Deacetylation of cortactin by SIRT1 promotes cell migration.

Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express less-acetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2alpha (the mouse homolog of SIRT1) migrated more slowly than did wild-type cells. The expression of SIRT1 in sir2alpha-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.

Activation of p38 mitogen-activated protein kinase is required for tumor necrosis factor-alpha -supported proliferation of leukemia and lymphoma cell lines.

To elucidate mechanisms of tumor necrosis factor alpha (TNF-alpha)-induced proliferation of a number of human leukemia and lymphoma cell lines, we examined the role of p38 mitogen-activated protein kinase (MAPK) in TNF-alpha signaling in Mo7e and Hut-78 cells. TNF-alpha-dependent p38 MAPK activation was detected in both Mo7e and Hut-78 cells and was blocked by the p38 MAPK inhibitor, SB203580. Ablation of p38 MAPK activity by SB203580 abrogated TNF-alpha-induced Mo7e cell proliferation and TNF-alpha-dependent autocrine growth of Hut-78. As we have shown previously that activation of the nuclear factor kappaB (NF-kappaB) is also required for TNF-alpha-induced Mo7e cell proliferation, the involvement of p38 MAPK in NF-kappaB activation was assessed. SB203580 did not affect TNF-alpha-signaled nuclear translocation and DNA-binding activity of NF-kappaB, and inhibition of NF-kappaB function did not affect TNF-alpha-induced p38 MAPK activation, indicating that these events are not dependent on each other. However, SB203580 depressed the expression of NF-kappaB-dependent genes, as monitored by a kappaB-driven reporter gene. Our findings demonstrate that activation of both p38 MAPK and NF-kappaB plays a critical role in TNF-alpha-mediated survival and proliferation of human leukemia and lymphoma cells, and p38 MAPK acts at least in part by facilitating the transcriptional activation function of NF-kappaB.

TNF-alpha-induced growth suppression of CD34+ myeloid leukemic cell lines signals through TNF receptor type I and is associated with NF-kappa B activation.

Conflicting results have been reported regarding the effect of TNF-alpha on the growth of human primitive hemopoietic cells. In this study, we have examined the effect of TNF-alpha on the proliferation of several CD34+/CD38+ (KG-1, TF-1) and CD34+/CD38- (KG-1a, TF-1a) myeloid leukemic progenitor cell lines. Our data show that TNF-alpha markedly inhibits the growth of these cells in both liquid and soft agar cultures. Addition of GM-CSF or IL-3 does not prevent TNF-alpha-induced growth inhibition. Flow cytometry analyses of propidium iodide-stained cells demonstrated cell death of all four cell lines, as judged by the presence of cells with hypodiploid DNA content after exposure of cells to TNF-alpha for 4 days. Annexin V assays detected apoptosis in TF-1, but not in TF-1a, KG-1, and KG-1a cells in terms of translocation of phosphatidylserine shortly after TNF-alpha treatment. Neutralizing anti-TNF receptor type I (TNFR-I; p55) Ab almost completely reversed TNF-alpha-induced growth inhibition in both liquid and soft agar cultures, whereas anti-TNFR-II (p75) Ab had only a marginal effect. TNF-alpha rapidly induced marked activation of nuclear transcription factor NF-kappa B in all 4 cell lines. The majority of this effect was abolished by the type I receptor Ab, whereas the type II receptor neutralizing Ab had no effect. Our data also show that TNF-alpha is incapable of inducing activation of the mitogen-activated protein kinase pathway in these leukemic cell lines.

Tumor necrosis factor-alpha-induced proliferation of human Mo7e leukemic cells occurs via activation of nuclear factor kappaB transcription factor.

Tumor necrosis factor-alpha (TNF-alpha) stimulates proliferation of Mo7e, CMK, HU-3, and M-MOK human leukemic cell lines. We report here the signal transduction pathway involved in TNF-alpha-induced Mo7e cell proliferation. Mo7e cells spontaneously die in the absence of growth factors, but treating the cells with interleukin (IL)-3, IL-6, thrombopoietin, granulocyte/macrophage colony-stimulating factor, or TNF-alpha promotes their survival and proliferation. Although most of these factors activate MAP kinase and Jun NH2-terminal kinase/signal transducer and activators of transcription signaling pathways, TNF-alpha fails to activate either pathway. When Mo7e cells were treated with TNF-alpha, nuclear factor kappaB (NF-kappaB) was activated transiently. The activated NF-kappaB consisted of heterodimers of p65 and p50 subunits. The degradation of IkappaBalpha coincided with activation of NF-kappaB in TNF-alpha-treated cells. To investigate the role of activated NF-kappaB in TNF-alpha-induced Mo7e proliferation, a cell-permeable peptide (SN50) carrying the nuclear localization sequence of p50 NF-kappaB was used to block nuclear translocation of activated NF-kappaB. Pretreating Mo7e cells with SN50 blocked TNF-alpha-induced nuclear translocation of NF-kappaB and inhibited TNF-alpha-induced Mo7e cell survival and proliferation. A mutant SN50 peptide did not affect TNF-alpha-induced Mo7e cell growth. SN50 had no effects on IL-3- or granulocyte/macrophage colony-stimulating factor-induced Mo7e cell proliferation. The results indicate that activation of NF-kappaB is involved in TNF-alpha-induced Mo7e cell survival and proliferation.

