Donna Y. Deng

BMP type I receptor inhibition reduces heterotopic ossification

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.

Structure–activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors

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.

Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation

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.

Bone Morphogenetic Protein (BMP) Type II Receptor Is Required for BMP-mediated Growth Arrest and Differentiation in Pulmonary Artery Smooth Muscle Cells

Bone morphogenetic protein (BMP) signals regulate the growth and differentiation of diverse lineages. The association of mutations in the BMP type II receptor (BMPRII) with idiopathic pulmonary arterial hypertension suggests an important role of this receptor in vascular remodeling. Pulmonary artery smooth muscle cells lacking BMPRII can transduce BMP signals using ActRIIa (Activin type II receptor). We investigated whether or not BMP signaling via the two receptors leads to differential effects on vascular smooth muscle cells. BMP4, but not BMP7, inhibited platelet-derived growth factor-activated proliferation in wild-type pulmonary artery smooth muscle cells, whereas neither ligand inhibited the growth of BMPRII-deficient cells. Adenoviral gene transfer of BMPRII enabled BMP4, as well as BMP7, to inhibit proliferation in BMPRII-deficient cells. BMP-mediated growth inhibition was also reconstituted by the BMPRII short isoform, lacking the C-terminal domain present in the long form. BMP4, but not BMP7, induced the expression of osteoblast markers in wild-type cells, whereas neither ligand induced these markers in BMPRII-deficient cells. Overexpression of short or long forms of BMPRII in BMPRII-deficient cells enabled BMP4 and BMP7 to induce osteogenic differentiation. Although signaling via BMPRII or ActRIIa transiently activated SMAD1/5/8, only BMPRII signaling led to persistent SMAD1/5/8 activation and sustained increases in Id1 mRNA and protein expression. Pharmacologic blockade of BMP type I receptor function within 24 h after BMP stimulation abrogated differentiation. These data suggest that sustained BMP pathway activation, such as that mediated by BMPRII, is necessary for growth and differentiation control in vascular smooth muscle.

Constitutively active ALK2 receptor mutants require type II receptor cooperation.

ABSTRACT Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease.

Circulating angiogenic modulatory factors predict survival and functional class in pulmonary arterial hypertension

The diagnosis of pulmonary arterial hypertension (PAH) is frequently delayed. We hypothesized that circulating angiogenic modulatory protein levels might correspond with vascular remodeling activity and serve as sensitive biomarkers of PAH. Levels of soluble endoglin (sEng), soluble vascular endothelial growth factor receptor-1 (sVEGFR1), N-terminal brain natriuretic peptide (NT-proBNP), C-reactive protein (CRP), and other biomarkers were measured in peripheral blood from 97 PAH patients, 16 first-degree relatives of idiopathic or heritable pulmonary arterial hypertension (HPAH) patients, and 56 controls, and correlated with disease, functional class, hemodynamic parameters, exercise capacity, and transplant-free survival. Endoglin expression was analyzed in lung tissues of six individuals with idiopathic or HPAH and four individuals without PAH. Levels of sEng, sVEGFR1, CRP, and NT-proBNP were elevated in Group I PAH of diverse etiologies, with sEng performing better than NT-proBNP in detecting PAH (receiver operator characteristic-area-under-the curve [ROC-AUC] of 0.82 ± 0.03 vs. 0.71 ± 0.05, P = 0.016). While sEng, sVEGFR1, and NT-proBNP correlated with New York Heart Association (NYHA) class, sEng levels were more sensitive than NT-proBNP in detecting NYHA Class I-II disease (ROC-AUC of 0.88 ± 0.05 vs. 0.67 ± 0.08, P = 0.028). sEng, sVEGFR1, CRP, and NT-proBNP predicted transplant-free survival by univariate Cox regression. After adjusting for NT-proBNP levels, each of the other three markers predicted transplant-free survival. In multivariate analysis, sEng and CRP were independent predictors of survival. Endoglin expression was markedly enhanced in the microvascular endothelium and endovascular lesions of PAH versus control lung tissues. Circulating angiogenic proteins sEng and sVEGFR1 are sensitive markers of prognosis and function in Group I PAH, including mildly symptomatic disease, and may provide unique noninvasive data reflecting underlying remodeling activity.