Hydroxyindole-O-methyltransferase mRNA Research Articles

Association of Tumor Hydroxyindole O-Methyltransferase and Serum 5-Methoxytryptophan with Long-Term Survival of Hepatocellular Carcinoma.

Simple Summary5-methoxytryptophan (5-MTP) is a tryptophan (Trp) metabolite synthesized by hydroxyindole O-methyltransferase (HIOMT). Expression of HIOMT is decreased in various tumors. However, whether HIOMT expression and serum 5-MTP concentration associate with prognosis of hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to analyze HCC tissue HIOMT mRNA and serum 5-MTP and determine their association with survival following therapeutic liver resection. We found a significant association of serum 5-MTP or tissue HIOMT and serum kynurenine (Kyn) with overall and relapse free (RF) survival of HCC. The combination of serum 5-MTP and Kyn is a potential prognostic biomarker of HCC.5-methoxytryptophan (5-MTP) is a recently discovered tryptophan (Trp) metabolite with anti-inflammatory and tumor-suppressing actions. Its synthesis is catalyzed by hydroxyindole O-methyltransferase (HIOMT). HIOMT levels were reported to be decreased in some patients with colorectal, pancreatic and breast cancer. It is unclear whether tissue HIOMT levels is altered in hepatocellular carcinoma (HCC). It is also unclear whether serum 5-MTP concentration is influenced by HCC. In this study, 150 HCC and adjacent normal liver tissues and serum samples were obtained from the HCC biobank established by a prospective multicenter study. Serum samples from 47 healthy subjects were included as a reference. HIOMT mRNA was measured by real time PCR. Serum 5-MTP and selected Trp metabolites were analyzed by quantitative LC-MS. HCC tissue HIOMT mRNA levels adjusted for adjacent normal tissue HIOMT mRNA levels was associated with overall and relapse-free (RF) survival. Combined serum 5-MTP or tissue HIOMT mRNA and serum kynurenine (Kyn) analysis predicted prolonged overall and RF survival following liver resection. A high serum 5-MTP or tissue HIOMT mRNA and low serum Kyn is associated with long-term survival. In conclusion, tumor tissue HIOMT mRNA and serum 5-MTP are potential biomarkers of HCC, especially when analyzed in combination with serum Kyn.

Interleukin-1 β Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study.

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1β (75 pg/mL), or (4) NE + IL-1β. It was found that IL-1β abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1β also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1β treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

The expanding roles of 1‐methyl‐tryptophan (1‐MT): in addition to inhibiting kynurenine production, 1‐MT activates the synthesis of melatonin in skin cells

Indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO1 is a leading target for interventions aimed at restoring melanoma immune surveillance. Here, in a scenario involving the tryptophan catabolism, we report that melatonin biosynthesis is driven by 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO1, in human fibroblasts, melanocytes and melanoma cells. In addition to melatonin biosynthesis, 1-MT induced the expression of tryptophan hydroxylase, arylalkylamine-N-acetyltransferase and hydroxyindole O-methyltransferase mRNA in fibroblasts and melanocytes. We observed a great variability in the levels of IDO1 mRNA expression and kynurenine release between skin cells and melanoma cell lines in response to interferon-γ, a classical IDO1 inducer. In this setting, melatonin was shown to downregulate kynurenine production. Furthermore, in a condition of low basal activity of IDO1, it was observed that 1-MT, as well melatonin, inhibited the proliferation of human melanoma cells. Taken together, our results suggest that 1-MT may serve as more than just a tool to disrupt tumor immune escape (via the inhibition of IDO1) because it was shown to act directly on the proliferation of human melanoma cells and induce melatonin biosynthesis in the tumor milieu. Moreover, 1-MT-mediated inhibition of IDO occurs in normal skin and melanoma cells, which addresses the possibility that all cells in the skin microenvironment can be targeted by 1-MT. Our findings provide innovative approaches into understanding tumor therapy related to the control of tryptophan metabolism by 1-MT.

