Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 beta: identification of miR-153 target genes with functions related to IA-2 beta in pancreas and brain

AIMS/HYPOTHESIS: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function. METHODS: A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. RESULTS: Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. CONCLUSIONS/INTERPRETATION: This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.status: publishe

Prostate specific membrane antigen (PSMA) regulates angiogenesis independently of VEGF during ocular neovascularization.

Aberrant growth of blood vessels in the eye forms the basis of many incapacitating diseases and currently the majority of patients respond to anti-angiogenic therapies based on blocking the principal angiogenic growth factor, vascular endothelial growth factor (VEGF). While highly successful, new therapeutic targets are critical for the increasing number of individuals susceptible to retina-related pathologies in our increasingly aging population. Prostate specific membrane antigen (PSMA) is a cell surface peptidase that is absent on normal tissue vasculature but is highly expressed on the neovasculature of most solid tumors, where we have previously shown to regulate angiogenic endothelial cell invasion. Because pathologic angiogenic responses are often triggered by distinct signals, we sought to determine if PSMA also contributes to the pathologic angiogenesis provoked by hypoxia of the retina, which underlies many debilitating retinopathies.Using a mouse model of oxygen-induced retinopathy, we found that while developmental angiogenesis is normal in PSMA null mice, hypoxic challenge resulted in decreased retinal vascular pathology when compared to wild type mice as assessed by avascular area and numbers of vascular tufts/glomeruli. The vessels formed in the PSMA null mice were more organized and highly perfused, suggesting a more 'normal' phenotype. Importantly, the decrease in angiogenesis was not due to an impaired hypoxic response as levels of pro-angiogenic factors are comparable; indicating that PSMA regulation of angiogenesis is independent of VEGF. Furthermore, both systemic and intravitreal administration of a PSMA inhibitor in wild type mice undergoing OIR mimicked the PSMA null phenotype resulting in improved retinal vasculature.Our data indicate that PSMA plays a VEGF-independent role in retinal angiogenesis and that the lack of or inhibition of PSMA may represent a novel therapeutic strategy for treatment of angiogenesis-based ocular diseases

Retinal vasculature of wild type mice undergoing OIR expresses PSMA.

<p><b>A</b>) A time line depicting the stages of retinal layer vascularization with diagrams of approximate vascular morphology of the retina at various time points. <b>B</b>) Conventional RT-PCR of RNA isolated from wild-type OIR retinas from P17 mice are positive for PSMA; PSMA wild-type kidney and TRAMP-C1 cells- PSMA positive controls, PSMA null retina- negative control. <b>C</b>) qRT-PCR for PSMA over time from retinal RNA isolated at the indicated time points relative to P12 levels. Paraffin embedded OIR retinas were immunostained for PSMA protein using the 3E2 antibody. Staining (red-brown) was observed on vascular tufts (arrows) of wild-type (<b>D</b>) but not PSMA-null (<b>E</b>) retinas. (n = 3 per group), **p,0.05, ***p,0.001.</p

Angiogenic growth factor production is normal in response to hypoxia in PSMA null mice.

<p>PSMA expression was measured by qRT-PCR from RNA isolated from wild type and PSMA null retinas daily, beginning at the initiation of tissue hypoxia (P12 through P17). <b>A</b>) Wild type and PSMA mice produce similar VEGF expression levels during hypoxia treatment. <b>B</b>) PSMA null mice show similar Ang-2 levels to wild type mice over time. (n = 3 per time point).</p

Inhibition of PSMA decreases pathologic angiogenesis in a mouse model of OIR.

<p><b>A</b>) Wild type mice undergoing OIR were treated systemically with either a single dose of 100mg/kg 2-PMPA on P14 (n = 3, <b>center</b>) or three daily doses from P14 through P16 (n = 6, <b>right</b>), controls received vehicle (PBS, <b>left</b>). Retinas were isolated on P17, vasculature was stained using GS Isolectin B4–A594 and the avascular area calculated using Image J software. <b>B</b>) Mice receiving a single dose on P14 showed a slight but not statistically significant decrease in avascular area, whereas mice treated from P14–16 showed a significant decrease (23%) in avascular area compared to vehicle treated controls (p = 0.0055). <b>C</b>) Wild type mice on the OIR protocol were treated once on P14 with 10mg intravitreal 2-PMPA (10mg/ml, 1mL) in one eye and 1mL Vehicle (PBS) in the contralateral eye. Retinas treated with intravitreal 2-PMPA showed a significant decrease (16.66%) in avascular area compared to control retinas.</p

Vascular pathology is decreased in PSMA null animals undergoing OIR.

<p><b>A</b>) Retinas were harvested from OIR mice at P12 (<b>left</b>), P15 (<b>center</b>) and P17 (<b>right</b>). Vasculature was stained using RCA-1-FITC (green, P12) or GS Isolectin B4-A594 (red, P15 and P17) and <b>B</b>) the central avascular area of each retina was measured using Image J. (n = 3 per group).</p

Retinal vasculature develops normally in PSMA null mice.

<p>Retinas from wild-type (<b>left</b>) and PSMA-null (<b>right</b>) mice (5X) raised in room air, harvested at P17 and perfused with FITC labeled <i>ricin communis</i> agglutinin 1 (RCA-1-FITC, green). Insets: higher (40X) magnification of the same retina and show normal radial branching pattern in animals of both genotypes. (Representative image, n = 3 per group).</p