Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human fetal skin fibroblasts (HFFF2) and in rat liver epithelial cells (WB-F344) Duygu Onat, Wilhelm Stahl, Helmut Sies* Institut fu ¨r Physiologische Chemie I, Heinrich-Heine-Universita ¨t Du ¨sseldorf, Postfach 101007, D-40001 Du ¨sseldorf, Germany Received 19 November 2000; accepted 28 May 2001 Abstract Gap junction channels maintain cell– cell communication and are essential for the coordination of tissues, playing a pivotal role in embryonal development. Gap junctional intercellular communication (GJIC), studied here in human fetal skin fibroblasts (HFFF2) and in rat liver epithelial cells (WB-F344), was almost doubled upon exposure to thalidomide (10 M) in the presence of NADH or NADPH (20 M). Neither in HFFF2 nor in WB-F344 cells did any detectable alteration in GJIC occur with the thalidomide analog EM 16 (10 M), known as a non-teratogenic compound. The thalidomide analog EM 364 (10 M) increased GJIC without prior metabolic activation. It is suggested that GJIC modification may be related to the pharmacological and toxicological properties of thalidomide. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Teratogen; Thalidomide; Gap junctions; Intercellular communication; Human fetal skin fibroblasts (HFFF2); Rat liver epithelial cells (WB-F344) 1. Introduction Gap junctional intercellular communication (GJIC) is a signaling pathway that involves the transfer of messenger compounds between adjacent cells [1]. Signaling is achieved via cell-to-cell channels that connect the cytosol of two neighboring cells, allowing for the exchange of low- molecular-mass compounds. A functional pore is formed from two half-channels, each consisting of a hexamer of proteins that belong to the gene family of connexins. Intercellular signaling can modulate gene expression at various levels. It has been suggested that untimely or chronic changes in gap junctional communication during embryonal or fetal development may lead to embryonic lethality or teratogenesis [2]. Gap junctional coupling has been demonstrated in embryonic tissues, and it is assumed that GJIC is involved in the process of pattern formation [3], probably providing a pathway for the formation of a puta- tive gradient of morphogens [4,5]. There is some evidence that GJIC plays a part in the patterning of the developing limb [5]. Various types of connexins have been detected within an organism, and there are differences in the expres- sion of connexin genes during embryonal development [6,7]. One of the most abundant connexin proteins is con- nexin43 (Cx43), which appears to be implicated in limb bud development [5,8,9]. GJIC is affected by various compounds, including reti- noic acid [10], retinoid derivatives [11], vitamin D [12,13], and tumor promotors such as phorbol esters [14]. It has been shown that thalidomide induces GJIC in human fibroblasts after metabolic activation [15]. Retinoic acid and thalido- mide are known as potent human teratogens. While these compounds induce different patterns of malformation, it is interesting to note that they both modify GJIC at the cellular level. The present study investigates the stimulation of GJIC by thalidomide in comparison to some of its structural analogs and retinoic acid in human fetal skin fibroblasts (HFFF2) and in rat liver epithelial cells (WB-F344) which show a high level of Cx43 expression [16]. * Corresponding author. Tel.: +49-211-811-2707; fax: +49-211-811- 3029. E-mail address: helmut.sies@uni-duesseldorf.de (H. Sies). Abbreviations: GJIC, gap junctional intercellular communication; PGA, 2-phthalimido glutaric acid; Cx43, connexin 43; Cx31, connexin 31; MTT, (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide); and THF, tetrahydrofuran. Biochemical Pharmacology 62 (2001) 1081–1086 0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0006-2952(01)00751-1