Angiotensin II stimulated transcription of cyclooxygenase II is regulated by a novel kinase cascade involving Pyk2, MEKK4 and annexin II.

Although it is known that MEKK4 regulates MKK6, and p38 MAP kinase, extracellular stimuli that activate the serine/threonine kinase, MEKK4, are unknown. The aim of this study was then to identify stimuli that regulate MEKK4. By using recombinant MEKK4, as bait to attract interacting proteins, the calcium binding protein, annexin II, was identified by mass spectrometry as interacting with MEKK4, suggesting that MEKK4 might be regulated by calcium. A calcium-dependent interaction between MEKK4 and annexin II was observed when MEKK4 was immunoprecipitated from rat aortic smooth muscle cells that were treated with angiotensin II. Additional studies using recombinant MEKK4 in a Far-Western immunoblot identified a protein of 120 kDa as interacting directly with MEKK4. Prior studies indicated that MEKK4 was phosphorylated on tyrosine in vivo, and in fact, Pyk2 interacts with MEKK4 in an angiotensin II dependent manner in rat aortic smooth muscle cells. Pyk2 phosphorylates MEKK4 in vitro and Pyk2-dependent phosphorylation further regulates MEKK4-dependent phosphorylation of MKK6. Finally, dominant-negative MEKK4 inhibits angiotensin II mediated transcription of a luciferase reporter construct containing the cyclooxygenase II promoter, demonstrating that MEKK4 functions in a calcium-dependent manner as a substrate for Pyk2 and regulates transcription of cyclooxygenase II.

Interferon-gamma-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin II and SHP2 in keratinocytes.

IFNgamma (interferon-gamma) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNgamma-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine-phosphorylated and the IFNgamma-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNgamma-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNgamma. Immunofluorescence imaging of HaCaT cells shows an IFNgamma-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNgamma-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNgamma-dependent manner and co-localizes with MEKK4 after IFNgamma stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNgamma signalling pathway.

Characterization of the DialGuard device for endotoxin removal in hemodialysis.

Bacterial pyrogens, capable of penetrating dialyzer membranes, are responsible for a systemic inflammatory reaction in hemodialysis patients. Dialyzer reuse, involving rinsing of the dialyzer with pyrogen-containing water, may exacerbate this situation. Studies of the mechanism of action of endotoxin suggest that it irreversibly damages the vascular endothelium. The novel endotoxin removal method described here, is based on affinity-binding of endotoxin by the adsorbent ClarEtox, a USP Class VI-certified resin that is the active component of the medical device DialGuard. Under standard hemodialysis operating conditions, challenge of DialGuard with Pseudomonas maltophilia supernatant-spiked dialysate, containing 35-193 EU/ml endotoxin, resulted in endotoxin levels below 0.05 EU/ml in the treated dialysate. DialGuard was able to decrease endotoxin concentrations in the dialysate from a range of 2.39-8.49 to <0.005 EU/ml. DialGuard supports high fluid velocities at low back pressures and can be sanitized using the heat sanitization cycle of hemodialysis machines. DialGuard offers a simple, user-friendly way to reduce the concentration of endotoxin in dialysate and water for dialysis at a low cost.