Cell-mediated Co-action of Transforming Growth Factors : Incubation of Type with Normal Rat Kidney Cells Produces a Soluble Activity that Prolongs the Ruffling Response to Type

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed N R K cells. In the present study, we examined the roles of the transforming growth factors TGF-a and TGF-~3 in the induction and maintenance of ruffling in untransformed N R K cells, and observed the following: TGF-a caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-a. TGF-B caused no ruffling and did not itself prolong TGF-a ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-~ with N R K cells for 6-h extended the duration of maximal TGF-a-induced ruffling by several-fold. This study demonstrates that TGF-a alone causes an EGF-like, transient ruffling response, but neither TGF-a or TGF-B alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-a acts in concert with a new, TGF-/3-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGFa-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy. S EVERAL nontransforming polypeptide growth factors, including insulin (8), epidermal growth factor (EGF) l (5, 6, 9, 10), platelet-derived growth factor (19), fibroblast growth factor (see companion paper by Myrdal and Auersperg), and nerve growth factor (6), cause transient ruffling on sparsely cultured cells. In all cases, ruffling subsides within ~ 1 h, even in the continued presence of the factor, indicating a cellular off-regulation mechanism that limits growth factor responses. This off-regulation is at least somewhat factor specific. For example, PC I2 cells previously exposed to, and therefore refractory to, EGF still ruffle when exposed to nerve growth factor (6). Similarly, normal rat kidney (NRK) cells respond to insulin after a saturating EGF exposure (unpublished observation). In a model presented previously (see companion paper) we suggested that ruffling of cultured cells may reflect a cellular mechanism by which nutrient transport molecules are inserted into the plasma ~ Ahbreviations used in this paper: EGF, epidermal growth factor: KNRK, Kirsten murine sarcoma virus-transformed NRK: NRK, normal rat kidney: TGF-~, transforming growth factor, type a; TGF-/L transforming growth factor. type #. membrane from intracellular storage sites. In untransformed cells, this mechanism appears to be tightly and specifically regulated. Numerous transformed cells (1, 12, 16), including Kirsten murine sarcoma virus-transformed NRK cells (KNRK) (14), ruffle continuously in sparse culture. We reported previously that KNRK cells secrete their own ruffling-stimulating agent(s) (companion paper), which cause ruffling in both untransformed NRK cells and in KNRK cells made quiescent by a pH increase. This ruffling does not cease for at least several hours and thus is not subject to the normal cellular off-regulation that limits the response of cells to nontransforming growth factors. Because this ruffling is similar to that caused by growth factors, except for the duration, and also because the previously described transforming growth factors TGF-a and TGF-~ (3, 20, 22; for review see reference 20) are secreted by KNRK cells (15), we have studied and characterized the roles of these two factors in the ruffling response. Those studies are described in this paper. Ruffling is used here as a rapid, real-time monitor of an early cellular response to growth factors. A significant advan© The Rockefeller University Press, 0021-9525/86/04/1230/05 $1.00 The Journal of Cell Biology. Volume 102. April 1986 1230-1234 1230 on A uust 7, 2017 jcb.rress. org D ow nladed fom tage of real-time monitors is that this kind of approach often permits dissection of component mechanisms that are obscured in an assay employing a longer, after-the-fact biological effect, such as growth in soft agar, thymidine uptake, or cell number increase. In particular, study of the ruffling response has permitted us to explore the phenomenon of normal cellular off-regulation of a growth factor-induced response, and the escape from this off-regulation by transforming growth factors. Materials and Methods NRK cells (7) were cultured in sparse monolayers (10% fetal bovine serum, Waymouth's medium MB752/1, 37"C, 5% CO2 atmosphere). On day 1 after passage, complete medium was replaced with nutrient deficient Waymouth's medium, made without glucose, amino acids, and vitamins (i.e., a buffer with the same salt composition as Waymouth's medium), After 2-4 h, live cells were examined by inverted phase microscopy using a 25× objective lens for total magnification of ~250 x. We have already detailed and validated the quantification method (companion paper). In brief, ruffling was scored just before and at intervals after addition of the ruffling agent. 60-100 cells were observed per count. Intervals between counts were 3-5 rain right after the agent was added and increased to 15-20 rain later. In this study the following modification was employed. Ruffling was quantified by visual scan of several areas, recording numbers of cells without any marginal ruffles (a), with < 10% of the free margin ruffled (b) and with >-10% of the free margin ruffled (c). A weighted percentage of ruffled cells was calculated according to the equation (b/n + c)/(a + b + c) x 100 = percent ruffled cells, with a, b, and c defined above and n a small integer. We found that n = 5 distinguished large responses from smaller responses without obliterating the smallest responses and that value was used in all the graphs presented below. Curves were drawn with the aid of an Apple lie computer programmed to convert the values a, b, and c of individual readings into weighted percentages as just described and to plot those values against time in minutes from addition of the ruffling agent. An averaging program generated composite curves from two or more repetitions of the same experiment. In brief this program functioned as follows. After the individual curves to be averaged were selected, they were plotted onto the same graph, The computer then scanned along the horizontal (time) axis until coming to a point at which any one of the curves had an actual data point. At that time point, the computer scanned vertically and averaged the points intersected by all of the curves being considered. Alternatively, the computer scanned the horizontal axis and averaged the points along the vertical axis at every minute. Both methods produced essentially the same composite curves, but the former was much faster, so it was used to produce the composites presented here. Composite curves rather than data point averages were determined because counts were obtained in rapid sequence, and, therefore, time points at which cells were counted differed somewhat from one experiment to another, so data could not be averaged at specific time points. Individual curves were usually quite similar to the composites obtained, and individual data points never varied from the composite by >10%. In all cases, when one experimental variable was compared with another, the composite curves within a comparison were all derived from the same set of experiments. The differences obtained among the experimental variables presented here were quantitatively reproducible, observed at least five times, and never failed to occur. EGF was obtained from Collaborative Research Inc., Lexington, MA. Rat TGF-c~ was purified from Snyder-Theillen feline sarcoma virus-transformed Fisher rat embryo cell (25) culture supernatants as previously described (13). TGF-~. lot 784, was a generous gift of Drs. H. L. Moses and R. Tucker. Antiserum to TGF-c~ was prepared against a synthetic peptide corresponding to the COOH-termina] ] 7 amino acids of rat TGF-a. The antiserum recognizes both small and large forms of rat and human TGF-a and distinguishes TGF-c~ from the functionally related peptides EGF and urogastrone (11). Total responses to factors, or combinations of factors, were quantified by measuring the area under the response curve over the period of interest with the prefactor (untreated) ruffling value used as the baseline. A Zeiss MOP-3 was used to measure areas.