In the last few years, the discovery of lysine and arginine methylation in histones and other proteins and the enzymes that carry out these posttranslational modifications has added a new dimension to the signal transduction field. In particular, there has been a huge surge in our understanding of how methylation of nucleosomal histones at specific lysine or arginine residues affects chromatin conformations and either facilitates or inhibits transcription from neighboring genes. It appears that the responsible methyltransferases can be targeted in some cases to specific genes and in other cases to broader regions of euchromatin or heterochromatin. Methylation of histones is mechanistically linked to other types of histone modifications, such as acetylation, phosphorylation, and monoubiquitylation; combinations of these modifications cooperate to regulate chromatin structure and transcription by stimulating or inhibiting binding of specific proteins. Although lysine methylation has thus far been observed almost exclusively on histones, arginine methylation has been observed on a variety of other proteins associated with gene regulation, including DNA-binding transcriptional activators, transcriptional coactivators, and many RNA binding proteins involved in RNA processing, transport, and stability. Thus, lysine and arginine methylation of proteins, like many other types of posttranslational modifications, are regulated steps of many specific signaling pathways.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pm) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated withl-N ^G-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition ofS-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.

Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators glucocorticoid receptor interacting protein 1, coactivator-associated arginine methyltransferase 1 (CARM1), and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltransferase activity of CARM1 but does not absolutely require the enzymatic activity of G9a and is specific to CARM1 and G9a among various protein methyltransferases. Reduction of endogenous G9a diminished hormonal activation of an endogenous target gene by the androgen receptor, and G9a associated with regulatory regions of this same gene. G9a fused to Gal4 DNA binding domain can repress transcription in a lysine methyltransferase-dependent manner; however, the histone modifications associated with transcriptional activation can inhibit the methyltransferase activity of G9a. These findings suggest a link between histone arginine and lysine methylation and a mechanism for controlling whether G9a functions as a corepressor or coactivator.

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pmrhVEGF₁₆₅ for 0–7 days. Western blot analysis showed a 3–5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2–3-fold increase in high affinity receptor number (5.0 × 10⁴/cellversus 2.4 × 10⁴/cell) without significantly affecting receptor binding affinity (K _d 76 pm versus 72 pm). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 μm sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.