Korean J Ophthalmol.  2007 Jun;21(2):111-119. 10.3341/kjo.2007.21.2.111.

Microarray for Genes Associated with Signal Transduction in Diabetic OLETF Keratocytes

• Lee JE
• Lee JS
• Hwang SH

• KMID: 1101914
• DOI: http://doi.org/10.3341/kjo.2007.21.2.111

Abstract

PURPOSE: The purpose of this study was to identify differences in signal transduction gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Normal and diabetic keratocytes were primarily cultured and treated with 20 ng/ml IL-1alpha and TNF-alpha for 6 h. cDNA was hybridized to an oligonucleotide microarray. Genes identified by the microarray were further evaluated by real-time PCR. RESULTS: Diabetic keratocytes over-expressed components of the MAPK and Notch pathways, and under-expressed components of the insulin, calcium, and TGF-beta pathways. Cytokine treated diabetic keratocytes differentially expressed components of the TGF-beta and MAPK pathways. After IL-1alpha and TNF-alpha treatment, nine genes were under-expressed, falling in the insulin, TGF-beta, and Toll-like receptor pathways. Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene. CONCLUSIONS: There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways. In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.

Keyword

Diabetes mellitus; Keratocyte; Microarray; Rat; Signaling transduction

MeSH Terms

Animals
Apoptosis
Cells, Cultured
Cornea/drug effects/*metabolism/pathology
DNA/*genetics
Diabetes Mellitus, Experimental/*genetics/pathology
Gene Expression Profiling
Insulin/genetics
Interleukin-1alpha/pharmacology
Mitogen-Activated Protein Kinase Kinases/genetics
Nuclear Proteins/genetics
Oligonucleotide Array Sequence Analysis/*methods
Phosphoric Monoester Hydrolases/genetics
Polymerase Chain Reaction
Prolactin/genetics
Rats
Rats, Long-Evans
Receptors, Notch/genetics
Signal Transduction/drug effects/*genetics
Transforming Growth Factor beta/genetics
Tumor Necrosis Factor-alpha/pharmacology
Ubiquitin-Protein Ligases/genetics

Figure

• 

  Fig. 1 Quantitative real-time PCR of three genes using GAPDH as an endogenous control. "*" indicates a significant difference between normal and diabetic rats stimulated with or without IL-1α or TNF-α IL-6 and TGF-β 2 showed no significant difference between untreated normal and diabetic rat, but the diabetic rat showed a significant down-regulation when cytokine treated. Ppm1a showed a significant up-regulation in all diabetic cases.

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