Health

Retinopathy of prematurity (ROP) is a major cause of blindness in children in the Western World. Premature birth disturbs normal vascular development. Children with more avascular retina have higher risk of developing pathologic neovascularization (NV) and
severe ROP. The present standard of care has been laser or cryoablation of the peripheral avascular retina in order to control the
pathologic neovascularization. Despite the undeniable benefit of these primitive yet time tested interventions, there is incomplete effectiveness and significant morbidity. Molecular understanding of normal and abnormal vascular development has highlighted the
role of VEGF and suggested a role of anti-VEGF therapy.1 The effectiveness of anti-VEGF therapy in AMD and other neovascular diseases has generated excitement about application of ‘‘gentler’’ anti-VEGF therapies for ROP. The Bevacizumab Eliminates the
Angiogenic Threat of Retinopathy of Prematurity study gave us excitement, but also caution about delayed normal vascular development and NV formation.(2)

It is natural to think that in a developing organ, the exact level of VEGF inhibition will be important in order to control pathologic NV, while at the same time allowing normal vascular development to proceed. Lutty et al.3 has demonstrated this principle in a dog model of ROP using an agent that blocks simultaneously VEGF-A, VEGF-B, and placental growth factor. In this issue, McCloskey et al.4 show similar findings using VEGF blockade alone. In a well controlled study in rodents, they show that more anti-VEGF blockade is not better but worse, leading to increased avascular retina and delayed atypical NV formation with activation of compensatory angiogenic signals that may not respond to subsequent VEGF blockade. They also showed that anti- VEGF therapy was associated with reduced weight gain. Their study highlights the need for more systematic research regarding the appropriate dose and monitoring of anti-VEGF in ROP babies in order to have a safer and more effective therapy than the current primitive ablative approaches.

The small GTP-binding protein Ras (Ha-, Ki-, and N-Ras) plays a central role in transmitting proliferative and differentiation signals downstream of cell surface receptors in mammalian cells. Ras has been demonstrated to relay signals from receptor tyrosine kinases (1), (e.g. EGF1 receptor), non-tyrosine kinase receptors (2) (e.g. T cell antigen receptor), and heterotrimeric G protein-coupled receptors (3).

The understanding of the biochemical mechanism by which Ras transmits signals in higher eucaryotic cells has been greatly clarified in recent years. Ras is located at the inner surface of the plasma membrane; activation of cell surface receptors promotes the exchange of Ras-GDP for GTP, thereby converting Ras to the active state. This activation results from GTP-induced conformational change, wherein two discrete Ras segments, called switch I (or the effector domain loop aa 32–40) and switch II (aa 60–72) exhibit a significant displacement as compared with the GDP-bound state. This conformational change renders Rasable to interact effectively with its downstream effectors or targets (4).

The first Ras effectors in mammalian cells to be identified are the protein kinases of the Raf family. GTP-bound Ras directly binds Raf primarily through an interaction between the switch I region and amino-terminal segment on Raf (amino acids 50–150).
The ability of Raf to bind to Ras in a GTP-dependent manner, in vitro and in situ, is the cardinal biochemical evidence in support of Raf’s role as a direct effector of Ras (5). The Raf-MEK-Erk pathway is the best characterized Ras effector pathway and is required for transformation of rodent fibroblasts by oncogenic Ras (6).

However, In recent years, many observations have indicated that Ras transmits signals into multiple effector pathways. For instance, constitutively active Ras and Raf both transform NIH3T3 fibroblasts, but only constitutively active Ras, but not Raf, can transform rat intestinal epithelial cells (RIE-1), thus pathways besides the Raf-MEK-Erk pathway need to be activated to transform RIE-1 cells (7).

Similarly, in PC-12 cells, activated Raf induces the expression of only a subset of genes which can be induced by oncogenic Ras or nerve growth factor (8). An elegant study demonstrated that in Hela cells and NIH3T3 fibroblasts, the increase in Ras-GTP charging achieved immediately after release from mitosis is much less than a second phase of Ras activation that occurred some 5 h later, in mid-G1.

Interestingly, only the first phase of Ras activation was accompanied by Erk activation, whereas the latter, much stronger Ras activation occurred without significant Erk activation (9). The biologic significance of Ras activation in mid-G1 phase, and the nature of the effectors recruited by activated Ras at that time is entirely unknown.

Following on the discovery of Raf as the initial Ras effector in higher eucaryotic cells, a number of candidate Ras effectors have been proposed based on the ability of these polypeptides to bind to Ras through its effector loop, and in a GTP-dependent fashion, including PI 3-kinase, members of the Ral-GDS family, Rin 1, AF-6, diacylglycerol kinases, PKC-z, MEKK1, etc. The standing of these polypeptides as candidate Ras effectors has been reviewed (10, 11). We used the yeast two-hybrid system to look for novel proteins that directly interact with Ras. We describe here the identification of a potential new Ras effector, which we have named Nore1.

EXPERIMENTAL PROCEDURES

Two-hybrid Screen—A cDNA encoding V12-Ha-Ras deleted of the last four amino acids was subcloned into vector pAS-CYH-II carboxyltermianl to the Gal-4 DNA binding domain to form the bait construct pAS-Ras. 100 mg of cDNA made from a mouse T cell library constructed in the GAL-4 DNA activation domain vector pACT was transformed into the yeasts expressing pAS-Ras, and the transformants were plated out on His2Leu2Trp2 selection plates. After 8 days, 20 large colonies appeared. X-gal filter assay was performed for all the colonies and all showed strong blue color.

cDNA Cloning of Nore1—The 2.5-kb cDNA encoding Nore1 from the initial two-hybrid screen was labeled with [a-32P]dCTP and used to
screen a cDNA library made from mouse brain (CLONTECH’s mouse brain 59-stretch plus cDNA library in l-gt 10 vector, catalog number ML 3000a). A positive clone, which contains a 3-kb insert, was isolated. Tissue and Cell Line Western Blot—Sprague-Dawley rats (65 g) were starved overnight, anesthetized with pentobarbital, and tissues were excised in the following order: gastrocnemius, testis, spleen, kidney, liver, lung, and heart. Brain was excised from other intact anesthetized animals after decapitation. Cell lines were gown to 80–90% confluence before harvesting. Both tissues and cell lines were disrupted and extracted in radioimmune precipitation buffer.