Calcium mediates high glucose-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells through CaMKII-CREB pathway.
OBJECTIVE
To investigate the effects of high glucose (HG) medium on expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in cultured rat retinal Müller cells and to determine the signaling pathways mediating the effects.


METHODS
Primary cultures of retinal Müller cells were prepared from Sprague-Dawley rats, and incubated in a medium containg HG (30 mmol/L) in the presence of the membrane-permeable Ca(2+) chelator BAPTA-AM (10 μmol/L) or the CaMKII inhibitor KN93 (10 μmol/L). The levels of CaMKII, p-CaMKII, CREB, p-CREB, HIF-1α, and VEGF proteins were measured with Western blotting, while HIF-1á and VEGF mRNA levels were determined using real-time RT-PCR.


RESULTS
The stimulation of retinal Müller cell with HG for 24 h remarkably increased the expression levels of HIF-1α and VEGF. These responses were significantly inhibited in the presence of BAPTA-AM or KN93. Both BAPTA-AM and KN93 also significantly inhibited HG-induced phosphorylation of CaMKII and CREB in the cultured retinal Müller cells. Transfection of the cultured retinal Müller cells with antisense CREB oligonucleotide (300 nmol/L) was similarly effective in blocking the HG-induced increase of HIF-1α and VEGF.


CONCLUSIONS
HG-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells depends on intracellular free Ca(2+) and activation of CaMKII-CREB pathway. The activation of CaMKII-CREB pathway by HG may be a possible mechanism underlying the pathogenesis of diabetic retinopathy.
Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai 200080, China.
Jun
J
Li
Shu-zhi
SZ
Zhao
Pei-pei
PP
Wang
Song-ping
SP
Yu
Zhi
Z
Zheng
Xun
X
Xu
D000818:Animals; D002118:Calcium / Q000502:physiology*; D065096:Calcium Chelating Agents / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000096:biosynthesis*; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis*; D063928:Ependymoglial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D005947:Glucose / Q000633:toxicity*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D065096:Calcium Chelating Agents; D017362:Cyclic AMP Response Element-Binding Protein; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D005947:Glucose; D002118:Calcium