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While extracellular acidification within solid tumors is well-documented, how reduced pH impacts regulation of insulin-like growth factor-I (IGF-I) has not been studied extensively. Because IGF-I receptor binding is affected by IGF binding proteins (IGFBPs), we examined how pH impacted IGFBP-3 regulation of IGF-I. IGF-I binding in the absence of IGFBP-3 was diminished at reduced pH. Addition of IGFBP-3 reduced IGF-I cell binding at pH 7.4 but increased surface association at pH 5.8. This increase in IGF-I binding at pH 5.8 corresponded with an increase in IGFBP-3 cell association. This, however, was not due to an increase in affinity of IGFBP-3 for heparin at reduced pH although both heparinase III treatment and heparin addition reduced IGFBP-3 enhancement of IGF-I binding. An increase in IGF-I binding to IGFBP-3, though, was seen at reduced pH using a cell-free assay. We hypothesize that the enhanced binding of IGF-I at pH 5.8 is facilitated by increased association of IGFBP-3 at this pH and that the resulting cell associated IGF-I is IGFBP-3 and not IGF-IR bound. Increased internalization and nuclear association of IGF-I at pH 5.8 in the presence of IGFBP-3 was evident, yet cell proliferation was reduced by IGFBP-3 at both pH 5.8 and 7.4 indicating that IGFBP-3-cell associated IGF-I does not signal the cell to proliferate and that the resulting transfer of bound IGF-I from IGF-IR to IGFBP-3 results in diminished proliferation. Solution binding of IGF-I by IGFBP-3 is one means by which IGF-I-induced proliferation is inhibited. Our work suggests that an alternative pathway exists by which IGF-I and IGFBP-3 both associate with the cell surface and that this association inhibits IGF-I-induced proliferation., (Copyright 2001 Wiley-Liss, Inc.)

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.

Insulin-like growth factor-I (IGF-I) increases extracellular acidification rate (ECAR), a measure correlated with proliferation for nonautocrine cells. To evaluate the effect of autocrine IGF-I secretion on cell responsiveness, a cell line that secretes IGF-I was tested. SV40-lGF-I cells also registered concentration-dependent increases in ECAR; however, unlike the parental cell line, signal attenuation upon repeat challenges was not evident. Furthermore, SV40-IGF-I cells did not proliferate in response to IGF-I. We investigated if lack of proliferation was due to differences in the protocols of the assays ([3H]thymidine incorporation and microphysiometry). We identified three key differences in the protocols: surface substrate, cell density, and fluid residence time. We found no increase in [3H]thymidine incorporation for cells on either tissue-culture plastic or polycarbonate transwells. Control levels of [3H]thymidine incorporation were cell-density-dependent, but IGF-I did not increase proliferation at any density studied. Finally, we investigated IGF-I stimulation for cells under microphysiometer flow conditions and found no proliferative response to IGF-I. We found that the cells do respond to IGF-I with increased amino acid uptake. These data suggest that IGF-I signaling is operational in the SV40-IGF-I cells, but the transduction pathway for IGF-I-induced proliferation is compromised, despite the fact that these cells respond to fetal bovine serum with increased growth. Ongoing studies are focused on identifying which elements in the signaling cascade are altered by autocrine secretion of IGF-I.

Binding of growth factors by cell-surface receptors is an essential means by which cells regulate normal tissue growth and differentiation. Exposure to growth factors is often transient, and our goal was to determine whether short-term exposure to insulin-like growth factor-1 (IGF-1) would lead to activation, assayed as cell proliferation, of mammary epithelial cells. The MAC-T cell line is an immortalized bovine mammary epithelial cell line, chosen as our model mammary cell line because of its known sensitivity to IGF-1. Using the Cytosensor Microphysiometer System, a biosensor capable of measuring extracellular acidification, we were able to measure activation of the cells owing to IGF-1 addition in real time and found that peak acidification occurred in only 14 min. We show that this rapid response to IGF-1 is dose dependent and specific for IGF-1. A significant increase in [3H]thymidine incorporation by cells after a similar short-term exposure to IGF-1 suggests that the measured increase in extracellular acidification following IGF-1 addition is physiologically relevant. This technology offers a new, novel, and rapid means for the study of IGF-1 activity, as well as the screening of IGF-1 inhibitors, in mammary epithelial cells.

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG., (Copyright 2000 Academic Press.)

Basic fibroblast growth factor (bFGF) binds to cell surface tyrosine kinase receptor proteins and to heparan sulfate proteoglycans. The interaction of bFGF with heparan sulfate on the cell surface has been demonstrated to impact receptor binding and biological activity. bFGF receptor binding affinity is reduced on cells that do not express heparan sulfate. The addition of soluble heparin or heparan sulfate has been demonstrated to rescue the bFGF receptor binding affinity on heparan sulfate deficient cells yet has also been shown to inhibit binding under some conditions. While the chemical requirements of the heparin-bFGF-receptor interactions have been studied in detail, the possibility that heparin enhances bFGF binding in part by physically associating with the cell surface has not been fully evaluated. In the study presented here, we have investigated the possibility that heparin binding to the cell surface might play a role in modulating bFGF receptor binding and activity. Balb/c3T3 cells were treated with various concentrations of sodium chlorate, so as to express a range of endogenous heparan sulfate sites, and [(125)I]bFGF binding was assessed in the presence of a range of heparin concentrations. Low concentrations of heparin (0.1-30 nM) enhanced bFGF receptor binding to an extent that was inversely proportional to the amount of endogenous heparan sulfate sites present. At high concentrations (10 microM), heparin inhibited bFGF receptor binding in cells under all conditions. The ability of heparin to stimulate and inhibit bFGF-receptor binding correlated with altered bFGF-stimulated tyrosine kinase activity and cell proliferation. Under control and chlorate-treated conditions, [(125) I]heparin was observed to bind with a high affinity to a large number of binding sites on the cells (K(d) = 57 and 50 nM with 3.5 x 10(6) and 3.6 x 10(6) sites/cell for control and chlorate-treated cells, respectively). A mathematical model of this process revealed that the dual functions of heparin in bFGF binding were accurately represented by heparin cell binding-mediated stimulation and soluble heparin-mediated inhibition of bFGF receptor binding.

