Generation of novel conditional and hypomorphic alleles of the Smad2 gene

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.     

Crystal structure of activin receptor type IIB kinase domain from human at 2.0 Angstrom resolution

Activin receptor type IIB (ActRIIB), a type II TGF-beta serine/threonine kinase receptor, is integral to the activin and myostatin signaling pathway. Ligands such as activin and myostatin bind to activin type II receptors (ActRIIA, ActRIIB), and the GS domains of type I receptors are phosphorylated by type II receptors. Myostatin, a negative regulator of skeletal muscle growth, is regarded as a potential therapeutic target and binds to ActRIIB effectively, and to a lesser extent, to ActRIIA. The high-resolution structure of human ActRIIB kinase domain in complex with adenine establishes the conserved bilobal architecture consistent with all other catalytic kinase domains. The crystal structure reveals that the adenine has a considerably different orientation from that of the adenine moiety of ATP observed in other kinase structures due to the lack of an interaction by ribose-phosphate moiety and the presence of tautomers with two different protonation states at the N9 nitrogen. Although the Lys217-Glu230 salt bridge is absent, the unphosphorylated activation loop of ActRIIB adopts a conformation similar to that of the fully active form. Unlike the type I TGF-beta receptor, where a partially conserved Ser280 is a gatekeeper residue, the AcRIIB structure possesses Thr265 with a back pocket supported by Phe247. Taken together, these structural features provide a molecular basis for understanding the coupled activity and recognition specificity for human ActRIIB kinase domain and for the rational design of selective inhibitors.     

Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro

Abstract

Background: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. Methods: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. Results: We observed a significant silencing (64–81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16–46% silencing with maximum of 46% by sh6 (p?=?0.0318) against endogenous ACVR2B whereas up to 66% (p?=?0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r?=?0.547; p?=?0.102), myogenin (r?=?0.517; p?=?0.126) and Myf5 (r?=?0.262; p?=?0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. Conclusions: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor

Background  

Bone morphogenetic proteins (BMPs) are key regulators in the embryonic development and postnatal tissue homeostasis in all animals. Loss of function or dysregulation of BMPs results in severe diseases or even lethality. Like transforming growth factors β (TGF-βs), activins, growth and differentiation factors (GDFs) and other members of the TGF-β superfamily, BMPs signal by assembling two types of serine/threonine-kinase receptor chains to form a hetero-oligomeric ligand-receptor complex. BMP ligand receptor interaction is highly promiscuous, i.e. BMPs bind more than one receptor of each subtype, and a receptor bind various ligands. The activin type II receptors are of particular interest, since they bind a large number of diverse ligands. In addition they act as high-affinity receptors for activins but are also low-affinity receptors for BMPs. ActR-II and ActR-IIB therefore represent an interesting example how affinity and specificity might be generated in a promiscuous background.     

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that “denticleless” is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.     

Production of bioactive extracellular domain of pig and chicken activin type IIB receptors in Pichia pastoris

Myostatin (MSTN) is a potent negative regulator for skeletal muscle growth, and binds to activin type IIB receptor (ActRIIB) for its cellular signal transduction. Administration of the extracellular domain of ActRIIB (ActRIIB-ECD) improved skeletal muscle growth in laboratory animals, suggesting that ActRIIB-ECD can be a useful pharmacological agent to improve skeletal muscle growth of meat-producing animals. In the current study, pig and chicken ActRIIB-ECDs were produced in the Pichia pastoris GS115, and the recombinant proteins were purified from induced culture media by Ni-NTA affinity chromatography. The digestion of pig and chicken ActRIIB-ECDs with PNGase F and glycoprotein staining demonstrated an N-linked glycosylation of these recombinant proteins. Glycoprotein staining also indicated an additional presence of glycosylation in chicken ActRIIB-ECD. Both the pig and chicken ActRIIB-ECDs were shown to inhibit MSTN activity in a reporter gene assay system in vitro. When MSTN-inhibitory potencies were compared by analyzing EC₅₀ values, no difference in MSTN-inhibitory potency was observed between the glycosylated and N-deglycosylated forms of pig or chicken ActRIIB-ECD, suggesting that glycosylation does not affect the bioactivity of ActRIIB-ECD. MSTN-inhibitory potency of chicken ActRIIB-ECD was greater (P < 0.01) than that of pig ActRIIB-ECD. Results of this study demonstrate that bioactive pig and chicken ActRIIB-ECDs can be produced from P. pastoris. In addition, the study indicates that the N-glycosylation status of ActRIIB-ECD does not affect its bioactivity in vitro.     

Pathophysiological consequences of isoform-specific IP3 receptor mutations

Ca²⁺ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca²⁺-signaling network is the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), a tetrameric channel that mediates Ca²⁺ release from the endoplasmic reticulum (ER) in response to IP₃. IP₃R function is controlled by a myriad of factors, such as Ca²⁺, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP₃R-mediated Ca²⁺ signaling is the result of the existence of three main isoforms (IP₃R1, IP₃R2 and IP₃R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP₃R1 is highly expressed in neurons, IP₃R2 in cardiomyocytes and hepatocytes and IP₃R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP₃R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP₃R1 to dysfunctional exocrine tissues and autoimmune diseases for IP₃R2 and -3. Over the past years, several IP₃R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP₃R isoform and the subsequent molecular mechanisms underlying the etiology of the disease.     

Bis-aryl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

3-Aryl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). They are active in both in vitro and an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.     

Discovery of potent and selective inhibitors of 11beta-HSD1 for the treatment of metabolic syndrome

High throughput screening efforts have identified a novel class of dichloroaniline amide 11β-HSD1 inhibitors. SAR studies initiated from dichloroaniline 4 focused on retaining the potency and selectivity profile of the lead.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.     

Immunocytochemical study of activin type IB receptor (XALK4) in Xenopus oocytes

Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria.     

Generation of Ppp2Ca and Ppp2Cb conditional null alleles in mouse

Protein phosphatase 2A (PP2A) is one of the most abundant serine/threonine phosphatases, with a critical role in embryonic development and human disease. There are two isoforms of the catalytic subunit of PP2A, Ppp2ca and Ppp2cb. Null mutation of Ppp2ca leads to early embryonic lethality at E6.5, hindering functional study of PP2A beyond this stage. We generated conditional null alleles of Ppp2ca and Ppp2cb by flanking with loxP sites exons 3 to 5 of Ppp2ca and exon 3 of Ppp2cb. Ppp2ca(fl/fl) mice did not display any visible phenotype. Homozygous mutants in which Cre-mediated excision resulted in global deletion of Ppp2ca displayed embryonic lethality and developmental defects similar to those previously reported. Ppp2cb(Δ/Δ) mice generated by the same strategy did not display any obvious morphological or physiological defects. These mouse strains can serve as important genetic tools to study the roles of PP2A during development and disease in a spatial- or temporal-specific manner.