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.

Inhibition of bone morphogenetic protein signaling reduces vascular calcification and atherosclerosis.

OBJECTIVE: The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. Although genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes. METHODS AND RESULTS: We tested the impact of pharmacological BMP inhibition on atherosclerosis and calcification in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice fed a high-fat diet developed abundant vascular calcification within 20 weeks. Prolonged treatment of LDLR-/- mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the antiatherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis. CONCLUSION: These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification.

Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease.

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-beta family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.

Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse.

INTRODUCTION: In this study we investigated the action of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF-κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. METHODS: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. RESULTS: RAP-031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP-031 with UDCA produced increases in FPT that were associated with an increase in WBT. CONCLUSIONS: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF-κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice.

Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.

Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70-80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.

A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurologic disease characterized by progressive weakness that results in death within a few years of onset by respiratory failure. Myostatin is a member of the TGF-beta superfamily that is predominantly expressed in muscle and acts as a negative regulator of muscle growth. Attenuating myostatin has previously been shown to produce increased muscle mass and strength in normal and disease animal models. In this study, a mouse model of ALS (SOD1(G93A) transgenic mice) was treated with a soluble activin receptor, type IIB (ActRIIB.mFc) which is a putative endogenous signaling receptor for myostatin in addition to other ligands of the TGF-beta superfamily. ActRIIB.mFc treatment produces a delay in the onset of weakness, an increase in body weight and grip strength, and an enlargement of muscle size whether initiated pre-symptomatically or after symptom onset. Treatment with ActRIIB.mFc did not increase survival or neuromuscular junction innervation in SOD1(G93A) transgenic mice. Pharmacologic treatment with ActRIIB.mFc was superior in all measurements to genetic deletion of myostatin in SOD1(G93A) transgenic mice. The improved function of SOD1(G93A) transgenic mice following treatment with ActRIIB.mFc is encouraging for the development of TGF-beta pathway inhibitors to increase muscle strength in patients with ALS.

Prevention of cachexia-like syndrome development and reduction of tumor progression in inhibin-deficient mice following administration of a chimeric activin receptor type II-murine Fc protein.

Inhibin is a secreted tumor suppressor, and inhibin alpha null mice develop gonadal sex cord-stromal tumors with 100% penetrance at an early age. Inhibin-deficient mice die of a severe wasting syndrome due to increased activin signaling through activin receptor type II. The current study was designed to assess the in vivo effects of an activin antagonist, a chimeric activin receptor type II fused to the Fc region of a murine IgG2a (ActRII-mFc), administered transiently to the inhibin-deficient mice. Results showed that the severe weight loss was prevented in the ActRII-mFc-treated mice, FSH levels were reduced, and an extended life span was observed for these mice compared with phosphate-buffered saline-treated controls. Although ActRII-mFc treatment did not seem to prevent the formation of gonadal tumors, tumors were smaller in the majority of experimentally treated mice and were characterized by the presence of variable numbers and sizes of cysts in contrast to the solid hemorrhagic tumors that typically developed in the controls. Moreover, the ActRII-mFc-treated mice were less anemic, and their livers and stomachs were histologically normal. In summary, this study demonstrated that in vivo administration of the activin antagonist, ActRII-mFc, not only prevents the cachexia-like symptoms in the inhibin-deficient mouse model, but also reduces tumor progression. These results support an essential role of activins in the cachexia-like syndrome development and implicate activins as growth-promoting factors in gonadal tumor progression. The current findings have potential implications in the design of new drugs or strategies for the treatment of ovarian and testicular tumors and other conditions where ligands signal through ActRII.