Differential effects on Xenopus development of interference with type IIA and type IIB activin receptors

One candidate for a mesoderm-inducing factor in early amphibian development is activin, a member of the TGF beta family. Overexpression of a truncated form of an activin receptor Type IIB abolishes activin responsiveness and mesoderm formation in vivo. The Xenopus Type IIA activin receptor XSTK9 differs from the Type IIB receptor by 43 and 25% in extracellular and intracellular domains respectively, suggesting the possibility of different functions in vivo. In this paper, we compare the Type IIA receptor with the Type IIB to test such a possibility. Simple overexpression of the wild-type receptors reveals minimal differences, but experiments with dominant negative mutants of each receptor show qualitatively distinct effects. We show that while truncated (kinase domain-deleted) Type IIB receptors cause axial defects as previously described, truncated type IIA receptors cause formation of secondary axes, similar to those seen by overexpression of truncated receptors for BMP-4, another TGF beta family member. Furthermore, in animal cap assays, truncated type IIB receptors inhibit induction of all mesodermal markers tested, while truncated type IIA receptors suppress induction only of ventral markers; the anterior/dorsal marker goosecoid is virtually unaffected. The suppression of ventral development by the type IIA truncated receptor suggests either that the truncated Type IIA receptor interferes with ventral BMP pathways, or that activin signaling through the Type IIA receptor is necessary for ventral patterning.     

The role of 12-lipoxygenase in pancreatic -cells (Review)

A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed. Cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium. The inclusion of 10(-7) M dexamethasone in the medium induced chondrogenic differentiation of cells within the aggregate as evidenced by the appearance of toluidine blue metachromasia and the immunohistochemical detection of type II collagen as early as 7 days after beginning three-dimensional culture. After 21 days, the matrix of the entire aggregate contained type II collagen. By 14 days of culture, there was also evidence for type X collagen present in the matrix and the cells morphologically resembled hypertrophic chondrocytes. However, chondrogenic differentiation was achieved in only approximately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth factor-beta 1 (TGF-beta 1), chondrogenesis was induced in all marrow cell preparations, with or without the presence of 10(-7) M dexamethasone. The induction of chondrogenesis was accompanied by an increase in the alkaline phosphatase activity of the aggregated cells. The results of RT-PCR experiments indicated that both type IIA and IIB collagen mRNAs were detected by 7 days postaggregation as was mRNA for type X collagen. Conversely, the expression of the type I collagen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochemical, and molecular evidence for the in vitro chondrogenic differentiation of adult mammalian progenitor cells derived from bone marrow.     

Use of tissue plasminogen activator factor for acute ischemic stroke

Recently, a new isoform of the type II transforming growth factor beta receptor (TGF-beta RII) was identified. This isoform (TGF-beta RII2) contains an insertion of 25 amino acids in the extracellular domain of the receptor. Using RT-PCR the authors demonstrated that both TGF-beta RII1 and TGF-beta RII2 are expressed by chondrocytes in murine and human articular cartilage. Bovine articular chondrocytes expressed TGF-beta RII1 mRNA but did not express detectable levels of TGF-beta RII2 mRNA, suggesting that the new isoform does not play an important role in normal bovine cartilage physiology. Because TGF-beta responses seem to be age related and differential TGF-beta responses have been described between normal cartilage and cartilage undergoing repair the authors studied if the relative mRNA expression between these isoforms is altered during cartilage repair and aging. No differences in the relative mRNA expression of the two isoforms of the type II TGF-beta receptor could be demonstrated in murine cartilage during aging or during the repair phase after mild PG depletion indicating that it is unlikely that age-related TGF-beta responses and differential TGF-beta responses between normal cartilage and cartilage undergoing repair are the result of differences in the relative expression of the two TGF-beta RII isoforms.     

The Marburg parent-child spelling training

Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.     

Truncated activin type II receptors inhibit bioactivity by the formation of heteromeric complexes with activin type I. receptors

Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP-lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors.     

Use of the Taylor Visagraph II system to evaluate eye movements made during reading

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson's trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform alpha-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.     

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.     

