Bone morphogenetic proteins signal via SMAD and mitogen-activated protein (MAP) kinase pathways at distinct times during osteoclastogenesis.

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII(fl/fl);lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.

A pair of transmembrane receptors essential for the retention and pigmentation of hair.

Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division.

Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain.

In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX(ΔE2) mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRX(ΔE2) mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRX(ΔE2) mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX(ΔE2) mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX(ΔE2) mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRX(ΔE2) mice may contribute to mental retardation syndromes seen in human patients.

BMPR-II is dispensable for formation of the limb skeleton.

Initiation of BMP signaling is dependent upon activation of Type I BMP receptor by constitutively active Type II BMP receptor. Three Type II BMP receptors have been identified; Acvr2a and Acvr2b serve as receptors for BMPs and for activin-like ligands whereas BMPR-II functions only as a BMP receptor. As BMP signaling is required for endochondral ossification and loss of either Acvr2a or Acvr2b is not associated with deficits in limb development, we hypothesized that BMPR-II would be essential for BMP signaling during skeletogenesis. We removed BMPR-II from early limb mesoderm by crossing BMPR-II floxed mice with those carrying the Prx1-Cre transgene. Mice lacking limb expression of BMPR-II have normal skeletons that could not be distinguished from control littermates. From these data, we conclude that BMPR-II is not required for endochondral ossification in the limb where loss of BMPR-II may be compensated by BMP utilization of Acvr2a and Acvr2b.

Reduced expression of the ATRX gene, a chromatin-remodeling factor, causes hippocampal dysfunction in mice.

Mutations of the ATRX gene, which encodes an ATP-dependent chromatin-remodeling factor, were identified in patients with α-thalassemia X-linked mental retardation (ATR-X) syndrome. There is a milder variant of ATR-X syndrome caused by mutations in the Exon 2 of the gene. To examine the impact of the Exon 2 mutation on neuronal development, we generated ATRX mutant (ATRX(ΔE2)) mice. Truncated ATRX protein was produced from the ATRX(ΔE2) mutant allele with reduced expression level. The ATRX(ΔE2) mice survived and reproduced normally. There was no significant difference in Morris water maze test between wild-type and ATRX(ΔE2) mice. In a contextual fear conditioning test, however, total freezing time was decreased in ATRX(ΔE2) mice compared to wild-type mice, suggesting that ATRX(ΔE2) mice have impaired contextual fear memory. ATRX(ΔE2) mice showed significantly reduced long-term potentiation in the hippocampal CA1 region evoked by high-frequency stimulation. Moreover, autophosphorylation of calcium-calmodulin-dependent kinase II (αCaMKII) and phosphorylation of glutamate receptor, ionotropic, AMPA 1 (GluR1) were decreased in the hippocampi of the ATRX(ΔE2) mice compared to wild-type mice. These findings suggest that ATRX(ΔE2) mice may have fear-associated learning impairment with the dysfunction of αCaMKII and GluR1. The ATRX(ΔE2) mice would be useful tools to investigate the role of the chromatin-remodeling factor in the pathogenesis of abnormal behaviors and learning impairment.

BMP type II receptor regulates positioning of outflow tract and remodeling of atrioventricular cushion during cardiogenesis.

Signaling of bone morphogenetic protein (BMP) via type I and type II receptors is involved in multiple processes contributing to cardiogenesis. To investigate the role of the BMP type II receptor (BMPRII) in heart development, the BMPRII gene was deleted throughout the embryo during gastrulation using a Mox2-Cre transgene. BMPRII(flox/-);Mox2-Cre mice exhibited cardiac defects including double-outlet right ventricle, ventricular septal defect (VSD), atrioventricular (AV) cushion defects, and thickened valve leaflets. To characterize the tissue-specific functions of BMPRII in cardiogenesis, a series of Cre transgenes (alphaMHC-, Tie2-, Wnt1-, and SM22alpha-Cre) was employed. Interestingly, myocardial development was normal when the BMPRII gene was deleted in myocardial cells using Mox2-Cre, alphaMHC-Cre, or SM22alpha-Cre transgenes, suggesting that signaling by other BMP type II receptors may compensate for the absence of BMPRII in the myocardial cells. AV cushion defects including atrial septal defect, membranous VSD, and thickened valve leaflets were found in BMPRII(flox/-);Tie2-Cre mice. Abnormal positioning of the aorta was observed in BMPRII(flox/-);Wnt1-Cre and BMPRII(flox/-);SM22alpha-Cre mice. Taken together, these results demonstrate that endocardial BMPRII expression is required for septal formation and valvulogenesis. Moreover, mesenchymal BMPRII expression in the outflow tract cushion is required for proper positioning of the aorta.

