Prostatic growth factor: purification and structural relationship to basic fibroblast growth factor

Prostatic growth factor (PrGF) was purified from alkaline homogenates of human benign prostatic hyperplastic tissue by a combination of ammonium sulfate precipitation, heparin affinity chromatography, and cation-exchange chromatography. The 17,600-dalton, basic (pI 10.2) PrGF is related to basic fibroblast growth factor (bFGF) since antisera raised against synthetic peptides with sequence homologies corresponding to an internal peptide and amino- and carboxyl-terminal peptides of bFGF react with the growth factor. The growth factor appears larger than bFGF, suggesting that additional amino-terminal sequences may be present as a result of alkaline extraction in the presence of protease inhibitors.     

Production of homogeneous basic fibroblast growth factor by specific enzymatic hydrolysis of larger microheterogeneous molecular forms.

The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies. However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus. The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E. coli. Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms. This process takes advantage of the protecting effect that heparin exerts on bFGF. Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form. Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF.     

Direct evidence for methylation of arginine residues in high molecular weight forms of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. Protein sequence analysis of bFGF isolated from tissue sources initially established that it is composed of 146 amino acids (apparent Mr 18,000). More recently larger apparent molecular weight forms have been identified and partially characterized. In addition, these high molecular weight forms (apparent Mr 22,000 and 25,000) have been shown to localize preferentially to nuclear fractions of transfected cells. In this report we demonstrate that the higher molecular weight, amino terminally extended forms of bFGF contain methylated arginine residues. The demonstration is based on 1) amino acid sequence analysis of a protein known to contain methylated arginine (myelin basic protein) and a comparison with amino acid sequence analysis of trypsin-derived fragments of the high molecular weight bFGF purified from guinea pig brain and 2) the ability to label in vivo the high molecular weight forms of bFGF with S-adenosyl-L-(methyl-3H)-methionine, the substrate of arginine-protein methylase I. These results are suggestive of a role of arginine methylation in directing nuclear localization of certain forms of bFGF.     

The hyaluronan-binding protease upregulates ERK1/2 and PI3K/Akt signalling pathways in fibroblasts and stimulates cell proliferation and migration

The hyaluronan-binding protease (HABP) is a serine protease in human plasma which is structurally related to plasminogen activators, coagulation factor XII and hepathocyte growth factor activator. It can in vitro activate the coagulation factor FVII, kininogen and plasminogen activators. The present study was initiated to gain a more complete picture of the cell-associated activities of this fibrinolysis-related protease. Treatment of lung fibroblasts with HABP lead to a rapid activation of signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway with c-Raf, MEK and ERK1/2. Additionally the activation of the PI3K/Akt pathway and of several translation-related proteins was found. Proliferation assays confirmed the assumption of a strong growth-stimulating effect of HABP on human lung and skin fibroblasts. Intracellular signalling and growth stimulation were strongly dependent on the proteolytic activity of HABP. Stimulation of signalling and proliferation by HABP involved the fibroblast growth factor receptor 1 (FGFR-1). HABP-stimulated proliferation of lung fibroblasts MRC-5 was accompanied by a significant intracellular increase in basic fibroblast growth factor (bFGF), the major ligand of FGFR-1; bFGF could however not be identified in the supernatant of HABP-treated cells. Though, the conditioned medium from HABP-treated cells showed a strong growth-promoting activity on quiescent fibroblasts, indicating the release of a yet unknown growth factor amplifying the initial growth stimulus. In a two-dimensional wound model HABP stimulated the invasion of fibroblasts into a scratch area, adding a strong pro-migratory activity to this plasma protease. In summary, HABP exhibits a significant growth factor-like activity on quiescent human lung and dermal fibroblasts. Our findings suggest that this fibrinolysis-related plasma protease may participate in physiologic or pathologic processes where cell proliferation and migration are pivotal, like tissue repair, vascular remodelling, wound healing or tumor development.     

HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.     

Endothelial proteoglycans inhibit bFGF binding and mitogenesis

Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG-A and PG-B. The larger sized fraction (PG-A) had greater inhibitory activity than did PG-B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG-A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG-B. Both proteoglycan fractions contained perlecan-like core proteins; however, PG-A contained an additional core protein (approximately 190 kDa) that was not observed in PG-B. Both proteoglycan fractions bound bFGF directly, and PG-A bound a significantly greater relative amount of bFGF than did PG-B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF-induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis. J. Cell. Physiol. 172:209–220, 1997. © 1997 Wiley-Liss, Inc.     

Combined administration of naked DNA vectors encoding VEGF and bFGF enhances tissue perfusion and arteriogenesis in ischemic hindlimb

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.     

Isolation of an amino terminal extended form of basic fibroblast growth factor

Extraction of bovine pituitaries in the presence of enzyme inhibitors (2 mM PMSF, 2 mM sodium tetrathionate, 15 microM pepstatin A, and 1 mM EDTA) resulted in the isolation of two distinct forms of basic fibroblast growth factor. Partial characterization of both molecules showed one form to be identical to basic FGF(1-146) which has already been reported by our laboratory. The second form was estimated by SDS-PAGE to have a molecular weight of 17,000 Daltons which is slightly larger than that of basic FGF(1-146). Amino acid analysis shows the presence of 8 new residues more than basic FGF(1-146) which accounts for the difference in molecular weight. Gas-phase sequencing of this molecule indicated that it bears a blocked amino terminus. Furthermore, this higher molecular weight form of basic FGF did not show immunoreactivity with antibodies specific for the amino terminus of basic FGF(1-146) but cross reacted with antibodies generated against midportion fragments of basic FGF(1-146), indicating that the molecule is amino terminally extended. Like basic FGF(1-146), the molecule is a potent mitogenic factor for vascular endothelial cells. Taken together these results demonstrate the existence of a precursor form of basic FGF which is extended by 8 residues at the amino terminus with the first residue being blocked.     

Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor

Monoclonal antibodies (McAbs) were developed that identify the complete (1-146 aa) and the NH2-terminal truncated (des 1-15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1-15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine-brain-derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2-terminal of complete bFGF. McAb 42 recognizes a "core" epitope found on both the complete and des 1-15 aa bFGFs. The McAbs are murine IgGs with affinity constants of 10(7)-10(8) liter/M for bovine-pituitary-derived bFGF. McAbs 8 and 42 have been used in a two-site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine-brain-derived aFGF. These McAbs should be useful in distinguishing the native and des 1-15 aa forms of bFGF from each other, and from aFGF and other growth factors.     

Divergence in Signaling Pathways Involved in Promotion of Cell Viability Mediated by bFGF,NGF, and EGF in PC12 Cells

We employed a series of inhibitors of intracellular cascade to disclose the precise molecular mechanisms by which basic fibroblast growth factor (bFGF) promotes viability of PC12 cells and compared with nerve growth factor (NGF) and epidermal growth factor (EGF). The MEK 1 and 2 inhibitors, U0126 and PD98059, significantly suppressed cell viability mediated by bFGF in a dose-dependent manner, and to a greater extent compared with EGF and NGF. The degree of MEK dependency for growth factor-mediated cell viability was estimated to be in the order of bFGF, EGF, and NGF. Rapamycin strongly inhibited the effect of NGF on cell viability, compared with bFGF and EGF. The mechanisms of action of NGF-mediated cell viability may depend largely on p70 S6 kinase-related signal transduction pathways comparing to bFGF and EGF. The present findings suggest that different signal transduction systems may be involved in the molecular mechanisms by which bFGF, NGF, and EGF mediate cell viability.     

Selective impairment of the synthesis of basic fibroblast growth factor binding domains of heparan sulphate in a COS cell mutant defective in N-sulphotransferase

N-Sulphation is a key step in the overall sulphation of heparansulphate. We have isolated a COS cell-derived mutant, CM-15,that is impaired in its ability to bind to basic fibroblastgrowth factor (bFGF) and has a 2- to 3-fold reduction in N-sulphotransferaseactivity [Ishihara et al., (1992a) Anal. Biochem., 206, 400–407].We now provide structural evidence that CM-15 is selectivelyimpaired in the synthesis of highly sulphated regions or ‘blocks’that display high-affinity binding to bFGF; these are completelyN-sulphated blocks of decasaccharide or greater length thatare enriched in O-sulphate groups. The synthesis of sulphatedblocks that did not show high affinity to the growth factorwas relatively unimpaired in the mutant cells; this includedfully N-sulphated octamer (or smaller) blocks and, unexpectedly,decasaccharide or larger blocks that were poorly O-sulphated.In the latter fraction, the failure to form high-affinity bindingregions was the result of a failure to stimulate O-sulphationrather than N-sulphation in CM-15 cells. In agreement with otherstudies, disaccharide analysis of the wild-type-derived sulphatedblocks suggested that 2-O-sulphation of iduronate residues inthe polymer was a necessary element to produce a high-affinitybinding sequence once N-sulphation was completed in the decasaccharideor larger fraction. These results suggest that a selective reductionin both N- and O-sulphation in the larger blocks produced byCM-15 cells is a consequence of the reduction of N-sulphotransferaseactivity. These data provide a potential mechanism for regulatingthe synthesis of high-affinity bFGF binding domains in the heparansulphate of mammalian cells. basic fibroblast growth factor COS cell mutant heparan sulfate N-sulphotransferase     

Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.     

