Ventromorphins: A New Class of Small Molecule Activators of the Canonical BMP Signaling Pathway.

Here, we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ∼600 000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them "ventromorphins." As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a genetic response similar to BMP4 treatment. Unlike isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 h of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 min of treatment and achieved peak activity within 1 h, indicating that their responses are consistent with directly activating BMP signaling.

Harnessing the BMP signaling pathway to control the formation of cancer stem cells by effects on epithelial-to-mesenchymal transition.

Cancer stem cells (CSCs) persist in tumors as a distinct population and may be causative in metastasis and relapse. CSC-rich tumors are associated with higher rates of metastasis and poor patient prognosis. Targeting CSCs therapeutically is challenging, since they seem to be resistant to standard chemotherapy. We have shown that a novel peptide agonist of bone morphogenetic protein (BMP) signaling, P123, is capable of inhibiting the growth of primary tumor cells by interacting with type I receptors selectively [activin receptor-like kinase 2 (ALK2) and ALK3, but not ALK6] and type II BMP receptors, activating SMAD 1/5/8 signaling and controlling the cell cycle pathway. Furthermore, the compound is capable of blocking transforming growth factor-ß induced epithelial-to-mesenchymal transition (EMT) in primary tumor cells, a critical step for tumor progression and metastasis. In addition, we have investigated the effects of P123 on self-renewal, growth, differentiation (reversal of EMT) and apoptosis of isolated human breast CSCs. We have shown that P123 and BMP-7 reverse the EMT process in human breast CSCs, and inhibit self-renewal and growth. Moreover, compared with single treatment with paclitaxel, co-treatment with paclitaxel and P123 showed an increase in cell apoptosis. Together, these findings suggest that P123 has the therapeutic potential to suppress both bulk tumor cells and CSCs. We believe that P123 represents a new class of drugs that have the potential to eliminate the primary tumor, prevent reoccurrence and metastasis, and enhance the treatment of breast cancer.

Early response of the human SOST gene to stimulation by 1α,25-dihydroxyvitamin D3.

The osteocyte expressed gene SOST encodes sclerostin, a potent negative regulator of bone formation and inducer of bone resorption. We have recently demonstrated that the human SOST gene is positively regulated in response to 1α,25-dihydroxyvitamin D3 (1,25D). Responsiveness may be mediated at least in part by a single classical DR3-type vitamin D response element (VDRE). In this study we examined the early responsiveness of the SOST gene to both 1,25D and to parathyroid hormone (PTH), a known repressor of SOST expression, in SaOS2 cells differentiated to an osteocyte-like stage of cell maturation. Both SOST mRNA levels and sclerostin protein levels increased in these cultures as early as 3h post-treatment with 1,25D and declined in response to PTH in the same timeframe. For 1,25D, the level of induced SOST appeared dependent on the extent, to which the degradative enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) was induced. Together with the observed rapid decrease in SOST/sclerostin levels in response to PTH, endocrine regulation of sclerostin production appears to be an important determinant of sclerostin levels. These findings confirm that the human SOST gene and sclerostin expression can be considered to be directly 1,25D-responsive in osteocytes.

Calcium induces pro-anabolic effects on human primary osteoblasts associated with acquisition of mature osteocyte markers.

Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.

Antagonism and synergy between extracellular BMP modulators Tsg and BMPER balance blood vessel formation.

Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously acts proangiogenically on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators, especially Tsg, on endothelial cell behaviour and compare them to BMPER. In Matrigel assays, BMP modulators chordin and noggin had no stimulatory effect; however, gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As the activation dynamics of Tsg were similar to those of BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse Matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro, dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.

Identification of small molecule activators of BMP signaling.

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.

Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.

Molecules associated with the transforming growth factor ß (TGF-ß) superfamily, such as bone morphogenic proteins (BMPs) and TGF-ß, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-ß1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

Secretion of bioactive hepcidin-25 by liver cells correlates with its gene transcription and points towards synergism between iron and inflammation signaling pathways.

