TY  - JOUR
A1  - Rauert-Wunderlich, Hilka
A1  - Siegmund, Daniela
A1  - Maier, Eduard
A1  - Giner, Tina
A1  - Bargou, Ralf C.
A1  - Wajant, Harald
A1  - Stühmer, Thorsten
T1  - The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NF kappa B Transcription Factors
JF  - PLoS ONE
N2  - Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.
KW  - signal inhibition
KW  - necrotic cell death
KW  - cell viability testing
KW  - cell death
KW  - small interfering RNAs
KW  - HT29 cells
KW  - phosphorylation
KW  - multiple myeloma
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130140
VL  - 8
IS  - 3
ER  - 
TY  - JOUR
A1  - Boschert, Verena
A1  - van Dinther, Maarten
A1  - Weidauer, Stella
A1  - van Pee, Katharina
A1  - Muth, Eva-Maria
A1  - ten Dijke, Peter
A1  - Mueller, Thomas D.
T1  - Mutational Analysis of Sclerostin Shows Importance of the Flexible Loop and the Cystine-Knot for Wnt-Signaling Inhibition
JF  - PLoS ONE
N2  - The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured \(\beta\)-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.
KW  - Wnt signaling cascade
KW  - binding analysis
KW  - osteoblasts
KW  - sequence motif analysis
KW  - biological locomotion
KW  - signal inhibition
KW  - cell binding assay
KW  - luciferase
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129862
VL  - 8
IS  - 11
ER  -