Syndecan-1 expression is upregulated in degenerating articular cartilage in a transgenic mouse model for osteoarthritis.

OBJECTIVE: Mice heterozygous for the Del1 transgene locus with a short deletion mutation in the type II collagen gene develop early-onset degenerative changes in the knee joints that progress to end-stage osteoarthritis by the age of 12-15 months. This study focuses on the expression and distribution of syndecan-1, a cell-surface heparan sulfate proteoglycan, during the development of osteoarthritic cartilage degeneration, to better understand its role in this disease. METHODS: Northern analyses of total RNA extracted from knee joints of transgenic Del1 mice and their nontransgenic controls were used to monitor changes in syndecan-1 mRNA levels during development, growth, ageing, and cartilage degeneration. Immunohistochemistry was used to study the distribution of syndecan-1 in the knee joints at different stages of cartilage degeneration. RESULTS: Syndecan-1 mRNA was present in knee joints throughout life, with the highest mRNA levels in ageing knee joints. In Del1 mice, a transient upregulation of syndecan-1 mRNA synthesis was observed at the age of 6 months coinciding with early stages of cartilage degeneration and a period of attempted repair. Immunostaining for syndecan-1 was most intense in chondrocytes of superficial and intermediate zones of articular cartilage adjacent to defect areas. Chondrocyte clusters also stained strongly for syndecan-1. CONCLUSION: The present temporospatial expression data on upregulation of syndecan-1 in articular cartilage during early stages of cartilage degeneration suggest that this molecule is involved in the attempted repair of cartilage fibrillations. Combined with the known role of syndecan-1 during skeletal development and wound healing, this interesting finding warrants further validation.

High syndecan-1 expression is associated with favourable outcome in squamous cell lung carcinoma treated with radical surgery.

Expression of syndecan-1 is down-regulated in many cellular transformation models. We studied the clinical significance of syndecan-1 expression in 116 squamous cell lung carcinomas treated with radical surgery. Paraffin-embedded tissue samples were immunostained with two antibodies against human syndecan-1 (B-B4 and 104-9). Syndecan-1 expression was higher in well differentiated cancers than in moderately or poorly differentiated cancers with either antibody (P=0.001 for B-B4, and P<0.0001 for 104-9), but no significant association was found with the primary tumour size (T-stage) or the clinical stage. When the median expression (10% of cancer cells positive in B-B4 staining) was used as the cut-off value, cancers with high expression were associated with more favourable survival than those with low expression (the 2-year survival rate corrected for intercurrent deaths 84% vs 61%, P=0.026). However, syndecan-1 expression was not an independent prognostic factor in a multivariate survival analysis. We conclude that syndecan-1 expression decreases in parallel with histological dedifferentiation in squamous cell carcinoma of the lung, and that low syndecan-1 expression is associated with unfavourable outcome.

Proximal promoter of the murine syndecan-1 gene is not sufficient for the developmental pattern of syndecan expression in B lineage cells.

Syndecan-1 (CD138) is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. The role of syndecan-1 in the control of cell growth and morphology has been illustrated by its altered expression in hematological malignancies such as multiple myeloma as well as some solid tumors. It has been reported that the expression of syndecan-1 in cells of the B lineage is developmentally regulated such that pre-B cells and plasma cells express syndecan-1 while mature B cells do not. Thus, we investigated whether the proximal promoter region of the murine syndecan-1 promoter was able to confer the observed on-off-on expression of syndecan-1 in cells of the B lineage as they develop from pre-B cells to plasma cells. Experiments carried out using deletion mutants of the proximal promoter cloned upstream of the CAT reporter gene transfected into murine cell lines, representing the above stages of B-cell development, such as BA/F3 (pro-B cell), 70Z/3 (pre-B cell), 2PK3 (late mature B cell), and MPC-11 (plasma cell), showed detectable levels of CAT expression. The WEHI-231 (mature B cell) cell lines did not show detectable levels of CAT reporter activity. The strong levels of expression were observed with a fragment of the proximal promoter spanning the region from -365 to -95 (from the translation start point). However, Northern analysis of RNA obtained from the five murine B-cell lines, representing various stages of B-cell development, showed that the 70Z/3, MPC-11 but not BA/F3, and 2PK3 cells expressed detectable levels of syndecan-1 mRNA. By FACS analysis, using a rat anti mouse syndecan-1 antibody, syndecan-1 expression on the cell surface was found to correlate with the observed mRNA expression patterns in these cell lines. Our results indicate that the proximal promoter of the murine syndecan-1 promoter is not sufficient for the observed developmental pattern of syndecan expression in B cells.

