJ consist of two hemichannels called connexons (one of each cell), each formed by six polypep­tides belonging to a family of transmembrane proteins called connexins (Cx; for review see Beyer et al. 1990; Richard 2000). Various experiments have demon­strated the existence of homotypic and heterotypic channels, formed by identical or different connexons. Moreover, connexons can consist of either identical or dif­ferent connexins (homomeric/heteromeric ). However, not all connexins and con­nexons are compatible (Elfgang et al. 1995; Stauffer 1995; Jiang and Goodenough 1996). The type of connexins forming GJ influences the selectivity of the channels and thereby controls the specificity of cell-cell communication (Brissette et al. 1994; Elfgang et al. 1995; Veenstra 1996).

Ten of the known 21 different connexins are expressed in human skin. GJ have been supposed to be involved in the regulation ofkeratinocyte growth, differentia-

K. I. Baumann et al. (eds.), The Merkel Cell© Springer-Verlag Berlin Heidelberg 2003

220 Haass et al.

tion and migration (Pitts et al. 1988; Salomon et al. 1993; Brissette et al. 1994; Goliger and Paul 1995). Moreover, Cx26 and Cx30 are induced during wound healing (Goliger and Paul 1995; Brandner, in prep.), psoriasis (Labarthe et al. 1998); and treatment with all-trans-retinoic-acid (Masgrau-Peya et al. 1997). Ge­netic studies have demonstrated the importance of epidermal gap junctions as mu­tations in their connexins are associated with autosomal dominant epidermal dis­eases.

There is evidence suggesting that connexins play a role in tumour biology (for review, see Krutovskikh and Yamasaki 1997; Li et al. 2002). Because of the shown changes of the expression pattern of Cx26, e.g., in hyperproliferative epi­dermis, we were interested in the distribution of Cx26 in skin tumours and their adjacent epidermis. We also investigated Cx30 because of its close relationship to the important, but until recently, not much understood Cx26. Because of the ubiq­uitous expression of Cx43 in normal skin, we were also interested in changes of its expression in skin tumours.

Recently, we have shown a change in the expression pattern of connexins in basal cell carcinoma, squamous cell carcinoma and malignant melanoma (Haass et al. 2001, 2002). There is not much known about the expression patterns of con­nexins in Merkel cell carcinoma and adjacent epidermis.

Material and Methods

Investigations were performed by immunofluorescence microscopy using pre­viously described specific antibodies. Antibodies specific for Cx30 and Cx43 were purchased from Zymed Laboratories, San Francisco, CA, USA; the polyclonal an­tibody specific for Cx26 was made in our laboratory; the monoclonal antibody specific for CK20 was purchased from Progen Biotechnik, Heidelberg, Germany. For nuclear staining, DAPI (Boehringer Mannheim, Mannheim, Germany) was used.

Immunofluorescence microscopy was performed with an Axiophot II (Carl Zeiss, Jena/Oberkochen, Germany), photographed with a CCD Camera (Hamamatsu Photonics, Hamamatsu City, Japan) and documented using the soft­ware Openlab (lmprovision, Lexington, MA, USA).

Results

As previously shown, both Cx30 and Cx26 are only weakly, if at all, expressed in normal interfollicular epidermis, while Cx43 is expressed ubiquitously in all cell layers.

Now we show that neither Cx30 (cf. Fig. 1a), Cx26 (cf. Fig. lb) nor Cx43 (cf. Fig. lc) occur in Merkel cell carcinoma. As expected, Cx43 is expressed through­out the interfollicular epidermis- adjacent to and also distant from the Merkel cell

Connexins in Merkel cell carcinoma 221

Fig. 1. a Immunofluorescence microscopy of a Merkel cell carcinoma and adjacent epi­dermis using antibodies against Cx30 (red) and CK20 (green), nuclear staining (DAPI, blue), overlaid with corresponding phase contrast. There is no expression of Cx30 in Merkel cell carcinoma cells, but note the expression of Cx30 in the epidermis adjacent to the Merkel cell carcinoma. Bar 6 11m. b Immunofluorescence microscopy of a Merkel cell carcinoma and adjacent epidermis using antibodies against Cx26 (red) and CK20 (green), nuclear staining (DAPI, blue), overlaid with corresponding phase contrast. There is no ex­pression of Cx26 in Merkel cell carcinoma cells, but note the expression of Cx26 in the epidermis adjacent to the Merkel cell carcinoma. Bar 6 f.lm. c Immunofluorescence micros­copy of a Merkel cell carcinoma using antibodies against Cx43 (red) and CK20 (green), nuclear staining (DAPI, blue), overlaid with corresponding phase contrast. There is no ex­pression ofCx43 in Merkel cell carcinoma cells. Bar 6 11m

carcinoma (not shown). Moreover, as expected, there is no expression of Cx30 and Cx26 in unaffected interfollicular epidermis distant from the Merkel cell car­cinoma (not shown). However, in striking contrast, both Cx30 (cf. Fig. la) and Cx26 ( cf. Fig. I b) are expressed in the interfollicular epidermis adjacent to Merkel cell carcinoma.

