Retinoic acid signaling cascade in differentiating murine epidermal keratinocytes: alterations in papilloma- and carcinoma-derived cell lines.

The retinoic acid (RA) signaling pathway was investigated by transient transfection of a chloramphenicol acetyltransferase (CAT) reporter gene construct containing the RA response element (RARE) of the murine (m) RAR beta 2 gene into murine primary epidermal keratinocytes (PEK), papilloma-derived SP1 cells, and carcinoma-derived 3P2 cells. Murine PEK transfected in a low-Ca2+ medium (0.05 mM Ca2+) exhibited a strong transactivation of the CAT gene after exposure of the cells to 0.1 mu M RA. Transactivation of the CAT gene could, however, also be achieved by shifting RARE beta 2-transfected low-Ca2+ PEK to high-Ca2+ conditions (0.15-1.2 mM Ca2+). Concomitantly, the Ca2+ raise also led to the induction of both cellular retinol (ROL)-binding protein I (CRBPI) and cellular RA-binding protein II (CRABPII), whereas expression of cellular RA-binding protein I (CRABPI) was not observed. Moreover, induction of in vitro differentiation also activated the ROL-->RA converting enzyme system in PEK. These findings suggest the following sequence of events involved in the high Ca2+-mediated activation of RARE beta 2. First, high Ca2+ induces the synthesis of mCRBPI, which binds ROL released from retinyl ester stores and makes it accessible to the ROL-->RA converting enzyme system. Enzymatically generated RA is taken over by mCRABPII and transported to the nucleus, where it acts as ligand for nuclear receptors, which complex with RARE beta 2 to activate the reporter gene. This hypothetical cascade of RA signaling was supported by our findings that inhibition of the ROL-->RA converting enzyme system by citral abolished the Ca2+-mediated transactivation of the CAT gene in a nontoxic manner. Studies in transformed murine cell lines revealed that Ca2+-induced activation of RARE beta 2 was essentially maintained in papilloma-derived SP1 cells, although all parameters of the Ca2+-dependent RARE beta 2 activation cascade were induced to a much lower extent. In contrast, strong RARE beta 2 activity was already observed in low-Ca2+ carcinoma-derived 3P2 cells. Low-Ca2+ 3P2 cells also expressed high levels of both mCRBPI and mCRABPII and possessed a highly active ROL-->RA converting enzyme system. Again, inhibition of the enzyme by citral abolished RARE beta 2 activity in low-Ca2+ 3P2 cells. Our data show that Ca2+-induced differentiation in cultured murine PEK entails a series of events that ultimately lead to the activation of RARE-containing genes. These properties are maintained in transformed epidermal keratinocytes. However, with increasing malignant potential of the cells, the respective signaling pathway becomes independent from a differentiation stimulus and leads to constitutive activation of RARE-controlled genes. (C) 1997 Wiley-Liss, Inc.