Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition.

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4-HS complexes, FR1c fails to bind FGF-4-HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.

The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity.

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG "donor" cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR "reporter" cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 1) differentially regulates the binding and signaling of FGFs 1 and 2 and 2) acts as a positive regulator of FGF-2 signaling.

Relative expression of epidermal growth factor receptor in placental cytotrophoblasts and choriocarcinoma cell lines.

The role of transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor (EGFR) interactions in regulating benign and malignant trophoblast proliferation were examined. Benign cytotrophoblast (CT) demonstrated mitogenic stimulation in response to TGF-alpha; BeWo and JAr choriocarcinoma cell lines failed to respond. EGFR levels in BeWo and JAr were determined by enzyme linked immunoassay (ELISA) to be at least 10-fold higher than those in benign CT. EGFR isolated from BeWo and JAr also demonstrated functional tyrosine kinase activity. Using a combination of immunoperoxidase (IP) and ELISA techniques, choriocarcinoma cells were found to produce significant quantities of TGF-alpha that were comparable with those reported previously by this laboratory for benign CT, and were felt to be stimulating their own proliferation in an autocrine fashion. EGFR blocking and TGF-alpha neutralizing antibodies inhibited JAr proliferation whereas an EGF neutralizing antibody did not. The data presented here and in our previous report indicate that a TGF-alpha-EGFR autocrine loop may regulate normal and malignant CT proliferation. Choriocarcinoma cells may be proliferating at a maximal rate due, in part, to EGFR overexpression and are therefore unable to respond further to exogenous growth factor. Thus, EGFR overexpression may contribute to the uncontrolled proliferation of choriocarcinoma cells in general.

A potential transforming growth factor alpha/epidermal growth factor receptor autocrine circuit in placental cytotrophoblasts.

A potential autocrine loop involving transforming growth factor alpha (TGF-alpha) and the epidermal growth factor (EGF) receptor (EGFR) in benign placental cytotrophoblasts was examined. EGFR were localized to villous cytotrophoblasts and syncytiotrophoblasts in frozen sections throughout gestation using immunoperoxidase (IP) and autoradiography with 125I-EGF. EGF and TGF-alpha stimulated uptake of [3H]-thymidine in cultured cytotrophoblasts from first and second trimester placentae, demonstrating both functional EGFR and the mitogenic ability of either growth factor in these cells. Using IP, TGF-alpha was localized consistently throughout gestation to cytotrophoblasts with little or no staining of syncytiotrophoblasts in formalin-fixed sections. Variable staining of villous stromal cells and intense staining of maternal decidua were also observed. TGF-alpha production by cultured cytotrophoblasts was confirmed in vitro via IP analysis of cytotrophoblasts cultured in serum-free media and enzyme-linked immunosorbent assay analysis of cytotrophoblast serum-free conditioned media. The results suggest that a TGF-alpha/EGFR autocrine loop stimulates proliferation of benign cytotrophoblasts.

Distribution and metabolism of the retinoid, N-(4-methoxyphenyl)-all-trans-retinamide, the major metabolite of N-(4-hydroxyphenyl)-all-trans-retinamide, in female mice.

The metabolism and disposition of N-(4-methoxyphenyl)-all-trans-retinamide (MPR), the major metabolite of N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), were investigated in female B6D2F1 (BDF) mice. Following a single oral dose of 10 mg/kg, MPR distributed to the serum, liver, mammary gland, urinary bladder, and skin. The highest levels of MPR were detected in the liver and mammary gland, and the largest values for AUC were in the mammary gland followed by the skin and liver. The t1/2 for MPR was 5.1 hr in liver, 5.6 hr in serum, 18.7 hr in urinary bladder, 23.1 hr in skin, and 26.6 hr in mammary gland. MPR and five metabolites were detected; levels varied between tissues. One metabolite was 4-HPR; the other four, which eluted at 7, 12, 13, and 18 min, remain unidentified. The major metabolite of MPR was the 18-min metabolite and comprised 17% of total retinoid in skin and 14% in mammary gland. 4-HPR was only a minor metabolite of MPR; 4-HPR was not detectable in serum or urinary bladder and accounted for less than 4% of total retinoid in the other tissues. In mice dosed with 10 mg/kg 4-HPR, the parent compound, MPR, a putative 4-HPR ester, and three of the MPR metabolites (7, 13, and 18 min) were found. These data suggest that the interconversion of 4-HPR and MPR greatly favors formation of MPR.(ABSTRACT TRUNCATED AT 250 WORDS)