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions.

The Cytosensor Microphysiometer, a biosensor developed by Molecular Devices, was used to assay rapid binding activity of IGF-I for bovine mammary epithelial cell lines. Insulin-like growth factor-I (IGF-I) is a potent mitogen for mammary epithelial cells and has been implicated in breast cancer cell proliferation, a leading cause of cancer death of women in the U.S. today. IGF-I acts by binding to cell surface receptors. We are interested in how autocrine secretion might alter the activity and regulation of IGF-I. Real-time changes in excretion of protons, which we assert results from IGF-I binding, are detected by the Cytosensor Microphysiometer and can be correlated with cellular activity. We present IGF-I dose-dependent responses as well as correlated binding data to detect cell surface receptor concentration and thymidine incorporation results to determine cell proliferation following IGF-I stimulation. We examine the effect of insulin-like binding protein-3 (IGFBP-3) both in the presence and absence of IGF-I. We believe comparison of autocrine and paracrine environments for IGF-I stimulation, and the components contributing to the binding of IGF-I to the cell membrane receptor may provide pertinent information for the development of intervention schemes to slow or interrupt IGF-I binding to tumor cells and therefore cancer growth.

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 large 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.

Autocrine ligands regulate important cell behavioral functions in both physiological and pathological conditions. Binding of these ligands to cell-surface receptors involves more subtle considerations than that of exogenous (endocrine or paracrine) ligands. Autocrine secretion leads to a release of molecules in the local microenvironment proximal to the cell surface, thus allowing interaction with receptors to compete directly with diffusive loss to the bulk extracellular medium. Complications in autocrine systems due to this binding vs. transport competition arise in at least three aspects: (i) experimental measurement of autocrine ligand secretion rates is compromised; (ii) kinetics of autocrine ligand binding to cell-surface receptors are difficult to follow; and (iii) inhibition by exogenous blockers of autocrine ligand binding to cell receptors is problematic. At the heart of all these complications is the need to determine the fractional distribution of the secreted autocrine ligand between cell-surface receptor capture and diffusive loss to the bulk media. In this paper we offer a theoretical treatment of this problem using Brownian dynamics simulation techniques to calculate the capture probability of the cell receptors for the autocrine ligand. A major result is that the capture probability is significantly lower than the predicted by the Berg-Purcell steady-state diffusion approach. Another is that the capture probability is essentially independent of release location. Implications of these results for the complications found in autocrine systems are discussed.

Stimulation of cell behavioral functions by ligand/receptor binding can be accomplished in autocrine fashion, where cells secrete ligand capable of binding to receptors on their own surfaces. This proximal secretion of autocrine ligands near the surface receptors on the secreting cell suggests that control of these systems by inhibitors of receptor/ligand binding may be more difficult than for systems involving exogenous ligands. Hence, it is of interest to predict the conditions under which successful inhibition of cell receptor binding by the autocrine ligand can be expected. Previous theoretical work using a compartmentalized model for autocrine cells has elucidated the conditions under which addition of solution decoys for the autocrine ligand can interrupt cell receptor/ligand binding via competitive binding of the secreted molecules (Forsten, K. E., and D. A. Lauffenburger. 1992. Biophys. J. 61:1-12.) We now apply a similar modeling approach to examine the addition of solution blockers targeted against the cell receptor. Comparison of the two alternative inhibition strategies reveals that a significantly lower concentration of receptor blockers, compared to ligand decoys, will obtain a high degree of inhibition. The more direct interruption scheme characteristic of the receptor blockers may make them a preferred strategy when feasible.

Autocrine ligands have been demonstrated to regulate cell proliferation, cell adhesion, and cell migration in a number of different systems and are believed to be one of the underlying causes of malignant cell transformation. Binding of these ligands to their cellular receptors can be compromised by diffusive transport of ligand away from the secreting cell. Exogenous addition of antibodies or solution receptors capable of competing with cellular receptors for these autocrine ligands has been proposed as a means of inhibiting autocrine-stimulated cell behavioral responses. Such "decoys" complicate cellular binding by offering alternative binding targets, which may also be capable of aiding or abating transport of the ligand away from the cell surface. We present a mathematical model incorporating autocrine ligand production and the presence of competing cellular and solution receptors. We elucidate effects of key system parameters including ligand diffusion rate, binding rate constants, cell density, and secretion rate on the ability of solution receptors to inhibit cellular receptor binding. Both plated and suspension cell systems are considered. An approximate analytical expression relating the key parameters to the critical concentration of solution "decoys" required for inhibition is derived and compared to the numerical calculations. We find that in order to achieve essentially complete inhibition of surface receptor binding, the concentration of decoys may need to be as much as four to eight orders of magnitude greater than the equilibrium disociation constant for ligand binding to surface receptors.