Valproic acid downregulates heme oxygenase-1 independently of Nrf2 by increasing ubiquitination and proteasomal degradation
Abstract
Aims: Heme oxygenase-1 (HO-1; HMOX1 in human, Hmoxl in mice) is an antioxidative enzyme affecting wide range of sub-cellular processes. It was shown to modulate tumor growth or vascular-related diseases, thus being putative molecular target for tailored therapies.
Therefore it is of importance to elucidate novel compounds regulating HO-1 activity/expression and to delineate mechanisms of their action. In the present study we aimed to understand mode of action of valproic acid (VA), an antiepileptic drug, on HO-1 expression.
Results: We demonstrated that HO-1 expression is decreased by VA at protein but not mRNA level in human alveolar rhabdomyosarcoma cell line CW9019. Nrf2 transcription factor, the activator of HO-1 expression through ARE sequence, was excluded as a mediator of HO-1 decrease, as VA down regulated Bachl, a Nrf2 repressor, concomitantly. upregulating ARE activation.
Also miRNA-dependent inhibition was excluded as a mechanism of HMOX1 regulation. However, co-immunoprecipitation assay showed a higher level of ubiquitinated HO-1 after VA treatment.
Accordingly, MG132, an inhibitor of proteasomal degradation, reversed the effect of VA on HO-1 suggesting that decrease in HO-1 expression by VA is through protein stability. The inhibitory effect of VA on HO-1 was also observed in murine cells including embryonic fibroblasts isolated from Nrf2-deficient mice, what confirms Nrf2-independent effect of the compound.
Importantly, VA decreased also HO-1 expression and activity in murine skeletal muscles in vivo. Conclusion: Our data indicate that VA downregulates HO-1 by acting through ubiquitin-proteasomal pathway leading to decrease in protein level.