Bone Morphogenetic Proteins

TGFβ1 and FGF2 are autocrine growth factors in prostatic stroma and are elevated in benign prostatic hyperplasia (BPH), a disease characterized by enlargement of the stromal compartment of the prostate. TGFβ1 has a biphasic effect on proliferation of prostatic stromal cells, inducing proliferation at low doses ( < 1 ng/ml), but inhibiting growth above 1 ng/ml. This study investigated the role of TGFβ1 and FGF2 on growth factor bioavailability and extracellular matrix (ECM) accumulation synthesis in cultured prostatic stromal cells. Real-Time-PCR showed that TGFβ1 expression is auto-inductive, whereas FGF2 is auto-repressive. FGF2 also induced TGFβ1 secretion in the absence of increased TGFβ1 mRNA expression. TGFβ1 and FGF2 have opposing actions on Type 1 collagen expression, a finding confirmed by Western blotting. The bioavailability of TGFβ1 regulated by FGF2 may represent part of a negative feedback mechanism controlling stromal growth, differentiation and ECM. Dysregulation of this pathway in favour of TGFβ1 bioactivity may exacerbate BPH.     

Alkali-Urea Extraction of Demineralized Bone Matrix Removes Noggin,an Inhibitor of Bone Morphogenetic Proteins

Demineralized bone matrix (DBM) and native bone morphogenetic protein (nBMP) are complex mixtures of non-collagenous bone proteins. These mixtures contain many of the BMPs that are available as recombinant molecules. Information regarding the presence in these materials of molecules that may affect the availability and activity of the BMPs is very limited. We have devised a simple chemical extraction of DBM using alkali-urea that produces a water soluble extractate that inhibits the osteogenic activity of DBM. We have demonstrated the presence of noggin, an extracellular BMP ligand antagonist, in this material. We conclude that differential chemical extraction may be a useful means of removing inhibitory molecules from DBM and nBMP.     

BMP家族的研究近况

骨形态发生蛋白是一组具有类似结构的高度保守的功能蛋白,能够在体内诱导骨和腱样组织形成,在肢体生长、软骨内骨化、骨折早期及肌腱修复时表达,从而发挥其骨发生、骨诱导、骨修复及肌腱修复作用。此外,骨形态发生蛋白对神经组织的形成以及在对神经元形成的类型和数量方面都起到一定的控制作用,并参与调节胚胎发育过程及造血祖细胞分化,某些成员还参与诱导细胞凋亡。     

Changes with Age in the Non-Collagenous Proteins of Human Bone

Little is known about the function of the non-collagenous proteins in the organic matrix of bone. Information about these proteins was obtained from studies of human cortical bone. Analysis of fractions of bone differing in density showed that 96% of the α₂HS-glycoprotein and 58% of the albumin was associated with the mineralized phase. A study of age-related changes in the composition of bone showed that the amounts present of α₂HS-glycoprotein, albumin, sialoprotein, soluble collagen and of EDTA-soluble protein were all higher in bone from children than in adults. In subjects aged over 60 y the content in bone of albumin, sialoprotein and of total protein was similar to that of children. These differences could not be correlated with age-related changes in the calcium/hydroxyproline ratio nor with previous reports of alterations in the crystal structure and the physical properties of human cortical bone.     

Characterization of Multiple Forms of Small Collagenous Apatite-Binding Proteins in Bone

A number of small (M_rs 25–28 kDa) collagenous apatite-binding (SCAB) proteins that stain blue with “Stains-All” have been isolated from fetal porcine bone by sequential extractions with 4M GuHCl (G1), followed by 0.5M EDTA (E), and again with 4M GuHCl (G2). Following purification under dissociative conditions, two types of SCAB proteins both with approximately one-third of their structure being collagenous, were identified in the EDTA extract. One type, which appears to be a novel protein, was revealed in two forms (SCABs 1 and 2, M_rs 25 and 28 kDa) that were recognized by a monoclonal antibody (MBP-322). The second type, SCAB 3, was also present in two forms; one form (SCAB 3a) having a lower affinity for hydroxyapatite than the other (SCAB 3b). These proteins were resistant to CNBr and displayed the chemical and immunochemical properties of the α1 pN-propeptide of type I collagen. A third form of the propeptide (G2-28K) was a prominent component of the second 4M GuHCl extract. The chromatographic properties of serum α1(I) pN-propeptide were similar to SCAB 3a, indicating that SCAB 3b and G2-28K are post-translationally modified forms of the propeptide produced by bone cells. These propeptides may provide a link between the hydroxyapatite and collagen fibrils, and also have the potential to suppress collagen synthesis during bone resorption.     

Protein Kinases of Cultured Chicken Osteoblasts That Phosphorylate Extracellular Bone Proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with M_rS 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of -9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialo-protein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5–1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabol-ically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Heterodimeric Bone Morphogenetic Proteins Show Enhanced Activity In Vitro and In Vivo

Abstract

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-β gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.     

