Abstract
Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H(2)O(2)-induced NF-kappaB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.
Highlights
Oxidative stress, as a result of hyperglycemia and diabetes, has been postulated to be a major metabolic abnormality causing a variety of cellular dysfunctions and vascular pathologies [1,2,3]
One of the earliest and most specific histopathological findings associated with hyperglycemia and diabetic retinopathy is the selective loss of retinal capillary pericytes, probably because of apoptosis [9]
Our results indicate that insulin activates Heme oxygenase (HO)-1 expression by increasing its promoter activity selectively by the IRS1/PI3K/ Akt2-dependent pathway
Summary
Reagents—Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin-streptomycin were obtained from Invitrogen. HO-1 expression was induced by adding 20 M hemin or different concentrations of insulin to the culture medium and incubating at 37 °C for various time points. Luciferase Reporter Constructs and Expression Plasmids—An HO-1 luciferase reporter plasmid, HO-1-(Ϫ4045/ϩ74), was amplified from mouse genomic DNA by PCR and subcloned into the pGL2 luciferase reporter vector (Promega, Madison, WI). This HO-1 promoter was described previously [33]. The cells were washed three times with cold PBS and lysed, and nuclear-specific proteins were isolated using the NucBuster protein extraction kit (Novagen, Madison, WI) according to the manufacturer’s instructions. HO-2, which is constitutively expressed in the cell, was not affected by the addition of insulin (Fig. 2, A and C) or hemin (data not shown)
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