ABSTRACT
All-trans retinoic acid (RA) compromises epidermal differentiation and causes keratinocyte hyperproliferation through mechanisms not completely understood, but may involve the regulatory matrix molecule hyaluronan. In this work, the influences of all-trans RA on epidermal morphology and hyaluronan metabolism were examined in organotypic and monolayer cultures of rat epidermal keratinocytes (REKs). All-trans RA treatment of organotypic REK cultures (10 days) increased the synthesis of hyaluronan, the expression of hyaluronan synthases Has2 and Has3, and the CD44 receptor, with hyperplasia of the epidermis. The hyperplasia and hyaluronan production induced by all-trans RA were blocked with (1) AG1478, an inhibitor of the EGFR; (2) UO126, an inhibitor of the MAPK/ERK kinase, and (3) GM6001, an inhibitor of the matrix metalloproteinases. These effects were consistent with the findings that all-trans RA upregulated heparin-binding epidermal growth factor-like growth factor mRNA expression and increased the phosphorylation of EGFR and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, the activation of EGFR and ERK1/2 was seen already 30 minutes after all-trans RA treatment, suggesting that the activation of this signaling pathway is a primary response to all-trans RA. These results indicate that the effects of all-trans RA on keratinocyte proliferation and hyaluronan synthesis are partly mediated through EGFR signaling.
Subject(s)
Epidermis/drug effects , Epidermis/pathology , ErbB Receptors/metabolism , Hyaluronic Acid/metabolism , Tretinoin/pharmacology , Animals , Cell Line , Enzyme Inhibitors/pharmacology , Epidermis/enzymology , Glucuronosyltransferase/antagonists & inhibitors , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Hyaluronan Receptors/analysis , Hyaluronan Receptors/metabolism , Hyaluronan Synthases , Hyaluronic Acid/analysis , Hyperplasia , Keratinocytes/drug effects , Keratinocytes/enzymology , Keratinocytes/pathology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Metalloproteases/antagonists & inhibitors , Metalloproteases/metabolism , Rats , Signal Transduction/drug effectsABSTRACT
Members of the reticulon gene family are endoplasmic reticulum (ER)-related proteins expressed in various human tissues, but their molecular functions are not understood. The reticulon 4 subfamily consists of three members, reticulon 4/Nogo-A, -B and -C. Reticulon 4-A is under intense investigation because of its inhibitory effect on neurite outgrowth, and reticulon 4-B has been suggested to induce apoptosis. Reticulon 4-C, the shortest member of this subfamily, is the least characterized. Reticulons are presumably guided to endoplasmic reticulum by a putative N-terminal retention motif. In this study the expressions of reticulon 4 subtypes in human chondrosarcoma cell line and in primary bovine chondrocytes were analyzed on mRNA level. These cell types, exposed to strong mechanical forces in vivo, were subjected to high hydrostatic pressure and mechanical stretch to study the possible mechanosensitivity of reticulon 4 genes. In addition, a green fluorescent protein-tagged reticulon 4-C and a fusion protein with mutated endoplasmic reticulum retention signal were used to study the significance of the C-terminal translocation signal (the di-lysine motif). As the result, both cell types expressed the three main isoforms of reticulon 4 family. The steady-state level of reticulon 4-B mRNA was shown to be up-regulated by pressure, but not by mechanical stretch indicating transcriptional barosensitivity. The reticular distribution pattern of reticulon 4-C was observed indicating a close association with endoplasmic reticulum. Interestingly, this pattern was maintained despite of the disruption of the putative localization signal. This suggests the presence of another, yet unidentified endoplasmic reticulum retention mechanism.
