Comparative characteristics of the composition and degree of sulfation of glycosaminoglycans in resting and actively proliferating tissues

A comparative study of the composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans isolated from normal animal tissues and from actively proliferating embryonic, tumour and regenerating tissues was carried out. Significant differences in the ratios of various types of sulfated GAG were revealed. The relative content of chondroitin sulfate AC in actively proliferating tissues was considerably increased. It was found that all types of sulfated GAG in actively proliferating tissues, with the exception of regenerating tissue, are characterized by a lower degree of sulfation as compared to GAG from resting tissues. The experimental results are discussed in terms of the role of proteoglycans in the organization of extracellular matrix and in control of cell proliferation.     

Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.     

Composition and degree of sulfation of glycosaminoglycans from tissues of different animal species: heterogeneity and tissue specificity of heparan sulfates

The composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans from various animal tissues were studied. It was shown that sulfated GAG contain chondroitin sulfates AC and B as well as heparan sulfates. The bulk of GAG in the majority of tissues under study is represented by 2-3 types of heparan sulfate molecules differing in the degree of sulfation. According to the degree of sulfation heparan sulfates from all tissues studied can be classified into three groups. Homologous tissues of various animal species are characterized by a similar composition and the degree of sulfation The data obtained are discussed in terms of the feasible role of proteoheparan sulfates in specific cell-to-cell interactions.     

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.     

Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein

Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.     

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).     

Heparan sulfate depletion within pulmonary fibroblasts: implications for elastogenesis and repair

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF-2 and glycosaminoglycans (GAG) from PG-depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF-2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.     

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1(-/-) to study the importance of proteoglycan desulfation in lung development. The Sumf1(-/-) mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1(-/-) as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1(-/-) mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1(-/-) mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.     

Serum regulation of Id1 expression by a BMP pathway and BMP responsive element

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.     

Bone Morphogenetic Protein Type I Receptor Antagonists Decrease Growth and Induce Cell Death of Lung Cancer Cell Lines

Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.     

Sulfation of glycosaminoglycans depends on the catalytic activity of lithium-inhibited phosphatase BPNT2 in vitro

Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3′-phosphoadenosine-5′-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.     

Changes in secreted and cell associated proteoglycan synthesis during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2: role of decorin in cell response to BMP-2

Proteoglycans have been identified within the extracellular matrices (ECM) of bone and are known to play a role in ECM assembly, mineralization, and bone formation. Bone morphogenetic protein-2 (BMP-2) specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells. Microarray analyses of the mouse myoblast cell line C2C12 and its differentiation into osteoblastic cells in response to BMP-2 have suggested the up-regulation of several proteoglycan species, although there is a lack of biochemical evidence for this response. In this study we have biochemically analyzed and characterized the proteoglycan populations that are induced in C2C12 cells upon osteoblastic differentiation produced by BMP-2. An important and specific increase in the synthesis of secreted decorin was observed in BMP-2-treated cells, as compared to untreated myoblasts and myoblasts induced to differentiate into myotubes. Decorin was seen to contain larger glycosaminoglycan (GAG) chains in induced than in non-induced cells. BMP-2 also produced an augment in the synthesis of different heparan sulfate proteoglycans such syndecan-2, - 3, glypican, and perlecan in detergent-soluble and non-soluble cellular fractions. We also examined whether the evident changes induced by BMP-2 in secreted decorin could have a functional role. BMP-2 signaling dependent as well as induction of alkaline phosphatase (ALP) activity was diminished in decorin null myoblasts compared to wild type myoblats although cell surface level of BPM-2 receptors was unchanged. These results are the first biochemical evidence and analysis for the effect of BMP-2 on the synthesis of proteoglycan during osteogenic conversion of myoblasts and suggest a role for decorin in cell response to BMP-2.     

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.     

Sulfated hyaluronan derivatives reduce the proliferation rate of primary rat calvarial osteoblasts

Glycosaminoglycans (GAG) and proteoglycans, which are components of the extracellular bone matrix, are also localized in and at the membrane of osteoblasts and in the pericellular matrix. Due to their interaction with several growth factors, water and cations these molecules play an important role in regulating proliferation and differentiation of osteoblasts and bone development. The aim of this study was to assess in vitro the effects of two chemically sulfated hyaluronan (HyaS) derivatives on the proliferation of rat calvarial osteoblasts and to compare with those of native hyaluronan (Hya) and natural sulfated GAG such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS) and heparan sulfate (HS). Moderately and highly sulfated HyaS derivatives caused a time-dependent reduction of osteoblast proliferation. The anti-proliferative effect of HyaS was accompanied by a cell cycle arrest in the G1 phase, but was not associated with cell death. Whereas non-sulfated high molecular weight (HMW)- and low molecular weight (LMW)-Hya as well as C4S, C6S, DS and HS showed no effect on the cell proliferation.     

An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP-2 (0.5-100ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24h vs. 3-6 days, respectively, of BMP treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGFbeta1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells.     

Quinoline Compound KM11073 Enhances BMP-2-Dependent Osteogenic Differentiation of C2C12 Cells via Activation of p38 Signaling and Exhibits In Vivo Bone Forming Activity

Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl) piperazino][1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]methanone (KM11073) strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.