OBJECTIVEKlotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys.RESEARCH DESIGN AND METHODSRenal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536.RESULTSRenal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding.CONCLUSIONSThese findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domain of RelA.

The PI3K/AKT signaling pathway is aberrant in a wide variety of cancers. Downstream effectors of AKT are involved in survival, growth, and metabolic-related pathways. In contrast, contradictory data relating to AKT effects on cell motility and invasion, crucial pro-metastatic processes, have been reported pointing to a potential cell type and isoform type-specific AKT driven function. By implication, study of AKT signaling should optimally be conducted in the appropriate intracellular environment. Prognosis in soft-tissue sarcoma (STS), aggressive malignancies of mesenchymal origin, is poor reflecting our modest abilities to control metastasis, an effort hampered by lack of insight into molecular mechanisms driving STS progression and dissemination. We examined the impact of the cancer progression relevant AKT pathway on the mesenchymal tumor cell internal milieu. We demonstrate that AKT1 activation induces STS cell motility and invasiveness at least partially via a novel interaction with the intermediate filament vimentin. The binding of AKT (tail region) to vimentin (head region) results in vimentin Ser39 phosphorylation enhancing the ability of vimentin to induce motility and invasion while protecting vimentin from caspase induced proteolysis. Moreover, vimentin phosphorylation was shown to enhance tumor and metastasis growth in vivo. Insights into this mesenchymal-related molecular mechanism may facilitate development of critically lacking therapeutic options for these devastating malignancies.

Reactive oxygen species (ROS) and elevated levels of p38 MAPK activity accelerate physiological aging. This emphasizes the importance of understanding the molecular mechanism(s) that link ROS production to activation of the p38 mediated promotion of aging, longevity, and resistance to oxidative stress. We examinedKlotho(-/-) (elevated ROS) and Klotho overexpressing mice (low ROS and resistance to ROS) to determine whether the ROS-sensitive apoptosis signal-regulating kinase (ASK1)-signalosome → p38 MAPK pathway plays a role in the accelerated aging of Klotho(-/-), and resistance to oxidative stress and extended lifespan in the Klotho overexpressing models. Our results suggest that increased endogenous ROS generated by Klotho(-/-) and resistance to oxidative stress in Klotho overexpression are linked to the regulation of ASK1-signalosome → p38 activity. We propose that (a) the ASK1-signalosome → p38 MAPK pathway is activated by oxidative stress due to ablation of the Klotho gene; (b) increased longevity by Klotho overexpression is linked to to suppression of the ASK1-signalosome-p38 MAPK activity; (c) the ROS-responsive ASK1-signalosome regulates physiological aging via its regulation of p38 MAPK, through a mechanism that balances the levels of inhibitory vs. activating ASK1-signalosomes. We conclude that the Klotho suppressor-of-aging activity is linked to the ASK1-signalsome, a physiological ROS-sensitive signaling center.

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SummaryThe antifolate combination pyrimethamine/sulphadoxine (PYR /SDX; Fansidar) is frequently used to combat chloroquine-resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR / SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.