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ANTICANCER RESEARCH 32: 4765-4772 (2012)

MTNR1A Receptor Expression in Normal and Pathological Salivary Glands

SALVADOR ARIAS-SANTIAGO1,2, JOSÉ ANEIROS-FERNÁNDEZ1, BORJA ARIAS-SANTIAGO3, MARÍA SIERRA GIRÓN-PRIETO1, MERCEDES CABA-MOLINA1, ANTONIO LÓPEZ-VALVERDE4, JOSÉ ANEIROS-CACHAZA1, ANTONIO CAMPOS2 and ANTONIO CUTANDO3

1Department of Pathology, San Cecilio University Hospital, Granada, Spain; 2Department of Histology, University of Granada, Spain; 3School of Dentistry University of Granada, Spain and 4School of Dentistry. University of Salamanca, Spain

Abstract. Aim To analyze and compare the expression of hormone that is primarily synthesized and secreted by the MTNR1A receptor in normal and pathological major and (1) and is widely distributed throughout the minor salivary glands. Materials and Methods: Twenty human body (2). It exerts chronobiotic, immunomodulatory, samples of major and minor salivary glands and 10 with oncostatic and actions, among others, via direct Warthin’s tumor were studied. Expression of the MTNR1A and indirect mechanisms (3). is also synthesized receptor (goat polyclonal antibody raised against a peptide in the digestive system by enterochromaffin cells of the mapping at the N-terminus of MEL-1A R of human origin) intestinal mucosa in response to food intake (4), sometimes was analyzed. Results: The excretory ducts of major salivary reaching concentrations up to 400-fold higher than those glands demonstrated intense intracytoplasmic positivity but produced by the pineal gland (5). scant cytoplasmic membrane positivity for MTNR1A. The After its release into the blood, melatonin enters the oral studied Warthin’s tumors showed intense cytoplasmic cavity by passive diffusion in the saliva. Melatonin positivity for MT1 receptor in all cylindrical epithelial cells concentrations in the saliva are 15-33% increased compared lining spaces and a less intense positivity in basal cells. The to those in plasma, since 70% of plasma melatonin is bound lymphoid component accompanying the tumor was negative to albumin and does not enter the saliva to any appreciable for MT1 receptor. Conclusion: Intense intracytoplasmic extent. Hence, salivary melatonin represents the percentage positivity for the MTNR1A receptor in the excretory ducts of of circulating melatonin that is not albumin-bound, i.e. free human major and minor salivary glands and Warthin’s melatonin. Evaluating the levels of salivary melatonin is a tumor was found. The intense expression of MTNR1A reliable technique for monitoring circadian rhythms (2). receptors observed in this study in the excretory ducts of Two types of melatonin receptor, MTNR1A and MTNR2A major and minor salivary glands may be related to salivary have been reported in the cytoplasmic membrane of human regulation. cells (6). The MTNR1A gene is located at and MTNR2A gene at (7). These receptors, Most saliva is produced by the major salivary glands, which which are coupled to heterotrimeric guanine nucleotide vary between different species in number, size, and location. binding proteins (G-proteins), are found in high Some hormones can be found in saliva, with physiological concentrations in the pituitary, the of consequences such as regulation of inflammatory processes, the , the , and ependymal cells of the the promotion of cell proliferation, and contribute to a rapid choroid plexus (8, 9). MTNR1A has been implicated in the wound healing in epithelia. inhibition of melatonin in the suprachiasmatic nucleus of the Melatonin (N-acetyl-5-methoxytryptamine) is a lipophilic hypothalamus and in the effect of melatonin on MCF-7 breast cancer cells (10). Experimental findings in rats suggest that the MTNR2A receptor mediates the action of melatonin in the retina and induces a phase shift of the Correspondence to: Salvador Arias-Santiago, MD, Ph.D., San in the suprachiasmatic nucleus of the hypothalamus (11). Cecilio University Hospital and Histology Department. University Results of an immunoblotting study of MTNR1A and of Granada, Spain. Av Dr. Oloriz 16. Granada. 18012. Spain. E- mail: [email protected] MTNR2A receptors in the parotid gland of rat suggested that melatonin may be involved in salivary regulation via direct Key Words: Melatonin, MTNR1A, normal salivary glands. action on its receptors and via nitric oxide (12). However, no

0250-7005/2012 $2.00+.40 4765 ANTICANCER RESEARCH 32: 4765-4772 (2012)

Table I. The expression pattern of MTNR1A for major normal and pathological salivary glands and minor salivary glands.