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate.

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4.

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.

Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate.

The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.

Expression and glycosylation studies of human FGF receptor 4.

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.

Proximal promoter-independent activation of the far-upstream FGF-inducible response element of syndecan-1 gene.

Far upstream enhancers are predicted to act by looping and activating general transcription factors on core promoters and to require proximal promoter sequences for appropriate gene activation in time and space. We have previously described an FGF-inducible response element (FiRE) located far upstream on the syndecan-1 gene. The FiRE is activated specifically by members of the fibroblast growth factor (FGF) family in NIH3T3 cells. Here we describe the requirements of syndecan-1 proximal promoter for the activation of FiRE by FGF-2. Transient and stable transfections revealed that neither proximal promoter SP1 sites nor TATA-box are required for the FGF-2 induced activation of FiRE. Notably, the enhancer is activated in both orientations by FGF-2 even in the absence of proximal promoter. Importantly, removal of the promoter did not affect the growth factor specificity of FiRE. Proximal promoter independent activation of syndecan-1 gene by FGF-2 might be required during development when syndecan-1 proximal promoter needs to be largely attenuated but simultaneous transient and rapid FGF-2 induced transcription is required.

Transcriptional targeting of adenoviral gene delivery into migrating wound keratinocytes using FiRE, a growth factor-inducible regulatory element.

Impaired cutaneous wound healing is a common complication in diabetes, ischemia and venous insufficiency of lower extremities, and in long-term treatment with corticosteroids or other immunosuppressive agents. In development of gene therapy for wound repair, expression of therapeutic transgenes should be precisely targeted and controlled. Here, we describe a recombinant adenovirus RAdFiRE-EGFP, in which a growth factor inducible element (FiRE) of the murine syndecan-1 gene controls the expression of enhanced green fluorescent protein (EGFP) reporter gene. Treatment of RAdFiRE-EGFP-transduced murine epidermal keratinocytes in culture with FiRE-activating growth factor markedly enhanced the expression of EGFP. In ex vivo organ culture of wounded murine skin transduced with RAdFiRE-EGFP, the EGFP expression was specifically detected in wound margin keratinocytes, but not in intact skin. Activity of EGFP was first detected 2 days after a single application of RAdFiRE-EGFP and persisted up to 10 days. Similarly, FiRE-driven EGFP expression was detected specifically in epidermal keratinocytes in the edge of incisional wounds in murine skin transduced with RAdFiRE-EGFP. In contrast, adenovirus-mediated lacZ expression driven by CMV promoter was detected scattered in epidermal, dermal and subcutaneous layers in ex vivo and in vivo wounds, as well as in intact skin. These data demonstrate the feasibility of FiRE as a tool for transcriptional targeting of adenovirus-mediated transgene expression to cutaneous wound edge keratinocytes.

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4.

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.

Involvement of protein kinase A in fibroblast growth factor-2-activated transcription.

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

Association of syndecan-1 with tumor grade and histology in primary invasive cervical carcinoma.

OBJECTIVES: The expression of syndecan-1, a cell surface heparan sulfate proteoglycan, is reduced during malignant transformation of squamous cells. Studies on squamous cell carcinoma of the head and neck have shown that syndecan-1-positive tumors are associated with longer overall and recurrence-free survival. The purpose of this study was to analyze syndecan-1 expression in invasive cervical carcinoma and to examine the association of syndecan-1 expression with prognostic factors and overall survival. METHODS: The study population consisted of 124 patients treated for primary invasive carcinoma of the uterine cervix at the Turku University Central Hospital during the years 1970-1988. The material consisted of 102 (82.3%) squamous cell carcinomas, 16 (12.9%) adenocarcinomas and 1 (0.8%) adenosquamous carcinoma, 1 (0.8%) small cell carcinoma, 1 (0. 8%) adenoid basal carcinoma, 1 (0.8%) carcinosarcoma, and 2 (1.6%) unclassified cervical carcinomas. Syndecan-1 expression was determined on paraffin-embedded tissue blocks using a human syndecan-1-specific monoclonal antibody B-B4 and immunohistochemistry. The expression of syndecan-1 was classified according to staining intensity as well as the percentage of positively stained tumor cells. RESULTS: Staining intensity was strong in 44 (36%) samples, while 24 (19%) specimens remained syndecan-1-negative. In 49 (40%) samples, the percentage of syndecan-1-positive cells was >/=90%. Syndecan-1 expression, as determined by >/=50% positively stained tumor cells, was associated with the grade of differentiation (P = 0.03) and squamous histology (P < 0.001), but was not associated with clinical stage (P = 0.16) or disease-free survival (P = 0.86). Age (P = 0.003) and clinical stage (P < 0.001) were significant prognostic factors, but syndecan-1 expression determined neither by percentage of positively stained tumor cells nor by staining intensity was associated with the outcome. CONCLUSIONS: In cervical carcinoma syndecan-1 is associated with histological differentiation grade and squamous histology, but does not predict clinical outcome.

