Expression and distribution of the adrenomedullin system in newborn human thymus.
Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.
Sara
S
De Martin
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.
Giovanna
G
Paliuri
Department of Molecular Medicine, University of Padova, Padova, Italy.
Annasandra
A
Belloni
Scientific Institute IRCCS Eugenio Medea, Conegliano, Treviso, Italy.
Genny
G
Orso
Scientific Institute IRCCS Eugenio Medea, Conegliano, Treviso, Italy.
Erica
E
Zanarella
Pediatric and Congenital Cardiac Surgery Unit, Department of Cardiac, Thoracic and Vascular Surgery, University of Padova, Padova, Italy.
Giovanni
G
Stellin
Department of Pediatrics, University of Padova, Padova, Italy.
Ornella
O
Milanesi
Department of Pediatrics, University of Padova, Padova, Italy.
Giuseppe
G
Basso
Department of Pediatrics, University of Padova, Padova, Italy.
Ezia Maria
EM
Ruga
Department of Pediatrics, University of Padova, Padova, Italy.
Chiara
C
Frasson
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.
Daniela
D
Gabbia
Department of Molecular Medicine, University of Padova, Padova, Italy.
Giada
G
Perdoncin
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.
Pietro
P
Palatini
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.
Sergio
S
Bova
D053607:Adrenomedullin / Q000378:metabolism*; D058286:Calcitonin Receptor-Like Protein / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D008407:Mast Cells / Q000166:cytology; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D058263:Receptor Activity-Modifying Protein 2 / Q000378:metabolism; D058264:Receptor Activity-Modifying Protein 3 / Q000378:metabolism; D060168:Thymocytes / Q000166:cytology; D013950:Thymus Gland / Q000254:growth & development* / Q000378:metabolism
C507862:ADM protein, human; C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D053607:Adrenomedullin; D000242:Cyclic AMP