Genetic ablation of the BMPR2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal lung disease of diverse origins. PAH is now further subclassified as idiopathic PAH, familial PAH, and associated PAH varieties. Heterozygous mutations in BMPR2 can be detected in 50% to 70% of patients with familial PAH and 10% to 40% of patients with idiopathic PAH. Although endothelial cells have been suspected as the cellular origin of PAH pathogenesis, no direct in vivo evidence has been clearly presented. The present study was designed to investigate whether endothelial Bmpr2 deletion can predispose to PAH. METHODS AND RESULTS: The Bmpr2 gene was deleted in pulmonary endothelial cells using Bmpr2 conditional knockout mice and a novel endothelial Cre transgenic mouse line. Wide ranges of right ventricular systolic pressure were observed in mice with heterozygous (21.7 to 44.1 mm Hg; median, 23.7 mm Hg) and homozygous (20.7 to 56.3 mm Hg; median, 27 mm Hg) conditional deletion of Bmpr2 in pulmonary endothelial cells compared with control mice (19.9 to 26.7 mm Hg; median, 23 mm Hg) at 2 to 7 months of age. A subset of mice with right ventricular systolic pressure >30 mm Hg exhibited right ventricular hypertrophy and an increase in the number and wall thickness of muscularized distal pulmonary arteries. In the lungs of these mice with high right ventricular systolic pressure, the expression of proteins involved in the pathogenesis of PAH such as serotonin transporter and tenascin-C was elevated in distal arteries and had a high incidence of perivascular leukocyte infiltration and in situ thrombosis. CONCLUSIONS: Conditional heterozygous or homozygous Bmpr2 deletion in pulmonary endothelial cells predisposes mice to develop PAH.

Histone H3-K9 methyltransferase ESET is essential for early development.

Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre- and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset(-/-) blastocysts showed defective growth of the inner cell mass and, in contrast to the approximately 65% recovery of wild-type and Eset(+/-) ES cells, no Eset(-/-) ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset(-/-) blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

B-cell translocation gene 2 (Btg2) regulates vertebral patterning by modulating bone morphogenetic protein/smad signaling.

Btg2 is a primary p53 transcriptional target gene which may function as a coactivator-corepressor and/or an adaptor molecule that modulates the activities of its interacting proteins. We have generated Btg2-null mice to elucidate the in vivo function of Btg2. Btg2-null mice are viable and fertile but exhibit posterior homeotic transformations of the axial vertebrae in a dose-dependent manner. Consistent with its role in vertebral patterning, Btg2 is expressed in the presomitic mesoderm, tail bud, and somites during somitogenesis. We further provide biochemical evidence that Btg2 interacts with bone morphogenetic protein (BMP)-activated Smads and enhances the transcriptional activity of BMP signaling. In view of the genetic evidence that reduced BMP signaling causes posteriorization of the vertebral pattern, we propose that the observed vertebral phenotype in Btg2-null mice is due to attenuated BMP signaling.

Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice.

BACKGROUND: Bone morphogenetic protein receptor-2 (BMPR2)-heterozygous, mutant (BMPR2(+/-)) mice have a genetic trait similar to that of certain patients with idiopathic pulmonary arterial hypertension (IPAH). To understand the role of BMPR2 in the development of IPAH, we examined the phenotype of BMPR2(+/-) mice and their response to inflammatory stress. METHODS AND RESULTS: BMPR2(+/-) mice were found to have the same life span, right ventricular systolic pressure (RVSP), and lung histology as those of wild-type mice under unstressed conditions. However, when treated with recombinant adenovirus expressing 5-lipoxygenase (Ad5LO), BMPR2(+/-) mice exhibited significantly higher RVSP than wild-type mice. The increase of RVSP occurred in the first 2 weeks after Ad5LO delivery. Modest but significant muscularization of distal pulmonary arterioles appeared in BMPR2(+/-) mice 4 weeks after Ad5LO treatment. Measurement of urinary metabolites of vasoactive molecules showed that cysteinyl leukotrienes, prostacyclin metabolites, and PGE2 were all increased to a similar degree in both BMPR2(+/-) and wild-type mice during 5LO transgene expression, whereas urinary endothelin-1 remained undetectable. Urinary thromboxane A2 metabolites, in contrast, were significantly higher in BMPR2(+/-) than in wild-type mice and paralleled the increase in RVSP. Platelet activation markers, serotonin, and soluble P-selectin showed a trend toward higher concentrations in BMPR2(+/-) than wild-type mice. Cell culture studies found that BMP treatment reduced interleukin-1beta-stimulated thromboxane A2 production in the pulmonary epithelial cell line A549. CONCLUSIONS: BMPR2(+/-) mice do not develop pulmonary hypertension spontaneously; however, under inflammatory stress, they are more susceptible to an increase in RVSP, thromboxane A2 production, and vascular remodeling than wild-type mice.

Compound disruption of smad2 accelerates malignant progression of intestinal tumors in apc knockout mice.