Expression and cellular localizaion of melatonin-synthesizing enzymes in rat and human salivary glands

Melatonin, discovered in 1958, is secreted by the pineal gland primarily during the night. Its secretion is controlled by the light/dark cycle of the environment. Melatonin is also produced in and secreted by various extrapineal organs, tissues and cells and its synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT) is expressed in various extrapineal organs, tissues and cells. Recently, it was reported that melatonin is present in saliva, but it is not certain where melatonin was synthesized and whether it was secreted into saliva and what function it may have in saliva. The present study was performed to investigate where melatonin was synthesized and whether it was secreted by salivary glands into saliva. We performed immunohistochemical analysis of the expression of AANAT in rat parotid, submandibular and sublingual glands and the expression of both AANAT and hydroxyindole-O-methyltransferase (HIOMT) in human submandibular glands. We evaluated the expression of AANAT and HIOMT mRNA in rat submandibular glands by quantitative reverse transcription-polymerase chain reaction. As a result, we observed expression of AANAT in epithelial cells of striated ducts in rat salivary glands and expression of AANAT, HIOMT and melatonin in epithelial cells of striated ducts in human submandibular glands. In addition, we also confirmed the expression of the most potent melatonin receptor, melatonin 1a receptor, in rat buccal mucosa. Our findings suggest that melatonin might be produced and secreted by salivary glands directly into saliva and that it might play some physiological role in the oral cavity.

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.

Hydroxyindole-O-methyltransferase, a Season-coding Enzyme for Melatonin Synthesis in the Pineal Gland of Rodents

The time giver hormone melatonin is secreted with both daily and seasonal rhythms in the mammalian pineal gland. We report here that the two main melatoninsynthesizing enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) are differently involved in the regulation of the daily and photoperiodic rhythms of melatonin synthesis. The activity of the unstable NAT enzyme is stimulated acutely at night by norepinephrine via cAMP-dependent transcriptional mechanisms, indicating that it triggers the nocturnal increase in melatonin synthesis and drives its duration according to the night length. In contrast, the activity of the stable HIOMT enzyme is regulated chronically by a norepinephrine-induced, 2-fold increase in HIOMT mRNA that occurs every night. Such regulation leads to a photoperiodic variation in HIOMT activity which is positively related to duration of the night. Whether in vivo or in vitro, at high NAT activity, HIOMT activity appears to limit the amount of melatonin release. This suggests that HIOMT activity may tune the amplitude of the nocturnal increase in pineal melatonin secretion with photoperiodic variations.

Demonstration of hydroxyindole‐O‐methyltransferase (HIOMT) mRNA expression in pineal parenchymal tumors: Histochemical in situ hybridization

The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors.

Cyclic AMP increases hydroxyindole- O-methyltransferase mRNA levels in the chicken pineal gland, but is not required for circadian rhythmicity of this transcript

Hydroxyindole- O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In the chicken pineal gland, HIOMT mRNA concentration exhibits a circadian rhythm with a threefold peak at midday. The present study sought to evaluate the possible role of cyclic AMP in this transcriptional rhythm. In cultured pineal glands from 4-day-old chicks, cyclic AMP analogs and the adenylate cyclase activator, forskolin, increased HIOMT mRNA levels twofold to threefold in a dose-dependent manner. Vasoactive intestinal polypeptide increased HIOMT mRNA levels by 50%. Actinomycin-D chase experiments indicated that cyclic AMP did not affect the stability of HIOMT mRNA, thus providing indirect evidence that the effect of cyclic AMP was exerted at transcriptional level. In cultured pineal glands from 11 days embryos, HIOMT mRNA levels failed to respond to cyclic AMP. However, a daily rhythm of HIOMT mRNA, with an endogenous component in constant darkness was clearly observed at this developmental stage. This observation indicates that cyclic AMP is not required for circadian rhythmicity of HIOMT gene transcription.

Photoperiodic control of the rat pineal arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase gene expression and its effect on melatonin synthesis.

