Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development.

Pax6 is a key transcription factor involved in eye, brain, and pancreas development. Although pax6 is expressed in the whole prospective retinal field, subsequently its expression becomes restricted to the optic cup by reciprocal transcriptional repression of pax6 and pax2 However, it remains unclear how Pax6 protein is removed from the eyestalk territory on time. Here, we report that Mid1, a member of the RBCC/TRIM E3 ligase family, which was first identified in patients with the X-chromosome-linked Opitz BBB/G (OS) syndrome, interacts with Pax6. We found that the forming eyestalk is a major domain of mid1 expression, controlled by the morphogen Sonic hedgehog (Shh). Here, Mid1 regulates the ubiquitination and proteasomal degradation of Pax6 protein. Accordantly, when Mid1 levels are knocked down, Pax6 expression is expanded and eyes are enlarged. Our findings indicate that remaining or misaddressed Pax6 protein is cleared from the eyestalk region to properly set the border between the eyestalk territory and the retina via Mid1. Thus, we identified a posttranslational mechanism, regulated by Sonic hedgehog, which is important to suppress Pax6 activity and thus breaks pax6 autoregulation at defined steps during the formation of the visual system.

A phosphorylation-acetylation switch regulates STAT1 signaling.

Cytokines such as interferons (IFNs) activate signal transducers and activators of transcription (STATs) via phosphorylation. Histone deacetylases (HDACs) and the histone acetyltransferase (HAT) CBP dynamically regulate STAT1 acetylation. Here we show that acetylation of STAT1 counteracts IFN-induced STAT1 phosphorylation, nuclear translocation, DNA binding, and target gene expression. Biochemical and genetic experiments altering the HAT/HDAC activity ratio and STAT1 mutants reveal that a phospho-acetyl switch regulates STAT1 signaling via CBP, HDAC3, and the T-cell protein tyrosine phosphatase (TCP45). Strikingly, inhibition of STAT1 signaling via CBP-mediated acetylation is distinct from the functions of this HAT in transcriptional activation. STAT1 acetylation induces binding of TCP45, which catalyzes dephosphorylation and latency of STAT1. Our results provide a deeper understanding of the modulation of STAT1 activity. These findings reveal a new layer of physiologically relevant STAT1 regulation and suggest that a previously unidentified balance between phosphorylation and acetylation affects cytokine signaling.

The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell-matrix adhesion.

Density-enhanced protein-tyrosine phosphatase-1 (DEP-1 also CD148) is a transmembrane molecule with a single intracellular PTP domain. It has recently been proposed to function as a tumor suppressor. We have previously shown that DEP-1 dephosphorylates the activated platelet-derived growth factor (PDGF) beta-receptor in a site-selective manner (Kovalenko et al. (2000). J. Biol. Chem. 275, 16219-16226). We analysed cell lines with inducible DEP-1 expression for cellular functions of DEP-1. Several aspects of PDGFbeta-receptor signaling were negatively affected by DEP-1 expression. These include PDGF-stimulated activation of inositol trisphosphate formation, Erk1/2, p21Ras, and Src. Activation of receptor-associated phosphoinositide-3 kinase activity and of Akt/PKB were weakly attenuated at early time points of stimulation. Inhibition of PDGF-stimulated signaling depended on DEP-1 catalytic activity. Importantly, DEP-1 inhibited PDGF-stimulated cell migration. The catalytically inactive DEP-1 C1239S variant enhanced cell migration and PDGF-stimulated Erk1/2 activation, suggesting a dominant negative interference with endogenous DEP-1. In contrast to cell migration, cell-substrate adhesion was promoted by active DEP-1 and delayed or suppressed by DEP-1 C1239S, correlating with positive effects of DEP-1 on adhesion-stimulated Src kinase. We propose that negative regulation of growth-factor stimulated cell migration and promotion of cell-matrix adhesion may be related to the function of DEP-1 as tumor suppressor.

Growth factors in the collateral circulation of chronic total coronary occlusions: relation to duration of occlusion and collateral function.

