The Mediator complex is an essential transcription regulator that bridges transcription factors with RNA polymerase II. This interaction is controlled by dynamic interactions between Mediator and the CDK8 module, but the mechanisms governing CDK8 module-Mediator association remain poorly understood. We show that Fbw7, a tumor suppressor and ubiquitin ligase, binds to CDK8-Mediator and targets MED13/13L for degradation. MED13/13L physically link the CDK8 module to Mediator, and Fbw7 loss increases CDK8 module-Mediator association. Our work reveals a novel mechanism regulating CDK8 module-Mediator association and suggests an expanded role for Fbw7 in transcriptional control and an unanticipated relationship with the CDK8 oncogene.

Objective Pro-inflammatory molecules promote osteoclast-mediated bone erosion by upregulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as TNFα plus IL-6, induce RANKL-independent osteoclastogenesis. This study sought to better understand TNFα/IL-6 induced osteoclast formation, and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. Methods Myeloid precursors from wild-type (WT) mice, or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12 or Fcrg, were treated with either RANKL, or TNFα plus IL-6. Osteoprotegerin, anti-IL-6 receptor (IL6R) and hydroxyurea were used to block RANKL, the IL6R and cell proliferation, respectively. Clinical scoring, histology, micro-computed tomography and qPCR were employed to evaluate K/BxN serum transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by performing osteoclast cultures. Results TNFα/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL6R, NFATc1, DAP12 and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNFα/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from RANK-deficient animals. Multiple IL-6 family members (IL-6, LIF, OSM) were upregulated in the synovium of arthritic mice. Conclusion The persistence of bone erosion and synovial osteoclasts in RANK-deficient mice, and the ability of TNFα/IL-6 to induce osteoclastogenesis, suggest more than one cytokine pathway exists to generate these bone resorbing cells in inflamed joints.

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC 50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed ϳ560,000 sites/ cell of ET A receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ET A receptor. ET-1 treatment did not induce phosphorylation of insulin receptor ␤-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 M), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ET A receptor.

Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors ( Erg and Ets1 ) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg’s key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.