Normal parotid Pathological parotid Submaxillary Sublingual Minor salivary gland (n=4) gland (n=10) gland (n=3) gland (n=3) gland (n=10)

Principal duct Diffuse ++ (4/4) – Diffuse ++ (3/3) Diffuse +++ (3/3) Diffuse ++ (8/10) Diffuse + (2/10) Interlobular/intralobular duct Diffuse +++ (4/4) – Diffuse +++ (3/3) Difusse +++ (3/3) Diffuse +++ (8/10) Diffuse ++ (2/10) Acini (serous, mucous) Granular + (4/4) – Granular + (3/3) Granular ++ (3/3) Granular ++ (6/10) Granular + (4/10) Epithelial component – Diffuse +++ (10/10) – – – Lymphoid component – Diffuse + (7/10) – – – Negative – (3/10)

data have been published on the immunohistochemical MTNR1A in thick clumps, with no evidence of positivity in expression of melatonin receptors in normal and pathological the cytoplasmic membrane. The myoepithelial cells human salivary glands. The objective of the present study surrounding the serous cells were not MTNR1A-positive. was to analyze the expression of MTNR1A receptors in The excretory ducts (lobar and lobular) demonstrated intense normal major and minor salivary glands and in Warthin’s intracytoplasmic positivity (+++) but scant cytoplasmic tumor from . membrane positivity for MTNR1A (Figure 1).

Materials and Methods Pathological parotid glands (Warthin’s tumor). The epithelial component. This presented intense (+++) positivity for Patients’ samples. The study included samples of major salivary MTNR1A receptor in the cytoplasm and a lesser positivity glands from 10 patients, minor salivary glands from 10 patients (+) in the cell membrane; positivity (++) was also observed and 10 Warthin’s tumor. Structures corresponding to the major in the cytoplasm of basal cells (Figures 2 and 3). (parotid, submaxillary, sublingual) and minor (palatal and labial) salivary glands were preserved in all cases and utilized in the study. The median age of patients was 44 years (range, 33-66 Lymphoid component. This did not stain for MTNR1A years). All surgical procedures were carried out between 9 am and although a slight positivity (+) was detected in some 1 pm. Written informed consent was obtained from all patients, histiocyte cells in lymphoid follicles; lymphocytes located and the study was approved by the Ethics Committee of our among epithelial cells were negative for MTNR1A. institution. Submaxillary gland. This gland comprises of mixed acini Immunohistochemical studies. Samples were fixed in 10% buffered with a predominance of serous cells. MTNR1A positivity in formalin for 24 h and embedded in paraffin. Paraffin-embedded 4-μm sections were then de-waxed, hydrated, and heat-treated at 95˚C for the serous component was similar to that in the parotid gland 20 min in 1 mM EDTA buffer, pH 8, for antigenic unmasking. (+). However, the mucous cells, which were less abundant, Sections were incubated for 30 min at room temperature with goat exhibited scant positivity in the cytoplasmic membrane and polyclonal antibody raised against a peptide mapping at the N- reticular positivity in the cytoplasm. The myoepithelial cells terminus of MTNR1A of human origin; it was applied at a dilution around the serous cells were not MTNR1A-positive. The of 1:500 (Santa Cruz Biothecnology, Santa Cruz, California, USA). excretory ducts (lobar and lobular) showed intense The immunohistochemical study was carried out on an automatic cytoplasmic positivity for MTNR1A (+++), with weaker immunostainer (Autostainer 480; LabVision Fremont, CA, USA), by an indirect polymer-peroxidase-based method followed by positivity in the cytoplasmic membrane. development with diaminobenzidine (Masvision; Master Diagnóstica, Granada, Spain). Sublingual gland. Mucous cells were more abundant and The MTNR1A cytoplasmic staining pattern was graded as exhibited MTNR1A positivity (++) in the cytoplasmic weakly positive (+), moderately-positive (++), or strongly-positive membrane and the peripheral portion of the cytoplasm, and (+++); samples of and retina tissue were used as positive reticular positivity in the cytoplasm. The myoepithelial cells controls. around the serous cells were not MTNR1A-positive. The Results excretory ducts (lobar and lobular) exhibited intense cytoplasmic positivity (+++) for MTNR1A and weaker Normal parotid gland. The serous cells that make up the positivity for MTNR1A in the cytoplasmic membrane acini exhibited focal intracytoplasmic positivity (+) for (Figure 4).

