Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.     

Enzymatic deconjugation of catecholamines in human and rat plasma and red blood cell lysate

We have developed a method for enzymatic hydrolysis of both sulfated and glucuronidated catecholamines in plasma and red blood cell lysate. Hydrolysis occurs in the course of the radioenzymatic assay for catecholamines. In human plasma, catecholamines are conjugated almost entirely with sulfate while, in rat plasma, glucuronides are the main conjugates of epinephrine and dopamine but not norepinephrine. Rat plasma contains less percent conjugated catecholamine than human plasma. Human red blood cell lysate contains less conjugated catecholamine than plasma, whereas free E in lysate exceeds that of plasma and free NE has same level both in plasma and lysate. This method is useful in detecting total (free + sulfated + glucuronidated) catecholamines and the nature of conjugated catecholamines.     

Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.     

Tyrosine O-sulfate ester in proteoglycans

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Recombinant human fibrinogen and sulfation of the gamma' chain

Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.     

Cultured human epidermis cells produce cell-associated interleukin 1-like prostaglandin E2- and collagenase-stimulating factors

In order to identify factors which may regulate the functions of dermal fibroblasts, cell lysates and conditioned media of cultured human epidermal cells were tested on dermal fibroblasts for the stimulation of prostaglandin E2- and collagenase-production. Both prostaglandin E2- and collagenase-stimulating activities appeared during epidermal cell culture: after 2 d they were detected in the cell lysate, and after 4 d of culture they were found also in the conditioned media. Molecular sieving chromatography of epidermal cell lysates led to the detection of two main peaks showing concomitant prostaglandin E2- and collagenase-stimulating activities at Mr approximately equal to 18 000 and Mr approximately equal to 10 000. A single peak of concomitant prostaglandin E2- and collagenase-stimulating activities were seen at Mr approximately equal to 10 000 in the epidermal cell conditioned media. This suggests that the cell-associated concomitant prostaglandin E2- and collagenase-stimulating activities are processed from a common precursor molecule and released. Collagenase-stimulating activity without accompanying prostaglandin E2 was also detected in the range of Mr approximately equal to 30 000-45 000.     

Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lystate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4- and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.     

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.     

Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections

Infections of body tissue by Staphylococcus aureus are quickly followed by degradation of connective tissue. Patients with rheumatoid arthritis are more prone to S. aureus-mediated septic arthritis. Various types of collagen form the major structural matrix of different connective tissues of the body. These different collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells, and inflammatory cells that are induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF). To determine the host's contribution in the joint destruction of S. aureus-mediated septic arthritis, we analyzed the MMP expression profile in human dermal and synovial fibroblasts upon exposure to culture supernatant and whole cell lysates of S. aureus. Human dermal and synovial fibroblasts treated with cell lysate and filtered culture supernatants had significantly enhanced expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, and MMP-11 compared with the untreated controls (p < 0.05). In the S. aureus culture supernatant, the MMP induction activity was identified to be within the molecular-weight range of 30 to >50 kDa. The MMP expression profile was similar in fibroblasts exposed to a combination of IL-1/TNF. mRNA levels of several genes of the mitogen-activated protein kinase (MAPK) signal transduction pathway were significantly elevated in fibroblasts treated with S. aureus cell lysate and culture supernatant. Also, tyrosine phosphorylation was significantly higher in fibroblasts treated with S. aureus components. Tyrosine phosphorylation and MAPK gene expression patterns were similar in fibroblasts treated with a combination of IL-1/TNF and S. aureus. Mutants lacking staphylococcal accessory regulator (Sar) and accessory gene regulator (Agr), which cause significantly less severe septic arthritis in murine models, were able to induce expression of several MMP mRNA comparable with that of their isogenic parent strain but induced notably higher levels of tissue inhibitors of metalloproteinases (TIMPs). To our knowledge, this is the first report of induction of multiple MMP/TIMP expression from human dermal and synovial fibroblasts upon S. aureus treatment. We propose that host-derived MMPs contribute to the progressive joint destruction observed in S. aureus-mediated septic arthritis.     