Smad2 is a receptor-regulated Smad that is activated specifically by transforming growth factor beta and activin signaling. We disrupted the mouse Smad2 gene by gene targeting. Homozygous Smad2 mutant mice died around E8.5 with impaired visceral endoderm function and deficiency of mesoderm formation. Heterozygotes were fertile and had no apparent abnormality up to at least 1 1/2 year of age. To examine the role of Smad2 inactivation in the process of carcinogenesis, we prepared compound heterozygous mice, which carry both Apc and Smad2 mutations on the same chromosome in the cis-configuration. Compound inactivation of Smad2 in heterozygous Apc mutant mice did not change the total number of intestinal tumors but increased sudden death from intestinal obstruction caused by extremely large tumors. Furthermore, histological examination revealed that Apc/Smad2 cis-compound heterozygotes developed multiple invasive cancers that had never been observed in Apc single heterozygotes. These results indicate that loss of Smad2 does not initiate tumorigenesis by itself but accelerates malignant progression of tumors to invasive cancer in the late stages of carcinogenesis.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Smad7-modified alleles by various gene-targeting strategies.

Signalling of the transforming growth factor-ß (TGF-ß) family is tightly regulated by various mechanisms including negative feedback by Inhibitory Sma- and Mad-related proteins (Smads) (I-Smads: Smad6 and Smad7). Smad6 preferentially inhibits bone morphogenetic protein (BMP) signalling, whereas Smad7 suppresses both TGF-ß and BMP signalling. To elucidate the roles of Smad7 in murine development and in TGF-ß signalling, several Smad7-deficient mouse strains have been generated. Tojo et al. (Smad7-deficient mice show growth retardation with reduced viability. J. Biochem. 2012;151:621-631.) demonstrated that Smad7 null mutation caused perinatal lethality on a C57BL/6 background. However, the Smad7-deficient mice on an ICR background survived to adulthood, but showing growth retardation. Unexpectedly, phosphorylation levels of Smad2 and Smad3 were slightly reduced in murine embryonic fibroblast (MEF) cells isolated from Smad7-deficient embryos compared with wild-type MEF cells. Together with other Smad7-mutant mouse strains, these mutant mice provide useful tools to understand important roles of Smad7 in the development of murine embryos and diseases.

A rapid and high-throughput quantitation assay of the nuclear factor κB activity using fluorescence correlation spectroscopy in the setting of clinical laboratories.

BACKGROUND: Transcription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites. METHODS: Non-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured. RESULTS: The percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes. CONCLUSIONS: This assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.

Deletion of the sequence encoding the tail domain of the bone morphogenetic protein type 2 receptor reveals a bone morphogenetic protein 7-specific gain of function.

The bone morphogenetic protein (BMP) type II receptor (BMPR2) has a long cytoplasmic tail domain whose function is incompletely elucidated. Mutations in the tail domain of BMPR2 are found in familial cases of pulmonary arterial hypertension. To investigate the role of the tail domain of BMPR2 in BMP signaling, we generated a mouse carrying a Bmpr2 allele encoding a non-sense mediated decay-resistant mutant receptor lacking the tail domain of Bmpr2. We found that homozygous mutant mice died during gastrulation, whereas heterozygous mice grew normally without developing pulmonary arterial hypertension. Using pulmonary artery smooth muscle cells (PaSMC) from heterozygous mice, we determined that the mutant receptor was expressed and retained its ability to transduce BMP signaling. Heterozygous PaSMCs exhibited a BMP7­specific gain of function, which was transduced via the mutant receptor. Using siRNA knockdown and cells from conditional knockout mice to selectively deplete BMP receptors, we observed that the tail domain of Bmpr2 inhibits Alk2­mediated BMP7 signaling. These findings suggest that the tail domain of Bmpr2 is essential for normal embryogenesis and inhibits Alk2­mediated BMP7 signaling in PaSMCs.

MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons.

Bone morphogenetic protein (BMP) signaling has emerged as an important regulator of sensory neuron development. Using a three-generation forward genetic screen in mice we have identified Megf8 as a novel modifier of BMP4 signaling in trigeminal ganglion (TG) neurons. Loss of Megf8 disrupts axon guidance in the peripheral nervous system and leads to defects in development of the limb, heart, and left-right patterning, defects that resemble those observed in Bmp4 loss-of-function mice. Bmp4 is expressed in a pattern that defines the permissive field for the peripheral projections of TG axons and mice lacking BMP signaling in sensory neurons exhibit TG axon defects that resemble those observed in Megf8 (-/-) embryos. Furthermore, TG axon growth is robustly inhibited by BMP4 and this inhibition is dependent on Megf8. Thus, our data suggest that Megf8 is involved in mediating BMP4 signaling and guidance of developing TG axons. DOI:http://dx.doi.org/10.7554/eLife.01160.001.

Basolateral BMP signaling in polarized epithelial cells.

Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-ß or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

Constitutively active ALK2 receptor mutants require type II receptor cooperation.

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.

Antagonism between Smad1 and Smad2 signaling determines the site of distal visceral endoderm formation in the mouse embryo.

The anterior-posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.