Constitutive RelB activation in v-Src-transformed fibroblasts: requirement for IkappaB degradation.

RelB, an NF-kappaB/Rel-related transacting factor, was initially identified as an immediate-early gene product in fibroblasts and subsequently shown to exhibit constitutive DNA binding activity in lymphoid cells. The data presented in this report show that RelB is also constitutively active, as monitored by electrophoretic mobility shift assay, in the v-Src-transformed fibroblast cell line, SR1. By contrast, nontransformed parental (3Y1) cells displayed inducible NF-kappaB activity; RelB activity was also observed, although to a lesser extent, in two additional v-Src-transformed fibroblast lines. RelB activation in SR1 cells did not require an increase in RelB expression or result from a decrease in the levels of IkappaB alpha or p105, proteins previously shown to bind to and inhibit the activity of the Rel proteins. Numerous studies have shown that stimulus-dependent Rel activation requires degradation of IkappaB alpha, p105 or other member of the IkappaB family, and that this process is precluded by agents that inhibit proteasome activity. We show that treatment of SR1 cells with proteasome inhibitors abolishes RelB activity and thus suggest that RelB in these cells is associated with IkappaB and that v-Src transformation activates RelB by accelerating IkappaB proteolysis. Additional data show that serum and tumor necrosis factor-alpha (TNF-alpha) increase RelB protein levels in 3Y1 cells and that this process is blocked by proteasome inhibitors.

Transactivation by expression of the hepatitis B virus X protein with an inducible system.

We describe here the conditional expression of the hepatitis B virus X protein using the inducible system controlled by a tet-responsive promoter. Induction of the X protein in Rat-2 fibroblasts activated transcription from a heterologous gene promoter and stimulated the DNA-binding activity of NFkB. The ability to produce this biologically active X protein in a stable cell line will accelerate the elucidation of the function and mechanisms of X and eventually help us understand the role of X in natural viral infection and carcinogenesis.

Inducible activation of RelB in fibroblasts.

RelB, a member of NF-kappaB/Rel family of transcriptional regulators, is an immediate-early gene product that mediates constitutive lymphoid-specific kappaB-directed gene expression. Data presented here show that RelB also functions as a mitogen-inducible DNA binding activity in fibroblasts. Activation of RelB was observed in three mouse fibroblast lines, and agents activating RelB in quiescent and cycling cells included platelet-derived growth factor, phorbol ester, and serum. Additional data implicate cyclic AMP in the regulation of RelB activity. These findings suggest that RelB may play different roles in different cell types.

Participation of reactive oxygen species in the lysophosphatidic acid-stimulated mitogen-activated protein kinase kinase activation pathway.

Recent evidence suggests that reactive oxygen species (ROS) may function as second messengers in intracellular signal transduction pathways. We explored the possibility that ROS were involved in lysophosphatidic acid (LPA)-induced mitogen-activated protein (MAP) kinase signaling pathway in HeLa cells. Antioxidant N-acetylcysteine inhibited the LPA-stimulated MAP kinase kinase activity. Direct exposure of HeLa cells to hydrogen peroxide resulted in a concentration- and time-dependent activation of MAP kinase kinase. Inhibition of catalase with aminotriazole enhanced the effect of LPA on induction of MAP kinase kinase. Further, LPA stimulated ROS production in HeLa cells. These findings suggest that ROS participate in the LPA-elicited MAP kinase signaling pathway.

Characterization of platelet-derived growth factor alpha receptor synthesis and metabolic turnover.

