Elevated 80K-H protein in breast cancer: a role for FGF-1 stimulation of 80K-H.

An increase in fibroblast growth factor-1 (FGF-1) is established as part of the cause of several important cancers including breast cancer, but the mechanisms by which it induces malignant behavior are not known. We now report that the protein 80K-H, a substrate for PKC, appears to be part of this mechanism and that it is increased in breast cancer and localizes to the nucleus as part of the mechanism. Our conclusion is based on an examination of a total of 58 biopsy specimens from human breast cancer patients for the presence of relationships between the 80K-H protein and the following: fibroblast growth factor receptor-1 (FGFR-1), tumor grade, microvessel counts (MVC), estrogen receptor (ER) and progesterone receptor (PgR) status. Based on histological grading and immunohistochemical (IHC) assays, we found strong direct relationships between 80K-H and FGFR-1 (r = 0.49, p = 0.003) and tumor grade (r = 0.42, p = 0.006). A trend for a direct relationship was observed with PgR (r=0.27, p=0.087). Notably, 80K-H immunostaining was largely limited to the epithelial cells of the mammary ducts. Subsequently, we studied the effects of FGF-1 on 80K-H in cultured human mammary carcinoma epithelial cells in order to establish a more direct relationship between these two molecules. We observed that FGF-1 treatment of MCF-7 cells stimulated translocation of 80K-H protein to the cell nucleus, as demonstrated by subcellular fractionation studies. Maximal intranuclear 80K-H was observed approximately 30 minutes following FGF-1 treatment. In addition, FGF-1 treatment of MCF-7 cells increased growth and invasion of MCF-7 cells, as demonstrated by cell proliferation and a modified Boyden chamber assay, respectively. Further support for 80K-H nuclearization was provided by the immunostaining of human breast cancer specimens and computer-assisted identification of a putative nuclear localization signal (NLS) near the amino terminus of 80K-H protein structure. These data support the existence of a previously unrecognized FGF-1/80K-H nuclear pathway in progression of human breast cancer and suggest that 80K-H may be useful for the assessment of breast tumor progression.

Construction, expression, and characterization of a baculovirally expressed catalytic domain of human matrix metalloproteinase-9.

We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration.

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta.

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.

Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.

Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery.

Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium-specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus-mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS-expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs. Nomega-Nitro-L-arginine (L-NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon-injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3-fold increase in vessel diameter) at 2 weeks compared with LXSN SMC-seeded arteries. Orally administered L-NA blocked these changes. Phosphorylation of vasodilator-stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC-seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo.

Metalloproteinase blockade by local overexpression of TIMP-1 increases elastin accumulation in rat carotid artery intima.

We have recently demonstrated that the blockade of matrix metalloproteinases by local overexpression of the intrinsic inhibitor tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) reduces intimal hyperplasia. We now report a major change in the elastin content of the intima of rat carotid arteries seeded with TIMP-1-overexpressing smooth muscle cells. To understand the mechanism responsible for elastin accumulation, synthesis and degradation of elastin in TIMP-1 and control cell-seeded rats were measured. There were no differences in elastin mRNA or elastin synthesis, as documented by 14[C]proline incorporation between TIMP-1 and control cell-seeded arteries. In contrast, there was an increase in cross-linked elastin in the TIMP-1 group. In addition, in TIMP-1 and control rats, an elastase activity of approximately 28 kD was detected by elastin zymography and was decreased in TIMP-1 cell-seeded vessels. The 28 kD elastolytic activity was inhibited by exogenously added TIMP-1 and EDTA but not by PMSF, suggesting that it was a metalloelastase. Therefore, we have demonstrated that a shift of the proteolytic balance toward protease inhibition by TIMP-1 overexpression does not change elastin synthesis but rather changes posttranslational processing, resulting in increased elastin accumulation.

Plasminogen activator inhibitor type 1 and tissue inhibitor of metalloproteinases-2 increase after arterial injury in rats.

Vascular injury induced by angioplasty causes smooth muscle cells to migrate, proliferate, and form a neointima. The neointima is further enlarged by the accumulation of matrix molecules synthesized by smooth muscle cells. Smooth muscle cell migration and matrix accumulation are associated with an increase in the expression of matrix-degrading enzymes and might be regulated by the balance of protease and anti-protease activity. We have studied the inhibitors of two major classes of matrix-degrading enzymes, the plasminogen activators and the matrix metalloproteinases (MMPs) to understand better the regulation of proteolytic activity following balloon catheter injury in the rat carotid artery. At various times after injury, protease inhibitor expression was analyzed by Northern blotting, reverse zymography, immunohistochemistry, and Western blotting. During the first month after injury, we found that the expression of two proteinase inhibitors (plasminogen activator inhibitor type 1 [PAI-1] and tissue inhibitor of metalloproteinases-2 [TIMP-2]) was modulated. PAI-1 mRNA expression reached a maximum 6 hours after injury before tapering off to baseline levels by 3 days. PAI-1 activity, as measured by reverse zymography, followed the same temporal profile. PAI-1, localized by immunohistochemistry, was expressed at low levels in the media of control arteries and was increased after injury primarily in the medial smooth muscle cells. TIMP-2 mRNA levels began to increase 24 hours after injury and reached a maximum at day 7. TIMP-2 activity, measured by reverse zymography, peaked at day 3 after injury. TIMP-2 protein was increased in the intima compared with the media and adventitia at day 7 after injury. The increase of PAI-1 and TIMP-2 after injury supports the hypothesis that changes in the proteolytic balance play an important role in smooth muscle cell migration after arterial injury.

Overexpression of tissue inhibitor of matrix metalloproteinase-1 inhibits vascular smooth muscle cell functions in vitro and in vivo.

Arterial smooth muscle cells (SMCs) are in a quiescent growth state under normal physiological conditions, but they can be stimulated to proliferate and migrate from one tissue compartment to another if the vessel is injured. This response might require a selective and focal increase in tissue degradation, which might be mediated through the increased production of matrix metalloproteinases (MMPs). Blockade of MMP activity might therefore inhibit the SMC response to injury. To test this hypothesis, we developed clones of rat SMCs that overexpress baboon tissue inhibitor of matrix metalloproteinase-I (TIMP-1), using retrovirally mediated gene transfer, and characterized the functional capacity of these cells in vitro and in vivo. SMCs transduced with the TIMP-1 vector (LTSN) grew more slowly and also migrated through a gel matrix in a Boyden chamber assay more slowly than the vector alone (LXSN) cells. The conditioned medium from LTSN cells completely inhibited the platelet-derived growth factor-BB-induced migration of normal SMCs across a matrix-coated filter, while the LXSN cell conditioned medium had no effect. The inhibitor activity in the LTSN conditioned medium could be neutralized with an antibody to TIMP-1. In vivo, local overexpression of TIMP-1 using LTSN cells implanted onto balloon-injured rat carotid artery inhibited intimal hyperplasia. Neutralizing antibodies against TIMP-1 suppressed the effect of LTSN cell seeding on intimal thickening. These data support the conclusion that the process of SMC activation leading to a thickened intima is dependent on MMP activity and that TIMP-1 could be utilized to inhibit this process.

Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus.

The traditional method of antibody (Ab) generation requires repeated injections of antigen (Ag). We have developed an alternative method that allows an investigator to generate a polyclonal antiserum with only a cDNA in hand. We cloned a cDNA encoding the coding frame for baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Fischer rat arterial smooth muscle cells (SMC) transduced with the baboon TIMP-1 using a replication-defective retrovirus were propagated in culture. TIMP-1 overexpressing rat SMC were seeded into de-endothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the presence of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-linked immunosorbent assay in 5 of 6 of the animals. The major portion of the Ab generated against baboon TIMP-1 during the 12-month monitoring period after the cell seeding was identified as belonging to the IgG1 subtype. More interestingly, the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab are its capacity to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab against a secreted protein can be obtained in response to continuous expression of the cDNA by vascular SMC. Purified Ag is not required.

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy.

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.

Cloning and characterization of a cDNA encoding the baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).

A baboon aortic smooth muscle cell (SMC) cDNA library was screened for the presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by polymerase chain reaction (PCR); oligodeoxyribonucleotide primers corresponding to the coding frame of the known human TIMP-1 gene were used as primers. Sequencing of the PCR-amplified baboon cDNA demonstrated only eight single-nucleotide (nt) mismatches, when compared with the coding frame of human TIMP-1. The authenticity of the PCR-amplified TIMP-1 cDNA was further confirmed by clonal screening of the library with the PCR probe and sequencing of positive clones. On Northern blots from cultured baboon SMC, the baboon cDNA hybridized to a TIMP-1-specific mRNA of 800 bp. Phorbol ester (PMA) treatment of cultured baboon SMC produced a 2.5-fold increase in TIMP-1 transcript. TIMP-1 transcripts were also demonstrated in cultures of endothelial cells and fibroblasts obtained from baboon arteries. Immunohistochemical analysis demonstrated that TIMP-1 protein is localized to the adventitial layer of baboon artery. We conclude that TIMP-1 is a conserved molecule across species and localized to the tunica adventitia of baboon vessels.

