Egr-1 induces DARPP-32 expression in striatal medium spiny neurons via a conserved intragenic element.

DARPP-32 (dopamine and adenosine 3\u27, 5\u27-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominant-negative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation

Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP

AbstractWe have studied the regulation of expression of the carbamoyl-phosphate synthetase II-aspartate transcarbamylase-dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady-state levels of CAD mRNA decreased by 7-fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady-state levels of β-actin mRNA between induced and uninduced cells. Despite a 7-fold decrease in the steady-state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid- and dibutyryl cyclic AMP-induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell-growth-dependent and its regulation in this system is at the posttranscriptional level

ROS via BTK-p300-STAT1-PPARγ signaling activation mediates cholesterol crystals-induced CD36 expression and foam cell formation

In understanding the mechanisms of cholesterol in the pathogenesis of atherosclerosis, previous studies from other laboratories have demonstrated that cholesterol crystals (CC) induce scavenger receptor CD36 expression and NLRP3-mediated inflammasome formation. In elucidating the mechanisms by which CC could enhance CD36 expression and foam cell formation, here we report that CC via NADPH and xanthine oxidases-mediated ROS production activates BTK, a non-receptor tyrosine kinase. In addition, CC induce p300 tyrosine phosphorylation and activation in a BTK-dependent manner, which in turn, leads to STAT1 acetylation and its interaction with PPARγ in CD36 expression, oxLDL uptake and foam cell formation. Furthermore, p300, STAT1 and PPARγ bound to a STAT binding site at −107 nt in CD36 promoter and enhanced its activity in ROS and BTK-dependent manner. Disruption of this STAT binding site by site-directed mutagenesis abolished CC-induced CD36 promoter activity. Together these results reveal for the first time that CC via producing ROS and activating BTK causes p300-mediated STAT1 acetylation and its interaction with PPARγ in CD36 expression, oxLDL uptake and foam cell formation

Activation of cytosolic phospholipase A2 downstream to Src-PLD1-PKCγ signaling axis is required for hypoxia-induced pathological retinal angiogenesis

Towards understanding the mechanisms of retinal neovascularization, previously we have reported that VEGF-induced pathological retinal angiogenesis requires activation of Src-PLD1-PKCγ signaling. In the present work, we have identified cytosolic phospholipase A2 (cPLA2) as an effector molecule of Src-PLD1-PKCγ signaling in the mediation of VEGF-induced pathological retinal angiogenesis based on the following observations: VEGF induced cPLA2 phosphorylation in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). VEGF also induced arachidonic acid (AA) release in a dose-, time- and cPLA2-dependent manner. Depletion of cPLA2 levels inhibited VEGF-induced HRMVEC DNA synthesis, migration and tube formation. In addition, exogenous addition of AA rescued VEGF-induced HRMVEC DNA synthesis, migration and tube formation from inhibition by downregulation of cPLA2. Inhibition of Src, PLD1 or PKCγ attenuated VEGF-induced cPLA2 phosphorylation and AA release. Consistent with these findings, hypoxia induced cPLA2 phosphorylation and activity in VEGF-Src-PLD1-PKCγ -dependent manner in a mouse model of oxygen-induced retinopathy (OIR). In addition, siRNA-mediated downregulation of cPLA2 levels in retina abrogated hypoxia-induced retinal endothelial cell proliferation and neovascularization. These observations suggest that cPLA2-dependent AA release is required for VEGF-induced Src-PLD1-PKCγ -mediated pathological retinal angiogenesis

Cyclic AMP Response Element Binding Protein Mediates Pathological Retinal Neovascularization via Modulating DLL4-NOTCH1 Signaling

Retinal neovascularization is the most common cause of moderate to severe vision loss in all age groups. Despite the use of anti-VEGFA therapies, this complication continues to cause blindness, suggesting a role for additional molecules in retinal neovascularization. Besides VEGFA and VEGFB, hypoxia induced VEGFC expression robustly. Based on this finding, we tested the role of VEGFC in pathological retinal angiogenesis. VEGFC induced proliferation, migration, sprouting and tube formation of human retinal microvascular endothelial cells (HRMVECs) and these responses require CREB-mediated DLL4 expression and NOTCH1 activation. Furthermore, down regulation of VEGFC levels substantially reduced tip cell formation and retinal neovascularization in vivo. In addition, we observed that CREB via modulating the DLL4-NOTCH1 signaling mediates VEGFC-induced tip cell formation and retinal neovascularization. In regard to upstream mechanism, we found that down regulation of p38β levels inhibited hypoxia-induced CREB-DLL4-NOTCH1 activation, tip cell formation, sprouting and retinal neovascularization. Based on these findings, it may be suggested that VEGFC besides its role in the regulation of lymphangiogenesis also plays a role in pathological retinal angiogenesis and this effect depends on p38β and CREB-mediated activation of DLL4-NOTCH1 signaling