Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments.

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 µM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Role of dimethylarginine dimethylaminohydrolases in the regulation of endothelial nitric oxide production.

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.