The circulatory system is the first organ system to develop in the vertebrate embryo and is critical throughout gestation for the delivery of oxygen and nutrients to, as well as removal of metabolic waste products from, growing tissues. Endothelial cells, which constitute the luminal layer of all blood and lymphatic vessels, emerge de novo from the mesoderm in a process known as vasculogenesis. The vascular plexus that is initially formed is then remodeled and refined via proliferation, migration and sprouting of endothelial cells to form new vessels from pre-existing ones during angiogenesis. Mural cells are also recruited by endothelial cells to form the surrounding vessel wall. During this vascular remodeling process, primordial endothelial cells are specialized to acquire arterial, venous, and blood-forming hemogenic phenotypes and functions. A subset of venous endothelium is also specialized to become lymphatic endothelium later in development. The specialization of all endothelial cell subtypes requires extrinsic signals and intrinsic regulatory events, which will be discussed in this review.

Calcium (Ca2+) is an essential ligand that binds its primary intracellular receptor Calmodulin (CaM) to trigger a variety of downstream processes and pathways. Central to the actions of Ca2+/CaM is the activation of a highly conserved Ca2+/CaM kinase (CaMK) cascade, which amplifies Ca2+ signals through a series of subsequent phosphorylation events. Proper regulation of Ca2+ flux is necessary for whole-body metabolism and disruption of Ca2+ homeostasis has been linked to a variety of metabolic diseases. Herein, we provide a synthesis of recent advances that highlight the roles of the Ca2+/CaM kinase axis in key metabolic tissues. An appreciation of this information is critical in order to understand the mechanisms by which Ca2+/CaM-dependent signaling contributes to metabolic homeostasis and disease.

Hepatic cancer is one of the most lethal cancers worldwide. Here, we report that the expression of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is significantly up-regulated in hepatocellular carcinoma (HCC) and negatively correlated with HCC patient survival. The CaMKK2 protein is highly expressed in all eight hepatic cancer cell lines evaluated and is markedly up-regulated relative to normal primary hepatocytes. Loss of CaMKK2 function is sufficient to inhibit liver cancer cell growth, and the growth defect resulting from loss of CaMKK2 can be rescued by ectopic expression of wild-type CaMKK2 but not by kinase-inactive mutants. Cellular ablation of CaMKK2 using RNA interference yields a gene signature that correlates with improvement in HCC patient survival, and ablation or pharmacological inhibition of CaMKK2 with STO-609 impairs tumorigenicity of liver cancer cells in vivo. Moreover, CaMKK2 expression is up-regulated in a time-dependent manner in a carcinogen-induced HCC mouse model, and STO-609 treatment regresses hepatic tumor burden in this model. Mechanistically, CaMKK2 signals through Ca2+/calmodulin-dependent protein kinase 4 (CaMKIV) to control liver cancer cell growth. Further analysis revealed that CaMKK2 serves as a scaffold to assemble CaMKIV with key components of the mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway and thereby stimulate protein synthesis through protein phosphorylation. Conclusion The CaMKK2/CaMKIV relay is an upstream regulator of the oncogenic mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway, and the importance of this CaMKK2/CaM-KIV axis in HCC growth is confirmed by the potent growth inhibitory effects of genetically or pharmacologically decreasing CaMKK2 activity; collectively, these findings suggest that CaMKK2 and CaMKIV may represent potential targets for hepatic cancer.

SUMMARY Delineating the mechanism(s) that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development, and demonstrate that cell cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2−/−) primordial endothelium induced Notch signaling and p27 expression, which restored cell cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling and p27-mediated cell cycle control.