ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis.

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin-proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3).

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Isoflurane post-conditioning down-regulates expression of aquaporin 4 in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/Smad1/5/8 signaling pathway.

AIM: Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining. RESULTS: Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05). CONCLUSION: Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.

Different Degrees of Iodine Deficiency Inhibit Differentiation of Cerebellar Granular Cells in Rat Offspring, via BMP-Smad1/5/8 Signaling.

Iodine deficiency (ID) during development results in dysfunction of the central nervous system (CNS) and affects psychomotor and motor function. It is worth noting that maternal mild and marginal ID tends to be the most common reason of preventable neurodevelopmental impairment, via a mechanism that has not been elucidated. Therefore, our aim was to study the effects of developmental mild and marginal ID on the differentiation of cerebellar granule cells (GCs) and investigate the activation of BMP-Smad1/5/8 signaling, which is crucial for the development and differentiation of cerebellum. Three developmental rat models were created by feeding dam rats with a diet deficient in iodine and deionized water supplemented with potassium iodide. Our results showed that different degrees of ID inhibited and delayed the differentiation of cerebellar GCs on postnatal day (PN) 7, PN14, and PN21. Moreover, mild and severe ID reduced the expression of BMP2 and p-Smad1/5/8, and increased the levels of Id2 on PN7, PN14, and PN21. However, marginal ID rarely altered expression of these proteins in the offspring. Our study supports the hypothesis that mild and severe ID during development inhibits the differentiation of cerebellar GCs, which may be ascribed to the down-regulation of BMP-Smad1/5/8 signaling and the overexpression of Id2. Furthermore, it was speculated that maternal marginal ID rarely affected the differentiation of cerebellar GCs in the offspring.

KMUP-1 Promotes Osteoblast Differentiation Through cAMP and cGMP Pathways and Signaling of BMP-2/Smad1/5/8 and Wnt/ß-Catenin.

Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP-1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso-enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP-1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3-E1 cells were examined. KMUP-1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3-E1 cells. KMUP-1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP-2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP-1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP-1 upregulated Smad1/5/8 phosphorylations with subsequent BRE-Luc activation confirmed by transient transfection assay. In addition, KMUP-1 inactivated glycogen synthase kinase-3ß (GSK-3ß), with associated nuclear translocation of ß-catenin. Co-treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP-1-induced activations of Smad1/5/8, ß-catenin, and Runx2. The findings demonstrate for the first time that KMUP-1 can promote osteoblast maturation and differentiation in vitro via BMP-2/Smad1/5/8 and Wnt/ß-catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP-1 may be a novel osteoblast activator and a potential new therapy for osteoporosis.

BMP-Smad 1/5/8 signalling in the development of the nervous system.

The transcription factors, Smad1, Smad5 and Smad8, are the pivotal intracellular effectors of the bone morphogenetic protein (BMP) family of proteins. BMPs and their receptors are expressed in the nervous system (NS) throughout its development. This review focuses on the actions of Smad 1/5/8 in the developing NS. The mechanisms by which these Smad proteins regulate the induction of the neuroectoderm, the central nervous system (CNS) primordium, and finally the neural crest, which gives rise to the peripheral nervous system (PNS), are reviewed herein. We describe how, following neural tube closure, the most dorsal aspect of the tube becomes a signalling centre for BMPs, which directs the pattern of the development of the dorsal spinal cord (SC), through the action of Smad1, Smad5 and Smad8. The direct effects of Smad 1/5/8 signalling on the development of neuronal and non-neuronal cells from various neural progenitor cell populations are then described. Finally, this review discusses the neurodevelopmental abnormalities associated with the knockdown of Smad 1/5/8.

Axonally translated SMADs link up BDNF and retrograde BMP signaling.

Axonal target-derived BMP and neurotrophin signaling are both known to regulate neuronal gene expression, differentiation, and axon growth. In this issue of Neuron, Ji and Jaffrey (2012) discovered that BMP-signaling endosomes depend on BDNF-induced axonal synthesis of SMADs to retrogradely regulate transcription in developing trigeminal neurons, providing a mechanism of integrating the two target-derived signals.

Intra-axonal translation of SMAD1/5/8 mediates retrograde regulation of trigeminal ganglia subtype specification.

In many cases, neurons acquire distinct identities as their axons navigate toward target cells and encounter target-derived signaling molecules. These molecules generate retrograde signals that activate subtype-specific gene transcription. Mechanisms by which axons convert the complex milieu of signaling molecules into retrograde signals are not fully understood. Here, we examine retrograde signaling mechanisms that specify neuronal identity in the trigeminal ganglia, which relays sensory information from the face to the brain. We find that neuron specification requires the sequential action of two target-derived factors, BDNF and BMP4. BDNF induces the translation of axonally localized SMAD1/5/8 transcripts. Axon-derived SMAD1/5/8 is translocated to the cell body, where it is phosphorylated to a transcriptionally active form by BMP4-induced signaling endosomes and mediates the transcriptional effects of target-derived BDNF and BMP4. Thus, local translation functions as a mechanism by which coincident signals are converted into a retrograde signal that elicits a specific transcriptional response.

Evidence for the prevention of enthesitis in HLA-B27/hß(2)m transgenic rats treated with a monoclonal antibody against TNF-α.

Transgenic rats with high expression of HLA-B27 and human ß(2) -microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF-α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF-α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9-week-old B27TR received an anti-TNF-α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9-week treatment with anti-TNF-α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF-α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte-like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k-treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti-TNF-α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti-TNF-α-treated animals. Smad1/5/8 signalling was not inhibited by late anti-TNF-α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF-α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.

An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney.

BACKGROUND: Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney. RESULTS: Mouse strains reporting on BMP pathway activation were generated by transgenically expressing beta-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips. CONCLUSION: Transgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.