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Publication
Journal: FASEB Journal
March/13/1990
Abstract
The molecular signals controlling liver regeneration are becoming rapidly defined. Control of growth in regenerating liver has advanced from elusive serum factors and nutrient effects to identification of entirely new growth factors with apparent liver specificity as well as establishment of meaningful gene expression patterns for growth factors already known. Based on studies with hepatocyte cultures and gene expression in regenerating liver, the substances EGF, TGF alpha, HBGF-1 (aFGF), and two new substances (HPTA/HGF and Hepatopoietin B) have been defined as complete mitogens for hepatocytes and implicated in control of liver growth. The amino acid sequence of HPTA/HGF recently became clear and revealed interesting structural homologies in a molecule that might become the largest known growth factor. The plasticity of growth responses seen in liver may be controlled by these factors as well as by comitogenic substances such as norepinephrine which, although nonmitogenic per se, can initiate growth in hepatocytes exposed to the above mitogenic growth factors or mitogenic inhibitors such as TGF beta. The role of the latter in cessation of DNA synthesis in liver regeneration will be discussed, presenting the positive and negative evidence that constitutes the TGF beta paradox of liver regeneration.
Publication
Journal: Science
February/20/1991
Abstract
A heparin binding mitogenic protein isolated from bovine uterus shares NH2-terminal amino acid sequence with a protein isolated from newborn rat brain. The cDNA's of the bovine, human, and rat genes have been isolated and encode extraordinarily conserved proteins unrelated to known growth or neurotrophic factors, although identity of nearly 50 percent has been found with the predicted sequence of a retinoic acid induced transcript in differentiating mouse embryonal carcinoma cells. Lysates of COS-7 cells transiently expressing this protein were mitogenic for NRK cells and initiated neurite outgrowth from mixed cultures of embryonic rat brain cells. RNA transcripts encoding this protein were widely distributed in tissues and were developmentally regulated. This protein, previously designated as heparin binding growth factor (HBGF)-8, is now renamed pleiotrophin (PTN) to reflect its diverse activities. PTN may be the first member of a family of developmentally regulated cytokines.
Publication
Journal: Physiological Reviews
November/20/1990
Abstract
This review tries to provide a general, and very speculative, view of growth control mechanisms that may be common to the development of blood vessels and to pathological processes including cell proliferation. From a developmental point of view, vascular growth is most likely to include local autocrine or paracrine mechanisms that permit the two cells of the vessel wall to grow, organize into the characteristic tubular and layered structures of the vessel wall, and eventually achieve a return to quiescence. The "real" mechanisms controlling growth in vivo are difficult to ascertain from studies in culture. For example, a large list of angiogenesis molecules must be able to generate endothelial replication, but in culture many of these molecules are inhibitory for each endothelial replication. Similarly, in culture, we have a long list of smooth muscle mitogens, but none of these have as of yet been proven to control smooth muscle growth in vivo. Endothelial growth control has been attributed to the presence of membrane molecules able to inhibit endothelial replication and to the actions of soluble growth factors and their receptors. Unfortunately for the former hypothesis we still lack specific molecules with the properties of contact inhibition of replication. The data discussed here, however, suggest that modulation of expression or function of cell-cell adhesive molecules could be critical both to morphogenic changes and to mitogenesis by release of cells from cell-cell contact. Moreover, our data and data from other laboratories suggest that angiogenic factors, including the HBGFs and TGF-beta, may function in angiogenesis by altering cell-cell and cell-cell substrate interactions rather than via a primary effect on cell replication. This view of angiogenesis is consistent with the absence of a mitogenic effect of some angiogenic factors. Although endothelial cell replication is obviously necessary to angiogenesis, the lack of mitogenic effect of some factors suggests a need for a more general explanation of the actions of angiogenic factors. Endothelial injury may be interrelated with smooth muscle growth. The simplest possibility is that a failure of the endothelial cell barrier function, due either to denudation or an increase in adhesivity for leukocytes, would permit access of platelets or leukocytes to the vessel wall. These extrinsic cells, in turn, would stimulate smooth muscle cell replication by release of growth factors. The second possibility is that the endothelial cell may itself release growth factors into the vessel wall.(ABSTRACT TRUNCATED AT 400 WORDS)
Publication
Journal: Journal of Cellular Physiology
August/18/1987
Abstract
Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.
