Abstract
Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O2). Melatonin was applied at 100 μM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced Sirt1 gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.
Highlights
Hypoxia-inducible factors (HIF’s), hypoxia-inducible factor-1 (HIF-1), HIF-2, and HIF-3, are the main mediators of metabolic responses to the state of hypoxia
In view of the earlier observations, we presumed that melatonin inhibitory action on HIF-1α expression in HK-2 cells involves its impact on the stability of this protein
We turned our attention to sirtuins, to sirtuin 1, known to be able to deacetylate many proteins, including HIF-1α (Joo et al, 2015; Ryu et al, 2019), and we proved that in HK-2 cells cultured under hypoxic conditions, both protein (Figure 2A) and mRNA (Figure 2B) levels of SIRT1 were significantly diminished upon the addition of melatonin to experimental media
Summary
Hypoxia-inducible factors (HIF’s), HIF-1, HIF-2, and HIF-3, are the main mediators of metabolic responses to the state of hypoxia. Hydroxylated HIF-1α is recognized by the von Hippel–Lindau (VHL) protein, ubiquitinated, and guided to be degraded in the proteasome (Nangaku et al, 2013; Chan et al, 2016; Shu et al, 2019). Because of this mechanism, the functional complex of HIF-1 practically does not occur under conditions of optimal oxygenation. In addition to regulating the stability of the protein by oxygen concentration, the ultimate activity of HIF-1 is controlled by a variety of other factors, including those affecting transcription, translation initiation, stability and nuclear translocation of HIF-1α subunit and, functional HIF-1 dimer activity [cf. Koyasu et al (2018) for review]
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