4766 Arias-Santiago et al: MTNR1A in Salivary Glands

Figure 1. Normal parotid gland, immunohistochemical study of MTNR1A expression in the excretory ducts (diffuse +++) and acini (granular +) (original magnification ×200).

Figure 2. Pathological parotid gland (Warthin’s tumor), immunohistochemical study of MTNR1A expression in the epithelial component (diffuse +++), (original magnification ×40).

4767 ANTICANCER RESEARCH 32: 4765-4772 (2012)

Figure 3. Pathological parotid gland (Warthin’s tumor), immunohistochemical study of MTNR1A expression in the epithelial component (diffuse +++), (original magnification ×400).

Figure 4. Normal sublingual gland, immunohistochemical study of MTNR1A expression in the acini (granular ++) (original magnification ×200).

4768 Arias-Santiago et al: MTNR1A in Salivary Glands

Figure 5. Normal lingual gland, immunohistochemical study of MTNR1A expression in the acini (granular ++) and the excretory ducts (diffuse ++) (original magnification ×200).

Minor salivary glands. These glands exhibit cell variability Discussion as a function of their localization: in the palate they are mucous glands, whereas in the tongue, lip and oral mucosa, The melatonin receptor, MTNR1A has been found in they are mixed salivary glands, with a variable proportion of multiple sites in the human body, and also in cancer of the serous-mucous components (predominantly serous in the prostate, breast, bone and , and melanoma. To the lip). Regardless of the site of these glands, the mucous and best our of knowledge, our study is the first report, which serous components exhibited the same pattern of positivity compares MTNR1A expression in Warthin’s tumor and in for MTNR1A. Mucous cells revealed variable positivity, normal major and minor salivary glands. ranging from light (+) to moderate intensity (++) in the The biological role and clinical relevance of the MTNR1A cytoplasmic membrane, with focal intracytoplasmic in normal and tumor tissues are poorly understood. MTNR1A positivity with a reticular pattern. In serous cells, positivity was found to modulate the proliferation of malignant cells and was granular and was found in thick intracytoplasmic was reported to mediate the effects of melatonin on growth clumps. The myoepithelial cells around the serous cells were suppression and gene modulation in breast cancer cells (13). not MTNR1A-positive. The system of ducts (lobar and Warthin’s tumor is a multicystic lesion containing liquid and lobular) exhibited variable positivity for MT1 (++), with less granular materials that are secreted by the tumor cells. The intense expression in intercalated ducts (+) (Figure 5). tumor cells exhibit intense expression of the cytokeratins