Structural identification of sulfated tyrosine in human urine

A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions.     

T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins.

Engagement of the T cell antigen receptor initiates signal transduction involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that Crk adapter proteins undergo activation-induced interaction with the zeta-chain associated protein (ZAP-70) tyrosine kinase in the human T cell line, Jurkat. Incubation of various glutathione S-transferase fusion proteins with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.     

The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels.

We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

Aspirin inhibits proliferation and promotes differentiation of neuroblastoma cells via p21Waf1 protein up‐regulation and Rb1 pathway modulation

Several clinical and experimental studies have demonstrated that regular use of aspirin (acetylsalicylic acid, ASA) correlates with a reduced risk of cancer and that the drug exerts direct anti‐tumour effects. We have previously reported that ASA inhibits proliferation of human glioblastoma multiforme‐derived cancer stem cells. In the present study, we analysed the effects of ASA on nervous system‐derived cancer cells, using the SK‐N‐SH (N) human neuroblastoma cell line as an experimental model. ASA treatment of SK‐N‐SH (N) dramatically reduced cell proliferation and motility, and induced neuronal‐like differentiation, indicated by the appearance of the neuronal differentiation marker tyrosine hydroxylase (TH) after 5 days. ASA did not affect cell viability, but caused a time‐dependent accumulation of cells in the G₀/G₁ phase of the cell cycle, with a concomitant decrease in the percentage of cells in the G₂ phase. These effects appear to be mediated by a COX‐independent mechanism involving an increase in p21^Waf1 and underphosphorylated retinoblastoma (hypo‐pRb1) protein levels. These findings may support a potential role of ASA as adjunctive therapeutic agent in the clinical management of neuroblastoma.     

Oligo(tyrosine sulfate)s as heparin pentasaccharide mimic: evaluation by surface noncovalent affinity mass spectrometry

Since the discovery of anti-HIV activity in oligo(tyrosine sulfate)s in our laboratory, we have been interested in their potential as heparin pentasaccharide mimics. In this study, we investigated their interactions with synthetic heparin-binding peptides, derived from human antithrombin III (hAT III) and heparin-interacting protein (HIP), using surface noncovalent affinity mass spectrometry. We compared binding affinities to those heparin-binding peptides between oligo(tyrosine sulfate)s and several known sulfated compounds and found that oligo(tyrosine sulfate)s bind to hAT III (123-139) more strongly than a heparin-derived hexasaccharide dp6. Moreover, we found longer oligo(tyrosine sulfate) has higher binding affinity to hAT III (123-139).     

Normal Human Fibroblasts Produce Membrane-Bound and Soluble Isoforms of FGFR-1

Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which αFGF and βFGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.     

WISP-1 binds to decorin and biglycan

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Structural and experimental evidence suggests that CCN family member activities are modulated by their interaction with sulfated glycoconjugates. To elucidate the mechanism of action for WISP-1, we characterized the specificity of its tissue and cellular interaction and identified binding factors. WISP-1 binding was restricted to the stroma of colon tumors and to cells with a fibroblastic phenotype. By using a solid phase assay, we showed that human skin fibroblast conditioned media contained WISP-1 binding factors. Competitive inhibition with different glycosaminoglycans and treatment with glycosaminoglycan lyases and proteases demonstrated that binding to the conditioned media was mediated by dermatan sulfate proteoglycans. Mass spectrometric analysis identified the isolated binding factors as decorin and biglycan. Decorin and biglycan interacted directly with WISP-1 and inhibited its binding to components in the conditioned media. Similarly, WISP-1 interaction with human skin fibroblasts was inhibited by dermatan sulfate, decorin, and biglycan or by treatment of the cell surface with dermatan sulfate-specific lyases. Together these results demonstrate that decorin and biglycan are WISP-1 binding factors that can mediate and modulate its interaction with the surface of fibroblasts. We propose that this specific interaction plays a role in the regulation of WISP-1 function.