Publication
Journal: Science
November/7/1990
Abstract
Heparin-binding growth factor-1 (HBGF-1) is an angiogenic polypeptide mitogen for mesoderm- and neuroectoderm-derived cells in vitro and remains biologically active after truncation of the amino-terminal domain (HBGF-1 alpha) of the HBGF-1 beta precursor. Polymerase chain reaction mutagenesis and prokaryotic expression systems were used to prepare a mutant of HBGF-1 alpha lacking a putative nuclear translocation sequence (amino acid residues 21 to 27; HBGF-1U). Although HBGF-1U retains its ability to bind to heparin, HBGF-1U fails to induce DNA synthesis and cell proliferation at concentrations sufficient to induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression. Attachment of the nuclear translocation sequence from yeast histone 2B at the amino terminus of HBGF-1U yields a chimeric polypeptide (HBGF-1U2) with mitogenic activity in vitro and indicates that nuclear translocation is important for this biological response.
Publication
Journal: Cell regulation
May/27/1991
Abstract
Human mononuclear cells were plated in culture, and the conditioned media of these cells were analyzed by heparin-Sepharose affinity chromatography. The fractions were tested for growth factor activity as measured by the stimulation of DNA synthesis in BALB/c 3T3 cells. After 2 d in culture, two peaks of heparin-binding growth factor (HBGF) activity were detected, one eluting with 0.5 M NaCl, which could be shown to be platelet-derived growth factor (PDGF)-like, and the other eluting with 1.0 M NaCl. After 7-11 d in culture, when monocytes had clearly differentiated into macrophages, greater than 95% of the HBGF activity in conditioned medium consisted of the 1.0 M NaCl elution peak. This activity, which was designated macrophage-derived HBGF (MD-HBGF), was found to be a cationic heat-resistant polypeptide with a molecular weight in the range of 14-25 kDa. Analysis using Western blots and specific neutralizing antisera, as well as comparative heparin affinity analysis, indicated that MD-HBGF was not identical to other heparin-binding 3T3 cell growth factors known to be produced by macrophages, such as PDGF (AB, AA, and BB forms), acidic fibroblast growth factor, and basic fibroblast growth factor. In addition to stimulating mitogenesis in 3T3 cells, MD-HBGF also stimulated the proliferation of vascular smooth muscle cells, but did not stimulate the proliferation of vascular endothelial cells.
Publication
Journal: Biochemical and Biophysical Research Communications
February/21/1991
Abstract
Hepatocyte growth factor/hepatopoietin A is a mitogen for primary hepatocytes and may mediate regeneration after liver damage. To date, the activity of this novel factor has been restricted to hepatocytes. We now show that the factor is also a mitogen for a number of primary epithelial cells but is inactive with human foreskin fibroblasts, human endothelial cells and HEP3B cells. The factor also substitutes for HBGF-2 (basic FGF) in stimulating the anchorage-independent growth of SV-40 transformed rat kidney epithelial cells. Therefore, hepatocyte growth factor/hepatopoietin A appears to act on a variety of epithelial, but not mesenchymal, cells which respond to HBGFs.
Publication
Journal: Journal of Clinical Investigation
April/13/2008
Abstract
Cells isolated from many types of human cancers express heparin-binding growth factors (HBGFs) that drive tumor growth, metastasis, and angiogenesis. The heparan sulfate proteoglycan glypican-1 (GPC1) is a coreceptor for HBGFs. Here we show that both cancer cell-derived and host-derived GPC1 are crucial for efficient growth, metastasis, and angiogenesis of human and mouse cancer cells. Thus downregulation of GPC1 in the human pancreatic cancer cell line PANC-1, using antisense approaches, resulted in prolonged doubling times and decreased anchorage-independent growth in vitro as well as attenuated tumor growth, angiogenesis, and metastasis when these cells were transplanted into athymic mice. Moreover, athymic mice that lacked GPC1 exhibited decreased tumor angiogenesis and metastasis following intrapancreatic implantation with either PANC-1 or T3M4 human pancreatic cancer cells and fewer pulmonary metastases following intravenous injection of murine B16-F10 melanoma cells. In addition, hepatic endothelial cells isolated from these mice exhibited an attenuated mitogenic response to VEGF-A. These data indicate that cancer cell- and host-derived GPC1 are crucial for full mitogenic, angiogenic, and metastatic potential of cancer cells. Thus targeting GPC1 might provide new avenues for cancer therapy and for the prevention of cancer metastasis.
