The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.     

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.     

Collagen VI Regulates Normal and Transformed Mesenchymal Cell Proliferationin Vitro

Suggestions exist that, in addition to traditional growth factors, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed fibroblasts because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell growth on an individual ECM protein were not correlated. To test if ECM can stimulate cell growth, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of 20 μg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human fibroblasts and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor α2β1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell growth via a pathway that is independent of the integrin α2β1 and that the stimulatory region appears to be within the native helical portion of the collagen.     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

PTH-related protein enhances LoVo colon cancer cell proliferation, adhesion, and integrin expression

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. Tumor cell adhesion to extracellular matrix (ECM) proteins plays a major role in the invasion and metastasis of tumor cells, and is mediated via integrin subunits. The LoVo human colon cancer cell line was used as a model system to study the effects of PTHrP on cell proliferation and adhesion to ECM proteins found in normal liver. Clones of LoVo cells engineered to overexpress PTHrP by stable transfection with a PTHrP cDNA showed enhanced cell proliferation vs. control (empty vector-transfected) cells. PTHrP-overexpressing cells also showed significantly higher adhesion to collagen type I, fibronectin, and laminin, and enhanced expression of the [symbol: see text] integrin subunits. These results indicate that PTHrP may play a role in colon cancer invasion and metastasis by increasing cell proliferation and adhesion to the ECM via upregulation of proinvasive integrin expression.     

Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family--amphiregulin and heparin-binding EGF-like growth factor (HB-EGF)-on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver-colonizing potential. The effect of amphiregulin and HB-EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte-derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB-EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte-derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB-EGF synergistically acted with hepatocyte-derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor-alpha (TGF-alpha) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte-derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF-alpha mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB-EGF stimulated expression of erb-B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte-derived ECM, amphiregulin inhibited erb-B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site-specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte-derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM.     

Effects of extracellular matrices derived from different cell sources on chondrocyte functions

Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM.     

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture

There is significant interest in the development of tissue-engineered skin analogues, which replace both the dermal and the epidermal layer, without the use of animal or human derived products such as collagen or de-epidermalised dermis. In this study, we proposed that alginate hydrogel could be used to encapsulate fibroblasts and that keratinocytes could be cultured on the surface to form a bilayered structure, which could be used to deliver the co-culture to a wound bed, initially providing wound closure and eventually expediting the healing process. Encapsulation of fibroblasts in 2 and 5% w/v alginate hydrogel effectively inhibited their proliferation, whilst maintaining cell viability allowing keratinocytes to grow uninhibited by fibroblast overgrowth to produce a stratified epidermal layer. It was shown that the alginate degradation process was not influenced by the presence of fibroblasts within the hydrogel and that lowering the alginate concentration from 5 to 2% w/v increased the rate of degradation. Fibroblasts released from the scaffold were able to secrete extracellular matrix (ECM) and thus should replace the degrading scaffold with normal ECM following application to the wound site. These findings demonstrate that alginate hydrogel may be an effective delivery vehicle and scaffold for the healing of full-thickness skin wounds.     

Approaches in extracellular matrix engineering for determination of adhesion molecule mediated single cell function

The native extracellular matrix (ECM) and the cells that comprise human tissues are together engaged in a complex relationship; cells alter the composition and structure of the ECM to regulate the material and biologic properties of the surrounding environment while the composition and structure of the ECM modulates cellular processes that maintain healthy tissue and repair diseased tissue. This reciprocal relationship occurs via cell adhesion molecules (CAMs) such as integrins, selectins, cadherins and IgSF adhesion molecules. To study these cell-ECM interactions, researchers use two-dimensional substrates or three-dimensional matrices composed of native proteins or bioactive peptide sequences to study single cell function. While two-dimensional substrates provide valuable information about cell-ECM interactions, three-dimensional matrices more closely mimic the native ECM; cells cultured in three-dimensional matrices have demonstrated greater cell movement and increased integrin expression when compared to cells cultured on two-dimensional substrates. In this article we review a number of cellular processes (adhesion, motility, phagocytosis, differentiation and survival) and examine the cell adhesion molecules and ECM proteins (or bioactive peptide sequences) that mediate cell functionality.     

