Histone demethylation has been suggested to play an important role in the lifespan of model organisms
[19, 20]. However, much of the evidence for this came from manipulations made using RNAi-mediated knockdown. Here we report additional evidence in favour of the inhibitory effects of the histone demethylase JMJD2 family by the small molecule PA. Previously, PA was known as a polyphenol that is naturally found in the fruiting bodies of Phellinus linteus, Ganoderma applanatum and Ranunculus sieboldii, the roots of Salvia miltiorrhiza, the leaves of Vitis vinifera, and grape and barley seeds
[21–27]. It was shown to inhibit the activities of tyrosinase, herpes simplex virus type-1 replication, tumour necrosis factor (TNF)-α-induced cell-surface expression of vascular adhesion molecule-1, aldose reductase, phosphatidylinositol kinase and advanced glycation end product-bovine serum albumin (BSA) formation
Several studies reported on the use of pharmacological manipulation with transcription factors and nucleosomal histone modification to inhibit adipocyte differentiation
[16, 17]. To gain further knowledge of relative efficacy, the inhibitory effects of PA were compared with a well-known JMJD2 family inhibitor, 2,4-PDCA. PA was approximately 2-fold less potent than 2,4-PDCA in affecting JMJD2A activity. Moreover, inhibitory changes by PA were sensitive to the JMJD2 family but insensitive to LSD1, suggesting that PA might involve 2-oxoglutarate oxygenase from the Jumonji domain-containing family, but not amine oxidase in lysyl demethylase. To better understand the property of PA, it is important to clarify the specificity of PA against different classes of histone demethylases. As 2,4-PDCA and the collagen proline hydroxylase inhibitor 3,4-dihydroxybenzoate bind to the 2-oxoglutarate binding site of prolyl 4-hydroxylase, inhibition of the 2-oxoglutarate co-substrate in the JMJD2 family is a potential mechanism
[12, 32]. Further studies are needed to elucidate the identity of the demethylation site that may be affected by PA. Crystallographic analyses of JMJD2A in complex with 2,4-PDCA have shown that it binds in a similar manner to the 2-oxoglutarate co-substrate
[15, 33]. In the present study, molecular computational modelling analysis showed that PA can bind to JMJD2A at the active site, in a similar manner to 2,4-PDCA.
In humans, about 30 JmjC proteins have been identified and grouped into eight distinct subfamilies: JHDM1, JHDM2, JMJD2, PHF2, PHF8, Jumonji(A + T)-rich interactive domain (JARID), ubiquitously transcribed tetratricopeptide repeat X/Y-linked (UTX/UTY) and JmjC-domain
. Han and colleagues observed that RNAi of the utx-1 gene extends the mean lifespan of Caenorhabditis elegans by about 30%
. By contrast, Li et al. recently reported that histone demethylase-inactive Lid flies are short lived
, whereas another study showed that disruption of Dmel/Kdm4A, a homologue of the human JMJD2 family, reduces male-specific longevity
. Lifespan is highly sensitive to genetic background and environmental conditions. Therefore, it is possible that the physiological situation is different between Kdm4A mutant flies and those treated with PA. Lifespan of Kdm4A mutant male was shorter than wild-type, while that of mutant females was unchanged. In contrast, PA extended the lifespan of female, but not male. Interestingly, its toxicity was more obvious in males than in females. Therefore, there is a consistency of sex difference: females were more tolerant to the reduced activity of Kdm4A compared to males. PA might have additional functions including inhibitory activity against other KDM4/JMJD2 demethylases. Further studies are needed to clarify the mechanism of lifespan extension by PA.
In the present study, Drosophila lifespan extension was specific to female individuals. Gender-specific lifespan extension has been reported in several Drosophila mutants, including the insulin substrate chico, kelch-like ECT-associated protein 1 (keap1), p53 and puckered (puc) mutants
Another goal of the present study was to gain insights into the genetic components affected by PA through a large-scale analysis of gene expression. In Drosophila S2 cells, 4E-BP was up-regulated in response to PA. 4E-BP has been reported to play an important role in lifespan extension following dietary restriction in Drosophila. Moreover, Demontis et al. recently reported that key roles of FOXO/4E-BP signaling are to preserve muscle function and extend the lifespan of Drosophila. Thus, extension of Drosophila lifespan by PA might involve, at least in part, the 4E-BP signal cascade. It is unclear at present whether PA induces 4E-BP via the insulin receptor/4E-BP pathway. It is reasonable hypothesis that PA might extend the lifespan through downregulating the insulin/IGF signaling pathway. Further studies are needed to elucidate whether the intracellular 4E-BP-dependent signaling pathway induced by PA might affect the lifespan extension of Drosophila.