Using the new approach of screening for stereotyped deviations in embryonic dorsoventral patterning, we have identified a molecule that selectively inhibits BMP signaling. This small-molecule inhibitor recapitulates with high specificity and low toxicity the phenotypic spectrum of embryos with genetic defects in the BMP signaling pathway. The functional specificity of dorsomorphin is demonstrated in vivo by its ability to compensate for the loss of the BMP antagonist chordin during dorsoventral axis formation and by its ability later in development to inhibit axial skeleton mineralization in the absence of other observable defects. Functional selectivity for BMP signaling is further supported by in vitro data demonstrating that dorsomorphin preferentially inhibits BMP signaling over TGF-β or activin signaling.
Despite the apparent selectivity of dorsomorphin for BMP signaling, it is important to consider the possibility that dorsomorphin, like all known kinase inhibitors, may affect other kinase targets, particularly at higher doses. Although dorsomorphin is structurally identical to a molecule previously shown to antagonize AMPK activity21
, AMPK inhibition does not seem to be responsible for the effects of dorsomorphin on dorsoventral axis formation or hepcidin regulation, because neither pharmacological nor morpholino-mediated inactivation of AMPK reproduces these effects. Nevertheless, when using dorsomorphin, the possibility of confounding effects from secondary targets needs to be considered, and the lowest effective concentrations should be used to maximize selectivity for BMP signaling.
Previously, in vivo
inhibition of BMP signaling in experimental therapy has been accomplished by administration of soluble receptor extracellular domains or ligand-specific neutralizing antibodies37
, or by gene transfer of endogenous antagonists such as noggin
. Because all of these strategies for inhibiting BMP signaling function by sequestering BMP ligands, they do not offer the possibility of differentiating between SMAD-dependent and SMAD-independent BMP signaling mechanisms. Dorsomorphin inhibits SMAD activation without compromising the ability of BMPs to activate MAPK p38 in PASMCs, which suggests that this function may be independent of SMAD activation. It is possible that adaptor proteins that transduce MAPK signals from BMP receptors40
do not require type I receptor kinase function. Dorsomorphin might help to elucidate the mechanisms by which SMAD-independent BMP signals are transduced, as well as the functional significance of such signals.
A small-molecule inhibitor provides great advantages in that it permits dissecting the function of BMP signals with exquisite temporal control while bypassing early embryonic lethality. Dorsomorphin inhibits osteogenic differentiation of myofibroblasts in vitro
and alters bone mineralization in zebrafish, which is consistent with inhibition of BMP signals critical for osteoblast commitment. In a similar fashion, time- and dose-controlled use of dorsomorphin may allow characterization of BMP functions throughout early and late development and in formation of multiple organ systems including the heart, kidneys and vasculature. Because BMPs also regulate regeneration of mature tissues and stem cell differentiation41,42
, modulating BMP signaling might also prove useful for the manipulation of progenitor cells.
Beyond development and cell fate determination, a potential role for BMP signaling in iron metabolism has recently emerged. The identification of Hfe2
gene mutations in juvenile hemochromatosis, which is characterized by low hepcidin levels and excessive iron accumulation, and the recognition that Hfe2
encodes the BMP co-receptor HJV, both suggest that BMPs have a role in iron regulation7,11
. The observation that BMPs and HJV induce hepatocytes to express hepcidin, which in turn inactivates ferroportin, provides a mechanism by which BMP signals may modify systemic iron metabolism. Recently it was reported that holotransferrin inhibits shedding of HJV from hepatoma cells, which reveals a potential mechanism by which iron might modulate BMP signaling43
. Here we provide the first experimental evidence that systemic iron administration rapidly activates BMP-responsive SMADs in the liver, and that iron-mediated hepcidin expression requires BMP signaling. The present data suggest that IL-6–induced hepcidin expression also requires intact BMP signaling in hepatocytes35
. The functional importance of BMP signaling in iron regulation is supported by the ability of dorsomorphin to inhibit hepcidin expression and raise serum iron levels in vivo
. Taken together, these findings demonstrate an essential role of BMP signaling in iron- and inflammation-mediated hepcidin expression ().
Whereas impaired hepcidin expression leads to iron overload, excessive hepcidin expression leads to hypoferremia and anemia. Anemia of chronic disease, a condition affecting almost half of all chronically ill people, is thought to be caused by maladaptive overexpression of hepcidin induced by chronic inflammation33,34,44
. Dorsomorphin’s ability to block hepatic BMP-responsive SMAD activation, inhibit hepcidin expression and increase serum iron concentrations strongly suggests that BMP type I receptor inhibition may be an effective therapeutic approach for reversing states of elevated hepcidin levels of diverse etiologies. Therefore, in addition to being a useful tool for fundamental studies of BMP signaling, dorsomorphin may lead to new therapies for treating anemia of chronic disease.
Until now, traditional methods for identifying small-molecule inhibitors have not yielded a specific BMP pathway inhibitor, perhaps because of the homology between BMP receptor kinases and other kinases. Kinase inhibitor discovery has been an area of intense interest, in part because of the therapeutic potential of kinase inhibitors in cancer chemotherapy. Despite this interest, identifying specific kinase inhibitors remains arduous, requiring coordinated selection for activity toward the target and selection against activity toward related kinases that might interfere with the desired effect. In this report, a rapid in vivo screening approach facilitated the identification of an inhibitor of the target pathway while excluding many nonspecific compounds with confounding toxicity. We screened for small molecules that could phenocopy genetic mutations in the BMP pathway without phenocopying genetic mutations in the TGF-β pathway. The selectivity of the resulting molecule for BMP over TGF-β signaling supports the notion that organism-based screening using well-defined phenotypes can identify previously elusive inhibitors of important target pathways.