Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

In vitro binding of retinoids to the nuclear retinoic acid receptor alpha

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RAR alpha. Transfection of cos-7 cells with the expression vector RAR alpha O produces a 100-fold increase in intracellular RAR alpha concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, [3H]CD367. Complete separation between RAR alpha and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RAR alpha.     

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells.

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA). RAC65 cells express a truncated RA receptor alpha (RAR alpha) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RAR alpha mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the defect in RAC65 RAR alpha results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.     

Modulation by retinoids of mRNA levels for nuclear retinoic acid receptors in murine melanoma cells

Retinoids inhibit the growth and enhance the differentiation of murine S91-C2 melanoma cells. Specific alterations in gene expression are a plausible mechanism for these effects. Since nuclear retinoic acid receptors (RAR) are likely mediators of retinoid-induced changes in gene expression, we used Northern blotting to analyze the expression of RAR alpha, RAR beta, and RAR gamma in S91-C2 cells. mRNA for both RAR alpha and RAR gamma was detected in these cells, but no RAR beta mRNA could be found. Treatment with 10(-7) and 10(-6) M beta-all-trans-retinoic acid (RA) for 24 h caused a 1.5- to 2-fold increase in RAR alpha and RAR gamma mRNA, whereas lower concentrations of RA were ineffective. RAR beta mRNA, which was undetectable in untreated cells, was detected after 24 h of treatment with a RA concentration as low as 10(-9) M, and its level increased with up to 10(-6) M RA. At the latter dose, RAR beta mRNA induction occurred by 4 h and increased progressively, reaching a plateau after 24 h of treatment. RAR beta mRNA induction at 4 h was not inhibited by cycloheximide at a concentration that suppressed protein synthesis by more than 90%. Several retinoids and related synthetic compounds, including 13-cis RA, TTNPB, Ch55, Am80, and the trifluoromethyl nonyloxyphenyl analog of RA, also induced RAR beta mRNA, whereas a 24-h treatment with 10(-6) M retinol, TTNP (a decarboxylated analog of TTNPB), or the phenyl analog of RA failed to induce RAR beta mRNA. With the exception of retinol and the trifluoromethyl nonyloxyphenyl analog of RA, the ability of the retinoids to induce RAR beta mRNA and their growth inhibitory effect were correlated. However, S91-C154, a RA-resistant mutant subclone derived from S91-C2 cells, showed mRNA levels of RAR alpha and RAR gamma and induction of RAR beta by RA similar to those detected in the sensitive S91-C2 cells. Like the S91 melanoma cells, two other mouse melanoma cell lines, K-1735P and B16-F1, constitutively expressed RAR alpha and RAR gamma mRNAs. The level of RAR beta mRNA was increased by RA only in B16-F1 cells, although the growth of both was inhibited by RA. These results demonstrate that RA can, directly and rapidly, induce the expression of mRNA for a high affinity nuclear receptor in some murine melanoma cells and that this induction is not sufficient to inhibit growth.     

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Modulation of retinoic acid receptor alpha activity by lysine methylation in the DNA binding domain

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys (347) trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys (109) and Lys (171) identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys (109), but not Lys (171), reduces RA-dependent activation. Methylation at Lys (109) plays a more dominant role than trimethylation at Lys (347) in terms of RA activation of the full-length receptor. Lys (109) is located in a homologous sequence (CEGC K GFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys (109) in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha.     

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.