Inducible nitric oxide synthase (iNOS) is an essential mediator in diabetic vascular lesions and known to be regulated by activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The aim of this study was to investigate whether CaMKII affects iNOS-mediated pericyte death in the retina of diabetic mice with early stage disease. Total- and phospho-CaMKII, iNOS, and active caspase-3 protein levels were assessed by Western blotting, and CaMKII activity was measured by kinase assay. iNOS-related pericyte death was assessed by double immunofluorescent staining for iNOS and α-smooth muscle actin, followed by the TUNEL assay. Autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was injected into the right vitreous 2 days before sacrifice of mice, to examine the effect of CaMKII inactivation in diabetic retinas. The levels of total- and phospho-CaMKII, iNOS, and active caspase-3 protein, and CaMKII activity were significantly increased in the diabetic retinas compared with those of control retinas. Furthermore, TUNEL-positive signals colocalized with iNOS-immunoreactive pericytes in the same retinas. However, inactivation of CaMKII by AIP treatment inhibited all these changes, which was accompanied by less pericyte loss. Our results demonstrate that CaMKII contributes to iNOS-related death of pericytes in the diabetic retina and that inactivation of this enzyme may be a potential treatment for retinal vascular lesion.

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Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions −806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl2, incubation under 1% O2 conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl2 and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.