Transcriptional regulation of Syndecan-1 expression by growth factors.

Syndecan-1 is a prototype member of a family of transmembrane heparan sulfate proteoglycans. Syndecan-1 binds extracellular matrix components and fibroblast growth factors (FGFs) and modifies the function of FGFs. Syndecan-1 is constitutively expressed by several epithelial cells, but expression is also induced during many biological phenomena, such as tissue regeneration and the epithelial-mesenchymal interactions during organ development. Growth factors have been the prime candidates to induce syndecan-1 expression in these situations. In fibroblasts syndecan-1 is induced by FGF-2 and in keratinocytes by epidermal growth factor (EGF) and keratinocyte growth factor (KGF). The search for cis-acting elements regulating the growth factor-induced syndecan-1 expression has led to identification of a novel FGF-inducible response element (FiRE). FiRE is activated in fibroblasts and keratinocytes by the same growth factors that induce syndecan-1 expression in these cells. In adult tissues the activation of FiRE is restricted to migrating keratinocytes of healing wounds. The composition of the transcription factor binding to FiRE differs depending on the cell type and the activating growth factor. The FiRE provides a powerful tool for studies on growth factor specificity and regeneration of tissues. Moreover, it implies a novel transcriptional link that creates an FGF action-controlling autoregulatory loop between the heparan sulfate proteoglycans and the heparin-binding FGFs.

Extracellular matrix-dependent activation of syndecan-1 expression in keratinocyte growth factor-treated keratinocytes.

Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Määttä, A., and Jalkanen, M. (1998) FASEB J. 12, 959-969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiRE-dependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix.

Syndecan-1 expression has prognostic significance in head and neck carcinoma.

The syndecans are a family of cell-surface heparan sulphate proteoglycans that regulate cell behaviour by binding extracellular matrix molecules such as growth factors. The syndecan family has four members, of which syndecan-1 is the most studied and best characterized. We have studied the prognostic significance of syndecan-1 expression in squamous cell carcinoma (SCC) of the head and neck treated with surgery and post-operative radiotherapy. Paraffin-embedded tissue samples taken from 175 patients with primary SCC, followed up from 2 to 15 years after surgery, were studied for expression of syndecan-1 by immunohistochemistry. A low number (< or =50%, the median value) of syndecan-1-positive tumour cells was associated with low histological grade of differentiation (P<0.0001), a large primary tumour size (T1-2 vs. T3-4, P = 0.02), positive nodal status (NO vs. N1-3, P = 0.0006), and high clinical stage (stage I or II vs. III or IV, P<0.0001). Low syndecan-1 expression was also associated with unfavourable overall survival in a univariate analysis (P = 0.001). In a multivariate survival analysis, the clinical stage and syndecan-1 expression were the only independent prognostic factors. We conclude that syndecan-1 is a novel prognostic factor in SCC of the head and neck treated with surgery and post-operative radiotherapy.

Immunohistochemical expression of syndecan-1 in human endometrial cancer cells.

There are indications of increased frequency of endometrial cancer, one of the most common malignancies in women. Tissue samples of normal and malignant endometria were obtained post operatively from 30 women. We noted expression of syndecan-1 in 40% of investigated cancers. The most differentiated cancers showed 75% of positively stained specimens, moderately differentiated 40% and poorly differentiated neoplasm did not stain at all. In normal endometrial tissue syndecan-1 expression was regular and distinct in each specimen, but immunoreactivity of the hyperplastic endometrial specimens was absent. The detection of syndecan-1 in endometrial cancer of different clinical and histological stages could be of prognostic value in clinical diagnosis.

The activation and composition of FiRE (an FGF-inducible response element) differ in a cell type- and growth factor-specific manner.

