Epithelial effects on limb chondrogenesis involve extracellular matrix and cell shape

Collagen gel cultures of limb bud mesenchymal cells are normally permissive for chondrogenesis but become inhibitory for chondrogenesis when they are preconditioned by limb ectoderm. This inhibition is specific for cartilage differentiation, inasmuch as myoblast differentiation is unaffected and flattened, fibroblastic cells are more numerous on conditioned gels. The antichondrogenic effect of ectoderm-conditioned gels is not blocked by agents that elevate intracellular cyclic AMP levels and that promote chondrogenesis under other conditions. In contrast, the inhibitory effect of the ectoderm is alleviated when cultures are treated with cytochalasin D, a cytoskeleton-disrupting agent that causes the cells to remain spherical. These results suggest that ectoderm-conditioned collagen gels inhibit chondrogenesis through an effect on cell shape.     

Extracellular matrix mediates epithelial effects on chondrogenesis in vitro

It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.     

Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development

Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.     

Inhibition of limb chondrogenesis by fibronectin

Abstract. This study compares the chondrogenic capacity of high density cultures prepared from either the develop-mentally younger, distal region or more advanced proximal region of stage 23/24 limb mesenchyme in high density cultures. Distal cultures undergo extensive chondrogenesis whether in F₁₂ medium supplemented with 10% fetal calf serum, 5% fetal calf serum, or fibronectin. On the other hand, proximal cultures fail to undergo chondrogenesis in medium containing 10% fetal calf serum or fibronectin, but do form cartilage in medium containing a decreased serum concentration or no serum. Furthermore, if the cells are cultured at low densities in native type I collagen gels, proximal cells have a reduced chondrogenic capacity in the presence of fibronectin, while chondrogenesis by distal cells is unaffected by the addition of fibronectin. The results demonstrate that proximal and distal cells respond differentially to serum and to fibronectin, and they suggest that the response of the cell to prevalent components of the extracellular matrix might change with development.     

Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.     

How Tumor Cells Make Use of CD44

A variant of CD44 containing exon v3 sequences is expressed in the apical ectodermal ridge of the limb bud during embryogenesis. This variant is modified by heparan sulfate moieties and acts as low affinity receptor for FGFs. These FGFs are presented by CD44 to mesenchymal cells which induces their proliferation and limb outgrowth. We suggest that a similar growthfactor presentation mechanism accounts for the function of CD44 variants on metastasizing tumor cells.     

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.     

Role of vitronectin in embryonic rat endocardial cell migration in vitro

Summary Vitronectin is one of the extracellular matrices that mediate cell spreading and attachment in vitro. In the present paper, we demonstrate the involvement of vitronectin in the migration of cushion mesenchymal cells of the embryonic rat heart. Immunohistochemistry established the localization of vitronectin in the myocardial cells and in some of the cushion mesenchymal cells of the truncus arteriosus and atrioventricular canal. In vitro, vitronectin, fibronectin, and collagen type-I revcaled significant stimulating activity for cushion mesenchymal cell migration. The distance migrated by cushion mesenchymal cells cultured on vitronectin, collagen type-I, or both vitronectin and fibronectin was similar, but that on fibronectin was significantly shorter. Following the addition of anti-vitronectin IgG to the medium, the migration distance of cushion mesenchymal cells on fibronectin was remarkably increased. Most explants on vitronectin or on both vitronectin and fibronectin became detached from dishes after the addition of the antivitronectin antibody. Immunostaining revealed that cushion mesenchymal cells cultured on substrata other than vitronectin synthesized vitronectin. From these results, it is suggested that vitronectin is synthesized by myocardial cells and some cushion mesenchymal cells, and that vitronectin inhibits cell movement on fibronectin. This feature of vitronectin may be important in the regulation of the migration of cushion mesenchymal cell in vivo.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

Retinoic acid enhances the displacement of newly synthesized hyaluronate from cell layer to culture medium during early phases of chondrogenesis

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid (1 microgram/ml or 3.3 X 10(-6) M). Since in control medium overt chondrogenesis begins on day 3, retinoic acid was introduced on day 2 so that the relationship between initial biosynthetic changes and inhibition of chondrogenesis could be studied. During the first 24 h of exposure the treated cells remained viable but suffered 10% inhibition in growth and synthesized [3H]glucosamine-labeled glycosaminoglycan at a level 24% below untreated cells. The amount of labeled hyaluronic acid released into the culture medium by the treated cells was, however, 2-fold greater, on a per cell basis, than that in the untreated cultures. It is suggested that the displacement of hyaluronate may play a role in the disruption of mesenchymal cell differentiation and of limb morphogenesis as observed in other systems.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Lectins influence chondrogenesis and osteogenesis in limb bud mesenchymal cells

The role of cell surface glycoproteins in cell behavior can be characterized by their interactions with plant lectins. This study was designed to identify the effects of lectins on chondrogenesis and osteogenesis in limb bud mesenchymal cells in vitro. Limb bud mesenchymal cells from mouse embryos were cultured in high-density micromass culture. Wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin (RCA) were added separately to the culture media. Cells were cultured for 5 or 9 days, and cell viability was assayed by neutral red on day 5. The micromasses were stained with alcian blue, alizarin red S and Von Kossa stains, and alkaline phosphatase assays were also done. Dolichos biflorus agglutinin induced an increase in chondrogenesis, calcium precipitation and proteoglycan production. ConA and PNA did not affect chondrocyte differentiation but induced chondrocytes to produce more proteoglycan. Wheat germ agglutinin reduced chondrification and ossification but induced mesenchymal cells to store lipid droplets. Ricinus communis agglutinin 1 was toxic and significantly reduced cell survival. In conclusion, DBA was the most effective inducer of ossification and chondrification. Wheat germ agglutinin induced adipogenesis instead. These assays showed that lectins play important roles in limb bud development.     

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.     

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.     

Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.     

Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.