Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12-O-tetradecanoylphorbol-13-acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants

12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated protein (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin-growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of our investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR-9 cell line is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Using [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, we found that growing cultures of 3T3 and TNR-9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. By contrast, the responses of quiescent TNR-9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. The changes in production of MRP in response to tumor promoters in quiescent and growing cells paralleled similar changes in the level of MRP mRNA. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulate DNA synthesis in quiescent 3T3 and TNR-9 cells. Evidently the biochemical condition that distinguishes TNR-9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR-9 cells occurs only when the cells are quiescent.     

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.     

Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell-shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts.     

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.     

Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH

Peptide growth factors selectively increase the amount of mitogen-regulated protein (MRP) and major excreted protein (MEP) released by mouse 3T3 cells. $\text{Balb}\text{c}$ 3T3 cells release mainly MEP and Swiss 3T3 cells release mainly MRP. Fibroblast growth factor, epidermal growth factor, nerve growth factor, serum, and concanavalin A increase the extracellular appearance of both MEP and MRP, but to different extents. Several agents that have been shown to, or would be expected to increase, intralysosomal pH also selectively increase the release of MEP and MRP from both $\text{Balb}\text{c}$ and Swiss 3T3 cells. The effective agents are monensin, nigericin, ammonium chloride, methylamine, chloroquine, and high extracellular pH.     

TGF-beta stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF-related peptides

Transforming growth factor-beta (TGF-beta) stimulates DNA synthesis in human foreskin fibroblasts after a prolonged lag period as compared with other growth factors. The mechanism of induction of DNA synthesis appears to be dependent on the synthesis and secretion of PDGF-related proteins as antibodies which are specific for PDGF can block the TGF-beta-induced DNA synthesis. Other growth factors such as PDGF, EGF, or FGF do not induce the synthesis of these PDGF-related proteins. Additionally, TGF-beta treatment of human foreskin fibroblasts induces the expression of the PDGF A-chain gene but not the B-chain gene. This phenomenon appears to function in vivo, as subcutaneous injection of TGF-beta in rat skin induces the expression of the PDGF A-chain gene. These data suggest that TGF-beta may stimulate the growth of fibroblastic cells via an autocrine production of PDGF-related proteins.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Specific induction of secreted proteins by transforming growth factor-beta and 12-O-tetradecanoylphorbol-13-acetate. Relationship with an inhibitor of plasminogen activator

To examine the mechanisms by which transforming growth factors (TGFs) regulate the proliferation of eukaryotic cells, five cell lines, from different species and tissues, were treated with three agents that inhibit DNA synthesis and proliferation: BSC-1 cell-derived growth inhibitor (GI/TGF-beta), platelet-derived transforming growth factor-beta (TGF-beta), and 12-O-tetradecanoylphorbol-13-acetate. The cell lines tested were mink lung CCL 64 epithelial cells, Maloney sarcoma virus-transformed CCL 64.1, monkey kidney BSC-1 epithelial cells, human epidermoid A431 cells, and mouse embryo AKR-2B (clone 84A) cells. All cell lines responded to one or more of these agents by synthesizing and secreting a 48 to 51-kDa protein (IIP48). The TGF-beta s and 12-O-tetradecanoylphorbol-13-acetate had little or no effect on the incorporation of [35S] methionine into other secreted proteins or on the pattern of [35S]methionine-labeled intracellular proteins analyzed by one-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximum increase in induction of IIP48 varied from 2-fold to greater than 800-fold compared with the controls and occurred within 6 h of adding GI/TGF-beta to CCL 64 cells. Actinomycin D, alpha-amanitin, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole selectively decreased both the control and induced levels of IIP48 even after as little as 6 h of incubation. Thus, it appears that IIP48 mRNA turns over rapidly. Induction of IIP48 was dissociated from the inhibition of DNA synthesis by GI/TGF-beta. However, we found that epidermal growth factor and GI/TGF-beta act synergistically to increase the secreted level of IIP48. Others have shown that epidermal growth factor and TGF-beta act synergistically to stimulate growth of cells in agar. IIP48 from CCL 64, BSC-1, and AKR-2B cells is specifically immunoprecipitated by antibody to bovine plasminogen activator inhibitor. We found previously that TGF-beta also inhibits the production of major excreted protein, a thiol protease. It is proposed that TGF-beta is able to promote anchorage-independent growth of untransformed cells because of its ability to inhibit the production of secreted proteases and to increase the production of protease inhibitors.     

Mitogen response and cell cycle kinetics of Swiss 3T3 cells in defined medium: differences from human fibroblasts and effects of cell density

The mitogen requirement and proliferative response of Swiss 3T3 cells in serum-free, chemically defined culture medium were compared with those of early-passage human diploid fibroblasts. The effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, transferrin, and dexamethasone on cell-cycle parameters were measured using 5'-bromo-deoxyuridine-Hoechst flow cytometry. Swiss 3T3 cells differ from human fibroblasts in several ways: (1) Swiss 3T3 cells showed a much higher dependence on PDGF than human fibroblasts; the growth of the latter, but not of the former, could be stimulated by the combination of EGF, insulin, and dexamethasone to the full extent of that when PDGF was present; (2) in the absence of PDGF, insulin was an absolute requirement for Swiss 3T3 cells to initiate DNA synthesis, while a substantial proportion of human fibroblasts could enter DNA synthesis without exogenous insulin or IGF-I; and (3) in the absence of PDGF, increasing insulin concentration increased the cycling fraction of Swiss 3T3 cells without an appreciable effect on the rate of cell exit from G0/G1, while under similar culture conditions, insulin showed its major effect on regulation of the G1 exit rate of human fibroblasts, without much effect on the cycling fraction. In addition, the proliferative response of high-density versus low-density, arrested Swiss 3T3 cells showed that the interaction of mitogens varied with cell density. At high cell density, the PDGF requirement was consistent with the "competence/progression" cell-cycle model. This growth response was not seen, however, when cells were plated at low density.     

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.     

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Inability of anti-epidermal growth factor receptor monoclonal antibody to block "autocrine" growth stimulation in transforming growth factor-secreting melanoma cells

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

The major excreted protein of transformed fibroblasts is an activable acid-protease

Malignant transformation of mouse cells by a variety of agents or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate or platelet-derived growth factor results in increased synthesis and secretion of a 39,000-dalton protein termed major excreted protein (MEP). We report here that secreted MEP is an acid-activable protease. The secreted precursor form of the protease is auto-activated at low pH and is able to digest a variety of proteins, including the extracellular matrix proteins fibronectin, collagen, and laminin. MEP protease activity has pH optimum of 3.3-3.6 and is temperature- and concentration-dependent. The activity is inhibited by sulfhydryl protease inhibitors such as leupeptin and iodoacetic acid and not by metallo-, seryl-, or carboxyprotease inhibitors. The MEP-derived protease has characteristics distinct from the cathepsins previously reported and thus may be a new acid-protease of mouse cells.     

Transforming growth factor β alters plasminogen activator activity in human skin fibroblasts

Adult human skin fibroblasts were used as a model to study the effects of transforming growth factor beta (TGFβ) on the secreted plasminogen activator (PA) activity of cultured cells. TGFβ, at nanogram concentrations, enhanced the secretion of pro-PA from two fibroblast strains in a time- and dose-dependent manner. The induced enzymatic activity was inhibited by anti-urokinase antibodies and it co-migrated with purified urokinase in polyacrylamide gels. The secretion of PA activity was abolished when cycloheximide (0.1 μg/ml) was added to the cultures. The activity was thus dependent on protein synthesis rather than just on direct activation of a plasminogen proactivator. TGFβ had only a slight mitogenic effect on the test cells. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were ineffective alone in inducing PA. Insulin, on the contrary, had an inhibitory effect on the TGFβ-induced PA activity. In addition to its effects on the secretion of PA, TGFβ enhanced the production of a proteinase inhibitor by these cells. The results suggest a role for TGFβ in the regulation of PA activity and pericellular proteolysis in fibroblastic cells.     

Modulation of the transport of a lysosomal enzyme by PDGF

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.