The expression of the heparan sulfate proteoglycan, syndecan-1, is induced both in keratinocytes and in fibroblasts during development and tissue regeneration. Here we report that in keratinocytes the syndecan-1 gene was stimulated by EGF but not by FGF-2. In fibroblasts it was stimulated by FGF-2 but not by EGF. Likewise, the recently discovered FGF-inducible response element (FiRE) on the gene of syndecan-1 was stimulated by FGF-2 in fibroblasts and by EGF in keratinocytes, but not vice versa. The FiRE has two binding sites for an activator protein-1 (AP-1), one for an FGF-inducible nuclear factor (FIN-1) and one for an upstream stimulatory factor-1 (USF-1). The growth factor-stimulated binding of these transcription factors, as well as their requirement for FiRE activation, varied between the two cell types. First, although AP-1s were required for activation of FiRE in both cell types, the binding of AP-1 to FiRE was increased by growth factor-stimulation only in fibroblasts and not in keratinocytes. Secondly, FiRE did not bind FIN-1 nor needed the FIN-1 binding site for EGF-stimulated activation in keratinocytes, in contrast to the FGF-stimulated activation of FiRE in fibroblasts. Thirdly, EGF, which did not activate FiRE in fibroblasts, failed to activate FIN-1 in these cells. Finally, an USF-1 binding site that was necessary for activation of FiRE in keratinocytes was not needed in fibroblasts. These data suggest mechanisms by which members of the EGF- and FGF-families can differentially stimulate transcription through AP-1 regulated elements in a cell type-specific manner.

Wound reepithelialization activates a growth factor-responsive enhancer in migrating keratinocytes.

Wound reepithelialization and keratinocyte migration require strictly ordered gene expression, which is assumed to be initiated by locally released mitogens and exposure of the cells to different matrix components. The mechanisms triggering gene expression specifically during reepithelialization are poorly understood. The far upstream AP-1-driven, FGF-inducible response element (FiRE) of the syndecan-1 gene was activated during cutaneous wound healing in transgenic mice. FiRE was induced selectively in migrating but not in proliferating keratinocytes at the wound edge. The activation was initiated at the start of the cell migration, was persistent throughout the merging and stratification phases, and was terminated after completion of reepithelialization. Although FiRE has been found within the gene of syndecan-1, the proximal promoter of syndecan-1 was not required for activation of FiRE in the migrating keratinocytes. The wounding induced activation was inhibited by blocking cell surface growth factor receptors with suramin. However, the activation of FiRE in resting skin required simultaneous growth factor- and stress-induced signals, but could also be elicited by the phosphatase inhibitor, okadaic acid. The activation by both wounding and chemical stimuli was blocked by inhibiting extracellular regulated kinase and p38 MAP kinases, suggesting the involvement of at least two parallel signal transduction pathways in wounding induced gene activation. As FiRE shows specificity for migrating keratinocytes only, it can be a useful tool for future wound healing studies and for targeting genes to injured tissues.

Biosynthesis of heparin/heparan sulfate. cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung.

Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-p. (1994) J. Biol. Chem. 269, 26953-26958). The present report describes the molecular cloning and functional expression of the lung enzyme. The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da. N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence. The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells. Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of 3H2O from a polysaccharide substrate containing C5-3H-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO3)n substrate resulted in conversion of approximately 20% of the GlcUA units into IdceA residues. Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant approximately 9-kilobase (kb) mRNA and a minor approximately 5-kb species. Mouse mastocytoma cells showed only the approximately 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.

Is the sensitivity of cells for FGF-1 and FGF-2 regulated by cell surface heparan sulfate proteoglycans?

We have examined the importance of cell surface heparan sulfate proteoglycans (HSPG) in fibroblast growth factor (FGF) signaling. 3T3 cells grown under conventional conditions were much more sensitive to FGF-2 compared to FGF-1. However, cells were equally sensitive to FGF-1 and FGF-2 using conditions which reduced the effect of endogenous HSPG. Addition of heparin, or treatment with chlorate, an inhibitor of proteoglycan sulfation, resulted in enhanced or reduced growth factor response, respectively, and eliminated the differences between FGF-1 and FGF-2. HSPGs isolated from trypsin digests of 3T3 cells had a much higher affinity for FGF-2 compared to that for FGF-1 when analyzed by affinity chromatography. Glycosaminoglycan chains or core protein fragments derived from the HSPG failed to show the same high apparent affinity for FGF-2, suggesting that an intact proteoglycan structure was important for the high FGF-2 affinity. Addition of HSPG ectodomains, isolated from cultured 3T3 cells or produced as recombinant molecules, to chlorate-treated cultures of 3T3 cells inhibited the mitogenic activity of FGF-2 and eliminated the effect of heparin as a potentiator of either growth factor. These results support the idea that the cell-associated HSPG is an integral component of the FGF signaling system and in 3T3 cells contributes to the increased sensitivity of these cells to FGF-2 compared to FGF-1. Since isolated ectodomains of HSPG inhibited rather than stimulated the mitogenic response of the FGFs, the proper anchoring of the HSPG in the cell membrane appears to be important for a stimulatory effect.