Control of squamous differentiation in tracheobronchial and epidermal epithelial cells: role of retinoids.

Hyperplasia and squamous differentiation in epidermal and tracheobronchial epithelial cells is a multistage process. In stage I, quiescent progenitor cells are recruited to reenter the cell cycle. Protein kinase C activators, retinoids, cytokines, and polypeptide growth factors have been identified to control this stage of hyperproliferation. In stage II, cells become committed to irreversible growth arrest, which in normal cells appears to be a prerequisite for the expression of the differentiated phenotype (stage III). Confluence or treatment with interferon gamma or phorbol esters are conditions that induce irreversible growth arrest and differentiation. Retinoids do not block stage II but specifically suppress the expression of stage III. The action of retinoids appears to be mediated by nuclear retinoic acid receptors. Studies understanding the mechanisms that regulate hyperplasia and squamous metaplasia may provide insight into the processes that lead to squamous cell carcinomas. Such studies may also provide new strategies for chemotherapy and chemoprevention

Regulation of retinoid-induced differentiation in embryonal carcinoma PCC4.aza1R cells: Effects of retinoid-receptor selective ligands

Retinoic acid (RA) is a potent inducer of differentiation of embryonal carcinoma PCC4.aza1R cells into mesenchymal stem cells, Induction of Hoxa-1, Hoxa-5, cellular retinoic acid-binding protein (CRABP) I and II, and retinoic acid receptor (RAR)-beta expression occurs early in this multistage program of differentiation. RA is also a potent inducer of these genes in the differentiation-defective mutant PCC4(RA)-1; however, RA is much less effective in the mutant cell line PCC4(RA)-2. the up-regulation of several of these genes by RA is, at least in part, due to increased transcription, It is likely that some of these changes are mediated either directly or indirectly by nuclear retinoid receptors, Previously, we characterized the expression of RARs in PCC4.aza1R and (RA)-1 and (RA)-2 cells, In this study, we show that these cells also express retinoid X receptor (RXR)-alpha, RXR-beta, and RXR-gamma and that RA treatment down-regulates the expression of RXR-gamma. No large differences were found in RXR mRNA expression between parental and mutant cell lines except that PCC4(RA)-1 cells expressed an 8-fold higher level of RXR gamma mRNA than the parental cells, To obtain more insight into the retinoid signaling pathways involved in the regulation of this pathway of differentiation, we examined the action of two retinoid receptor-selective agonists and one antagonist, The RAR-selective retinoid SRI-6751-84 is a very effective inducer of transactivation of beta RARE-tk-LUC, but not of RXRE-tk-CAT, in PCC4.aza1R cells and is a very potent inducer of morphological differentiation and Hoxa-1, Hoxa-2, CRABP II, and RAR-beta expression. In contrast, the RXR-selective retinoid SR11,217, which transactivates the RXRE-tk-CAT effectively, but beta RARE-tk-LUC poorly, is unable to induce differentiation and has little effect on the expression of these early genes, The RAR-alpha-selective antagonist Ro 41-5253, which inhibits RARE-dependent transcriptional activation, has by itself no effect on the differentiation of PCC4.aza1R cells. However, this antagonist is able to block the induction of morphological differentiation by the RAR-selective retinoid as well as the expression of Hoxa-1, Hoxa-5, CRABP II, and RAR-beta. Our data suggest that the activation of RAR signaling pathways is important in initiating the cascade of changes in gene expression that result in the differentiation of PCC4.aza1R into mesenchymal stem cells. In addition, we demonstrate that the two mutant cell lines, PCC4(RA)-1 and PCC4(RA)-2, are defective at different stages of the differentiation program

EXPRESSION OF NUCLEAR RETINOIC ACID RECEPTORS IN WILD-TYPE AND MUTANT EMBRYONAL CARCINOMA PCC4.AZA1R CELLS

Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR beta mRNA levels in PCC4.aza1R cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS