Divergent regulation of the Osteopontin promoter by the estrogen receptor-related receptors is isoform- and cell context dependent.

We sought to determine whether the estrogen receptor-related receptor gamma (mEsrrg) regulated the Osteopontin (Opn) promoter through the same AP1/CAAT box element that we have previously described for mEsrra. In HeLa cells mEsrrg used an additional site present in the 5'UTR, while in ROS17/2.8 cells the AP1/CAAT site was not used, but a completely novel site surrounding the transcription start site was used. We also find that in ROS17/2.8 cells mEsrra repressed, while mEsrrg activated the Opn promoter. None of the sites identified conform to established Esrr response elements (ERREs). Additionally, the two reported mEsrrg protein isoforms showed differences in their activation potential. Mutations in the activation function 2 (AF2) of mEsrra, predicted to abolish activation, surprisingly turned mEsrra into a better activator. In contrast, similar AF2 mutations in Esrrg2 abolished its ability to activate the Opn promoter. Mutation of the DNA binding domain of mEsrra/g2 abolished transcriptional activity in HeLa and ROS17/2.8 cells. Our data indicate, first, that the two Esrr isoforms regulate Opn in a cell context-dependent manner. Second, they suggest that although the DNA binding domains of mEsrra and mEsrrg are 93% identical and required for regulation, the receptors bind to distinct Opn promoter elements, suggesting that the two isoforms may co-regulate Opn, and perhaps other genes, without competing for the same site in the promoter. Finally, the results suggest that each isoform interacts differently with co-activators and co-repressors, as highlighted by the AF2 mutation that turns mEsrra into a better activator but abolishes activity of Esrrg2.

The orphan nuclear estrogen receptor-related receptor-alpha regulates cartilage formation in vitro: implication of Sox9.

We report for the first time the expression of estrogen receptor-related receptor (ERR)-alpha in fetal and adult rat chondrocytes in growth plate and articular cartilage and the rat chondrogenic cell line C5.18 cells in vitro. ERRalpha mRNA and protein were expressed from proliferating chondrocyte to mature chondrocyte stages. We show that overexpressing ERRalpha in C5.18 cell cultures induces an increase in Sry-type high-mobility-group box transcription factor (Sox)-9 expression, a master gene in cartilage formation. In parallel, we report Sox9 promoter regulation by ERRalpha in C5.18 cells. To assess a functional role for ERRalpha in chondrogenesis, its expression was blocked by antisense oligonucleotides in C5.18 cell cultures, and this led to inhibition of cartilage formation associated with down-regulation of Sox9 and Indian hedgehog expression and maturation of proliferating chondrocytes into hypertrophic chondrocytes in vitro. Together these results implicate ERRalpha in the formation and maintenance of cartilage and also suggest that agonists and antagonists of ERRalpha may be useful as therapeutic agents in a wide variety of diseases affecting cartilage and joints.

Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.