Transforming growth factor-β inhibits cellular adenylate cyclase activity in cultured human arterial endothelial cells

Summary Stimulation of human arterial endothelial cells with heparin-binding growth factor-1 (HBGF-1) resulted in a 40% to 60% increase in the cellular adenylate cyclase activity and intracellular cAMP content. The stimulatory effect of HBGF-1 was effectively suppressed by pretreating the cells with transforming growth factor-β (TGF-β), an endothelial cell growth inhibitor. The inhibition of the adenylate cyclase activity precedes growth inhibition by at least 24 h. The half maximal inhibitory dose was calculated to be 0.2 ng/ml for the inhibition of both cyclase activity and cell growth. The possible role of the adenylate cyclase suppression in growth inhibition by TGF-β is discussed. This work was supported in part by grants from NCI (CA 37589), RJR Nabisco, Inc. and Kyowa Hakko Kogyo, Co., Ltd. Editor's Statement The observation that heparin-binding growth factor activates adenylate cyclase in endothelial cells and TGF beta lowers cAMP levels in endothelial cells treated with heparin-binding growth factor raises the possibility that growth control may be mediated, at least partially, through cyclic nucleotides in this system, as well as raising questions about relationships between activities of these peptide growth factors and G protein activation.     

Potentiation of the activity of mouse liver-derived HBGF-1-like growth factor by heparin and dithiothreitol: evidence for the involvement of a plasma factor.

The potentiation of mouse liver derived heparin binding growth factors 1 and 2 (HBGF-1, HBGF-2) activity has been investigated. It was found that both heparin and various sulfhydryl reagents (such as dithiothreitol, DTT) markedly potentiated HBGF-1 activity, but not HBGF-2 activity. Further studies with HBGF-1 indicated that the growth factor would interact with a plasma factor, in a temperature-dependent manner, to become inactive, and that sulfhydryl reagents would reverse this inactivation. Inactivation would not occur either in the presence of heparin or DTT, indicating that heparin and DTT can protect the growth factor from plasma inactivation. When assayed in the absence of plasma, both heparin and DTT were required to reactivate plasma inactivated HBGF-1-ML. A model is presented to explain these data. This model predicts that either DTT or heparin can block the plasma induced inactivation process, but that once inactivation has occurred only sulfhydryl reagents can restore activity. Furthermore, heparin is thought to activate growth factor activity in the absence of plasma by blocking non-productive growth factor binding to the extracellular matrix. The identification of a plasma inactivating factor for mouse liver derived HBGF-1 has important implications for understanding the regulation of extracellular growth factor activity.     

Differential inhibitory effects of TGF-beta on EGF-, PDGF-, and HBGF-1-stimulated MG63 human osteosarcoma cell growth: possible involvement of growth factor interactions at the receptor and postreceptor levels

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.     

Receptor phenotype underlies differential response of hepatocytes and nonparenchymal cells to heparin-binding fibroblast growth factor type 1 (aFGF) and type 2 (bFGF)

Summary Heparin-binding fibroblast growth factors (HBGF) have been implicated in the regeneration of both parenchymal and nonparenchymal cells of the liver. The response to and phenotype of hepatocyte receptors for HBGF-1 (acidic fibroblast growth factor) and HBGF-2 (basic fibroblast growth factor) were compared to keratinocytes, fibroblasts, and endothelial cells. HBGF-1 stimulated DNA synthesis in hepatocytes, keratinocytes, fibroblasts, and endothelial cells whereas activity of HBGF-2 was limited to fibroblasts and endothelial cells. HBGF-2 antagonized the mitogenic activity of HBGF-1 for hepatocytes and keratinocytes. Hepatocytes and keratinocytes exhibited both high- and low-affinity, nonmatrix receptor sites for HBGF-1, but only low-affinity sites for HBGF-2. The mesenchymal cells displayed only high-affinity sites for both HBGF-1 and HBGF-2. Northern blot and immunochemical analysis revealed that the expression of HBGF receptor genesbek andflg are partitioned between normal hepatocytes and nonparenchymal cells, respectively. Expression of epithelial cell-specific, mesenchymal cell-derived HBGF-7 (keratinocyte growth factor) mRNA in regenerating liver tissue was undetectable relative to HBGF-1. The results support a multifunctional role of HBGF-1 acting through different receptor phenotypes in hepatocyte and nonparenchymal cells during liver regeneration.     

Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding.

A range of chemically modified heparins was examined for their ability to bind heparin-binding growth factor 1 (HBGF-1; acidic fibroblast growth factor) and potentiate the in vitro mitogenic and neurotrophic activity of HBGF-1. It was found that carboxyl-reduced heparin bound HBGF-1 as effectively as the native heparin molecule. Totally desulfated heparin and N-desulfated heparin lack HBGF-1-binding capacity, and substitution of the exposed amino group with acetyl or acetoacetyl groups only partially restored binding capacity, indicating that N-sulfates only play a limited role in growth factor binding. However, the failure of totally desulfated, N-resulfated heparin to interact with HBGF-1 demonstrated that N-sulfates alone are insufficient and ester sulfates are absolutely essential for HBGF-1 binding. In contrast, the ability of the modified heparins to potentiate the mitogenic activity of HBGF-1 correlated only to a limited extent with their affinity for HBGF-1. Thus, the carboxyl-reduced molecule which displayed similar affinity for HBGF-1 as native heparin was consistently less potent in augmenting mitogenesis. Similarly, the N-acetylated and the N-acetoacetylated species, which had much lower affinity for HBGF-1 than the carboxyl-reduced molecule, conferred similar biological activity to HBGF-1 whereas N-desulfated heparin, which was unable to bind growth factor, potentiated the mitogenic activity of HBGF-1 for both 3T3 and HUVE cells. In contrast, the neurotrophic activity of HBGF-1 was potentiated by modified heparin species which failed to bind HBGF-1 and were without activity in the mitogenic assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

High and low affinity binding of heparin-binding growth factor to a 130-kDa receptor correlates with stimulation and inhibition of growth of a differentiated human hepatoma cell

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.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

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.     

Fibronectin,not laminin,mediates heparin-dependent heparin-binding growth factor type I binding to substrata and stimulation of endothelial cell growth

Summary Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [¹²⁵I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [¹²⁵I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [¹²⁵I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors. This work was carried out in the laboratory of Dr. W. L. McKeehan and supported in part by grants CA37589, DK35310 and DK38639 from the Public Health Service, Department of Health and Human Services, Washington, DC.     

Regulatory effects of platelet-derived growth factor-AA homodimer on migration of vascular smooth muscle cells.

Migration of medial smooth muscle cells (SMC) into the intima is important in intimal thickening of atherosclerotic tissues. To study the functions of three isoforms of platelet-derived growth factor (PDGF) in atherosclerosis, we investigated their effects on SMC migration by Boyden's chamber method. Although PDGF-AB and PDGF-BB enhanced SMC migration dose-dependently, PDGF-AA did not enhance SMC migration, but instead inhibited SMC migration induced by PDGF-AB or PDGF-BB. PDGF-AA also inhibited SMC migration induced by two other migration factors, fibronectin and SMC-derived migration factor. PDGF-AA is considered to be coexpressed with transforming growth factor (TGF)-beta 1 in atherosclerotic tissues. Treatment of SMC with TGF-beta 1 reduced an autocrine migration activity from SMC. Studies using anti-PDGF antibody revealed that an increased secretion of PDGF-AA by TGF-beta 1 caused the reduced migration activity. cAMP increase by forskolin and dibutyryl cAMP suppressed SMC migration, whereas cAMP decrease by pertussis toxin had no effects on PDGF-AA-suppressed migration. In contrast, staurosporine, an inhibitor of protein kinase C, enhanced SMC migration and neutralized the inhibitory effect of PDGF-AA. These findings suggest that PDGF-AA regulates SMC migration in intimal thickening in atheroma formation and that protein kinase C may play an important role in the inhibitory mechanism of PDGF-AA.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

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.     

Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells

Cultured rat bladder smooth muscle cells (SMC) were grown oncollagen-coated silicone membranes and subjected to continuous cyclesof stretch-relaxation. Semiquantitative RT-PCR analysis revealed atime-dependent increase in heparin-binding epidermal growth factor(EGF)-like growth factor (HB-EGF) mRNA levels after stretch, withmaximal levels appearing after 4 h. Immunostaining for proHB-EGFrevealed higher levels of HB-EGF protein in the stretched than in thenonstretched SMC. The ANG II receptor type 1 antagonist losartanmarkedly suppressed stretch-activated HB-EGF expression. ANG II levelswere 3.3-fold higher in the stretch- than in thenon-stretch-conditioned media. Stretch stimulation of bladder SMC thathad been transiently transfected with an HB-EGF promoter-luciferaseexpression construct resulted in an 11-fold increase in reporteractivity. Mechanical stretch induced a 4.7-fold increase in tritiatedthymidine incorporation rate, and this was reduced by 25% in thepresence of losartan. We conclude that mechanical stretch activatesHB-EGF gene expression in bladder SMC and that this is mediated in partby autocrine ANG II secretion.

     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

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.     

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

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.     

Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis.

We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1). The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1. In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells. Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus. Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants. These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.