To determine whether there is an association between vascular phosphodiesterase type 5 (PDE-5) and NADPH oxidase (NOX) in cavernosal vascular smooth muscle cells (CVSMCs), and to study the actions of the PDE-5 inhibitor sildenafil; the pro-erectile actions of nitric oxide (NO) are reduced by PDE-5 which hydrolyses cGMP to inactive GMP, thus an up-regulation of PDE-5 and over-production of O(2)(-) derived from NOX might promote erectile dysfunction (ED).To study the effects of nicotine and tumour necrosis factor-alpha (TNF-alpha) on superoxide (O(2)(-)) production and PDE-5 expression, CVSMCs from rabbit penis were incubated with nicotine or TNF-alpha, and superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (NADPH inhibitor) for 16 h. The expression of PDE-5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blotting. O(2)(-) was measured spectrophotometrically.After a 16-h incubation, both nicotine (maximal at 10 microm) and TNF-alpha (10 ng/mL) significantly increased O(2)(-) formation in CVSMCs; this effect was blocked by co-incubating with SOD, catalase, and sildenafil (1 microm). Apocynin also inhibited O(2)(-) formation when added after 16-h incubation with nicotine (10 microm) or TNF-alpha. PDE-5 expression was also significantly increased in CVSMCs incubated with nicotine and TNF-alpha. This effect was negated by 16-h co-incubation with SOD, catalase, apocynin, and sildenafil.Nicotine and TNF-alpha up-regulate PDE-5 expression in CVSMCs through an a priori up-regulation of NOX and formation of O(2)(-). As PDE-5 hydrolyses cGMP, this effect might 'blunt' the pro-erectile actions of NO. Sildenafil inhibits O(2)(-) formation, and 'normalizes' PDE-5 expression. This represents a novel pathogenic mechanism underlying ED, and a novel mechanism of action of sildenafil.
Aims: Osteopontin (OPN) is an ubiquitous pro-metastasis gene associated with poor prognosis of colorectal, breast and lung cancer.Our aims were to identify any novel OPN suppressor gene and investigate its role in metastasis.Methods: Novel OPN interacting proteins were sought by bacterial and mammalian two-hybrid screens of a human cDNA library using full length rat OPN as ''bait''.Novel genes were cloned, sequenced and tested for OPN interaction by co-immunoprecipitation.OPN binding domains were mapped by sequence deletion experiments.Benign R37 mammary cells were rendered invasive by stable transfection with OPN.Effects of novel genes on OPN-mediated metastasis were investigated in vitro.Results: Of 25 different OPN interacting genes, ApoD (apolipoprotein D) showed maximal suppression of OPN expression.Transfection of invasive cells with ApoD inhibited OPN transactivation, reduced cell colony formation in soft agar from 207•8 ± 5•5 to 64•0 ± 5•5 (P < 0•001) and cell invasion from 70•5% to 47•3% (P < 0•001).Inhibition of ApoD by antisense RNA promoted OPN expression and enhanced cell invasion by parental R37 cells. Conclusion:This work shows for the first time that ApoD is a suppressor of OPN and inhibits metastasis, in a model system.These findings may provide a scientific basis for novel specific therapy to inhibit metastasis.
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