Photoperiodic changes of pineal melatonin (MEL) profile are accompanied by parallel changes of arylalkylamine-N-acetyltransferase (AA-NAT) activity. In the present study, the authors investigated, for the first time, whether two other important variables of pineal metabolism, AA-NAT and hydroxyindole-O-methyltransferase (HIOMT) gene expression, also may be affected by the photoperiod. Evening rises in AA-NAT and HIOMT mRNA and in circulating MEL occurred concomitantly with an increased delay from dark onset as scotophase shortened. On the opposite, the morning declines of all three variables occurred with different kinetics but were locked to light onset. These observations demonstrate that the daily rhythms in AA-NAT and HIOMT gene expression are modulated by the photoperiod and bring further evidence in favor of nor adrenaline as the possible link between the endogenous clock and MEL. Interestingly, the duration of the nocturnal peak in HIOMT mRNA was positively correlated with HIOMT activity. In conclusion, this study adds two important links to the chain of mechanisms involved in the photoperiodic control of pineal metabolism. First, photoperiodic modulation of the MEL rhythm primarily results from changes in the AA-NAT gene expression. Second, the photoperiodic regulation of HIOMT activity occurs at the transcriptional level.

Ontogenesis of hydroxyindole- O-methyltransferase gene expression and activity in the rat pineal gland

Postnatal development of hydroxyindole- O-methyltransferase (HIOMT) mRNA expression, HIOMT activity and melatonin content was investigated in the rat pineal gland from birth to adulthood (62-day old). For each age, animals were sacrificed at two different time-points: midday and midnight. HIOMT mRNA was first detectable one day after birth and maximal diurnal levels were reached at the end of the first week. A 2-fold nocturnal increase appeared significantly 11 days after birth. HIOMT activity was detectable from 5 days of life and significant day/night variations did not appear before 21 days after birth. Appearance of melatonin synthesis and rhythm followed that of HIOMT mRNA. The 10-day delay observed between the appearance of the nocturnal increase in HIOMT activity and expression is discussed in term of differential regulation of both HIOMT mRNA expression and HIOMT activity.

Distribution of hydroxyindole-O-methyltransferase mRNA in the rat brain: an in situ hybridisation study.

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes.

Retinoic acid increases hydroxyindole-O-methyltransferase activity and mRNA in human Y-79 retinoblastoma cells.

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all-trans-, 13-cis-, and 9-cis-RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.

Hydroxyindole- O-methyltransferase in Y-79 cells: regulation by serum

Hydroxyindole- O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of melatonin. In the present study, the regulation of HIOMT expression was examined in the human Y-79 retinoblastoma cell line, Cells were grown in suspension culture using medium supplemented with 10% fetal calf serum (FCS). HIOMT activity and mRNA were strongly reduced when FCS was substituted with 0.1% bovine serum albumin (BSA), and were restored by addition of FCS. The effect of FCS on HIOMT expression was relatively selective, because the abundance of mRNA encoding actin, G3PDH or interphotoreceptor retinoid-binding protein did not change following serum deprivation. However, S-antigen (arrestin) mRNA was regulated by serum coordinately with HIOMT mRNA, suggesting that S-antigen expression is also controlled by a serum factor. The effect of serum on HIOMT expression was not duplicated by treatment with a series of known differentiating factors, nor was it reduced by dialysis or stripping procedures which remove steroids, growth factors and thyroid hormones.

Hydroxyindole-O-methyltransferase in Y-79 cells: regulation by serum

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of melatonin. In the present study, the regulation of HIOMT expression was examined in the human Y-79 retinoblastoma cell line, Cells were grown in suspension culture using medium supplemented with 10% fetal calf serum (FCS). HIOMT activity and mRNA were strongly reduced when FCS was substituted with 0.1% bovine serum albumin (BSA), and were restored by addition of FCS. The effect of FCS on HIOMT expression was relatively selective, because the abundance of mRNA encoding actin, G3PDH or interphotoreceptor retinoid-binding protein did not change following serum deprivation. However, S-antigen (arrestin) mRNA was regulated by serum coordinately with HIOMT mRNA, suggesting that S-antigen expression is also controlled by a serum factor. The effect of serum on HIOMT expression was not duplicated by treatment with a series of known differentiating factors, nor was it reduced by dialysis or stripping procedures which remove steroids, growth factors and thyroid hormones.

Hydroxyindole- O-methyltransferase gene expression in the pineal gland of chicken embryo: development of messenger RNA levels and regulation by serum

Hydroxyindole- O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The present study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controled by serum factors.

