Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

Epithelial-mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme

Tenascin, a mesenchymal extracellular matrix glycoprotein, has been implicated in epithelial-mesenchymal interactions during fetal development (Chiquet-Ehrismann, R., E. J. Mackie, C. A. Pearson, T. Sakakura, 1986, Cell, 47:131-139). We have now investigated the expression of tenascin during embryonic development of the mouse kidney. In this system, mesenchymal cells convert into epithelial cells as a result of a tissue interaction. By immunofluorescence, tenascin could not be found in the mesenchyme until kidney tubule epithelial began to form. It then became detectable around condensates and s-shaped bodies, the early stages of tubulogenesis. In an in vitro culture system, tenascin expression by the mesenchyme is tightly coupled to the de novo formation of epithelial, and does not occur if tubulogenesis is suppressed. The results strongly suggest that the formation of the new epithelium stimulates the expression of tenascin in the nearby mesenchyme. During postnatal development, the expression of tenascin decreases and the spatial distribution changes. In kidneys from adult mice, no tenascin can be found in the cortex, but interspersed patches of staining are visible in the medullary stroma. The results strongly support the view that tenascin is involved in epithelial-mesenchymal interactions. It could therefore be crucial for embryonic development.     

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.     

Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation

Syndecan is an integral membrane proteoglycan that behaves as a matrix receptor by binding cells to interstitial matrix and associating intracellularly with the actin cytoskeleton. Using immunohistology, we have now localized this proteoglycan during the morphogenesis of various derivatives of the surface ectoderm in mouse embryos. Syndecan is expressed on ectodermal epithelia, but is selectively lost from the cells that differentiate into the localized placodes that initiate lens, nasal, otic and vibrissal development. The loss is transient on presumptive ear, nasal and vibrissal epithelia; the derivatives of the differentiating ectodermal cells that have lost syndecan subsequently re-express syndecan. In contrast, syndecan is initially absent from the mesenchyme underlying the surface ectoderm, and is transiently expressed when the surface ectoderm loses syndecan. These results demonstrate that expression of syndecan is developmentally regulated in a distinct spatiotemporal pattern. On epithelia, syndecan is lost at a time and, location that correlates with epithelial cell differentiation and, on mesenchyme, syndecan is acquired when the cells aggregate in proximity to the epithelium. This pattern of change with morphogenetic events is unique and not duplicated by other matrix molecules or adhesion receptors.     

Molecular cloning of syndecan, an integral membrane proteoglycan

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).     

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.     

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Intracellular localization and surface expression of a stage-specific embryonic glycoprotein

The intracellular and cell surface localization of an embryonic glycoprotein antigen (BL) has been investigated in preimplantation mouse embryos using ultrastructural immunocytochemistry. Several interesting points have emerged: (1) BL antigens are exclusively localized subjacent to the plasma membrane in the cortical region of cells, whereas antigens detected by a control antibody against mouse L cells are distributed throughout the embryo. (2) The distribution of BL antigens is polarized beginning with the first cleavage, with expression confined to the cortex underlying the free or apical portions of cells. No antigen is present underlying regions of cell contact. (3) Although embryonic synthesis of BL antigens does not begin until the two-cell stage, BL antigens are observed in unfertilized eggs, a fact verified by immunoblotting.     

Shh‐proteoglycan interactions regulate maturation of olfactory glomerular circuitry

The olfactory system relies on precise circuitry connecting olfactory sensory neurons (OSNs) and appropriate relay and processing neurons of the olfactory bulb (OB). In mammals, the exact correspondence between specific olfactory receptor types and individual glomeruli enables a spatially precise map of glomerular activation that corresponds to distinct odors. However, the mechanisms that govern the establishment and maintenance of the glomerular circuitry are largely unknown. Here we show that high levels of Sonic Hedgehog (Shh) signaling at multiple sites enable refinement and maintenance of olfactory glomerular circuitry. Mice expressing a mutant version of Shh (Shh^Ala/Ala), with impaired binding to proteoglycan co‐receptors, exhibit disproportionately small olfactory bulbs containing fewer glomeruli. Notably, in mutant animals the correspondence between individual glomeruli and specific olfactory receptors is lost, as olfactory sensory neurons expressing different olfactory receptors converge on the same glomeruli. These deficits arise at late stages in post‐natal development and continue into adulthood, indicating impaired pruning of erroneous connections within the olfactory bulb. In addition, mature Shh^Ala/Ala mice exhibit decreased proliferation in the subventricular zone (SVZ), with particular reduction in neurogenesis of calbindin‐expressing periglomerular cells. Thus, Shh interactions with proteoglycan co‐receptors function at multiple locations to regulate neurogenesis and precise olfactory connectivity, thereby promoting functional neuronal circuitry. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1255–1267, 2014     

The expression of NG2 proteoglycan in the developing rat limb

NG2 is a chondroitin sulfate proteoglycan previously found to be expressed by glial progenitor cells of the O2A lineage. We have examined the expression of NG2 in the developing rat limb by immunohistochemistry and northern blot analysis. Staining of embryonic day 14 (E14) rat limb bud sections with polyclonal and monoclonal anti-NG2 antibodies reveals reactivity in the precartilaginous mesenchymal condensation. The staining intensity increases with the differentiation of chondrocytes until E16. NG2 staining is not detected in the mature hypertrophic chondrocytes of E17 and postnatal day 3 (P3) limbs even after treatment of the sections with hyaluronidase or collagenase. Immuno-precipitations with anti-NG2 antibody using 125I-labeled limb cells in culture showed a 400 to 800 x 10(3) Mr proteoglycan species with a core protein size of 300 x 10(3) Mr, comparable to NG2 from O2A cells and neural cell lines. Northern blot analysis reveals the expression of an 8.9 kb mRNA in E16 limbs and at a lower level in P1 cartilage. The northern blot analyses also show that NG2 is distinct from the large aggregating proteoglycan of the cartilage. Our results indicate that in the developing limb cartilage, as in the differentiating oligodendrocytes, NG2 is present on immature cells in the process of differentiating, but its expression is downregulated as terminal differentiation of chondrocytes takes place.     

Syndecan, a cell surface proteoglycan, exhibits a molecular polymorphism during lung development

Syndecan, a cell surface proteoglycan, binds multiple extracellular ligands, and is developmentally regulated in epithelial and mesenchymal tissues. The branching morphogenesis of embryonic lung is dependent on epithelial-mesenchymal interactions and, based on studies with inhibitors, on proteoglycan synthesis. To assess the role of syndecan in lung development, we examined the structure and distribution of syndecan in Day 12 to 18 embryonic mouse lungs using monoclonal antibody 281-2 for histology, immunopurification, and Western blots. At Day 12, syndecan localizes mainly on epithelial cell surfaces, but also stains mesenchymal cells near the epithelium. By Day 14, syndecan is expressed predominantly on epithelia and by Day 18, syndecan remains on airway epithelia but is absent from the alveolar pneumocytes. This change in expression correlates with a change in syndecan structure; the relative mass of syndecan gradually falls from Day 12 to Day 18 without a change in relative mass of the core protein. The difference is due to a developmental reduction in the size of the glycosaminoglycan chains; heparan sulfate chains on syndecan from Day 14 lungs were nearly twofold larger than those from Day 18 lungs. Newly synthesized syndecan in the lungs had the same relative mass as total syndecan, indicating that the change in mass is due to a developmental change in the nature of the syndecan synthesized. The alteration in syndecan structure could alter the function of this proteoglycan during lung development.     

Glial organization and chondroitin sulfate proteoglycan expression in the developing thalamus

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30–P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20–E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action.

Monocyte/macrophages play important roles in regulating tissue growth and angiogenesis through the controlled release of heparin-binding growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin binding epidermal growth factor. The action of these potent growth mediators is known to be regulated by adsorption to heparan sulfate proteoglycans (HSPGs) on the surface and within the extracellular matrix of other neighboring cells, which respectively promote or restrict interactions with their signal-transducing receptors on target cells. Here we report on the nature of HSPGs inducibly expressed on the surface of macrophages that confer these cells with the capacity to regulate endogenous growth factor activity. We reveal that activated human macrophages express only a single major 48-kDa cell surface HSPG, syndecan-2 (fibroglycan) as the result of de novo RNA and protein synthesis. In addition, we demonstrate this macrophage HSPG selectively binds the macrophage-derived growth factors FGF-2, vascular endothelial growth factor and heparin binding EGF and can present FGF-2 in a form that transactivates receptor-bearing BaF32 cells. These results define a novel and unique proteoglycan profile for macrophages and imply a key role for syndecan-2 in the delivery of sequestered growth factors by inflammatory macrophages for productive binding to their appropriate target cells in vivo.     

Evidence for transsynaptic regulation of neuronal cell surface heparan sulfate proteoglycan in developing rat superior cervical ganglion

The effect of neonatal deafferentation on the expression of a neuronal cell surface heparan sulfate proteoglycan (HeS-PG) was investigated in the developing rat superior cervical ganglion. Two monoclonal antibodies, one directed against the core protein of HeS-PG, and one to a determinant associated with a heparan sulfate side-chain, were used to monitor postnatal increases of HeS-PG by radioimmunoassay. Following neonatal deafferentation by section of the cervical sympathetic trunk, total protein per ganglion was slightly reduced at survival times of 7, 14, and 30 days. Expression of the core protein determinant on HeS-PG was not altered in deafferented ganglia. In contrast, levels of side-chain determinant were significantly reduced at 14 and 30 days. These results suggest that processing of HeS-PG side-chains by principal ganglionic neurons is partially regulated by transsynaptic influences during development. Transsynaptic regulation of neuronal development may be a more general process than was believed previously, with effects not limited to molecules associated with synaptic development.     

Induced expression of syndecan in healing wounds

We have studied the expression of an integral cell surface proteoglycan, syndecan, during the healing of cutaneous wounds, using immunohistochemical and in situ hybridization methods. In normal mouse skin, both syndecan antigen and mRNA were found to be expressed exclusively by epidermal and hair follicle cells. After incision and subsequent suturing, remarkably increased amounts of syndecan on the cell surfaces of migrating and proliferating epidermal cells and on hair follicle cells adjacent to wound margins were noted. This increased syndecan expression was shown to be a consequence of greater amounts of syndecan mRNA. Induction was observed already 1 d after wounding, was most significant at the time of intense cell proliferation, and was still observable 14 d after incision. The migrating cells of the leading edge of the epithelium also showed enhanced syndecan expression, although clearly less than that seen in the proliferating epithelium. The merging epithelial cells at the site of incision showed little or no syndecan expression; increased syndecan expression, however, was detected during later epithelial stratification. When wounds were left unsutured, in situ hybridization experiments also revealed scattered syndecan-positive signals in the granulation tissue near the migrating epidermal sheet. By immunohistochemical analysis, positive staining in granulation tissue was observed around vascular endothelial cells in a subpopulation of growing capillaries. Induction of syndecan in granulation tissue both at the protein and mRNA levels was temporally and spatially highly restricted. Granulation tissue, which formed in viscose cellulose sponge cylinders placed under the skin of rats, was also found to produce 3.4 and 2.6 kb mRNA species of syndecan similar to that observed in the normal murine mammary epithelial cell line, NMuMG. These results suggest that syndecan may have a unique and important role as a cell adhesion and a growth factor-binding molecule not only during embryogenesis but also during tissue regeneration in mature tissues.     

Ammonium chloride interferes with a distinct step in the biosynthesis and cell surface expression of human melanoma-type chondroitin sulfate proteoglycan

Human melanoma cells synthesize a cell-associated chondroitin sulfate-rich proteoglycan, whose core protein is recognized by monoclonal antibody 9.2.27. We report that the core protein is present on the surface of melanoma cells in two forms, either free or modified by the addition of chondroitin sulfate chains, suggesting that the addition of glycosaminoglycan chains may not be a prerequisite for cell surface expression of the proteoglycan core protein. Free core protein found at the cell surface does not seem to represent an overflow of the proteoglycan synthetic pathway, since experiments using a beta-D-xyloside acceptor suggest that core protein is, in fact, limiting proteoglycan synthesis. NH4Cl inhibits the synthesis of melanoma-type proteoglycan, shifting the balance of surface core protein toward the free form. The inhibition of proteoglycan synthesis is apparently not due to a disruption of enzymes and precursors involved in glycosaminoglycan synthesis, since cells treated with NH4Cl retain their ability to initiate and elongate chondroitin 4-sulfate chains on a beta-D-xyloside acceptor. In contrast, the divalent ionophore monensin inhibited core protein maturation and synthesis of glycosaminoglycan chains. The effects of both NH4Cl and monensin were reversible; thus, experiments using the drugs sequentially indicated that monensin temporally precedes NH4Cl in interfering with proteoglycan biosynthesis. Since the NH4Cl and monensin share the property of inhibiting the acidification of intracellular vesicles within cells, the present findings raise the possibility that the accessibility of proteoglycan core protein to the Golgi site of glycosaminoglycan addition is regulated in melanoma cells by acidification of intracellular compartments.