Mineral Induction by Polyanionic Dentin and Bone Proteins at Physiological Ionic Conditions

Polyanionic proteins of calcified tissues have been postulated to provide calcium ion-binding sites which initiate mineral formation, even though it is known that such proteins in solution may inhibit apatite induction and growth. In the studies reviewed here, it was shown that minute amounts of non-collagenous macromolecules from dentin and bone, such as phosphoprotein and proteoglycan, are capable to induce apatite at physiological ion concentrations in vitro, when immobilized on a stable support.     

The Isolation and Detection of Non-Collagenous Proteins from the Compact Bone of the Dinosaur Iguanodon

This report describes the isolation of guanidinium chloride extractable protein from deminer-alised bone extracts obtained from the 125–130 mya dinosaur Iguanodon. Protein products were isolated in the Mr. range 5,000–66,000 using SDS-PAGE and represent the first electro-phoretically defined proteins isolated from dinosaur tissues. The levels of glycine, aspartate and serine tentatively suggest the presence of phosphoproteins. Hydroxylysine and hydroxy-proline were not detected, confirming the presence of non-collagenous material. In addition the absence of ornithine confirmed lack of bacterial contamination. The relatively high level of leucine in the 2MNaCl NaCl fractions together with the abolition of alcian blue reactivity following protease-free chondroitinase digestion suggests the presence of proteoglycans. The study is of interest in describing the early proteins laid down in mineralised tissues for epitac-tic crystal growth and may provide evidence on evolutionary aspects of bone proteins.     

Immunohistochemical Method for the Simultaneous Demonstration of Three Proteins in EDTA Decalcified Paraffin Embedded Bone Sections

Abstract

Immunohistochemistry was used to demonstrate the presence of three proteins (actin, osteocalcin and alkaline phosphatase) within the same ethylenediaminetetraacetic acid (EDTA) decalcified and paraffin embedded bone tissue section. EDTA chelated calcium ions, causing endogenous alkaline phosphatase (ALP) to be inactivated. By ablating endogenous ALP activity through chelation, we were able to use an ALP based detection system before reactivation of the endogenous enzyme with magnesium chloride. After reactivation, endogenous ALP itself was demonstrated immunohistochemically. (The J Histotechnol 15:93, 1992)     

不同蛋白涂层在脱细胞牛心包片上对骨髓间充质细胞黏附生长的影响

为观察和评价不同蛋白涂层对骨髓间充质细胞在脱细胞牛心包片上黏附生长的影响,采用酶消化结合去污剂洗涤法制作脱细胞牛心包片,在其表面用不同蛋白(纤维连接蛋白、明胶蛋白、型胶原蛋白)均匀涂层。提取并培养大鼠骨髓间充质细胞,将其接种到预涂层的牛心包片上,未涂层组作为对照。在不同时间点采用Hochest染色观察细胞贴附生长情况,48h采用MTT法定量分析。纤维连接蛋白涂层及明胶蛋白涂层与型胶原蛋白涂层和不涂层组相比,对骨髓间充质细胞贴附增殖能力均有显著提高(P<0.001);纤维连接蛋白涂层和明胶蛋白涂层之间(P>0.05)以及型胶原蛋白涂层和不涂层对照组之间(P>0.05)对细胞的贴附生长没有显著差异。结果表明:脱细胞牛心包片上纤维连接蛋白涂层及明胶蛋白涂层能明显提高骨髓间充质细胞的贴附增殖能力,而型胶原蛋白涂层对骨髓间充质细胞的贴附增殖没有明显的改善。     

Dentin Extracellular Matrix (ECM) Proteins: Comparison to Bone ECM and Contribution to Dynamics of Dentinogenesis

Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.     

Glycosylation of Therapeutic Proteins

During their development and administration, protein-based drugs routinely display suboptimal therapeutic efficacies due to their poor physicochemical and pharmacological properties. These innate liabilities have driven the development of molecular strategies to improve the therapeutic behavior of protein drugs. Among the currently developed approaches, glycoengineering is one of the most promising, because it has been shown to simultaneously afford improvements in most of the parameters necessary for optimization of in vivo efficacy while allowing for targeting to the desired site of action. These include increased in vitro and in vivo molecular stability (due to reduced oxidation, cross-linking, pH-, chemical-, heating-, and freezing-induced unfolding/denaturation, precipitation, kinetic inactivation, and aggregation), as well as modulated pharmacodynamic responses (due to altered potencies from diminished in vitro enzymatic activities and altered receptor binding affinities) and improved pharmacokinetic profiles (due to altered absorption and distribution behaviors, longer circulation lifetimes, and decreased clearance rates). This article provides an account of the effects that glycosylation has on the therapeutic efficacy of protein drugs and describes the current understanding of the mechanisms by which glycosylation leads to such effects.