The mice lacking MKK4 or MKK7 are embryo nic lethal suggesting the two kinases are non redundant and serve distinct functions. Some studies selleck kinase inhibitor suggest that these differences might be Inhibitors,Modulators,Libraries due to selective regulation by extracellular stimuli, distinct tissue distri bution and different biochemical properties. Thus, an alternative approach targeting the MKKs instead of JNK could suppress signaling responses Inhibitors,Modulators,Libraries that contribute to inflammatory arthritis but spare a subset of host defense or homoeostasis pathways. Our previous studies showed that MKK4 and MKK7 are expressed and phosphorylated in RA synovium and both are activated by cytokines in RA FLS. Surpris ingly, cytokine induced JNK activation and MMP pro duction are strictly dependent on MKK7 in cytokine stimulated Inhibitors,Modulators,Libraries FLS and do not require MKK4.
There fore, we evaluated whether selective targeting of MKK7 using anti sense oligonucleotides would block arthritis associated JNK activation and decreased arthri tis severity in K BxN serum transfer arthritis. The data indicate that blockade MKK7 mimics the effect of JNK deficiency and suppresses Inhibitors,Modulators,Libraries inflammatory arthritis. Materials and methods Oligonucleotides A series of uniform chimeric 20 mer phosphorothioate oligonucleotides containing 2 O methoxyethyl chimeric groups at positions 1 to 5 and 15 to 20 tar geted to murine MKK7 were synthesized and purified as described. Three ASOs complementary to murine MKK7 were ASO treatment in normal mice All animal protocols received prior approval by the institutional review board.
Pathogen free male C57BL 6 mice were purchased from The Jackson Laboratory and MKK7 or control ASOs were administered to mice based upon Inhibitors,Modulators,Libraries body weight by intra venous injection. Three days after injection of ASOs, mice were sacrificed and various tis sues evaluated for MKK7 gene expression. K BxN serum transfer arthritis and ASO treatment To induce K BxN serum transfer arthritis, serum samples were pooled from arthritic adult K BxN mice and injected intraperitoneally as previously described. C57BL 6 mice received PBS, MKK7 ASOs or control ASO i. v. twice a week beginning on Day 8 and then administered 100 ul of K BxN serum on Day 0. Clinical arthritis scores were eval uated using a scale of 0 to 4 for each paw for a total score of 16. Ankle thickness was measured with a cali per placed across the ankle joint at the widest point.
Histopathologic assessment was performed using a semi quantitative scoring system as previously described, including synovial inflammation, bone erosion and cartilage damage. Navitoclax clinical Quantitative real time PCR Ankle joints were collected at study termination, dis sected to remove extra articular tissue and snap frozen in liquid nitrogen. The specimens were pulverized and total RNA was isolated using Rneasy Lipid Tissue kit per manufacturers protocol.