Microarray reveals complement components are regulated in the serum-deprived rat retinal ganglion cell line.
OBJECTIVE
Glaucoma is a progressive eye disease that leads to blindness due to loss of retinal ganglion cells (RGCs). There are difficulties in using primary cultures of purified RGC to study this pathophysiology. RGC-5, a transformed not RGC line, expresses several markers characteristic of the RGCs. The aim of this study was to generate a genome-wide gene expression of RGC-5 following serum deprivation and to identify candidate genes that may be involved in the signal transduction pathways.


METHODS
Apoptosis in the transformed rat RGC-5 was induced by serum deprivation for 0, 8, 24, 48, and 96 h. Briefly, 400 ng of RNA from each sample was reverse transcribed and labeled with Cy3 dye. Fragmented fluorescent cRNA was mixed with hybridization buffer and incubated at 60 degrees C for 16 h. Labeled cRNA was hybridized to Rat Genome Oligonucleotide Arrays. These arrays contain 22,775 transcripts with one oligonucleotide per transcript (60-mer). Gene expression from scanned images was quantified and analyzed using ArrayVision software. Reproducibility among triplicate arrays was determined by ANOVA statistical analysis. Significant differences in gene expression between apoptotic and nonapoptotic cells were determined based on p-values.


RESULTS
Of the 22,775 transcripts present on the arrays (Agilent rat genome, 60-mer), 713 (8 h), 1,967 (24 h), 1,011 (48 h), and 1,161 (96 h) were differentially expressed relative to the 0 h time point (p-values <0.05). Twenty-three transcripts were common to 8, 24, 48, and 96 h and 130 transcripts were common to the 24, 48, and 96 h time points. The two most highly upregulated genes were Fdft1 and Lgals3 (8 h), C3 and Fcgrt (24 h), C and Lcn2 (48 h), and Mgp and C3 (96 h). A subset of the differentially expressed genes identified in microarray data (Ftl1, C3, C1s, Neu1, Polr2g, Acadm, Nupr1, Gch, Dia1, DNase1, Tgfb2, and Cyr61) were validated using quantitative real time polymerase chain reaction (QRT-PCR). Here we show that complement factor H (CFH), the major inhibitor of the alternative complement pathway is downregulated in serum-deprived RGC-5. CFH protein was detected within RGC-5 cells as well as the rat retina with the aid of immunocytochemistry and confocal microscopy.


CONCLUSIONS
This study was undertaken to generate a genome-wide gene expression profile of RGC-5 after serum deprivation, and to identify candidate and novel genes that may be involved in the signal transduction pathways leading to apoptosis. RGC-5 serum deprivation revealed up-and downregulation in gene expression profiles. The data gathered from this study was the first report that the genes identified in microarray data and validated by real-time RT-PCR may play an important role in RGC-5 cell death. Among the validated genes, C3 and C1s showed significant upregulation of the complement component pathway. The results further indicate that components of the complement pathway are present in neurons of the rat retina. The data indicated that complement factors are likely involved in the pathway leading to ganglion cell death in the serum-deprivation paradigm, which may be similar to the mechanism of cell death in glaucoma.
Khalyfa
Abdelnaby
A
Department of Anatomical Sciences and Neurobiology, University of Louisville, School of Medicine, Louisville, KY 40292, USA. a.khalyfa@louisville.edu <a.khalyfa@louisville.edu>
Chlon
Timothy
T
Qiang
He
H
Agarwal
Neeraj
N
Cooper
Nigel G F
NG
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002461:Cell Line, Transformed; D003172:Complement C1 / Q000378:metabolism; D003176:Complement C3 / Q000378:metabolism; D017242:Complement Factor H / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism*; D003199:Computer Systems; D016895:Culture Media, Serum-Free; D015536:Down-Regulation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D051381:Rats; D015203:Reproducibility of Results; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014018:Tissue Distribution; D015854:Up-Regulation
D003172:Complement C1; D003176:Complement C3; D016895:Culture Media, Serum-Free; D017242:Complement Factor H; D003165:Complement System Proteins