Myogenic cells express multiple myosin isoforms

In vivo and in vitro, proliferating motile myoblasts form aligned groups of cells, with a characteristic bipolar morphology, subsequently become post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members of the myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin isoforms, from at least five different classes of the myosin superfamily (classes I, II, V, VII and IX), using RT-PCR and degenerate primers to conserved regions of myosin. All of these myosin isoforms were expressed most highly in myoblasts and their expression decreased as they differentiated into mature myotubes, by RNAse protection assays, and Western analysis. However, only myosin I alpha, non-muscle myosin IIA and IIB together with actin relocalize in response to the differentiative state of the cell. In single cells, myosin I alpha was found at the leading edge, in rear microspikes and had a punctate cytoplasmic staining, and non-muscle myosin was associated with actin bundles as previously described for fibroblasts. In aligned groups of cells, all these proteins were found at the plasma membrane. Co-staining for skeletal myosin II, and myosin I alpha showed that myosin I alpha also appeared to be expressed at higher levels in post-mitotic myoblasts that had begun to express skeletal myosin prior to fusion. In early myotubes, actin and non-muscle myosin IIA and IIB remained localized at the membrane. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform of myosin I (mouse homologue to myr2) showed a punctate cytoplasmic staining which did not change as the myoblasts differentiated. In conclusion, although we found that myoblasts express many different isoforms of the myosin superfamily, only myosin I alpha, non-muscle myosin IIA and IIB appear to play any direct role in myogenesis.     

Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes

Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants.     

Alternatives to platelet transfusion]

It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro.     

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.     

Studies of kinetics and equilibrium membrane binding of class A and class L model amphipathic peptides

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-beta) as well as a 290 kDa form containing latent TGF-beta binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-beta in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-beta. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-beta1 were found in these digests by TGF-beta1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-beta, in addition to LTBP1 that is associated with the 100 kDa latent TGF-beta complex. These studies show for the first time that latent TGF-beta is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-beta, even at sites distant from the cell, in a relatively avascular environment.     

Insulin-like growth factors counteract the effect of interleukin 1 beta on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes

Interleukin 1 beta was found to stimulate arachidonic acid release, and the synthesis and secretion of type II phospholipase A2 by rabbit articular chondrocytes in vitro. Interleukin 1 beta had no effect on the level of cytosolic phospholipase A2 mRNA. Insulin-like growth factors, which help stabilize the cartilage matrix, reduced the effect of interleukin 1 beta on type II phospholipase A2 activity and mRNA level, and decreased the Interleukin 1 beta-stimulated arachidonic acid release to the basal values. This suggests that type II phospholipase A2 plays a key role in arachidonic acid release from rabbit articular chondrocytes and that insulin-like growth factors counteract the effect of interleukin 1 beta. They may therefore be considered as potential antiinflammatory agents.     

The conformational equilibrium of human growth hormone

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

Parathyroid hormone-related protein is required for tooth eruption

Parathyroid hormone (PTH)-related protein (PTHrP)-knockout mice die at birth with a chondrodystrophic phenotype characterized by premature chondrocyte differentiation and accelerated bone formation, whereas overexpression of PTHrP in the chondrocytes of transgenic mice produces a delay in chondrocyte maturation and endochondral ossification. Replacement of PTHrP expression in the chondrocytes of PTHrP-knockout mice using a procollagen II-driven transgene results in the correction of the lethal skeletal abnormalities and generates animals that are effectively PTHrP-null in all sites other than cartilage. These rescued PTHrP-knockout mice survive to at least 6 months of age but are small in stature and display a number of developmental defects, including cranial chondrodystrophy and a failure of tooth eruption. Teeth appear to develop normally but become trapped by the surrounding bone and undergo progressive impaction. Localization of PTHrP mRNA during normal tooth development by in situ hybridization reveals increasing levels of expression in the enamel epithelium before the formation of the eruption pathway. The type I PTH/PTHrP receptor is expressed in both the adjacent dental mesenchyme and in the alveolar bone. The replacement of PTHrP expression in the enamel epithelium with a keratin 14-driven transgene corrects the defect in bone resorption and restores the normal program of tooth eruption. PTHrP therefore represents an essential signal in the formation of the eruption pathway.