Publication
Journal: Journal of Biological Chemistry
September/4/1997
Abstract
Uterine growth factors are potential effector molecules in embryo growth signaling pathways. Pig uterine luminal flushings contained a heparin-binding growth factor (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8. This factor, which was heat- and acid-labile and of Mr 10,000 as assessed by gel filtration, stimulated DNA synthesis in fibroblasts and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent Mr of 10, 000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EENIKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKXIRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247-262 and 248-259 of the 349-residue predicted primary translation product of porcine connective tissue growth factor (pCTGF). 10-kDa CTGF-mediated fibroblast DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and 20 kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel growth factor in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.
Publication
Journal: Proceedings of the National Academy of Sciences of the United States of America
November/24/1987
Abstract
The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.
Publication
Journal: Journal of Biological Chemistry
March/11/1986
Abstract
Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.
Publication
Journal: Molecular and Cellular Biology
September/25/1990
Abstract
Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.
Publication
Journal: Journal of Cell Biology
May/10/1990
Abstract
The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.
Publication
Journal: Journal of Cellular Physiology
July/9/1991
Abstract
Basic fibroblast growth factor (bFGF) is a member of the heparin-binding growth factor (HBGF) family that includes at least seven species. These proteins are potent regulators of a number of cellular processes, including cell division and angiogenesis. Multiple forms of bFGF exist differing only in the length of their NH2-terminal extensions. These species of bFGF also have unique subcellular distributions. The smallest form (18 kD) occurs predominantly in the cytosol, while the higher molecular weight forms (22, 22.5, 24 kD) are associated with the nucleus and ribosomes. Here we report that the nuclear localization of the higher molecular weight forms of bFGF derives specifically from the amino acid sequences within the NH2-terminal extension. This has been demonstrated by constructing a chimeric protein containing the NH2-terminal extension of the highest molecular weight form of bFGF fused to beta-galactosidase (beta-gal). After transfection in a transient expression system, the chimeric protein accumulated in the nuclei of transfected cells, while the wild-type beta-gal was found predominantly in the cytoplasm.
Publication
Journal: Proceedings of the National Academy of Sciences of the United States of America
November/8/1989
Abstract
Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation.
Publication
Journal: Biochemical and Biophysical Research Communications
February/12/1990
Abstract
We have purified to near homogeneity a novel 17 kD growth factor from bovine uterus which we designated heparin-binding growth factor-8 (HBGF-8). The growth factor binds tightly to cation exchange resins and to Heparin-Sepharose and is stable to acetone precipitation and labile in acid. Based upon total activity in acetone extracts of bovine uterus stimulating 3H-thymidine incorporation into DNA of serum-starved NIH 313 cells, a 6940 fold purification was achieved with an overall yield of HBGF-8 activity of 0.4%, using extraction of acetone powders and chromatographic separations at neutral pH. Approximately 18 micrograms protein was obtained from 1.2 kg wet weight of tissue. HBGF-8 was clearly separated from 17.5 kD bovine uterus basic fibroblast growth factor (bFGF) by purification and its N-terminal amino acid sequence analyzed. A polypeptide with a unique 25 N-terminal amino acid sequence was found. HBGF-8 was as active as acidic fibroblast growth factor (aFGF) and slightly less active than bFGF in the mouse NIH 3T3 fibroblast mitogenic assay system with an intrinsic specific activity of 5000 dpm/ng under standard assay conditions.
Publication
Journal: Journal of Biological Chemistry
September/15/1988
Abstract
The differentiated human hepatoblastoma-derived cell line, HepG2, displayed two classes of specific membrane receptors for heparin-binding growth factor type 1 (HBGF-1). Specific membrane receptors were distinguished from nonreceptor heparin-like binding sites. Receptors with an apparent Kd of 9.2 +/- 0.9 pM and present at 15,000 +/- 900/cell correlated with HBGF-1 stimulation of HepG2 growth. Receptors with an apparent Kd of 2 +/- 0.4 nM and present at 180,000 +/- 18,000/cell correlated with inhibition of growth and changes in secretory products. Other hepatoma cell lines exhibited a simple positive mitogenic response to HBGF-1 and a single class of high affinity binding sites. HBGF-1 covalently cross-linked to hepatoma cell surface polypeptides of apparent mean molecular mass of 130 kilodaltons. At 37 degrees C, receptor-bound HBGF-1 was internalized (t 1/2 = 45 min) but not degraded for up to 6 h. The display of receptors decreased with increased cell density and expression of HBGF-1 mRNA and HBGF-1-like activity in the culture medium. Proliferating normal human hepatocytes also exhibited two classes of binding sites with affinities for HBGF-1 and apparent molecular weight similar to HepG2 cells. These results implicate HBGF-1 or homologues in human hepatoma cell growth and normal liver cell regeneration.
Publication
Journal: Journal of Cell Biology
April/7/1987
Abstract
Heparin-binding growth factor-2 (HBGF-2; also known as basic fibroblast growth factor) is mitogenic for most anchorage-dependent cells. It is shown here that HBGF-2 stimulates cell-substratum adhesion and neurite extension in the sympathetic nerve cell line PC12. When HBGF-2 is adsorbed to artificial extracellular matrices consisting of heparin or chondroitin sulfate, it causes the formation of cellular aggregates or circles of cells, respectively. HBGF-2 is also a nerve cell survival molecule, for it potentiates the survival of primary cultures of embryonic chick ciliary ganglion cells but not of embryonic neural retina cells. Finally, a series of synthetic peptides from the HBGF-2 sequence is described that selectively alter the biological effects of HBGF-2. The amphiphilic nature of one of these peptides is discussed with respect to its ability to stimulate cell adhesion.
Publication
Journal: Clinical and Experimental Metastasis
January/3/2006
Abstract
Perlecan (Pln) is a major heparan sulfate proteoglycan (HSPG) of extracellular matrices and bone marrow stroma. Pln, via glycosaminoglycans in domains I and V, acts as a co-receptor for delivery of heparin binding growth factors (HBGFs) that support cancer growth and vascularization. Specifically, glycosaminoglycans bind HBGFs and activate HBGF receptors, including those for FGF-2 and VEGF-A. The contribution of Pln to prostate cancer growth was tested using a ribozyme approach to knockdown Pln expression levels. Transfection into the androgen-independent, bone targeted prostate cancer line, C4-2B, and efficient stable knockdown of Pln was demonstrated by quantitative PCR, immunohistochemistry and immunoblotting. Three individually isolated subclones with 75-80% knockdown in Pln mRNA, protein expression and secretion into ECM were used to study in vitro growth responses to FGF-2 and VEGF-A. While cells with normal Pln levels responded to both HBGFs, knockdown cells responded poorly. All lines responded to serum growth factors and IGF-I. Anchorage-independent growth assays showed reduced colony size and cohesiveness by all Pln deficient subclones compared to parental C4-2B cells. In vivo effects of Pln knockdown were measured by inoculating knockdown and control ribozyme transfected cell lines into athymic mice. A reduced growth rate, smaller tumor size, diminished vascularization and failure to elevate serum PSA characterized mice bearing Pln knockdown C4-2B cells. Poor vascularization correlated with reduced levels of VEGF-A secreted by Pln knockdown lines. We conclude that Pln is an essential ECM component involved in growth responses of metastatic prostate cancer cells to HBGFs deposited in local and metastatic microenvironment.
Publication
Journal: Journal of Biological Chemistry
October/3/1991
Abstract
A novel Mr 17,000 heparin-binding protein was purified from culture medium conditioned by A431 human epidermoid carcinoma cells. This protein, designated HBp17, was found to bind the heparin-binding peptide growth factors HBGF-1 and HBGF-2 in a noncovalent, reversible manner. In addition HBp17 was found to inhibit the biological activities of both HBGF-1 and HBGF-2. Both the binding and inactivation of HBGF-1 and HBGF-2 by HBp17 were abolished by heparin. Full-length 1163-base pair HBp17 cDNA was cloned and sequenced by using the polymerase chain reaction technique. The deduced primary structure of HBp17 consisted of 234 amino acids including each of five partial peptide sequences obtained from proteolytic fragments of purified HBp17. The encoded protein included a 33-residue N-terminal signal sequence for secretion and a single potential N-linked glycosylation site. No homology with any known protein was found for the deduced primary structure of HBp17. The expression of HBp17 mRNA was found to occur preferentially in normal human keratinocytes and in squamous cell carcinomas. This pattern of HBp17 gene expression suggests that this binding protein for HBGFs 1 and 2 has a physiological role in squamous epithelia.
Publication
Journal: Proceedings of the National Academy of Sciences of the United States of America
January/31/1990
Abstract
Two monoclonal antibodies (mAbs) against bovine heparin-binding growth factor II (HBGF-II)/basic fibroblast growth factor (bFGF) were obtained from mouse hybridoma cell lines. They were highly specific for bFGF from bovine, human, and mouse sources and did not cross-react with bovine heparin-binding growth factor I (HBGF-I)/acidic fibroblast growth factor (aFGF). The immunoglobulin class and subclass of these mAbs were IgG1, K. The apparent dissociation constant (Kd) for bFGF of these mAbs ranged from 10(-9) to 10(-10) M. One mAb (bFM-2) also cross-reacted with heat-inactivated bFGF, while the other mAb (bFM-1) did not, suggesting that bFM-1 recognized the conformation of the bFGF molecule necessary for its biological activity. These mAbs inhibited growth of cultured bovine capillary endothelial cells in both the presence and absence of exogenous bFGF, indicating the autocrine action of this growth factor in in vitro growth of these cells. On the other hand, injection of these hybridoma cell lines s.c. into the backs of athymic mice resulted in development of highly vascularized solid tumors and a sustained high level of anti-bFGF activity in the blood of the tumor-bearing mice. These findings suggest that bFGF is not essential as an autocrine or paracrine growth factor for angiogenesis in vivo. These mAbs should be useful in further studies on the physiological role and the conformation-function relationship of bFGF because they block its biological activity.
Publication
Journal: Oncogene
August/30/2012
Abstract
Pancreatic ductal adenocarcinomas (PDACs) exhibit multiple molecular alterations and overexpress heparin-binding growth factors (HBGFs) and glypican-1 (GPC1), a heparan sulfate proteoglycan that promotes efficient signaling by HBGFs. It is not known, however, whether GPC1 has a role in genetic mouse models of PDAC. Therefore, we generated a GPC1 null mouse that combines pancreas-specific Cre-mediated activation of oncogenic Kras (Kras(G12D)) with deletion of a conditional INK4A/Arf allele (Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox);GPC1(-/-) mice). By comparison with Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox) mice that were wild type for GPC1, the Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox);GPC1(-/-) mice exhibited attenuated pancreatic tumor growth and invasiveness, decreased cancer cell proliferation and mitogen-activated protein kinase activation. These mice also exhibited suppressed angiogenesis in conjunction with decreased expression of messenger RNAs encoding several pro-angiogenic factors and molecules, including vascular endothelial growth factor-A (VEGF-A), SRY-box containing gene (SOX17), chemokine C-X3-C motif ligand 1 (CX3CL1) and integrin β3 (ITGB3). Moreover, pancreatic cancer cells isolated from the tumors of GPC1(-/-) mice were not as invasive in response to fibroblast growth factor-2 (FGF-2) as cancer cells isolated from wild-type mice, and formed smaller tumors that exhibited an attenuated metastatic potential. Similarly, VEGF-A and FGF-2 did not enhance the migration of hepatic endothelial cells and immortalized murine embryonic fibroblasts isolated from GPC1 null mice. These data demonstrate in an oncogenic Kras-driven genetic mouse model of PDAC that tumor growth, angiogenesis and invasion are enhanced by GPC1, and suggest that suppression of GPC1 may be an important component of therapeutic strategies in PDAC.
Publication
Journal: Tissue engineering
September/27/2006
Abstract
Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.
Publication
Journal: Molecular and Cellular Biology
August/17/1989
Abstract
Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.
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