Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism

Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells’ ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration.These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Dermatan sulfate exerts an enhanced growth factor response on skeletal muscle satellite cell proliferation and migration

Skeletal muscle regeneration is a complex process in which many agents are involved. When skeletal muscle suffers an injury, quiescent resident myoblasts called satellite cells are activated to proliferate, migrate, and finally differentiate. This whole process occurs in the presence of growth factors, the extracellular matrix (ECM), and infiltrating macrophages. We have shown previously that different proteoglycans, either present at the plasma membrane or the ECM, are involved in the differentiation process by regulating growth factor activity. In this article, we evaluated the role of glycosaminoglycans (GAGs) in myoblast proliferation and migration, using C2C12, a satellite cell-derived cell line. A synergic stimulatory effect on myoblast proliferation was observed with hepatocyte growth factor (HGF) and fibroblast growth factor type 2 (FGF-2), which was dependent on cell sulfation. The GAG dermatan sulfate (DS) enhanced HGF/FGF-2-dependent proliferation at 1-10 ng/ml. However, decorin, a proteoglycan containing DS, was unable to reproduce this enhanced proliferative effect. On the other hand, HGF strongly increased myoblast migration. The HGF-dependent migratory process required the presence of sulfated proteoglycans/GAGs present on the myoblast surface, as inhibition of both cell sulfation, and heparitinase (Hase) and chondroitinase ABC (Ch(abc)) treatment of myoblasts, resulted in a very strong inhibition of cell migration. Among the GAGs analyzed, DS most increased HGF-dependent myoblast migration. Taken together, these findings showed that DS is an enhancer of growth factor-dependent proliferation and migration, two critical processes involved in skeletal muscle formation.     

Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.     

Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling

Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling.     

The greatly increased amounts of accumulated versican and decorin with specific post-translational modifications may be closely associated with the malignant phenotype of pancreatic cancer

Pancreatic carcinoma (PC) is a cancer type with highly malignant growth and dissemination pattern of which the mechanisms are poorly understood. However, the malignant phenotype is closely linked to extracellular matrix (ECM) of which proteoglycans (PGs) and hyaluronan (HA) play a crucial role in the control of tumor progression and metastasis. In this study, we demonstrated that versican and decorin, two different PGs with contradictory roles and functions in the pathobiology of cancer, were the main matrix PGs in PC presenting a great increase 27- and 7-fold, respectively, in comparison to normal pancreas (NP). PC was characterized by the disproportional increase of versican compared to decorin, about 4 to 1, with a concurrent increase of HA, which may be closely associated with the growth and aggressiveness of this carcinoma. Significant specific post-translational modifications were also observed in both versican and decorin regarding the type, hydrodynamic size, sulfation pattern and extent of uronate epimerization of their glycosaminoglycan chains (GAGs). In particular, chondroitin sulphate (CS) was the predominant GAG type in both PC-associated versican and decorin. The CS of PC-decorin was increased 11-fold, compared to NP in which dermatan sulfate (DS) was the predominant GAG type in both PGs. The sulfation pattern of GAG chains was significantly altered in PC, since 6-sulfated disaccharides predominated in both versican and decorin with a marked presence of non-sulfated disaccharides accompanied by lower hydrodynamic sizes of both CS and DS chains compared to NP. In conclusion, all these findings agree with the highly malignant phenotype of this cancer and, thus, more studies need to be addressed on the roles of the post-translational modifications of versican and decorin in the biology of cancer.     

Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro

We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells.     

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparin's efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin-binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF-2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF-2 binding to endothelial cells and stripped pre-bound FGF-2 from cells. SOS also regulated FGF-2 stimulated proliferation. Further, SOS facilitated FGF-2 diffusion through Descemet's membrane, a heparan sulfate-rich basement membrane from the cornea, suggesting a possible role in FGF-2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study.     

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondro?tin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.     

Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2

The interaction of heparan sulfate (HS) (and the closely related molecule heparin) with FGF-1 is a requirement for enabling the growth factor to activate its cell surface tyrosine kinase receptor. However, little is known about the regulatory role of naturally occurring cell surface HS in FGF-1 activation. We have addressed this issue by utilizing a library of HS oligosaccharides, which are defined in both length and sulfate content. Mitogenic activation assays using these oligosaccharides showed that HS contained both FGF-1 activatory and inhibitory sugar sequences. Further analysis of these oligosaccharides showed a clear correlation between FGF-1 promoting activity and their 6-O-sulfate content. The results, in particular with the dodecasaccharide sequences, suggested that specific positioning of 6-O-sulfate groups may be required for the promotion of FGF-1 mitogenic activity. This may also be true for 2-O-sulfate groups though the evidence was not as conclusive. Differential activation of FGF-1 and FGF-2 was also observed and found to be mediated by both oligosaccharide length and sulfation pattern, with different specific O-sulfate positioning being implicated for the promotion of different growth factors. These results suggest that variation and tight control of the fine structure of HS may allow cells to not only control their positive/negative responses to individual FGFs but also to change specificity towards promotion of different members of the FGF family.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

Glycosaminoglycans inhibit the adherence and the spreading of osteoclasts and their precursors: role in osteoclastogenesis and bone resorption

The bone microenvironment (e.g. glycosaminoglycans (GAGs), growth factors) plays a major role in bone resorption, especially in the formation of osteoclasts which differentiate from the hematopoietic lineage in the presence of RANKL. Previous studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial, some studies showing an inhibitory effect of GAGs on osteoclastic differentiation whereas others demonstrated a stimulatory effect. To clarify their activities, we investigated the effect of 5 families of GAGs in three different models of human/mouse osteoclastogenesis. The present data revealed that heparin inhibited osteoclastogenesis in these three models, which was confirmed by a decrease in mRNA expression of osteoclastic markers and by an inhibition of the bone resorption capacity. We also demonstrated in RAW 264.7 cells that other families of GAGs different from heparin inhibited RANKL-induced osteoclastogenesis, and that this inhibition was dependent on the length and the level of sulfation of GAGs. In the present work, heparin did not bind to RANKL and did not modulate RANKL signaling. Heparin acted at 2 distinct steps of osteoclastogenesis from human CD14(+) cells: first, heparin strongly decreased the adherence of osteoclast precursors, and secondly inhibited osteoclasts to spread and to be active. Furthermore, the second action of heparin was reversible as the removal of heparin at the end of the culture time allowed the condensed cells to spread out and showed the formation of morphological active osteoclasts. The present work clearly evidences that GAGs inhibit osteoclastogenesis in vitro and strengthens the therapeutic interest of defined GAGs in osteolytic diseases.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Biophysical characterization of glycosaminoglycan-IL-7 interactions using SPR

Glycosaminoglycans (GAGs) interact with a number of cytokines and growth factors thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and the regulation of the tissue distribution of cytokines/growth factors. In the present study, we employed surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between GAGs and murine and human forms of interleukin-7 (IL-7). SPR results revealed that heparin binds with higher affinity to human IL-7 than murine IL-7 through a different kinetic mechanism. The optimal oligosaccharide length of heparin for the interactions to human and murine IL-7 involves a sequence larger than a tetrasaccharide. These results further demonstrate that while IL-7 is principally a heparin/heparan sulfate binding protein, it also interacts with dermatan sulfate, chondroitin sulfates C, D, and E, indicating that this cytokine preferentially interacts with GAGs having a higher degree of sulfation.     

Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein

Fibroblast growth factor-binding protein (FGF-BP) 1 is a secreted protein that can bind fibroblast growth factors (FGFs) 1 and 2. These FGFs are typically stored on heparan sulfate proteoglycans in the extracellular matrix in an inactive form, and it has been proposed that FGF-BP1 functions as a chaperone molecule that can mobilize locally stored FGF and present the growth factor to its tyrosine kinase receptor. FGF-BP1 is up-regulated in squamous cell, colon, and breast cancers and can act as an angiogenic switch during malignant progression of epithelial cells. For the present studies, we focused on FGF-1 and -2 and investigated interactions with recombinant human FGF-BP1 protein as well as effects on signal transduction, cell proliferation, and angiogenesis. We show that recombinant FGF-BP1 specifically binds FGF-2 and that this binding is inhibited by FGF-1, heparan sulfate, and heparinoids. Furthermore, FGF-BP1 enhances FGF-1- and FGF-2-dependent proliferation of NIH-3T3 fibroblasts and FGF-2-induced extracellular signal-regulated kinase 2 phosphorylation. Finally, in the chicken chorioallantoic membrane angiogenesis assay, FGF-BP1 synergizes with exogenously added FGF-2. We conclude that FGF-BP1 binds directly to FGF-1 and FGF-2 and positively modulates the biological activities of these growth factors.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.     

Exogenously added fibroblast growth factor 2 (FGF-2) to NIH3T3 cells interacts with nuclear ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner

Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.     

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.     

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A_L and B_L) or short (A_S and B_S) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA_L and PDGF-BB_S isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.