Biochemical properties of amphibian bone morphogenetic protein-4 expressed in CHO cells

Suzuki, Atsushi; Nishimatsu, Shin‐ichiro; Shoda, Akihito; Takebayashi, Kimiko; Murakami, Kazuo; Ueno, Naoto

doi:10.1042/bj2910413

Cited by 13 publications

(5 citation statements)

References 33 publications

(33 reference statements)

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“…Two immunoreactive bands, one at 18 kDa (the monomeric form) and the other at 36 kDa (the dimeric form) were detected for BMP-2. A single immunoreactive band at 36 kDa was detected for BMP-4, probably representing a dimeric conformation of the mature peptide (Suzuki et al 1993). For BMP-6, multiple bands were detected at 21 kDa (also detected in recombinant BMP-6), 23 kDa, 36 kDa, z45 kDa, 69 kDa, and 90 kDa in cultured osteoclasts, as previously reported in other cells (Boden et al 1997;Rickard et al 1998;Akiyoshi et al 2004).…”

Section: Western Blot Analysessupporting

confidence: 81%

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Garimella

Tague

Zhang

et al. 2008

J Histochem Cytochem.

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Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

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Section: Western Blot Analysessupporting

confidence: 81%

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Garimella

Tague

Zhang

et al. 2008

J Histochem Cytochem.

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“…30,31 According to the dextran diffusion analysis, the molecular weight cutoff of the microcapsules was most likely around 40 kDa ( Figure 2). Therefore, LIF can go through the capsules and the large secreted factors such as BMP4 30,32,33 can be accumulated inside. Although further analysis is required, the fact that primitive endoderm layer did not appear in aggregate in hydrogel capsules is important because there were few reports about how Alg-Ca capsules were able to keep PSCs undifferentiated.…”

Section: Discussionmentioning

confidence: 99%

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production

Horiguchi

Chowdhury

Sakai

et al. 2014

Biotechnology Progress

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Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel-based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium-alginate (Alg-Ca) hydrogel-based microcapsules; (i) Alg-Ca capsules without further treatment (Naked), (ii) Alg-Ca capsules with poly-l-lysine (PLL) coating (Coated), and (iii) Alg-PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10-13-fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT-PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel-based encapsulation culture. These results are helpful in further optimizing hydrogel-based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture.

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“…To examine the biological activity of sBMPR, ALP activity of MC3T3-E1 cells was examined. Various concentrations of sBMPR were added to the medium on day 1 of the culture, and ALP activity was determined on day 6 of the culture, using an established technique (34). ST2 cells were grown in the presence of rxBMP-4 (100 ng/ml) and various concentrations of sBMPR.…”

Section: Construction Of Sbmpr and Xenopus Bmp-4 (Xbmp-4)-mentioning

confidence: 99%

Interaction between Soluble Type I Receptor for Bone Morphogenetic Protein and Bone Morphogenetic Protein-4

Natsume¹,

Tomita

Iemura³

et al. 1997

Journal of Biological Chemistry

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Bone morphogenetic proteins (BMPs) are multifunctional proteins that comprise the largest subfamily of the transforming growth factor-␤. These proteins bind to types I and II serine/threonine kinase receptors. Ligand-induced heteromeric dimerization of these receptors is the key event in initiation of biological responses. We report here large-scale expression and purification of extracellular domain of the type I receptor for BMP-2/4, using a silkworm expression system. This soluble form of BMP receptor (sBMPR) was in monomer form in solution and bound to BMP-4 but not to activin or transforming growth factor-␤1. Surface plasmon resonance studies showed that kinetic parameters of sBMPR for BMP-4 consisted of a relatively rapid association rate constant (k a ‫؍‬ 3.81 ؎ 0.19 ؋ 10 4 s ؊1 M ؊1 ) and an extremely slow dissociation rate constant (k d ‫؍‬ 3.69 ؎ 0.26 ؋ 10 ؊4 s ؊1 ). From these two kinetic parameters, affinity was determined to be similar to that of the intact membrane-associated receptor expressed on COS cells. sBMPR inhibited the alkaline phosphatase activity in BMP responsive cell lines such as mouse osteoblastic cell MC3T3-E1 and bone marrow stromal cell ST2. These data indicate that the extracellular domain of type I receptor for BMP-2 and BMP-4 is sufficient for high-affinity binding to its ligands and should prove useful in understanding the role of BMP-2/4 in vivo, because a suitable high-affinity anti-BMP antibody has yet to be developed.Bone morphogenetic proteins (BMPs), 1 originally identified as proteins to induce endochondral bone formation in ectopic extraskeletal sites in vivo (1), are the largest subfamily of the transforming growth factor-␤ (TGF-␤). Of over a dozen of these BMP members (2, 3), BMP-2 and BMP-4 (vertebrate ortholog of Drosophila decapentaplegic) appear to play important roles in embryogenesis and body patterning (4). We reported that a Xenopus homologue of BMP-2 and BMP-4 present in developing Xenopus embryos (5-8) regulates dorsal-ventral patterning during mesoderm induction (9). In addition to the dorsal-ventral specification, BMP-2 and BMP-4 are also involved in later stages of development, e.g. differentiation of neural cells (10 -13), regulation of patterning in the limb bud (14, 15), and epithelial-mesenchymal interactions during organogenesis (16).Receptors for BMPs are a family of transmembrane serine/ threonine kinases (17). These receptors are divided into two distinct classes, type I (18 -20) and type II receptors (21, 22). Like receptors for other TGF-␤-related proteins, heteromeric dimerization of these receptors, induced by binding to their ligands, is responsible for initiating biological responses (22-25). However, a cross-linking study showed that when transiently expressed, these type I receptors are capable of binding to BMP-2 and BMP-4, without co-expression of the type II receptor (18 -20, 26). The type II receptor for BMPs bound to ligands weakly, but the binding was facilitated by the presence of type I receptors for BMPs (21,22,24). When the domi...

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Biochemical properties of amphibian bone morphogenetic protein-4 expressed in CHO cells

Cited by 13 publications

References 33 publications

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production

Interaction between Soluble Type I Receptor for Bone Morphogenetic Protein and Bone Morphogenetic Protein-4

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