Mechanisms of Has2 regulation and hyaluronan signaling during embryonic development

The cardiovasculature is the first functional system in the developing embryo and as such, it plays a crucial role in the proper nourishment and formation of all other body regions and organs. Malformation or malfunction of this system can lead to a series of conditions collectively known as cardiovascular diseases, which are a major cause of death around the world, claiming 17.5 million human lives a year. A detailed understanding the mechanisms that regulate cardiac morphogenesis is necessary to provide us with clues of what goes awry in disease states and to develop possible strategies for diagnostics and treatment of these problems.;There is a wide variety of processes and molecules that have been identified to date as important players for the proper formation of the cardiovasculature. One of these molecules is Hyaluronan synthase 2 (Has2), a membrane protein in charge of assembling the glycosaminoglycan hyaluronan (HA). Mouse embryos lacking Has2 do not produce HA and display abnormalities such as absence of endocardial cushions, a disarrayed vascular network, and growth retardation, leading to death by embryonic day E 9.5. Thus, Has2 and HA are necessary for the early stages of heart formation, but many questions remain to be answered in regards to their mechanism of action and their role in later events such as the formation of the coronary vessels. Our current study addresses these questions in vitro employing two cell lines. NIH-3T3 cells are used as a model of mesenchymal endocardial cushion cells while epicardial cells are used to assess Has2 and HA function in embryonic cells with an epithelial phenotype.;Here we show that HA induces biological activity in embryonic cells in a manner that is dependent on its molecular size, with high molecular weight HA (HMW-HA), but not low molecular weight HA (LMW-HA), being able to affect cellular behavior. HMW-HA induces invasion of NIH-3T3 cells while it promotes differentiation and invasion of epicardial cells.;We also demonstrate that stimulation of cells with HMW-HA promotes the association of MEKK1 with the HA receptor CD44 and induces MEKK1 phosphorylation. This leads to the activation of two distinct pathways, one ERK-dependent and another NFkappaB-dependent. Although both cells lines show activation of these cascades, the ERK-dependent pathway is more prominent in epicardial cells while the NFkappaB-dependent pathway is favored in NIH-3T3 cells. Blockade of CD44, transfection with a kinase inactive MEKK1 construct or the use of ERK1/2 and NFkappaB inhibitors significantly abrogates the cellular response to HMW-HA. Together, these findings suggest an important role for HA in the regulation of embryonic cell fate via activation of MEKK1 signaling mechanisms.;Finally, we have elucidated a novel functional connection between growth factor signaling, endogenous HA production and the regulation of cellular responses. Specifically, we show that both TGFbeta2 and EGF induce Has2 expression and/or phosphorylation, although distinct intracellular signals are activated for each growth factor. While TGFbeta2 governs Has2 via MEKK3-dependent mechanisms, EGF does not require MEKK3 and does not induce Has2 expression as robustly as TGFbeta2. Increased Has2 activity as a result of TGFbeta2 and EGF stimulation leads to enhanced HA synthesis. These increased endogenous levels of HA are coincident with enhanced cellular differentiation and invasion. Taken together, these findings underscore how EGF, TGFbeta2 and HA signals are integrated to form highly complex networks that are crucial for the proper formation of organs and tissues during development.