Human hydroxyindole-O-methyltransferase in pineal gland, retina and Y79 retinoblastoma cells

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) was studied in extracts of human pineal gland, retina and Y79 retinoblastoma cells. HIOMT enzyme activity and immunoreactive protein (approximately 42 kDa) were undetectable in the human retina; very low levels of HIOMT mRNA were detected using a highly sensitive RT-PCR/Southern blot method, as has been reported. Analysis of extracts of Y79 cells indicated that HIOMT enzyme activity, immunoreactivity (approximately 42 kDa) and mRNA (approximately 1.3 kb) were detectable at approximately 1/5-1/40 the levels found in the pineal gland. This unambiguously establishes that the HIOMT gene is expressed in Y79 cells. Kinetic analysis of Y79- and pineal-derived HIOMT indicates that the enzyme is generally similar in both tissues; one difference, however, is that substrate inhibition by N-acetylserotonin is greater with the Y79-derived enzyme. These studies show that Y79 cells represent a valid model to study the regulation of human HIOMT protein and mRNA; the differences detected may reflect the existence of tissue-specific regulatory mechanisms or differential patterns of expression of HIOMT isoforms.     

Cloning of a novel retinoic acid-inducible mRNA from human osteoblast-like cells

Retinoids have long been known to influence skeletal development and bone remodeling. Cells of the osteoblastic lineage play a key role in these processes. In this study we have used the differential display PCR technique to identify retinoic acid (RA)-induced mRNAs in human osteoblast-like cells. We report the cloning and sequencing of one such mRNA, AT-RA 6, which was specifically induced by all-trans RA both in normal human osteoblast-like cells and in MG-63 osteosarcoma cells. Maximal expression was found after 60 min, suggesting that this may be an early response gene. Expression was found in all tissues examined. No homology to known mRNA sequences was detected.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Effects of retinoic acid and estrogens on oxytocin gene expression in the rat uterus: in vitro and in vivo studies

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo administration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor alpha and RA receptor beta are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.     

Role of TGF-beta in RA-induced cleft palate in CD-1 mice

Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. RA exposure on gestational day (GD) 12 in CD-1 mice results in delayed palatal shelf elevation and subsequent clefts in the secondary palate. Given the dynamic and complex nature of palate development, it is not surprising that this system is susceptible to changes in retinoid levels. There is evidence that experimental manipulation of retinoid status during development alters normal transforming growth factor-beta (TGF-beta) status. To study the role of perturbation in TGF-beta levels in RA-induced cleft palate, gravid CD-1 mice were treated with 70 mg/kg RA on GD 12. We examined changes in TGF-beta proteins and the steady-state level of TGF-beta mRNA within the first 24 hr after exposure. The interactions between RA and TGF-beta s were very complex. RA differentially regulated the mRNA and protein levels of TGF-beta 1. Changes in mRNA steady-state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation was evident, because RA treatment resulted in an increase in TGF-beta 1 mRNA steady levels followed by a decrease in the intracellular and extracellular forms of TGF-beta 1 protein. Moreover, the patterns of localization and levels of TGF-beta 2 and TGF-beta 3 proteins were not dramatically affected, although there was an increase in TGF-beta 3 mRNA steady-state levels. The increases in mRNA steady-state levels for TGF-beta 2 and TGF-beta 3, as for TGF-beta 1, were rapid and transient in nature, again arguing for direct mediation by RA. These data provide evidence for interactions between RA and TGF-beta s, and indicate that RA is capable of differentially regulating TGF-beta isoforms through processes involving different stages of TGF-beta synthesis and secretion. Further, changes in TGF-beta isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.     

Characterization of the effects of retinoic acid on vasoactive intestinal polypeptide gene expression in neuroblastoma cells

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.     

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.     

The protective effect of metallothionein against lipid peroxidation caused by retinoic acid in human breast cancer cells

The treatment of breast cancer by retinoic acid (RA) may be mediated by lipid peroxidation. Expression of metallothionein (MT) in cancer cells, however, can protect against lipid peroxidation by scavenging hydroxyl radicals. In this study, a two-by-six factorial design was used to investigate the interactive effects of all-trans-RA and zinc (Zn)-induced MT on the growth of two human breast cancer cell lines differing in basal expression of MT and estrogen receptors; MCF7 cells express estrogen receptor, BT-20 cells do not. Cells were treated with Zn to induce MT and then treated with six RA concentrations. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly increased by Zn treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. Elevated RA concentrations increased lipid peroxidation as measured by thiobarbituric acid reactive substances. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by Zn pretreatment in BT-20 cells but not in MCF7 cells. RA increased MT levels in both cell lines, which suggests that RA may generate free radicals which will induce MT mRNA expression. Glutathione did not appear to be a significant factor. Therefore, induction of MT by Zn may modulate the growth inhibitory effects of RA in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation. Induction of MT by RA may be one explanation for acquired RA resistance in cancer.     

Tunicamycin in combination with retinoic acid synergistically inhibits cell growth while decreasing palmitoylation and enhancing retinoylation of proteins in the human breast cancer cell line MCF-7

All-trans-Retinoic acid (RA) induces differentiation and inhibits growth of many tumor types. Whereas the RA nuclear receptors mediate genomic effects of RA, there also are many nongenomic effects that do not have defined mechanisms. Some nongenomic effects of RA may involve retinoylation (RA acylation), a posttranslational modification of proteins occurring in many eukaryotic cell lines including the human breast cancer cell line MCF-7. To gain further knowledge of the role(s) of retinoylation, we studied the effects of tunicamycin (TM), an inhibitor of both protein N-glycosylation and palmitoylation, on growth and retinoylation in MCF-7 cells. We found that RA or TM alone inhibited growth of MCF-7 cells. Combinations of RA and TM inhibited growth synergistically. TM increased retinoylation and decreased palmitoylation. These results suggest that increased retinoylation and decreased glycosylation and palmitoylation may play a role in the synergistic inhibition of cell growth by combinations of TM and RA in MCF-7 cells. Furthermore, our results suggest that combinations of TM and RA may have clinical utility.     

Autoimmunity to cartilage link protein in patients with rheumatoid arthritis and ankylosing spondylitis

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) express cellular immunity to cartilage link protein (LP). METHODS: LP was purified from human fetal epiphyseal and bovine adult nasal cartilage. It was used in proliferation assays with the peripheral blood lymphocytes (PBL) isolated from 83 patients with RA, 21 patients with AS, and 30 healthy controls. RESULTS: Patients with RA (34%) and AS (71%) expressed a significantly higher prevalence of cellular immune responses to human LP compared with the healthy control group (13%). Such significant differences were not observed for bovine LP. Half the patients with RA responding to LP exhibited cellular immunity to both human and bovine protein. In the AS group, PBL from a majority of responders to LP recognized only human LP. CONCLUSION: These data suggest that LP is a potential autoantigen in the development of RA and AS and that cellular immune reactivity to common and distinct LP epitopes in patients with RA and AS may play a role in the pathogenesis of these diseases.