4769 ANTICANCER RESEARCH 32: 4765-4772 (2012) associated with columnar differentiation and it has been References reported that activation of MTNR1A increases phosphorylation of mitogen-activated protein kinase and MEK1-2 and ERK 1/2, 1 Claustrat B, Brun J and Chazot G: The basic physiology and probably leading to induction of synthesis of filamentous pathophysiology of melatonin, Sleep Med Rev 9: 11-24, 2005. structures of non-neuronal tissues (14). We suggest that the 2 Reiter RJ: Pineal melatonin: cell biology of its synthesis and of MTNR1A may actively participate in synthesizing cytokeratin its physiological interactions. Endocr Rev 12: 151-180, 1991. in Warthin’s tumor cells 3 Reiter RJ: Melatonin: clinical relevance. Best Pract Res Clin Endocrinol Metab 17: 273-285, 2003. Melatonin is not only produced by the pineal gland, and 4 Bubenik GA: Gastrointestinal melatonin, localization, function its synthesis at other sites, such as the gastrointestinal and clinical relevance. Dig Dis Sci 47: 2336-2348, 2002. apparatus, can be up to 400-fold higher (15). Melatonin 5 Raikhlin NT, Kvetnoy IM and Tolkachev VN: Melatonin may be levels increase considerably with food intake (16), which has synthesised in enterochromaffin cells. Nature 255: 344-345, 1975. been related to the stimulation of duodenal production of 6 Dubocovich ML, Rivera-Bermudez MA, Gerdin MJ and Masana bicarbonate as a defence mechanism against gastric MI: Molecular pharmacology, regulation and function of chlorhydric acid, increased pancreatic amylase (17, 18) and mammalian melatonin receptors. Front Biosci 8: 1093-1108, 2003. 7 Slaugenhaupt SA, Roca AL, Liebert CB, Gusella JF and Repert cholecystokinin (19, 20). The action of melatonin in the oral SM: Mapping of the gene for the Mel1a-melatonin receptor to cavity would appear to be related to the increase in human chromosome 4 (MTNR1A) and mouse chromosome 8 melatonin produced by enterochromaffin cells during food (MTNR1A). Genomics 27: 355-357, 1995. intake rather than to the physiological circadian cycle of 8 Reppert SM, Weaver DR and Godson C: Melatonin receptors melatonin production (21-22). step into the light: cloning and classification of subtypes. Trends The presence of melatonin increases the secretion of Pharmacol Sci 17: 100-102, 1996. salivary amylase by direct action, with no mediation of the 9 Vanĕcek J: Melatonin binding sites. J Neurochem 51: 1436- 1440, 1988. sympathetic or parasympathetic nervous systems nor of 10 Lissoni P, Tancini G, Barni S, Paolorossi F, Ardizzoia A, Conti A intestinal peptides (12). However, melatonin has not been and Maestroni G: Treatment of cancer chemotherapy-induced associated with an increase in salivary volume. The intense toxicity with the pineal hormone melatonin. Support Care expression of MTNR1A observed in this study in the Cancer 5: 126-129, 1997. excretory ducts of major and minor salivary glands may be 11 Hunt AE, Al-Ghoul WM, Gillette MU and Dubocovich ML: related to salivary regulation. Melatonin may be involved in Activation of MT(2) melatonin receptors in rat suprachiasmatic the secretion of sodium, potassium, chloride and bicarbonate nucleus phase advances the . Am J Physiol Cell Physiol 280: 110-118, 2001. ions in salivary ducts, similar to its regulatory role in the 12 Aras HC, Ekström J: Melatonin-evoked in vivo secretion of production of duodenal bicarbonate. The finding of a more protein and amylase from the parotid gland of the anaesthetised intense expression of MTNR1A in the ducts than in the acini rat. J Pineal Res 45: 413-421, 2008. suggests that the antioxidant effect of melatonin is of little 13 Shiu SY, Law IC, Lau KW, Tam PC, Yip AW and Ng WT: importance in the ducts as it is independent of the presence Melatonin slowed the early biochemical progression of of receptors. However, MTNR1A expression is weaker in the hormone-refractory prostate cancer in a patient whose prostate acini, where the antioxidant effect may therefore be more tumor tissue expressed MT1 receptor subtype. J Pineal Res 35: 177-182, 2003. intense, especially in the mucous component. It has been 14 Witt-Enderby PA, MacKenzie RS, McKeon RM, Caroll EA, reported that MTNR1A expression is inversely-related to the Bordt SL and Melan MA: Melatonin induction of filamentous concentration of melatonin and is in fact higher in its structures in non-neuronal cells that is dependent on expression absence (23, 24). Accordingly, the present finding that of the human mt1 melatonin receptor. Cell Motil Cytoskeleton MTNR1A positivity is high in excretory ducts and low in 46: 28-42, 2000. acini would suggest a lower concentration of melatonin in 15 Huether G: The contribution of extrapineal sites of melatonin ducts than in acini. synthesis to circulating melatonin levels in higher vertebrates. Experientia 49: 665-670, 1993. In conclusion, although further immunohistochemical 16 Bubenik GA: Gastrointestinal melatonin, localization, function studies are warranted to elucidate the role of melatonin and and clinical relevance. Dig Dis Sci 47: 2336-2348, 2002. its receptors in salivary secretion and their influence as 17 Sjoöblom M, Jedstedt G and Flemström G: Peripheral melatonin inflammatory or tumorous diseases of the oral cavity (25- mediates neural stimulation of duodenal mucosal bicarbonate 27), melatonin may participate in the modulation of secretion. J Clin Invest 108: 625-633, 2001. absorptive and/or secretory processes of salivary secretion 18 Jaworek J, Nawrot K, Konturek J, Leja-Szpak A, Thor P and and in the pathophysiology of Warthin’s tumor. Pawlik WW: Melatonin and its precursor, L-tryptophan: influence on pancreatic amylase secretion in vivo and in vitro. J Pineal Res 36: 155-164, 2004. Conflicts of Interest 19 Li Y, Hoa Y and Owyang C: High affinity CCK-A receptors on the vagus nerve mediate CCK-stimulated pancreatic secretion in None. rats. Am J Physiol 273: G679-G685, 1997.

