Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

Abstract

One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining K m values of 4.5, 1.0, and 0.8 M, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117-140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

“…The most intense signals were found in brain and in testis (Pitkanen et al 2001). This was recently conWrmed by Lopez-Contreras et al (2008a) who analyzed mouse tissues by real-time RT-PCR, and found the highest level of AZIN2 expression in testicular tissue. We have produced a rabbit antiserum speciWc to AZIN2.…”

Antizyme and antizyme inhibitor, a regulatory tango

“…ODCp was demonstrated to lack ornithine decarboxylase and arginine decarboxylase activity, but is able to rescue ODC from degradation and to inhibit the antizyme-mediated inhibition of polyamine uptake; therefore, it was termed antizyme inhibitor 2 [109][110][111][112][113]. Like antizyme inhibitor 1, antizyme inhibitor 2 is also rapidly degraded in a ubiquitin-dependent manner [111,113]; it stimulates ODC activity, polyamine uptake and cellular proliferation although less efficiently than antizyme inhibitor 1 [113].…”

An exercise in brain genoarchitectonics: Analysis of AZIN2‐Lacz expressing neuronal populations in the mouse hindbrain

“…These are paralogs of ODC that lack enzymatic activity and, due to their structural resemblance to ODC, inhibit AZ1‐3 by binding to them, thus lowering the access of antizymes to ODC and polyamine uptake‐related binding sites. This action enhances ODC‐mediated synthesis of polyamines and polyamine uptake, increasing cytoplasmic polyamine levels (López‐Contreras et al, ; López‐Contreras et al, ; López‐Contreras et al, ; Ramos‐Molina et al, ). It appears that polyamines indirectly promote cell growth.…”