The bilateral symmetry of the mouse embryo is broken by leftward fluid flow in the node. However, it is unclear how this directional flow is then translated into the robust, left side-specific Nodal gene expression that determines and coordinates left-right situs throughout the embryo. While manipulating Nodal and Lefty gene expression, we have observed phenomena that are indicative of the involvement of a self-enhancement and lateral-inhibition (SELI) system. We constructed a mathematical SELI model that not only simulates, but also predicts, experimental data. As predicted by the model, Nodal expression initiates even on the right side. These results indicate that directional flow represents an initial small difference between the left and right sides of the embryo, but is insufficient to determine embryonic situs. Nodal and Lefty are deployed as a SELI system required to amplify this initial bias and convert it into robust asymmetry.

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Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR)and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HBdel/del) and secretion-deficient(HBuc/uc) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HBdel/del eyelids. Crosses between HBdel/del mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HBdel/del embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFα-null mice and in HB-EGF and TGFα double null mice revealed that HB-EGF and TGFα contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFα is required for leading edge extension in epithelial sheet migration during eyelid closure.

In mouse, left-right (L-R) patterning depends on asymmetric expression of Nodal around the node, leading to Nodal expression specifically in the left lateral plate mesoderm (LPM). Bone morphogenetic protein (BMP) signaling is also involved, but the mechanistic relationship with Nodal expression remains unclear. We find that BMP signal transduction is higher in the right LPM, although Bmp4, which is required for L-R patterning, is expressed symmetrically. By contrast, the BMP antagonists noggin (Nog) and chordin (Chrd) are expressed at higher levels in the left LPM. In Chrd;Nog double mutants, BMP signaling is elevated on both sides, whereas Nodal expression is absent. Ectopic expression of Nog in the left LPM of double mutants restores Nodal expression. Ectopic Bmp4 expression in the left LPM of wild-type embryos represses Nodal transcription, whereas ectopic Nog in the right LPM leads to inappropriate Nodal expression. These data indicate that chordin and noggin function to limit BMP signaling in the left LPM, thereby derepressing Nodal expression. In the node, they promote peripheral Nodal expression and proper node morphology, potentially in concert with Notch signaling. These results indicate that BMP antagonism is required in both the node and LPM to facilitate L-R axis establishment in the mammalian embryo.

SUMMARYHB-EGF, a member of the EGF family of growth factors, plays an important role in cardiac valve development by suppressing mesenchymal cell proliferation. Here, we show that HB-EGF must interact with heparan sulfate proteoglycans (HSPGs) to properly function in this process. In developing valves, HB-EGF is synthesized in endocardial cells but accumulates in the mesenchyme by interacting with HSPGs. Disrupting the interaction between HB-EGF and HSPGs in an ex vivo model of endocardial cushion explants resulted in increased mesenchymal cell proliferation. Moreover, homozygous knock-in mice (HB hb/hb ) expressing a mutant HB-EGF that cannot bind to HSPGs developed enlarged cardiac valves with hyperproliferation of mesenchymal cells; this resulted in a phenotype that resembled that of Hbegf-null mice. Interestingly, although Hbegf-null mice had abnormal heart chambers and lung alveoli, HB hb/hb mice did not exhibit these defects. These results indicate that interactions with HSPGs are essential for the function of HB-EGF, especially in cardiac valve development, in which HB-EGF suppresses mesenchymal cell proliferation.

CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G 2 checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G 2 checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G 2 -M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G 2 -M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G 2 -M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca 2þ . Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K d ¼ 4.62 Â 10 À8 mol/L) that is reversed by Ca 2þ , whereas the K d for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca 2þ independent. We conclude that CaM inhibition contributes to CBP501 0 s activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.

The anti-cancer agent CBP501 binds to calmodulin (CaM). Recent studies showed that migration and metastasis are inhibited by several CaM antagonists. However, there is no available evidence that CBP501 has similar effects. Here we found that CBP501 inhibits migration of non-small cell lung cancer (NSCLC) cells in vitro, even in the presence of migration inducing factors such as WNT, IL-6, and several growth factors. CBP501 also inhibited epidermal growth factor (EGF) enhanced invasion and the epithelial-to-mesenchymal transition (EMT), and this inhibition was accompanied by (i) suppression of Akt and ERK1/2 phosphorylation, and (ii) suppression of expression of transcription factor Zeb1 and the mesenchymal marker Vimentin. A pull down analysis performed using sepharose-immobilized CaM showed that CBP501 blocks the interaction between CaM and KRas. Furthermore, EGF induced Akt activation and cell migration was effectively suppressed by KRas down-regulation in NSCLC cells. Stable knockdown of KRas also made cells insensitive to CBP501’s inhibition of growth factor-induced migration. Taken together, these results indicate that CBP501 inhibits binding of CaM with KRas and thereby suppresses the PI3K/AKT pathway, migration, invasion and EMT. These findings have identified a previously unrecognized effect of CBP501 on downstream KRas signaling mechanisms involving EMT and invasion, and provide support for the further clinical development of this agent.

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis in vivo using ErbB1-and ErbB4-deficient mice, and an ex vivo model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in ERBB4-null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation.