Synthesis of osteonectin by human retinal pigment epithelial cells is modulated by cell density.
OBJECTIVE
To determine whether human retinal pigment epithelial (HRPE) cells are able to synthesize the antiadhesive protein osteonectin, also known as secreted protein, acidic and rich in cysteine (SPARC). Additionally, because locally produced SPARC may modulate cellular behavior during tissue repair, to ascertain whether HRPE SPARC production and HRPE proliferation, migration, and/or differentiation are associated, in a simple HRPE wound-healing model.


METHODS
Immunohistochemical and Western blot analyses of SPARC protein expression by low- and high-density cultured HRPE cells were undertaken. Total RNA extracted from cultures was studied by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. Western and Northern blot analyses were evaluated by densitometry. Experiments were repeated with HRPE cells cultured in the presence of 1, 10, or 100 microM of the differentiating agents butyric acid (BA) and retinoic acid (RA).


RESULTS
HRPE cell cultures exhibited SPARC immunoreactivity. Western blot analysis of cell lysates and conditioned media showed a 43-kDa protein. RT-PCR and Northern blot analysis confirmed the presence of SPARC mRNA (with transcripts at 2.2 and 3.0 kb). Protein and mRNA transcript band densitometry revealed a higher proportion of SPARC protein and mRNA in high-density HRPE cell culture than in low-density culture. Neither BA nor RA (at the concentrations assessed) had a significant effect on SPARC production by HRPE cells in high- or low-density culture.


CONCLUSIONS
HRPE can synthesize SPARC. Although the findings do not support an invariable association between SPARC production by HRPE and HRPE proliferation, migration, or differentiation, they demonstrate that synthesis of SPARC by HRPE is modulated by cell density.
Department of Medicine, University of Liverpool, United Kingdom.
R M
RM
Magee
S
S
Hagan
P S
PS
Hiscott
C M
CM
Sheridan
J A
JA
Carron
J
J
McGalliard
I
I
Grierson
D015152:Blotting, Northern; D015153:Blotting, Western; D020148:Butyric Acid / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015676:Osteonectin / Q000096:biosynthesis* / Q000235:genetics; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000494:pharmacology
D017931:DNA Primers; D005136:Eye Proteins; D015676:Osteonectin; D012333:RNA, Messenger; D020148:Butyric Acid; D014212:Tretinoin