Hepcidin is a small liver-derived peptide central in the regulation of systemic iron homeostasis. Although the gene regulation has been extensively studied at transcriptional level, the corresponding effects on the production of bioactive peptide are largely unknown. We therefore applied a proteomics-based approach by combining immunocapture with time-of-flight mass spectrometry to characterize hepcidin-25 produced by hepatocyte-derived cell lines. Similar to its transcriptional regulation, mature hepcidin-25 was strongly secreted upon stimulation with BMPs and IL-6. The immunocaptured peptide down-modulated iron-exporter ferroportin on the monocyte/macrophage surface. Further mass spectrometry-based analyses indicated that hepcidin-25 in its bioactive conformation was very stable in serum and urine and not converted into its smaller isoforms. Hepcidin-25 was processed in the Golgi apparatus from its precursor, while the unprocessed prohepcidin was secreted only when furin-like protease activity was intracellularly inhibited. Furthermore, the amounts of hepatocytic secretion of hepcidin-25 are highly correlated with the gene transcript levels. An unexpected observation was the synergistic effect of BMPs and IL-6 on hepcidin-25 secretion, which points towards cross-talk between iron and inflammatory stimuli. The study underscores hepcidin-25 quantification as a valuable tool to unravel regulatory pathways in iron metabolism.

Bone morphogenetic protein-7 (BMP7) in chronic kidney disease.

Bone morphogenetic protein-7 (BMP7) is a member of the BMP-subfamily of perhaps a dozen proteins within the TGFbeta-superfamily of cysteine-knot fold cytokine-growth factors. BMP7 has pivotal functions during renal and eye development. In adult organisms, BMP7 is heavily expressed in kidney, specifically in podocytes, distal tubules and collecting ducts. The activity of BMP7 is reduced by inhibitors including some members of the dan-cerberus group and CTGF but can be enhanced by endoglin and KCP. Renal BMP7 disappears early in fibrogenic renal diseases which may facilitate progression. Exogenous administration of rhBMP7 or transgenic overexpression reduces renal fibrogenesis and apoptosis as well as transdifferentiation of epithelial cells. BMP7 improves maintenance of nephron function and structural integrity. These antifibrogenic activities result from inhibition of the nuclear translocation of TGFbeta-activated smad3 by smad6 downstream of BMP7-activated smad5. Although at present the beneficial effects of BMP7 have only been studied in rodent models of chronic renal diseases, there is promise for therapeutic utility of rhBMP7 or small molecule BMP7 agonists in patients.

Mammalian twisted gastrulation is essential for skeleto-lymphogenesis.

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.

Interdigital cell death can occur through a necrotic and caspase-independent pathway.

Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.

Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds.

Outgrowth and patterning of the vertebrate limb are controlled by reciprocal interactions between the posterior mesenchyme (polarizing region) and a specialized ectodermal structure, the apical ectodermal ridge (AER). Sonic hedgehog (SHH) signalling by the polarizing region modulates fibroblast growth factor (FGF)4 signalling by the posterior AER, which in turn maintains the polarizing region (SHH/FGF4 feedback loop). Here we report that the secreted bone-morphogenetic-protein (BMP) antagonist Gremlin relays the SHH signal from the polarizing region to the AER. Mesenchymal Gremlin expression is lost in limb buds of mouse embryos homozygous for the limb deformity (Id) mutation, which disrupts establishment of the SHH/FGF4 feedback loop. Grafting Gremlin-expressing cells into ld mutant limb buds rescues Fgf4 expression and restores the SHH/FGF4 feedback loop. Analysis of Shh-null mutant embryos reveals that SHH signalling is required for maintenance of Gremlin and Formin (the gene disrupted by the ld mutations). In contrast, Formin, Gremlin and Fgf4 activation are independent of SHH signalling. This study uncovers the cascade by which the SHH signal is relayed from the posterior mesenchyme to the AER and establishes that Formin-dependent activation of the BMP antagonist Gremlin is sufficient to induce Fgf4 and establish the SHH/FGF4 feedback loop.