Cellular responses to the AA isoform of platelet-derived growth factor (PDGF-AA) are mediated via the PDGF alpha receptor. Several studies suggest this receptor may signal pathways distinct from those activated by the PDGF beta receptor. Because alpha receptors are less well characterized than are beta receptors, and because the quantity of cell surface PDGF receptors governs the extent and perhaps type of PDGF-stimulated response, we examined the synthesis and degradation of alpha receptors in BALB/c-3T3 cells. Our data show that the ligand-independent half-life of alpha receptors is 3 h and that optimal turnover of alpha receptors requires protein synthesis. In the presence of ligand, the half-life of alpha receptors markedly decreases and is independent of protein synthesis. Although PDGF-AA accelerated the rate of alpha receptor turnover, pretreatment of cells with PDGF-AA and essentially complete down-regulation of alpha receptors did not correspondingly increase the level of alpha receptor synthesis. These findings indicate that the number of cell surface PDGF alpha receptors is regulated by the rate of internalization of these receptors. Lastly, we report that the recovery of PDGF-AA binding following down-regulation of alpha receptors is not affected by inhibition of RNA synthesis. Thus, repopulation of cell surface PDGF alpha receptors may not necessitate an increase in the level of PDGF alpha receptor mRNA.

Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.

Transforming growth factor beta 1 and cAMP inhibit transcription of epidermal growth factor- and oncogene-induced transin RNA.

Transin mRNA encodes a secreted metalloprotease which is transcriptionally induced in Rat-1 cells by epidermal growth factor (EGF) and a number of oncogenes. A role for transin in tumor progression is suggested by its overexpression in malignant and metastatic tumors compared to their benign counterparts. In an effort to elucidate mechanisms by which elevated transin expression may be inhibited, it has been determined that both transforming growth factor type beta 1 (TGF beta 1) and increased levels of intracellular cyclic 5'-adenosine monophosphate (cAMP) inhibit EGF and oncogene induction of transin mRNA. The inhibition of transin mRNA occurred at the level of transcription as demonstrated by nuclear run-on assays. EGF binding studies in Rat-1 cells showed no significant effect of cAMP or TGF beta 1 on EGF receptor number or affinity. We have also examined the effects of cAMP and TGF beta 1 on oncogene-induced transin using Rat-1 cells transformed by temperature-sensitive mutants of v-src and K-ras oncogenes. Both inhibitors prevented the induction of transin RNA as well as decreased the levels of transin once elevated at the permissive temperature. Despite the similarities in the actions of TGF beta 1 and cAMP on transin gene expression, TGF beta 1 treatment did not significantly elevate intracellular cAMP levels, thus making it unlikely that cAMP is a second messenger system for TGF beta 1 action. These studies suggest that the inhibitory effects of cAMP and TGF beta 1 occur by distinct pathways at the level of gene regulation.

Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylmethylxanthine.

Epidermal growth factor (EGF) stimulated the formation of inositol trisphosphate, inositol bisphosphate, and inositol phosphate in density-arrested BALB/c/3T3 cells pretreated for 1.5-4 h with cholera toxin, a potent activator of adenyl cyclase, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Concomitant addition of cholera toxin, IBMX, and EGF to cells did not increase inositol phosphate levels, and pretreatment with both agents was more effective than pretreatment with either alone. Pre-exposure of cells to cholera toxin and IBMX also enhanced the increase in inositol phosphates occurring in response to platelet-derived growth factor (PDGF). Preincubation of cells with cholera toxin and IBMX in the presence of cycloheximide abolished the effects of these agents on EGF- and PDGF-stimulated inositol phosphate production as well as the lesser increase in inositol phosphate formation produced by cholera toxin and IBMX in the absence of hormone. Preincubation of cells with cycloheximide did not affect EGF binding or the ability of PDGF to stimulate inositol phosphate formation. Cycloheximide also precluded EGF-induced inositol phosphate production when presented to cells 3 h after addition of cholera toxin and IBMX. These findings show that, under the appropriate conditions, EGF is capable of stimulating inositol phosphate formation in a nontransformed cell line.

Control of late G0/G1 progression and protein modification by SmC/IGF I.

Somatomedin C/insulin-like growth factor I (SmC/IGF I) mediates traverse of late G0/G1 in density-arrested BALB/c-3T3 cells from a distinct growth arrest point in mid-G0/G1 (the V point) to the initiation of DNA synthesis. As a prelude to future studies aimed at defining the mechanism of action of SmC/IGF I, we investigated the level (e.g., transcriptional, translational) at which SmC/IGF I modulates V to S traverse. The post-V point progression of cells arrested at the V point by amino acid starvation and released into amino acid-replenished medium containing SmC/IGF I, insulin, or platelet-poor plasma (PPP) did not require either mRNA synthesis or an increase in the overall level of protein synthesis. Although two-dimensional gel analysis of proteins prepared from SmC/IGF I-treated cells did not reveal any preferentially synthesized proteins, several SmC/IGF I-induced protein modifications, which result in an increase in isoelectric point (pI) and occur in the absence of mRNA synthesis, were evident. These findings suggest that SmC/IGF I modulates late G0/G1 progression by a posttranscriptional process that may involve protein modification.

Heterologous regulation of EGF receptors in fibroblastic cells.

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.