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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.

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.

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.

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.

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.

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.

Platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) decrease high affinity binding of 125I-labeled epidermal growth factor (EGF) and potentiate mitogenesis in BALB/c 3T3 cells, and both have been shown to induce the phosphorylation of the EGF receptor at threonine residues. These similarities suggest that the actions of PDGF on EGF binding may be mediated by protein kinase C, the cellular effector of PMA. We show that in density-arrested BALB/c 3T3 cells PDGF and PMA induce a rapid, transient, cycloheximide-independent loss of EGF binding activity. As has been previously shown for PDGF, the ability of PMA to reduce EGF binding was enhanced by cholera toxin, a potent activator of adenylate cyclase. In contrast to PMA, however, PDGF induced a further reduction in EGF binding that was strictly dependent upon continued protein synthesis. Furthermore, PDGF effectively reduced EGF binding in cells refractory to PMA. Cells desensitized to PMA, presumably due to the loss of protein kinase C activity, also remained mitogenically responsive to PDGF. These data suggest that the mechanism by which PDGF modulates EGF binding differs from that of PMA and thus, at least in part, is independent of protein kinase C.

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 (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.