Structural analysis of the human hydroxyindole-O-methyltransferase gene. Presence of two distinct promoters.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the metabolic pathway that synthesizes the hormone melatonin. We have found HIOMT mRNA present in small amounts in human retina and in relatively high abundance in the pineal gland. Two distinct 5' ends were found in human retina using a solid-phase 5'-rapid amplification of cDNA ends technique. The two 5' regions appear to originate from two distinct putative promoters. Although many similarities exist between the two promoters, they contain distinctive elements. Putative promoter A, for example, contains a recently discovered photoreceptor-conserved element (PCE-1, CAATTAAG) at -27 not found in promoter B, while promoter B contains an Ap1 site (ATGAGTCAA) at -166 and an octamer site (ATGCAAT) at -59 not found in promoter A. The HIOMT messages are also alternatively spliced in between exons 6 and 8, generating three distinct messages. One of the alternatively spliced messages contains a line-1 repetitive element that is spliced into the mRNA precisely as exon 6. Importantly, the downstream open reading frame is not altered by any of these splicing combinations. The gene is approximately 35 kilobases long containing either 9 or 10 exons (including the line-1 element) depending on which promoter is active. All of the splice sites follow the GT/AG rule. The dual promoters and opportunities for alternative splicing suggest a variety of mechanisms for control of HIOMT expression and biological activity in different tissues not previously recognized.

Analysis of the heterogeneity within bovine pineal gland by immunohistochemistry and in situ hybridization

In the present study, we demonstrate a cortical and medullary arrangement of parenchymal cells in the bovine pineal gland by using antibodies for neuron-specific enolase, synaptophysin, and hydroxyindole O-methyltransferase (HIOMT) as markers of pinealocytes, and glial fibrillary acidic protein (GFAP) as a marker of interstitial (glial) cells. Furthermore, by means of probes specific for HIOMT mRNA, we have examined possible differences in melatonin synthesis between the cortex and the medulla. Immunoreactive pinealocytes for each antigen investigated are more densely distributed in the cortex than in the medulla. In the cortex, GFAP-positive interstitial cells have large intenselystained somata endowed with several long, thin cytoplasmic processes, whereas in the medulla, they display smaller, less intensely labeled perikarya from which numerous fine short processes emerge. Golgi staining has confirmed these morphological differences between the interstitial cells in the cortex and those in the medulla. An analysis using confocal laser microscopy together with in situ hybridization for HIOMT mRNA has shown that the expression of mRNA transcripts in the cortex is more intense than that in the medulla. The expression of the HIOMT gene in a cluster of cells in the medial habenular nucleus is lower than that in pinealocytes of the pineal organ proper.

Regulation of hydroxyindole-O-methyltransferase gene expression in the pineal gland and retina

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin biosynthesis and appears to be specifically expressed in the pineal gland and in the retina. This review deals with the regulation of HIOMT by environmental light and with the developmental aspects of HIOMT expression in chicken and rat. Early studies based on HIOMT activity measurements and more recent studies involving cDNA hybridization to HIOMT mRNA are taken into consideration. Together, the data reveal that long term regulation of HIOMT by light would rely on a day/night rhythm of HIOMT gene transcription, coupled to a slow turnover of the protein. Rapid changes in HIOMT mRNA levels and early expression during embryonic development suggest that further studies on this gene may shed light on the molecular mechanisms involved in the differentiation of the melatoninergic function and in its regulation by light, both in the pineal gland and in the retina.

Localization of mRNA encoding the indolamine synthesizing enzyme, hydroxyindole- O-methyltransferase, in chicken pineal gland and retina by in situ hybridization

Hydroxyindole- O-methyltransferase (HIOMT) is the enzyme that catalyzes the final step in the synthesis of the hormone melatonin. We have examined the localization of expression of the mRNA encoding HIOMT by in situ hybridization in the 3-day-old and adult chicken retina and pineal gland. The riboprobe utilized for this study was transcribed from the complete coding region of HIOMT cDNA synthesized from retina RNA and amplified by polymerase chain reaction (PCR). High levels of HIOMT mRNA were present in the pinealocytes of the pineal gland. In the retina, the hybridization signal was localized to the photoreceptors. The retinal photoreceptors of the 3-day-old chick displayed a much lower level of hybridization than did the photoreceptors of the adult chicken. This study strongly suggests that the photoreceptors are the sites of melatonin synthesis in the retina.