A novel mechanism in maggot debridement therapy: protease in excretion/secretion promotes hepatocyte growth factor production

Maggot debridement therapy (MDT) is effective for treating intractable wounds, but its precise molecular mechanism, including the association between MDT and growth factors, remains unknown. We administered MDT to nine patients (66.3 ± 11.8 yr, 5 male and 4 female) with intractable wounds of lower extremities because they did not respond to conventional therapies. Significant increases of hepatocyte growth factor (HGF) levels were observed in femoral vein blood during 48 h of MDT (P < 0.05), but no significant change was found for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). We conducted NIH-3T3 cell stimulation assay to evaluate the relation between HGF and protease activity in excretion/secretion (ES) derived from maggots. Compared with the control group, HGF was significantly higher in the 0.05 μg/ml ES group (P < 0.01). Furthermore, protease inhibitors suppressed the increase of HGF (P < 0.05). The HGF expression was increased in proportion to the ES protein concentration of 0.025 to 0.5 μg/ml. In fact, ES showed stronger capability of promoting HGF production and less cytotoxicity than chymotrypsin or bromelain. HGF is an important factor involved in cutaneous wound healing. Therefore, these results suggest that formation of healthy granulation tissue observed during MDT results from the increased HGF. Further investigation to identify molecules enhancing HGF expression by MDT will contribute greatly to drug target discovery for intractable wound healing therapy.     

Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines.

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.     

Characterization of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells

The receptor for basic fibroblast growth factor (bFGF) was characterized in highly enriched cultures of fetal hippocampal neurons. Two major components of binding could be distinguished. One component comprising about 70% of total binding was removed by 2 M NaCl, and by analogy to other cells could be presumptively attributed to glycosaminoglycans. The remaining 30% of binding which was resistant to 2 M NaCl reflects a high affinity receptor. Scatchard analysis indicated that the two components have Kd values of 500 and 120 pM, and densities of 165,000 and 35,000-60,000 sites/neuron, respectively. Cross-linking 125I-bFGF to neuronal cultures with disuccinimidyl suberate labeled a major membrane protein of 135 kDa and a minor protein of 85 kDa. Examination of neuronal cultures derived from multiple brain regions and membrane preparations from fetal brain suggested that the larger protein was widely distributed. The neuronal bFGF receptor was not blocked by heparin at concentrations up to 100 micrograms/ml. Twelve synthetic peptide fragments of bFGF were examined to determine the domain of bFGF interacting with the neuronal receptor. Inhibition was observed chiefly with a peptide including the sequence 103-146. The properties of the neuronal bFGF receptor were compared directly to those of the receptors characterized on BHK cells and other mesenchymal cells. Specific differences observed between neuronal and mesenchymal bFGF receptors are discussed.     

Glucosylceramide Synthesis Is Required for Basic Fibroblast Growth Factor and Laminin to Stimulate Axonal Growth

Abstract: To test the hypothesis that neuronal growth requires the synthesis and supply of new membrane components to the growing neurite, we have examined the relationship between the synthesis of sphingolipids and the ability of two growth factors, basic fibroblast growth factor (bFGF) and laminin, to stimulate axonal growth in cultured hippocampal neurons. Both bFGF and laminin stimulate axonal growth by approximately fourfold, but the stimulatory effects of both factors can be abolished completely by two inhibitors of sphingolipid synthesis, fumonisin B₁ and d - threo -1-phenyl-2-decanoylamino-3-morpholino-1-propanol. By using these inhibitors, together with two stereoisomers of short acyl chain derivatives of ceramide, only one of which is metabolized to glucosylceramide, we demonstrate that ongoing synthesis of glucosylceramide, the simplest glycosphingolipid, is a prerequisite for both bFGF and laminin to stimulate axon growth. These data imply that the ability of a growth factor to stimulate neuronal growth is dependent on the synthesis of an essential membrane lipid.     

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.     

Aberrant Expression of Basic Fibroblast Growth Factor in NIH-3T3 Cells Alters Drug Resistance and Gene Amplification Potential

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.     

COX inhibitors modulate bFGF-induced cell survival in MCF-7 breast cancer cells

Basic fibroblast growth factor (bFGF) serves as a modulator of survival in breast cancer cells. The mechanisms by which bFGF transduces the anti-apoptotic signal and interacts with COX inhibitors were investigated. bFGF reduced apoptosis in MCF-7 breast cancer cells and up-regulated the expression of mitocondrial Bcl-2, whereas COX inhibitors meloxicam (selective COX-2) and aspirin (non-selective), induced apoptosis. bFGF up-regulated survivin protein expression and induced cdc-2 phosphorylation moderately at early (2-6 h), and substantially at late (24 h), time-points. Survivin mRNA expression was up-regulated only at the later time-point. COX inhibitors prevented up-regulation of survivin protein expression at both 2 and 24 h and prevented early modest increases in cdc-2 phosphorylation. Up-regulation of survivin mRNA was not found to be modulated by the COX-2 inhibitor meloxicam. bFGF regulation of survivin expression was found to be ERK1/2 kinase dependent and bFGF-induced phosphorylation of c-raf was prevented by the COX-2 inhibitor. bFGF was, however, unable to induce COX-2 protein expression or modulate COX-2 activity in MCF-7 cells as evidenced by unaltered PGE(2) production. These results indicate that bFGF regulates survivin expression in MCF-7 breast cancer cells by signaling through an ERK1/2 dependent pathway. COX-2 inhibitors can modulate bFGF-induced survivin expression in a COX-2 independent manner.     

Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming growth factor-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the fibroblast-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.