人参总皂甙对人GM-CSF和GM-CSFR表达的调控

为探讨人参调控粒细胞发生的生物学机制,采用造血祖细胞和骨髓基质细胞体外培养、造血生长因子生物学活性检测、免疫细胞化学、核酸分子原位杂交、免疫沉淀和蛋白印迹等现代生物学技术,研究人参总皂甙(total saponins of Panax ginaeng,TSPG)对人粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和粒-巨噬细胞集落刺激因子受体α(GM-CSFRα)表达的影响。结果：(1)经TSPG(50μg／m1)诱导制备的骨髓基质细胞、胸腺细胞、脾细胞、血管内皮细胞和单核细胞条件培养液可显著提高粒单系造血祖细胞(CFU-GM)的集落产率;(2)经TSPG(50μg/ml)诱导后,上述细胞的GM-CSF蛋白(诱导24h)和mRNA(诱导12h)表达显著提高;(3)经TSPG(50μg/ml)诱导24h骨髓造血细胞的GM-CSFRα蛋白表达增强;(4)经TSPG(50μg/ml)刺激后2min,GM-CSFRα和Shc发生酪氨酸磷酸化,5min时达高峰,随后去磷酸化。上述结果表明,TSPG可能通过直接和／或间接途径促进淋巴细胞与骨髓基质细胞合成与分泌GM-CSF,诱导骨髓造血细胞表达GM-CSFRα,并刺激GM-CSFRα和Shc的酪氨酸可逆磷酸化,从而通过调控GM-CSF的信号转导过程,促进CFU-GM的增殖。     

Different cell cycle responses of wound healing protagonists to transient in vitro hypoxia

Polyploidization is a process present in cells of many different human tissues. Since it is also prominent in human wound healing in vivo and in vitro, we focused on the influence of hypoxia on the cells proliferation and polyploidization response. The proliferation response of two major cell types, involved in human wound healing, human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) was quite similar in the in vitro setup: proliferation significantly decreased under the influence of 18 h of hypoxia and was reinitiated after 72 h of reoxygenation. The cells response concerning their tendency towards the development of polyploidy was different: NHDF did not generate any polyploid cells, which stands in contrast to former in vitro studies with human wound-derived fibroblasts, but HDMEC were characterized by the presence of both mononuclear and binuclear tetraploid cells. The number of tetraploids was downregulated during hypoxia and increased during reoxygenation, accompanied by proliferation onset. The immunomicroscopic survey of HDMEC opened up a cell cycle model, which might be useful in the future to evaluate cell cycle modulations leading to polyploidy without the need to apply any additional cell cycle inhibitors.     

Autoantibodies to fibrillin-1 activate normal human fibroblasts in culture through the TGF-beta pathway to recapitulate the "scleroderma phenotype"

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.     

Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro

To elucidate the effect of extracellular matrices (ECMs) and related and nonrelated-limbal feeder cells as substitutes for the in vivo niche on the phenotype and genotype of the limbal stem cell (SC) expansion in vitro, human limbal SCs were used. The limbus explants were expanded on human amniotic membrane (AM), commercial ECMs including matrigel (MAT), collagen (COL), and control (no ECM) in presence and absence of feeder cells including human limbal fibroblasts (LFs), a limbus-specific cell and mouse embryonic fibroblasts (MEFs). Proliferation, cell death, immunocytochemistry, expression of specific genes, ultrastructural characteristics, and size and granularity of expanded human limbal SCs in different groups were evaluated. The growth, cell proliferation, and survival of limbal SCs were enhanced by AM and MAT matrices. Ultrastructure and expression of stemness markers revealed that there was no significance difference between AM and MAT. However, flow cytometric analysis showed that the size and granularity of cultured cells increased in the presence of MAT and COL as well as in no ECM group. Moreover, co-culturing of limbal explants with LFs and MEFs on AM and MAT groups, enhanced the expansion and survival of cultured cells in comparison with others. In conclusion, the cultivation of human limbal explants on AM co-culturing with human LFs promises to be a good model for preparing undifferentiated epithelial sheets suitable for transplantation.     

Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts

Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.     

Amphiregulin and hepatocyte‐derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver‐colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family—amphiregulin and heparin‐binding EGF‐like growth factor (HB‐EGF)—on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver‐colonizing potential. The effect of amphiregulin and HB‐EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte‐derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB‐EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte‐derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB‐EGF synergistically acted with hepatocyte‐derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor‐α (TGF‐α) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte‐derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF‐α mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB‐EGF stimulated expression of erb‐B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte‐derived ECM, amphiregulin inhibited erb‐B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site‐specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte‐derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM. J. Cell. Biochem. 76:332–340, 1999. © 1999 Wiley‐Liss, Inc.     

Extracellular matrix proteins influence phenotype and cytokine expression in human breast cancer cell lines

Tumor growth and invasion are not only the result of malignant transformation but are also dependent on environmental influences from surrounding stroma, extracellular matrix (ECM), local cytokines and systemic hormones. We have investigated the influence of ECM components on three human breast cancer cell lines of different malignant potential: MCF-7, T47D and MDA-MB-231 were cultured on collagen I, collagen IV, laminin, fibronectin or poly-D-lysine, and we analyzed the proliferation rate and cytokine expression pattern. Among the three cell lines investigated we observed a distinct response to each ECM component. We hypothesize that ECM may have a significant modulatory effect on malignant behavior in vivo which might depend on individual responses and on the differentiation state of tumor cells. This study also shows that the surface on which cells are cultured greatly influences cell kinetics and the cytokine expression pattern.     

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 μg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.     

Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs. J. Cell. Physiol. 177:187–195, 1998. © 1998 Wiley-Liss, Inc.