Keratinocyte growth factor and fibroblast growth factor action on DNA synthesis in rat and human hepatocytes: modulation by heparin

Keratinocyte growth factor (KGF/FGF-7) is a member of the fibroblast growth factor (FGF) superfamily. Unlike other members of the family, the biological activity of KGF appears to be restricted to epithelial cells. Here we have tested the activity of KGF, acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) on normal adult rat and human hepatocytes and their modulation by heparin. Although more modest than the growth response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF), recombinant KGF enhanced DNA synthesis in rat hepatocytes by two- to threefold. This stimulation occurred in the absence of serum and of other exogenous growth factors. Addition of heparin inhibited the KGF response. Although basic FGF showed little activity on rat hepatocytes, acidic FGF stimulated DNA synthesis by approximately twofold and was substantially enhanced by heparin. In contrast to rat cells, human hepatocytes consistently failed to respond to KGF, aFGF, or bFGF with or without heparin, under conditions where EGF and HGF stimulated DNA synthesis up to sixfold. These results indicate that KGF is capable of acting as a complete mitogen for rat hepatocytes in culture and that the activity is consistent with expression by these cells of a type II FGF receptor subtype, the KGF receptor. These observations suggest that KGF/aFGF together with proteoglycans may help regulate rat but not human liver growth.     

Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7)

A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung. The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7). The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells. In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria. The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM. Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts. rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms. The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody. These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix.     

Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development

Mesenchymal-epithelial tissue interactions are important for development of various organs, and in many cases, soluble signaling molecules may be involved in this interaction. Hepatocyte growth factor (HGF) is a mesenchyme-derived factor which has mitogenic, motogenic and morphogenic activities on various types of epithelial cells and is considered to be a possible mediator of epithelial-mesenchymal interaction during organogenesis and organ regeneration. In this study, we examined the role of HGF during lung development. In situ hybridization analysis showed HGF and the c-met/HGF receptor gene to be respectively expressed in mesenchyme and epithelium in the developing lung. In organ cultures, exogenously added HGF apparently stimulated branching morphogenesis of the fetal lung. In contrast, HGF translation arrest or neutralization assays resulted in clear inhibition of epithelial branching. These results suggest that HGF is a putative candidate for a mesenchyme-derived morphogen regulating lung organogenesis. We also found that HGF is involved in epithelial branching, in collaboration with fibroblast growth factor (FGF) family molecule(s). In mesenchyme-free culture, HGF alone did not induce epithelial morphogenesis, however, addition of both HGF and acidic FGF (aFGF) or keratinocyte growth factor (KGF), ligands for the KGF receptor, induced epithelial branching more extensively than that was observed in explants treated with aFGF or KGF alone. In addition, the simultaneous inhibition of HGF- and FGF-mediated signaling using neutralizing antibody and antisense oligo-DNA resulted in drastic impairment of epithelial growth and branching. Possible interactions between HGF and FGFs or other growth factors in lung development is given consideration.     

Endogenous basic fibroblast growth factor is essential for cyclin E-CDK2 activity in multiple external cytokine-induced proliferation of AIDS-associated Kaposi's sarcoma cells: dual control of AIDS-associated Kaposi's sarcoma cell growth and cyclin E-CDK2 activity by endogenous and external signals

AIDS-associated Kaposi's sarcoma (KS) cell, a key element for development of KS lesions, proliferates in response to external cytokines, such as oncostatin M, the soluble IL-6R-IL-6 complex, TNF-alpha, and IL-1beta. In addition, the KS cell-produced basic fibroblast growth factor (bFGF) was reported to function as an autocrine growth factor. However, little is known of the exact roles of these external growth factors and endogenous bFGF on proliferation of KS cells, and underlying intracellular events have remained to be defined. We obtained evidence that anti-bFGF Ab abolished growth of KS cells by preventing S phase entry of the cell cycle, even in the presence of the external growth factors. Blockade of the FGF action profoundly inhibited cyclin E expression and cyclin-dependent kinase-2 (CDK2) activity, but not D-type cyclin expression and CDK4 activity. Exogenously added acidic FGF (aFGF), which generated a rapid tyrosine phosphorylation of FGFR1 and FGFR2 on KS cells, reversed the inhibitory effects of anti-bFGF Ab. Thus, FGF actions are essential for cyclin E-CDK2 activity and S phase entry. We also observed that the presence of external growth factors markedly induced cyclin E-CDK2 activity and S phase entrance, while the addition of aFGF or bFGF alone was insufficient to induce these responses. All this evidence shows that integration of the activities of external growth factors and endogenous bFGF is required for full activation of cyclin E-CDK2 activity and KS cell proliferation.     

Acidic and basic fibroblast growth factors in human breast tissue

Previously we have reported changes in fibroblast growth factors (FGF) in conditioned medium (CM) derived from rat mammary tumours undergoing remission. We have used a similar approach to assay for the presence of FGFs in human breast tissue and cell lines. The majority of cancer tissues (35/50), benign tissues (8/9) and all cancer adjacent normal tissues (20/20) released heat labile, NR6 transforming activity which coeluted from heparin with acidic FGF (aFGF) at 0.9-1.1 M NaCl and was neutralised by antibodies to aFGF. The conclusion that the majority of breast cancers contain active aFGF was supported by immunoblotting. The CM of a minority (15/50) of cancers and one benign tissue had highly transforming activity for NR6 cells, and was mitogenic for a breast cancer cell line, was heat labile, and strongly heparin binding, eluting at 1.5-2.0 M salt. It was not immunoreactive with antibodies to aFGF, basic FGF (bFGF) or Kaposi's FGF (kFGF) and its activity was reduced by the presence of aFGF, suggesting competition for the same receptor. Very little aFGF was observed in the CM of these tumours, and neither aFGF nor other FGF activity was detected in CM of breast cell lines.     

Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.     

Heparin stimulates the proliferation of bovine aortic endothelial cells probably through activation of endogenous basic fibroblast growth factor

We investigated the effect of heparin on the proliferation of cultured bovine aortic endothelial (BAE) cells. Heparin increased DNA synthesis in BAE cells in a concentration-dependent manner. The DNA synthesis increased by 2 to 2.5-fold with 1 mg/ml of heparin after 48 h incubation without serum and exogenous fibroblast (heparin-binding) growth factors. The stimulating effect of heparin decreased with the diminishing number of monosaccharide units which constitute heparin. By the addition of a neutralizing antibody to basic fibroblast growth factor (bFGF), the stimulating effect of heparin decreased, whereas an antibody to acidic fibroblast growth factor (aFGF) had no effect. The culture medium conditioned by heparin-treated BAE cells stimulated DNA synthesis in Balb/3T3 fibroblasts that proliferate in response to bFGF. The mitogenic activity of the conditioned medium was suppressed by the antibody to bFGF. However, heparin did not increase bFGF mRNA level in BAE cells. These results suggest that heparin stimulates the proliferation of BAE cells by the activation of endogenous bFGF, but not by the induction of its synthesis.     

Growth factor effects on apoptosis of rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric epithelium that show increased density during chronic atrophic gastritis. The current study determined cell number and apoptosis of isolated rat ECL cells in response to several growth factors. Isolated ECL cells from fundic mucosa (enrichment >90%) were grown in serum-free medium over 2-5 days. Cell number was determined by mitochondrial formazan production; apoptosis was measured by Tdt-mediated dUTP nick end labeling reaction and DNA fragmentation-based enzyme-linked immunosorbent assay. Immunocytochemistry and RT-PCR demonstrated the presence of epidermal growth factor receptor, neuronal growth factor receptor (type 1), and fibroblast growth factor (FGF) receptor (type 1). Gastrin (EC50, approximately 2 pM), transforming growth factor-alpha (TGF alpha; 10-30 ng/ml), and basic FGF (bFGF; 1-10 ng/ml) increased the total number of cultured ECL cells. bFGF augmented the gastrin (1 pM)-induced response. Beta-neuronal growth factor (10 ng/ml) and bFGF (2 ng/ml) decreased the programed death of ECL cells. Interleukin-1beta (100 pg/ml, 24 h) stimulated apoptosis 2- to 3-fold in ECL cells, and simultaneous incubation with TGF alpha (20 ng/ml) or bFGF (2 ng/ml) significantly inhibited this effect. ECL cells express specific receptors for gastrin, epidermal growth factor, neuronal growth factor, and FGF. bFGF prolonged ECL cell survival by inhibiting spontaneous apoptosis. Our data further indicate that TGF alpha and bFGF increase ECL cell number by inhibiting cytokine-induced programed cell death.     

Ovarian function in ewes made hypogonadal with GnRH antagonist and stimulated with FSH in the presence or absence of low amplitude LH pulses

Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situ hybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo.     

Sinusoidal capillarization of human hepatocellular carcinoma: possible promotion by fibroblast growth factor

Expression of acidic and basic fibroblast growth factors (aFGF and bFGF) and von Willebrand factor (vWF) was immunohistochemically investigated in 55 nodules of human hepatocellular carcinoma (HCC), 15 nodules of adenomatous hyperplasia (AH) of the liver and 10 cirrhotic livers (LC). AH, a putative preneoplastic lesion in the cirrhotic liver, was subdivided into ordinary and atypical types: the former was characterized by little cellular and structural atypia and the latter had some atypia equivocal as to benignity and malignancy. The positive rates of FGF (aFGF and/or bFGF) were as follows: 0% in LC, 20% in AH (ordinary and atypical) and 42% in HCC, whereas the positivity of vWF was 10% in LC, 20% in ordinary AH, 30% in atypical AH and 40% in HCC. There was no correlation between the expression of FGF or vWF and the size of HCC. No correlation was also found between the positivity of FGF and that of vWF in HCC and atypical AH. While vWF was not constantly expressed in the vicinity of FGF-positive HCC cells, capillarized sinusoids were significantly more numerous in FGF-positive cases than in FGF-negative cases (p < 0.01). These data indicate that FGF may be pathogenetically linked to the multistep development of HCC in relation to sinusoidal capillarization.     

Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth

Primordial germ cells (PGCs) are the embryonic progenitors of mature germ cells. During their proliferative stage, murine PGCs may be transiently cultured on mitotically inactive feeder layers. This culture system has permitted identification of several growth factors active toward PGCs. We and others have previously identified basic fibroblast growth factor (bFGF) as a powerful mitogen in this system. Here we characterize some of the functions of bFGF in PGC culture. Our data demonstrate that fibroblast growth factor (FGF) receptors I and II are present in the developing gonad and are consistent with expression of these receptors by PGCs. Moreover, PGCs can bind radiolabeled bFGF in vitro, demonstrating that the factor can act directly on these cells. While mitotic PGCs of either sex are shown to bind radiolabeled bFGF, oogonia that are undergoing meiotic arrest exhibit reduced bFGF binding, indicating potential developmental regulation of an FGF receptor.     

Regulation of astroglial-derived dopaminergic neurotrophic factors by interleukin-1 beta in the striatum of young and middle-aged mice

Interleukin-1 beta (IL-1 beta) can induce dopaminergic axonal sprouting in the denervated striatum of parkinsonian animals. In order to determine whether IL-1 beta effects on dopaminergic axonal sprouting are mediated by the induction of astroglial-derived dopaminergic neurotrophic factors, effects of IL-1 beta treatment on acidic and basic fibroblast growth factor (aFGF and bFGF) and glial cell line-derived growth factor (GDNF) gene expression were examined in primary striatal astrocyte cultures and after in vivo administration. We found a selective induction of bFGF mRNA synthesis but not aFGF or GDNF mRNA after IL-1 beta treatment both in vitro and in vivo. This suggests that bFGF may be the putative endogenous dopaminergic neurotrophic factor mediating lesion-induced plasticity of dopamine neurons. In addition, to determine why recovery from injury becomes reduced with age, we examined whether there was an aging-associated decline in the ability of IL-1 beta to induce the synthesis of neurotrophic factors in middle-aged animals compared to young mice. Interestingly, IL-1 beta stimulated a greater induction in bFGF mRNA levels in the middle-aged mice compared to young mice. These results suggest that the regulation of bFGF and possibly its receptor signaling efficacy may vary as the brain ages.     

The ContiNet of the International Continence Society

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4 degrees C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-beta 1. Scatchard analysis revealed the presence of approximately 1.0 x 10(5) FGF-2 binding sites per cell with an apparent Kd of 1.2 x 10(-10) M. Interestingly, the binding of [125I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.     

Transforming activity of a newly cloned androgen-induced growth factor

Our previous study demonstrated that androgen-dependent growth of mouse mammary carcinoma cells (SC-3) was mediated through an induction of heparin-binding growth factor, termed as androgen-induced growth factor (AIGF). Here, we report that NIH3T3 cells stably transfected with AIGF expression vector exhibit the abilities of tumor formation in nude mice, focus formation in monolayer culture and colony formation in soft agar. Thus, this newly cloned growth factor can be categorized as an oncogene. In addition, androgen-induced enhancement of DNA synthesis in SC-3 cells can be blocked by simultaneous incubation with AIGF antisense oligonucleotides. The possibility is also addressed that AIGF exerts its biological activity through an interaction with fibroblast growth factor (FGF) receptor. Transfection of expression vector encoding a variant form of FGF receptor-1 cloned from SC-3 cells into FGF receptor-negative L6 cells results in AIGF-dependent inhibition of differentiation. These results demonstrate that the ability of androgen to elicit transformed phenotype of SC-3 cells is mediated through AIGF induction and its interaction with FGF receptor-1.     

Identification of p90, a prominent tyrosine-phosphorylated protein in fibroblast growth factor-stimulated cells, as 80K-H

Tyrosine phosphorylation of cellular proteins occurs rapidly upon treatment of fibroblasts with acidic or basic fibroblast growth factors (aFGF, bFGF), suggesting a role for protein phosphorylation in the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins, of which the most prominent has been designated as p90. The phosphorylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N-terminal amino acid sequence which corresponded to a protein previously named as 80K-H. Polyclonal antibodies raised against the predicted C terminus of 80K-H recognized p90 on all Western blots. p90 was found to bind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphotyrosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not from control fibroblasts. The biological function of 80K-H is yet unknown. However, from this study and a previous observation of the obligatory dependence of p90 phosphorylation on FGF receptor occupation, it appears that 80K-H is involved in FGF signaling.     

Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development

To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.