Tyrosinase biosynthesis and trafficking in adult human retinal pigment epithelial cells.
BACKGROUND
Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whether it is synthesized in human postnatal retinal pigment epithelium (RPE). In this study, we investigated if phagocytosis of rod outer segments (ROS) can increase tyrosinase expression in vitro.


METHODS
Primary cultures of human RPE cells were fed with isolated ROS from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase presence and activity with several independent methods: (1) immunocytochemistry with anti-tyrosinase antibodies and (2) ultrastructural as well as light microscopic DOPA histochemistry; (3) mRNA was isolated from human RPE before incubation with ROS and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by real-time reverse transcription polymerase chain reaction (RT-PCR), and the tyrosinase activity was investigated by measuring tyrosine hydroxylase activity using [(3)H]tyrosine.


RESULTS
Tyrosinase was found in fed RPE cells using these methods, but was absent without feeding. Furthermore, we showed co-localization of rhodopsin and tyrosinase in the fed RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS, decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding.


CONCLUSIONS
Our study does not present new evidence that de novo melanogenesis takes place in the adult differentiated RPE. However, in contrast to the classic hypothesis, which states that tyrosinase is only detected in embryos, we provide evidence with several independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured human adult RPE by phagocytosis of ROS.
Julien
Sylvie
S
Section for Experimental Vitreoretinal Surgery, University Eye Hospital Tuebingen, Tuebingen, Germany. Sylvie.Julien@med.uni-tuebingen.de
Kociok
Norbert
N
Kreppel
Florian
F
Kopitz
Jürgen
J
Kochanek
Stefan
S
Biesemeier
Antje
A
Blitgen-Heinecke
Petra
P
Heiduschka
Peter
P
Schraermeyer
Ulrich
U
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics; D010587:Phagocytosis / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000201:enzymology* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D012333:RNA, Messenger; D004295:Dihydroxyphenylalanine; D012243:Rhodopsin; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase