p38 inhibition and not MK2 inhibition enhances the secretion of chemokines from TNF-α activated rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVES: For many years the p38 MAP kinase (MAPK) has been a major anti-inflammatory target for the development of an oral therapy for rheumatoid arthritis (RA). However, disappointing results from Phase II clinical studies suggest that adaptations may occur, which allow escape from blockade of the p38 pathway. In this study we investigated whether p38 inhibition mediated JNK activation represents such an escape mechanism. METHODS: Interaction between the JNK and p38 pathways was studied in TNF-α stimulated THP-1 monocytes, primary macrophages and fibroblast-like synoviocytes from OA and RA patients using pharmacological inhibitors and siRNAs. RESULTS: TNF-α induced phosphorylation of JNK and c-Jun was sustained by p38 inhibitors in monocytes, primary macrophages and FLS. Upregulation of Mip1α, Mip1ß and IL-8 mRNAs and protein were observed upon p38 inhibition. More importantly, inhibition of MK2, the substrate of p38 did not sustain JNK activation upon TNF-α activation and did not elevate Mip1α, Mip1ß and IL-8 chemokines as compared to TNF-α alone. In this study, TNF-α or IL-1ß induced JNK activation is sustained by p38 inhibition, resulting in enhanced chemokine secretion. CONCLUSIONS: Based on the suggested role of these chemokines in RA pathogenesis, the upregulation of these chemokines may provide an explanation for the lack of efficacy of p38 inhibitors in Phase II. The absence of any effect of MK2 inhibition in our models on this mechanism, while coming with similar efficacy on blocking p38, provides support for further investigations to reveal the potential of MK2 inhibition as a novel treatment of RA.

Human progesterone receptor A and B isoforms in CHO cells. II. Comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats.

The human progesterone receptor A and B isoforms (hPR-A and hPR-B) were stably transfected in Chinese Hamster Ovary (CHO) cells in the presence or absence of the mouse mamma tumor virus (MMTV) promoter and luciferase (LUC) reporter gene. In this way four stably transfected CHO cell lines, i.e. hPR-A, hPR-B, hPR-A-MMTV-LUC and hPR-B-MMTV-LUC cells, were prepared. hPR-A and -B isoforms were compared by binding and transactivation analysis for 14 progestagens and 7 antiprogestagens. Thereby Org 2058 was used as standard in both agonistic and binding assays and Org 31710 in antagonistic assays. The obtained data were compared with relative binding affinities (RBA) for both hPR-A and -B, which are present in human breast tumor MCF-7 cells, and with biopotency estimations with McPhail tests in rabbits and ovulation inhibition and pregnancy interruption tests in rats. The relative binding affinities of 14 progestagens and 7 antiprogestagens towards hPR-A, hPR-B or hPR-A/B of either CHO or MCF-7 cells were highly correlated with respect to ranking. This was also shown by the high correlation coefficients between the RBA's of hPR-B and hPR-A in CHO cells (r = 0.983) and between those of hPR-B of CHO and hPR A/B of MCF-7 cells (r = 0.957). The transactivation data of the 14 progestagens and 7 antiprogestagens for the hPR-B-MMTV-LUC cells were compared with those for hPR-A-MMTV-LUC cells and showed no differences between both cell lines with exception of the progestagens Org 32704 and 33766 and the antiprogestagen Org 33245. Therefore only the relative agonistic activities (RAA) and relative antagonistic activities (RANTA) of hPR-B-MMTV-LUC cells were compared with RBA values of hPR-B, showing a high similarity in ranking for the tested compounds, and high correlation coefficients of r = 0.91 and r = 0.97, respectively. Remarkably, RBA's were 1.6 fold higher than RAA's and RANTA's. These in vitro RBA, RAA and RANTA values for hPR-B were checked for their pharmacological relevance by in vivo biopotency measurements with the 14 progestagens and 7 antiprogestagens in rabbits and rats. The in vitro binding and transactivation potencies of progestagens appeared to be very predictive for in vivo analysis on endometrium proliferation in rabbits in the McPhail test with correlation coefficients of r = 0.81 and r = 0.87, while ovulation inhibition in rats correlated less well with r = 0.516 and r = 0.65. On the other hand, the antiprogestagenic potencies found with binding and transactivation assays had a good correlation with the potencies in the pregnancy interruption test in rats for all antiprogestagens tested, being r = 0.849 and r = 0.744, respectively. In conclusion, the binding and transactivation potencies for the tested compounds in hPR-A and -B containing cell lines showed in general a good resemblance. The transactivation studies with hPR-B-MMTV-LUC cells indicated that ranking of compounds was fairly identical to binding analysis and could be used for pre-screening of the (anti)-progestagenic bioactivity in the McPhail test in rabbits, the ovulation inhibition test and the pregnancy interruption test in rats. Therefore this transactivation assay can replace binding assays. Moreover, direct pre-screening of agonists, antagonists and partial antagonists is even possible in this in vitro bioassay, making transactivation assays for a particular class of chemical compounds to a valuable pre-screening tool for in vivo studies.

Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus.

In cytosol of the hippocampus corticosterone displays highest affinity for the sites that remain available for binding in the presence of excess RU 26988, which is shown to be a "pure" glucocorticoid. A rather high affinity (greater than or equal to 25%) was found for 11 beta-hydroxyprogesterone, 21-hydroxyprogesterone, 5 alpha-corticosterone, 19-nor-deoxycorticosterone, 11-deoxycorticosterone and cortisol. A moderate affinity (greater than 5% and less than 25%) was displayed by about 14 steroids among which progesterone, aldosterone, 9 alpha-fluorocortisol and dexamethasone. Corticosterone also shows highest affinity to plasma transcortin and thymus cytosol in the presence of RU 26988. However, the rank-order in affinity by the competing steroids was distinctly different from that observed in the hippocampus; cf. aldosterone and dexamethasone displaced [3H]corticosterone from sites unoccupied by RU 26988 in the hippocampus but not from transcortin or sites in thymus cytosol. In thymus cytosol some potent glucocorticoids have higher affinity for the [3H]dexamethasone labeled sites than dexamethasone. The binding of [3H]dexamethasone in thymus cytosol is completely abolished in the presence of a 100-fold excess of RU 26988. We conclude that our data support the evidence for RU 26988 as a selective ligand for glucocorticoid receptors. RU 26988 leaves binding sites available with highest affinity for corticosterone in hippocampus cytosol that are distinct from transcortin-like sites as found in thymus cytosol or from plasma transcortin.