Discussion

These results clearly show an induction of Cx30 and Cx26 in normal interfol­licular epidermis adjacent to Merkel cell carcinoma. Previously, we demonstrated similar results investigating gap junction proteins in malignant melanoma (Haass et al. 2002) and malignant epithelial skin tumours (Haass et al. 2001), but there was no such induction in the epidermis adjacent to semi-malignant (basal cell car­cinoma) and benign lesions (melanocytic nevi). Therefore, one might suggest that the change of the composition of gap junctions in the epidermis adjacent to malig­nant skin tumours may play a role in the metastasation of these tumours.

222 Haass et al.

References

Beyer EC, Paul DL, Goodenough DA (1990) Connexin family of gap junction proteins. J Membr Biolll6:187-194

Brissette JL, Kumar NM, Gilula NB, Hall JE, Dotto GP (1994) Switch in gap junction pro­tein expression is associated with selective changes in junctional permeability during keratinocyte differentiation. Proc Natl Acad Sci USA 91:6453-6457

Elfgang C, Echert R, Lichtenbert-Frate H, Butterweck A, Traub 0, Klein RA, Hiilser DF, Willecke K (1995) Specific permeability and selective formation of gap junction chan­nels in connexin-transfected HeLa cells. J Cell Bioll29:805-817

Goliger JA, Paul DL (1995) Wounding alters epidermal connexin expression and gap junc­tion-mediated intercellular communication. Mol Bioi Cell6:1491-1501

Haass NK, Brandner JM, von den Driesch P, Kief S, Wladykowski E, Houdek P, Moll I (2001) Induction of gap junction proteins in epithelial skin tumours and adjacent epi­dermis. J Invest Dermatol117:789 (Abstract)

Haass NK, Brandner JM, von den Driesch P, Wladykowski E, Kief S, Houdek P, Moll I (2002) Expression patterns of connexins in basal cell carcinoma, squamous cell carci­noma, malignant melanoma and adjacent epidermis. Arch Dermatol Res (Offprint) 294:72 (P229)

Jiang JX, Goodenough DA (1996) Heteromeric connexons in lens gap junction channels. Proc Natl Acad Sci USA 93:1287-1291

Krutovskikh V, Yamasaki H (1997) The role of gap junctional intercellular communication (GJIC) disorders in experimental and human carcinogenesis. Histol Histopathol 12:761-768

Labarthe MP, Bosco D, Saurat JH, Meda P, Salomon D (1998) Upregulation of Connexin 26 between keratinocytes of psoriatic lesions. J Invest Dermatol Ill :72-76

Li G, Satayamoorthy K, Herlyn M (2002) Dynamics of cell interactions during melanoma development. Crit Rev Oral Bioi Med 2002 13:62-70

Loewenstein WR (1981) Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev 61:829-913

Masgrau-Peya E, Salomon D, Saurat JH, Meda P (1997) In vivo modulation of connexins 43 and 26 of human epidermis by topical retinoic acid treatment. J Histochem Cyto­chem 45:1207-1215

Pitts JD, Finbow ME, KamE (1988) Junctional communication and cellular differentiation. Br J Cancer 9 (Suppl):52-57

Richard G (2000) Connexins: a connection with the skin. Exp Dermatol 9:77-96 Salomon D, Masgrau E, Vischer S, Chanson M, Saurat JH, Spray DS, Meda P (1993) Gap

junction proteins and communication in human epidermis. Progr Cell Res 3:255-231 Spray DC (1994) Physiological and pharmacological regulation of gap junction channels.

In: Citi S ( ed) Molecular mechanisms of epithelial cell junctions: from development to disease. R GLandes Company, Austin, TX, pp 195-215

Stauffer KA (1995) The gap junction proteins jH-connexin (connexin-32) and j32-connexin (connexin26) can form heteromeric hemichanne1s. J Bioi Chern 270:6768-6772

Veenstra RD (1996) Size and selectivity of gap junction channels formed from different connexins. J Bioenerg Biomembr 28:326-337