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) RARbeta2 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 CATgene after exposure of the cells to 0.1 microM RA. Transactivation of the CATgene could, however, also be achieved by shifting RAREbeta2-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 RAREbeta2. 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 RAREbeta2 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 RAREbeta2 was essentially maintained in papilloma-derived SP1 cells, although all parameters of the Ca2+-dependent RAREbeta2 activation cascade were induced to a much lower extent. In contrast, strong RAREbeta2 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 RAREbeta2 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.
Vettermann
O
O
German Cancer Research Center, Division of Tumor Cell Regulation, Heidelberg.
Siegenthaler
G
G
Winter
H
H
Schweizer
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002277:Carcinoma / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D002454:Cell Differentiation / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D010212:Papilloma / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D012867:Skin / Q000166:cytology; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D012879:Skin Physiological Phenomena; D015533:Transcriptional Activation; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology / Q000502:physiology*; D014801:Vitamin A / Q000378:metabolism
D018168:Receptors, Retinoic Acid; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; C082925:retinoic acid receptor beta; D014801:Vitamin A; D014212:Tretinoin; D015500:Chloramphenicol O-Acetyltransferase; D002118:Calcium