Abstract: Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers.
Abstract: Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.
Abstract: Vertebrate segmentation is regulated by the "segmentation clock", which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth. Formation of cervical, thoracic and lumbar somites/vertebrae, but not sacral and adjacent tail somites/vertebrae, depends on caudal, cyclic Lfng expression. Indeed, the sacral region segments normally in the complete absence of Lfng and shows a reduced requirement for another oscillating gene, Hes7, indicating that the architecture of the clock alters as segmentation progresses. We present evidence that Lfng controls dorsal-ventral axis specification in the tail, and also suggest that Lfng controls the expression or activity of a long-range signal that regulates axial extension.
Abstract: Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.
Abstract: A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t(1/2)=1.6h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.
Abstract: Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.
Abstract: Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A-P) patterning to identify the target of DTAB, a compound that caused A-P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors gamma and beta. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules.
Abstract: Myogenic precursor cells known as satellite cells persist in adult skeletal muscle and are responsible for its ability to regenerate after injury. Quiescent satellite cells are activated by signals emanating from damaged muscle. Here we describe the rapid activation of two genes in response to muscle injury; these transcripts encode LPS-inducible CXC chemokine (LIX), a neutrophil chemoattractant, and Tristetraprolin (TTP), an RNA-binding protein implicated in the regulation of cytokine expression. Using a synchronized cell culture model we show that C2C12 myoblasts arrested in G0 exhibit some molecular attributes of satellite cells in vivo: suppression of MyoD and Myf5 expression during G0 and their reactivation in G1. Synchronization also revealed cell cycle dependent expression of CD34, M-cadherin, HGF and PEA3, genes implicated in satellite cell biology. To identify other genes induced in synchronized C2C12 myoblasts we used differential display PCR and isolated LIX and TTP cDNAs. Both LIX and TTP mRNAs are short-lived, encode molecules implicated in inflammation and are transiently induced during growth activation in vitro. Further, LIX and TTP are rapidly induced in response to muscle damage in vivo. TTP expression precedes that of MyoD and is detected 30 minutes after injury. The spatial distribution of LIX and TTP transcripts in injured muscle suggests expression by satellite cells. Our studies suggest that in addition to generating new cells for repair, activated satellite cells may be a source of signaling molecules involved in tissue remodeling during regeneration.
