A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

Bone morphogenetic protein-6 induces castration resistance in prostate cancer cells through tumor infiltrating macrophages.

Bone morphogenetic protein (BMP) is a pleiotropic growth factor that has been implicated in inflammation and prostate cancer (CaP) progression. We investigated the potential role of BMP-6 in the context of macrophages and castration-resistant prostate cancer. When the androgen-responsive murine (Tramp-C1 and PTENCaP8) and human (LNCaP) CaP cell lines were cocultured with macrophages in the presence of dihydrotestosterone, BMP-6 increased androgen-responsive promoter activity and cell count significantly. Subsequent studies revealed that BMP-6 increased the expression level of androgen receptor (AR) mRNA and protein in CaP cell lines only in the presence of macrophages. Simultaneously, the AR antagonists bicalutamide and MDV3100 partially or completely blocked BMP-6-induced macrophage-mediated androgen hypersensitivity in CaP cells. Abolishing interleukin-6 signaling with neutralizing antibody in CaP/macrophage cocultures inhibited the BMP-6-mediated AR upregulation in CaP cells. Using Tramp-C1 and PTENCaP8 cells with a tetracycline-inducible expression of BMP-6, the induction of BMP-6 in vivo resulted in a significant resistance to castration. However, this resistance was blocked after the removal of macrophages with clodronate liposomes. Taken together, these results show that BMP-6 induces castration resistance by increasing the expression of AR through macrophage-derived interleukin-6.

An in vivo role of bone morphogenetic protein-6 in aldosterone production by rat adrenal gland.

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex. We previously reported the presence of a functional BMP system including BMP-6 in human adrenocortical cells. BMP-6 contributes to Ang II-induced aldosterone production by activating Smad signaling, in which endogenous BMP-6 action is negatively controlled by Ang II in vitro. In the present study, we examined the in vivo role of BMP-6 in regulation of aldosterone by neutralizing endogenous BMP-6 in rats treated with immunization against BMP-6. Three-week-old male rats were actively immunized with rat mature BMP-6 antigen conjugated with keyhole limpet hemocyanin (KLH). The immunization treatment had no effect on bilateral adrenal weight or its ratio to body weight. Urinary aldosterone excretion was time-dependently increased during the 8-week observation period in the control group. Of note, the level of urinary aldosterone excretion in BMP-6-KLH-immunized rats was significantly reduced compared to that in the control group, suggesting that endogenous BMP-6 contributes to the induction of aldosterone production in vivo. Moreover, the level of urinary aldosterone/creatinine after 8-week treatment was significantly lowered by treatment with BMP-6-KLH. In contrast, with chronic Ang II treatment, urinary aldosterone and creatinine-corrected values at 8 weeks were not significantly different between the two groups, suggesting that the effects of BMP-6-KLH were impaired under the condition of chronic treatment with Ang II. The mRNA levels of Cyp11b2, but not those of Star, P450scc and 3ßhsd2, were significantly decreased in adrenal tissues isolated from BMP-6-KLH-immunized rats after 8-week treatment. Furthermore, the ratio of plasma aldosterone level to corticosterone was significantly decreased by immunization with BMP-6-KLH. Collectively, the results indicate that endogenous BMP-6 is functionally linked to aldosterone synthesis by the zona glomerulosa in the adrenal cortex in vivo.

Bone morphogenetic proteins inhibit CD40L/IL-21-induced Ig production in human B cells: differential effects of BMP-6 and BMP-7.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily. TGF-ß can affect class switch recombination in human B cells, but whether BMPs also play a role have not been tested. We investigated the functional effects of exogenously added BMPs on CD27(-) naive and CD27(+) memory B cells from healthy donors. BMP-2, -4, -6 and -7 inhibited CD40L/IL-21-induced production of IgM, IgG and IgA. BMP-6 reduced Ig production by 70% in memory B cells and more than 55% in naive B cells, whereas the other BMPs were slightly less potent. We observed a striking difference in functional effects between the structurally similar BMP-6 and BMP-7, as BMP-6 mainly inhibited plasmablast differentiation, and BMP-7 mainly induced apoptosis. In memory B cells, BMP-6 upregulated expression of DNA-binding protein inhibitor genes, but potently inhibited CD40L/IL-21-induced upregulation of the transcription factor XBP1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells.

Endogenous bone morphogenetic proteins mediate 1alpha, 25-dihydroxyvitamin D(3)-induced expression of osteoblast differentiation markers in human dermal fibroblasts.

Human dermal fibroblasts are generally considered to be restricted to a fibroblastic lineage. Although dermal fibroblasts do not typically express markers of osteoblastic differentiation, they have previously been shown to undergo osteoinduction when stimulated with bone morphogenetic proteins (BMPs) or vitamin D(3). However, involvement of BMP signaling in vitamin D(3)-mediated osteoinduction has not been reported. In this study, human dermal fibroblasts were cultured in chemically defined medium containing vitamin D(3), in the presence of the BMP antagonist noggin or neutralizing antibodies specific for BMP-4 or BMP-6, and characterized for markers of osteoblastic differentiation. Treatment of dermal fibroblasts with vitamin D(3) induced expression of BMP-4 (1.2 +/- 0.2, 1.7 +/- 0.2, and 1.8 +/- 0.2 relative fold increase) and BMP-6 (9.1 +/- 0.3, 23.3 +/- 2.1, and 30.4 +/- 3.0 relative fold increase) at 3, 14, and 21 days, respectively. Vitamin D(3) was also shown to induce the expression of the osteoblast-specific markers, alkaline phosphatase and osteocalcin, in a dose-dependent manner in human dermal fibroblasts. Addition of noggin, BMP-4 antibodies, and BMP-6 antibodies resulted in a downregulation of alkaline phosphatase activity (by 42%, 22%, and 20%, respectively) and secreted osteocalcin (by 20%, 31%, and 49%, respectively) after 21 days in culture. However, blocking BMP signaling did not result in complete recovery of a fibroblastic phenotype. Taken together, these results suggest that BMP signaling plays a role in the induction of an osteoblastic phenotype in human dermal fibroblasts in response to vitamin D(3) stimulation.