How endoglin modulates the hepatocytes' response to BMP-9

Bone morphogenetic protein (BMP)-9, a member of the TGFbeta family of cytokines is constantly produced in the liver and circulates in an active conformation. Like other members of this family BMP-9 transduces its signal through binding to type I and II receptors on the cell surface. The receptors with the highest affinity to BMP-9 are believed to be the type I receptor Alk1 and the type II receptor ActRIIB. However with somewhat lower affinity BMP-9 can also bind to and signal through Alk2 and the type II receptors BMPRII and ActRIIA. BMP signaling is additionally modulated by the presence of co-receptors and extracellular regulators. Among the co-receptors, endoglin seems to be of special relevance, although the precise nature of this relation is not well understood. Whereas the response of normal hepatocytes to BMP-9 is rather stabilizing, several HCC cell lines respond with enhanced proliferation, migration and EMT. Aim of the present study was to investigate how the presence or absence of endoglin changes the BMP-9 responses in healthy as well as malignant hepatocytes.

We first determined the basal expression patterns of the different BMP-9 receptors and endoglin in two HCC lines (HLE and Hep3B) as well as quasi primary upcyteHC® by real-time PCR. Hep3B showed higher basal Smad1 activity and stronger BMP-9 mediated Smad1 phosphorylation than HLE. Both lines responded oppositely to BMP-9 stimulation with Hep3B resembling more the non-malignant, healthy hepatocyte and expressing almost no endogenous Alk1. We therefore tested if transfection with an Alk1 overexpressing plasmid would change the BMP-9 responses towards a more malignant one. Similar experiments were performed with the non-malignant upcyte®HC. We found that overexpression of Alk1 alone had little effect and tested next the additional stimulation with recombinant soluble endoglin +/- BMP-9. Surprisingly the combination of Alk1 and soluble endoglin induced massive cell death in Hep3B as well as upcyte®HC even in the absence of BMP-9. Endoglin alone did not cause this but led to a decreased (but not blocked) induction of BMP-9 target genes like Id1 and hepcidin.

In conclusion high Alk1 expression seems to mediate essential survival pathways in HCs that are inhibited in the presence of soluble endoglin. Such effects of Alk1 might even be independent of endogenous BMP-9 signalling. The individual combination of receptors/co-factors expressed in a given cell determines the cellular vitality in general and the nature of the BMP-9 response. Future studies will include the effects of overexpressing the wild-type, membrane-bound Endoglin.