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20 Leja-Szpak A, Jaworek J, Nawrot-Porabka K, Palonek M, Mitis- 25 Cutando A, Aneiros-Fernández J, López-Valverde A, Arias- Musioł M, Dembiński A, Konturek SJ and Pawlik WW: Santiago S, Aneiros-Cachaza J and Reiter RJ: A new perspective Modulation of pancreatic enzyme secretion by melatonin and its in oral health: Potential importance and actions of melatonin precursor; L-tryptophan. Role of CCK and afferent nerves. J receptors MT1, MT2, MT3, and RZR/ROR in the oral cavity. Phys Pharm 55: 33-46, 2004. Arch Oral Biol 56: 944-950, 2011. 21 Cutando A, Gomez-Moreno G, Villalba J, Ferrera MJ, Escames 26 Cutando A, Aneiros-Fernández J, Aneiros-Cachaza J and Arias- G and Acuna-Castroviejo D: Relationship between salivary Santiago S: Melatonin and cancer: Current knowledge and its melatonin levels and periodontal status in diabetic patients. J application to oral cavity tumours: J Oral Pathol Med 40: 593- Pineal Res 35: 239-244, 2003. 597, 2011. 22 Fischer TW, Sweatman TW, Semak I, Sayre RM, Wortsman J 27 Cutando A, López-Valverde A, Arias-Santiago S, DE Vicente J and Slominski A: Constitutive and UV-induced metabolism of and DE Diego RG: Role of melatonin in cancer treatment. melatonin in keratinocytes and cell-free systems. FASEB J 20: Anticancer Res 32: 2747-2753, 2012. 1564-1566, 2006. 23 Barrett P, MacLean A, Davidson G and Morgan PJ: Regulation of the Mel 1a melatonin receptor mRNA and protein levels in the ovine pars tuberalis: Evidence for a cyclic adenosine 3’,5’- monophosphate-independent Mel 1a receptor coupling and an autoregulatory mechanism of expression. Mol Endocrinol 10: 892-902, 1996. 24 Dillon DC, Easley SE, Asch BB, Cheney RT, Brydon L, Jockers R, Winston JS,Brooks JS, Hurd T and Asch HL: Differential expression of high-affinity melatonin receptors (MT1) in normal Received July 23, 2012 and malignant human breast tissue. Am J Clin Pathol 118: 451- Revised October 4, 2012 458, 2002. Accepted October 5, 2012

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