BACKGROUND: Despite extensive animal experimental evidence, there are few data on the relation of growth factors and collateral function in humans. METHODS AND RESULTS: In 104 patients with a chronic total coronary occlusion (CTO; >2 weeks' duration), collateral function was assessed invasively during recanalization by intracoronary Doppler and pressure recordings. A collateral resistance index, R(Coll), was calculated. Blood samples were drawn from the distal coronary bed supplied by the collaterals and from the aortic root to measure basic fibroblast growth factor (bFGF), monocytic chemotactic protein-1 (MCP-1), transforming growth factor-beta (TGF-beta), placenta growth factor (PlGF), and tumor necrosis factor-alpha (TNF-alpha). The bFGF concentration in the collateralized artery was higher than in the aortic root (34+/-20 versus 18+/-14 pg/mL; P<0.001). bFGF was highest in recent occlusions (2 to 12 weeks) with the highest R(Coll). Higher collateral concentrations were also observed for MCP-1, TGF-beta, and PlGF, but without a close relation to the duration of occlusion. TNF-alpha was not increased in collaterals compared with the systemic circulation. MCP-1, PlGF, and TGF-beta were significantly increased in small collaterals with the highest shear stress. Diabetic patients had lower bFGF and higher MCP-1 levels than nondiabetics. CONCLUSIONS: In CTOs, the continuous release of bFGF into collaterals showed a close relation to the duration of occlusion and collateral function, which underscores its therapeutic potential. Other factors influencing growth factor release appeared to be shear stress for MCP-1, TGF-beta, and PlGF and the presence of diabetes.

Stent-based release of a selective PDGF-receptor blocker from the bis-indolylmethanon class inhibits restenosis in the rabbit animal model.

Long-term success of modern therapies for myocardial ischemia is limited by restenosis, with proliferation and migration of vascular smooth muscle cells (VSMC) as key events. Since findings in recent years indicate, that the Platelet Derived Growth Factor (PDGF) is an important selective factor in mitogenic and motogenic pathways of VSMC, different concepts for reducing restenosis by inhibiting PDGF signaling have been investigated, with local delivery of small receptor kinase inhibitors looking most promising. We tested the stent-based delivery of the PDGF-receptor inhibitor D-65495, a bis(1H-2-indolyl)methanone, in the rabbit iliac artery model of restenosis. New Zealand white rabbits underwent balloon dilation of iliac arteries for implantation of D-65495-coated or non-coated (solvent, either DMSO or 90%THF / 10% DMSO) coronary stents. After 4 weeks stents were removed and neointima formation in medial and proximal/ distal stent sections was histomorphometrically and immunohistochemically analyzed. Arteries with D-65495 eluting stents showed an up to 50% reduced restenosis compared to control stents. Also, the neointimal area was reduced, but there were no significant differences in injury score. Importantly, endothelialization was similar for control stents as well as for D-65495-coated stents, suggesting absence of a general cytostatic effect of the inhibitor. The impact of D-65495 on PDGF-receptor signaling in the vessel wall was indirectly assessed by immunohistochemical staining for activated protein kinase Akt, and PCNA as a proliferation marker and revealed some reduction for the inhibitor-treated vessels. In conclusion, the application of D-65495 caused a significant decrease in neointima formation, further supporting the concept of using locally released PDGF-receptor kinase inhibitors as anti-restenotic agents.

Cardiotrophin-1 induces monocyte chemoattractant protein-1 synthesis in human umbilical vein endothelial cells.

In chronic heart failure (CHF) cardiotrophin-1 (CT-1) and monocyte chemoattractant protein-1 (MCP-1) plasma concentrations are elevated. CT-1 is a cytokine of the interleukin-6 (IL-6) superfamily. Most members of the IL-6 family are able to activate human umbilical vein endothelial cells (HUVEC) but so far there are no data which demonstrate that CT-1 can activate HUVEC. Because MCP-1-as a marker of endothelial activation-is elevated in CHF we examined whether CT-1 will induce MCP-1 production in HUVEC. MCP-1 mRNA levels were determined by real time PCR, RT-PCR and northern blot analysis and MCP-1 protein concentrations in the supernatant by ELISA. Signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (pSTAT3) were investigated by western blot analysis. Incubation of HUVEC with different CT-1 concentrations for various time periods induced time and concentration dependent MCP-1 mRNA. Maximal MCP-1 mRNA was reached after 6h. After 24h CT-1 caused a significant induction of MCP-1 protein in the supernatant compared to control. CT-1 induced concentration dependent phosphorylation of STAT3 without any change in total-STAT3 concentration. Piceatannol-a specific blocker of STAT3 phosphorylation-inhibited CT-1 induced MCP-1 induction completely. AG490-a blocker of the JAK2 pathway-was also able to inhibit CT-1 induced MCP-1 upregulation, indicating that the JAK2 pathway is also necessary for MCP-1 induction. Parthenolide-a blocker of NFkappaB-inhibited CT-1 induced MCP-1 expression, completely. Our data show that CT-1 induces in a concentration and time dependent manner MCP-1 mRNA and protein in HUVEC. STAT3 phosphorylation, the activation of JAK2 and NF-kappaB are involved in this pathway. In CHF, CT-1 may be able to induce MCP-1 which might be responsible for progression of heart failure either by recruiting inflammatory cells within the myocardium or by a direct modulation of myocyte function.