Angiogenesis-directed implantation of genetically modified endothelial cells in mice.

By virtue of their location within blood vessels and their ability to express foreign genes, endothelial cells are attractive vehicles for the delivery of therapeutic molecules in vivo. We wished to determine whether i.v.-injected, genetically modified endothelial cells can become incorporated into sites of active angiogenesis in vivo. To do so, we studied the fate of i.v.-injected, lacZ-expressing human umbilical vein endothelial cells in athymic nude mice bearing lethally irradiated NIH 3T3 murine fibroblast cells transfected with a sp-hst/KS3:fibroblast growth factor-1 chimera that forces the secretion of the angiogenic protein, fibroblast growth factor-1. Following i.v. injection, lacZ-labeled human umbilical vein endothelial cells accumulated at sites of fibroblast growth factor-1-induced angiogenesis, persisting for at least 4 weeks. These results suggest that i.v.-administered, genetically modified endothelial cells can migrate into and survive within an angiogenic site. This strategy may be useful for delivery of therapeutic molecules to sites of pathological angiogenesis during tumor metastasis.

Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo.

The prototype members of the heparin-binding fibroblast growth factor (FGF) family, acidic FGF (FGF-1) and basic FGF (FGF-2), are among the growth factors that act directly on vascular cells to induce endothelial cell growth and angiogenesis. In vivo, the role of the FGF prototypes in vascular pathology has been difficult to determine. We report here the introduction, by direct gene transfer into porcine arteries, of a eukaryotic expression vector encoding a secreted form of FGF-1. This somatic transgenic model defines gene function in the arterial wall in vivo. FGF-1 expression induced intimal thickening in porcine arteries 21 days after gene transfer, in contrast to control arteries transduced with an Escherichia coli beta-galactosidase gene. Where there was substantial intimal hyperplasia, neocapillary formation was detected in the expanded intima. These findings suggest that FGF-1 induces intimal hyperplasia in the arterial wall in vivo and, through its ability to stimulate angiogenesis in the neointima, FGF-1 could stimulate neovascularization of atherosclerotic plaques. Potentially, gene transfer of FGF-1 could also be used as a genetic intervention to improve blood flow to ischaemic tissues in selected clinical settings.

Differential transforming abilities of non-secreted and secreted forms of human fibroblast growth factor-1.

Fibroblast growth factor (FGF)-1(1-154), the precursor for acidic FGF-1(21-154), is a potent angiogenic polypeptide, the structure of which lacks a signal peptide sequence for secretion. To investigate the biological significance of this structural feature, we have attempted forced secretion of FGF-1 through fusion of the entire FGF-1 coding frame with the signal peptide (sp) from the hst/KS3 gene, a secretory member of the heparin-binding growth factor family. We also studied the transforming ability of the signal-less forms of FGF-1 comprising FGF(1-154) and FGF-1(21-154). The presence of a soluble and biologically active form of FGF-1 was readily detected in the conditioned medium of NIH 3T3 cells transfected with sp-hst/KS3:FGF-1(1-154) as demonstrated by Western blot analysis and DNA synthesis assays, whereas sp-hst/KS3:FGF-1(21-154) was not detectable in conditioned medium even though the protein was detected in cellular extracts. The secreted form of sp-hst/KS3:FGF-1(1-154) stimulated the proliferation of human umbilical vein endothelial cells in vitro and was able to induce receptor-mediated tyrosine phosphorylation. Furthermore, the forced secretion of biologically active FGF-1 resulted in NIH 3T3 cell transformation as demonstrated by altered morphology in vitro, the formation of discrete colonies in soft agarose, growth under serum-free conditions, and ability to rapidly form highly vascular tumors in vivo. Interestingly, sp-hst/KS3:FGF-1(21-154) also mediated the transition to a transformed phenotype despite the inability to detect extracellular FGF-1 in the media conditioned by these NIH 3T3 cell transfectants. Although the transfection of FGF-1(21-154) yielded similar NIH 3T3 cell morphologic changes, these transfectants did not grow under serum-free conditions or yield colonies in soft agarose, and formed tumors in vivo with delayed kinetics. Furthermore, the FGF-1(1-154) NIH 3T3 cell transfectants did not exhibit morphologic changes, and this may be due to the inability of mRNA to express protein. These data suggest that although non-sp forms of FGF-1 may alter the monolayer phenotype of NIH 3T3 cells in vitro, the ability of FGF-1 to transform NIH 3T3 cells requires the function of a sp-directed secretory pathway and suggests that this pathway increases tumorigenicity in vivo.

Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells.

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen.

Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure.

Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield. However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter.