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T-Kininogen Inhibits Fibroblast Proliferation in the G1 Phase of the Cell Cycle
Experimental Cell Research 269, 171–179 (2001) doi:10.1006/excr.2001.5299, available online at http://www.idealibrary.com on
T-Kininogen Inhibits Fibroblast Proliferation in the G 1 Phase of the Cell Cycle Claudio Torres, Min Li, Robin Walter, 1 and Felipe Sierra 1,2 MCP-Hahnemann University, 2900 Queen Lane, Philadelphia, Pennsylvania 19129; and Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile
By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblastderived cell lines that express biologically active Tkininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G 0 cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G 0 through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G 1 and S phases of the cell cycle. © 2001 Academic Press
Key Words: proliferation; cell cycle; T-kininogen; cysteine proteinases; MAP kinase.
INTRODUCTION
Cysteine proteinases provide a small percentage of the cell’s total proteolytic capabilities, and their virtually complete inhibition results in only a minor decline in total proteolytic rates. Nevertheless, several studies have indicated that they are implicated in the degra1
Current addresses: Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096, and Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile. 2 To whom reprint requests should be addressed. Fax: (562) 7355580. E-mail: [email protected].
dation of key regulatory factors including c-Fos [1], PKC [2], PKA [3], calmodulin [4], integrins [5], p53 [6], and pRb [7]. While some of these molecules are also degraded by the proteasome, alternative pathways, most likely involving calpain, appear to exist. Inhibition of their degradation could potentially lead to aberrant accumulation of these proteins and to a severe disturbance of important cellular functions, such as signal transduction and progression through the cell cycle. Indeed, several reports in the literature indicate that inhibition of cysteine proteinase activity results in inhibition of cell proliferation [8 –12]. Calpains appear to be the cysteine proteinases most likely involved in arresting cell cycle progression late in G 1 [13]. Furthermore, it has been shown that both addition of growth factors and malignant transformation of fibroblasts result in an induction of the cysteine proteinase, cathepsin L [14]. In many cancers, malignancy is closely paralleled by increases in the activity of cysteine proteinases such as cathepsin B [15, 16], and in some cases, a decrease in their inhibitors [17, 18]. Taken together, these data suggests that active cell proliferation requires cysteine proteinase activity. The precise role of cysteine proteinases in the control of cell proliferation is presently unclear. The activity of cysteine proteinases is primarily controlled by specific endogenous inhibitors, of which there are three major classes: stefins, cystatins, and kininogens [19]. Therefore, it is clear that any physiological or pathological circumstances that result in a change in the steady state level of these inhibitors could potentially result in a dysregulation of regulatory pathways involved in the control of cell proliferation. By differential screening of a cDNA library constructed from liver mRNA from old Sprague–Dawley rats, we have identified T-KG 3 as a major gene whose 3 Abbreviations used: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MKP-1, MAP kinase phosphatase-1; T-KG, T-kininogen; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; pRB, retinoblastoma protein; EGF, epidermal growth factor; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; TCA, trichloroacetic acid.
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0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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expression increases with age in this tissue [20 –23]. Further studies have demonstrated that intrahepatic T-KG retains its biological activity as an inhibitor of cysteine proteinases and, in fact, is primarily found complexed with intracellular cysteine proteinases [24]. In both Sprague–Dawley and in (F344x Brown Norway) F1 rats, we have observed that the increase in serum T-KG consistently occurs 2.5 to 4 months before the death of the animal, irrespective of the age at which this event occurs [22, 23]. Therefore, T-KG represents a good candidate for a biomarker of the aging process in rats. The fact that serum levels of highmolecular-weight kininogen (HMW-KG) also increase with age in humans [25] as well as in rats [26] further supports the notion that overexpression of kininogens is a relevant age-related phenomenon. Because of these observations, we reasoned that inhibition of intracellular cysteine proteinases during aging could lead to a decreased proliferative capacity. While the liver is primarily a postmitotic tissue, an age-related decrease in the ability of the liver to regenerate after partial hepatectomy has been reported [27, 28], and we reasoned that this decreased proliferative capacity could result, at least in part, because of inhibition of cysteine proteinases by the increase in intracellular T-KG. As a model to test this idea, in the present study we used fibroblasts in culture to investigate the ability of T-KG to influence proliferative homeostasis. Our results indicate that expression of T-KG leads a diminished increase in cell number in logarithmically growing cultures. Furthermore, both FACS analysis and measurement of the ability of cells to incorporate radioactive precursors into DNA suggest that T-KG-producing cells are not capable of reaching the S phase of the cell cycle in response to addition of serum. However, biochemical analysis of cyclin levels, MAPK activation and expression of early response genes indicate that cells are capable of sensing growth stimulatory signals. We conclude that T-KG-expressing cell lines are arrested in their progression through the cell cycle, and this inhibition could have a significant impact in proliferative homeostasis in the aged. MATERIALS AND METHODS Materials. Media and serum were purchased from Life Technologies Inc. [ 3H]TdR and [ 3H]CdR were from NEN. Antibodies against cyclin D3, cyclin A, and c-Fos were purchased from Transduction Laboratories. Myelin basic protein was from Gibco-BRL. Aprotinin, pepstatin, leupeptin, and PMSF were from Boehringer, and most other chemicals were from Sigma, unless specified. Plasmid construction and stable transfection. The isolation and characterization of full-length T-KG cDNA clones has been described elsewhere [20]. The cDNA used in this study extends from ⫹57 (in between cap sites 2 and 3) to 14 bp past the polyadenylation site AAUAAA. This cDNA was cloned downstream of the mouse metallothionein I promoter (from ⫺151 to ⫹61) in the vector pMT1-Cas, a kind gift of Dr. Phillip Shaw (CHUV, Lausanne, Switzerland), re-
sulting in the plasmid pMT-Kin. In some experiments, the cassette containing the T-KG cDNA under the control of the mouse metallothionein promoter was subcloned into pUC19, to give rise to plasmid pMT-Kin.pUC, which is devoid of Neo resistance and its associated SV40 early promoter region. No difference in expression or inducibility was observed when comparing these two constructs in transient assays. Stable transfections were performed by standard methods, and transfected cells were selected in 400 g/ml G418. As controls, Balb/c 3T3 cells were cotransfected with the empty vectors pCas-Neo and pMT1-Cas. No comparable control was prepared in the L TK- background, and in this case, the parental untransfected cell line was used as a control. Antibiotic selection gave rise to several clones, some of which have been further analyzed in this report. L MK6 cells are based on an L TK- background, while from Balb/c 3T3 cells we established B 2.3 cells, whose vector alone control are B MCN-4 cells, and B K.4 cells, whose control are B PUC-1 cells. Cell culture. Both L TK- and L MK-6 cells were maintained in minimum alpha medium (␣-MEM), while Balb/c 3T3, B 2.3, B MCN-4, B K.4, and B PUC-1 cells were maintained in Dulbecco’s MEM (DMEM). Both media were supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 g/ml). Cells were routinely plated at 1 ⫻ 10 4 cells/cm 2 and grown in a humidified 5% CO 2 atmosphere at 37°C. Unless otherwise noted, T-KG expression was induced 24 h after seeding, by addition of 75 M ZnSO 4, administered in two doses (25 and 50 M, respectively) 4 h apart. Expression of the protein was allowed to proceed for 24 h before harvesting the cells for analysis. Cultures in G 0 were obtained by incubation in medium containing 0.5% of FBS for 3 days, at which point most of the cells were quiescent as determined by loss of [ 3H]TdR incorporation. Expression of T-KG in G 0 cells was attained by incubation in the presence of 1.25 M CdCl 2 during the last 12 h of the serum starvation period, and serum stimulation was performed by refeeding in fresh medium containing 10% serum. It should be pointed out that the change of inducers (ZnSO 4 in logarithmically growing cells, and CdCl 2 in G 0 cells) is due to the high toxicity we observed when G 0 cells were treated with ZnSO 4. This effect was independent of the presence of T-KG. DNA synthesis. The level of DNA synthesis was measured by incorporation of [ 3H]TdR (Balb/c 3T3, B MCN-4, B 2.3, B PUC-1, and B K.4 cells) or [ 3H]CdR (L TK- and L MK-6 cells) into acid-insoluble material. For this, G 0 cells were stimulated to proliferate by addition of 10% FBS. Twelve hours later, 4 Ci/ml of [ 3H]TdR or [ 3H]CdR was added, and incubation was continued for 4 h. Cells were harvested (Packard, Harvester Filtermate 196, Meriden, CT), lysed, and fixed with 7% trichloroacetic acid. The acid-insoluble material was collected on glass filters (Skatron), dried, and counted in triplicate in a liquid scintillation counter (Packard, Matrix 9600, Meriden, CT). Flow cytometry. Quiescent cells induced with 1.25 M CdCl 2 for 12 h were stimulated for 0, 16, and 24 h with medium containing 10% FBS plus CdCl 2. Cells were prepared according to Vindelov et al. [29]. Briefly, washed cells were lysed in 250 mM sucrose, 40 mM trisodium citrate, pH 7.6, 0.1% NP-40, 1.5 mM spermine, 0.5 mM Tris, pH 7.6, 5% DMSO. The resulting nuclei were diluted 1:10 in the same buffer and incubated for 10 min at room temperature with 30 mg/ml trypsin, followed by 10 min in 0.5 mg/ml trypsin inhibitor, 100 g/ml RNAase A. DNA staining was accomplished by incubating the nuclei in 1 vol of 400 g/ml propidium iodide containing 1 g/ml spermine for 30 min, at 4°C in the dark. DNA fluorescence was measured with a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cells were excited at 488 nm, and detection was at 585 ⫾ 20 nm. Percentages of cells in G 0 plus G 1, S, and G 2 plus M phases of the cell cycle were determined using the FACScan CellFit program [30]. Western blot analysis. Total cellular extracts were prepared by washing the cell monolayers twice in ice-cold PBS, followed by lysis for 30 min at 4°C with rocking in a modified Hibi buffer [31] consist-
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FIG. 1. Basal and induced levels of T-KG expression in different transfected cell lines. Logarithmically growing cells were kept for 24 h in the absence (⫺) or presence (⫹) of 50 M ZnSO 4. Cells were then collected, lysed, and 25 g of total cellular proteins were analyzed by Western blot, using an anti-rat T-KG polyclonal antibody (a kind gift of Dr. J. Gauldie, University of Ontario, Canada), and developed by the ECL method (Amersham). L TK- are the parental cells from which the L MK-6 cell line was derived. All other cell lines are in a Balb/c 3T3 background. B PUC-2 and B MCN-4 were stably transfected with empty vector corresponding to the T-KG expression vectors used to produce B K.4 and B 2.3 cells, respectively.
ing of 25 mM Hepes, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl 2, 0.2 mM EDTA, 0.1% Triton X-100, 2 mM Na pyrophosphate, 0.5 mM DTT, 20 mM -glycerophosphate, 0.1 mM Na orthovanadate, 10 g/ml leupeptin, 10 g/ml aprotinin, and 100 g/ml PMSF. Particulate matter was removed by centrifugation at 4°C, and protein concentration was determined by the Bradford method as commercialized by BioRad. A 30 to 50 g aliquot of each sample was separated by 10% SDS–PAGE and electrotransferred to nitrocellulose membranes (Schleicher and Schuell). Western blotting was performed under standard conditions using 5% nonfat milk as a blocking agent, horseradish peroxydaseconjugated goat anti-rabbit or anti-mouse IgG (Transduction Laboratories) as secondary antibodies, and the ECL system (Amersham) for detection. In all cases, data from Western blot analysis were normalized by stripping and re-probing the membranes with an anti- actin monoclonal antibody (ICN). MAP kinase “in gel” assay. ERK activity was determined by an ‘in-gel’ kinase assay, with myelin basic protein as substrate, according to the method described by Kameshita and Fujisawa [32] with the following modifications. Ten micrograms of protein was electrophoresed on a 12.5% polyacrylamide minigel containing 0.5 mg/ml of myelin basic protein. After SDS elimination, denaturation, and renaturation, the gel was equilibrated in kinase buffer, and the kinase reaction was initiated by placing the gel in fresh kinase buffer containing 30 M ATP and 100 Ci [␥- 32P]ATP for 1 h at room temperature with gentle agitation. After extensive washes in 5% (v/w) TCA, 1% sodium pyrophosphate, the gel was dried, and subjected to autoradiography: ERK activities (p44 ERK1 and p42 ERK2) were identified according to their molecular weight and the ability to phosphorylate myelin basic protein. Data analysis. Autoradiograms were scanned and quantified by using the ImageQuant image analysis software (Molecular Dynamics). All Western blot data were normalized against -actin as a control for gel loading (not shown). All experiments were performed at least three times, and statistical significance was determined by two-tailed Student t test.
RESULTS
T-KG inhibits cell proliferation. In order to examine the effect of T-KG expression on cell proliferation, we made constructs in which the full-length T-KG cDNA was under the control of either the strong constitutive CMV promoter, or under the control of the
weak inducible (though leaky) MT-1 promoter. While both constructs efficiently produce immunoreactive T-KG when transiently transfected into a variety of cell lines (data not shown), stable transfectants were obtained only with the weaker, inducible MT-1 promoter constructs. Our inability to obtain stable cell lines using the strong constitutive CMV promoter suggested the possibility that high levels of T-KG expression might be incompatible with cell growth. To test this hypothesis, we have made use of the cell lines that express T-KG under the control of the weaker MT-1 promoter. Figure 1 shows T-KG expression in three independent cell lines, both in the presence and in the absence of the inducer, ZnSO 4. Due to the leakiness of the promoter, all subsequent experiments were performed under induced conditions, and using untransfected and vector-alone transfected cell lines as controls. As a first approach to investigate the effect of T-KG on cell proliferation, we simply measured the increase in cell numbers during a 48 h-period in control and T-KG-producing cells. For this, cells were plated as described under Materials and Methods, and 24 h later, the cultures were induced for 24 h with 75 M ZnSO 4. Figure 2 shows that, while parental cells were able to increase their cell number to approximately 5 times the original seeding density, T-KG-expressing cells failed to grow significantly during the time of the assay. In order to confirm our observations made in logarithmically growing cells, we used serum deprivation to synchronize all the cell lines under study, and measured their ability to mount a proliferative response to the addition of 10% serum. Figure 3 shows that, while all control cells responded to the presence of serum by vigorous growth, T-KG-expressing cells have a negligible capacity to increase their numbers within the time of the experiment (24 h after serum stimulation).
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FIG. 2. T-KG inhibits proliferation in logarithmically growing cells. Cell lines were seeded at 1 ⫻ 10 4 cells/cm 2. The following day, the cultures were treated with 75 M ZnSO 4 as indicated under Materials and Methods, and cells were allowed to grow for a further 24 h. At the end of this period, cells were trypsinized and counted in a Coulter counter. Results are expressed as cells/cm 2/10 ⫺3 and represent the mean of triplicate determinations ⫾ SE. Proliferation of T-KG-expressing cells is statistically diminished relative to either the parental cells and vector-alone transfected cells.
T-KG inhibits cell entry into the S phase of the cell cycle. In an effort to identify the stage(s) of the cell cycle where inhibition might occur, we measured the ability of serum stimulated cells to enter the S phase of the cell cycle. For this, cells in G 0, induced with CdCl 2 for 12 h, were induced to proliferate by addition of 10% FBS. After 12 h, 4 Ci/ml of [ 3H]TdR or [ 3H]CdR (for the thymidine kinase negative cell lines) were added, and incubation was continued for a further 4 h. The results shown in Fig. 4 indicate that T-KG inhibits cell entry into the S phase of the cell cycle. These results strongly suggest the existence of at least one point, located between G 0 and S, which is susceptible to inhibition by T-KG. These results were further confirmed by FACS analysis performed at 0, 16, and 24 h after serum stimulation. Figure 5 shows that, while the parental cell line (L TK-) responded to the addition of serum by a normal progression into S and G 2/M, the T-KG expressing cell line, L MK6, failed to leave the G 0/G 1 stage. Similar results have been obtained with B PUC-1/B K.4 cells (data not shown). In both of the T-KG-expressing cell lines (L MK6 and B K.4), we observe a rate of release from G 0/G 1 that reaches only less than 2% of the value observed in
control cell lines. We currently do not know why the inhibition of entry into S phase appears more pronounced when measured by FACS analysis than when measured by incorporation of radioactive precursors into DNA. Obviously, further experimentation will be required to solve this issue. T-KG-expressing cells are capable of sensing the stimulus given by serum. The data described thus far indicate that at least one point of inhibition of cell proliferation by T-KG occurs pre-S phase. We have previously observed that T-KG expression leads to a severe inhibition of the basal level of activity of the ERK pathway of signal transduction [33]. We hypothesized therefore that one possible explanation for our current observations could be an inability of T-KGexpressing cells to sense the presence of growth factors in the medium, due to a defect in signal transduction. Thus, we tested the ability of cells synchronized in G 0 to activate the ERK pathway in response to serum. Surprisingly, Fig. 6 shows that T-KG-expressing cells are capable of inducing ERK activity with kinetics and intensity comparable to that observed in control cell lines. The results were confirmed in a separate exper-
FIG. 3. T-KG inhibits cell proliferation in response to serum stimulation. Cells were seeded at 1 ⫻ 10 4 cells/cm 2, and the following day they were induced into G 0 by serum depletion. Two days later, the cultures were treated with 1.25 M CdCl 2 for 12 h. At that time, cells were either counted or induced to enter the cell cycle by addition of 10% FBS for a further 24 h. At the end of this period, cells were trypsinized and counted in a Coulter counter. Results are expressed as increase in cell number (cells/cm 2 24 h after serum stimulation minus cells/cm 2 before addition of serum). The results represent the mean of triplicate determinations ⫾ SEM. Proliferation of T-KG-expressing cells is statistically diminished relative to either the parental cells and vector-alone transfected cells.
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FIG. 4. T-KG inhibits entry into S phase. Cell lines were seeded and treated as in Fig. 3, except that 12 h after serum stimulation, they were given a 4-h pulse with 4 Ci/ml of either [ 3H]TdR or [ 3H]CdR (L TK- and L MK6 cells). Radioactivity incorporated into DNA was determined by binding to glass filters as described under Materials and Methods. Results are expressed as a fraction of the radioactivity incorporated in control cells (either transfected with vector alone or, in the case of L TK- cells, the parental cell line), in the presence of heavy metal induction.
iment by Western blot using anti-active MAPK antibodies (NEBiolabs, data not shown). Furthermore, Fig. 7 indicates that the cells can respond to that stimulation, by activating the expression of the early response genes c-fos and c-jun. To establish more exactly the site of inhibition, we determined the effect of T-KG expression at the molecular level, by following the fate of some cell-cycle-regulated molecules as a function of time after release from serum starvation. In order to confirm that the T-KG-expressing cells are unable to enter the S phase, we probed for their ability to induce the S-phase-specific cyclin A. Figure 8 shows that cyclin A is induced by 16 h in the parental cell line as expected. In contrast,
no significant expression of cyclin A was observed in either of the T-KG cell lines tested. Moreover, we studied the effects of T-KG on the normal degradation of both cyclin D3 and p27 at the end of the G 1 phase. Figure 8 shows that in Balb/c 3T3 cells, both p27 and cyclin D3 levels decline at approximately the time cells enter the S phase (10 –12 h after serum stimulation), and the protein remains undetectable after that. In contrast, in both of the T-KG-expressing cell lines tested, p27 and cyclin D3 levels fail to decline. For all cell-cycle-regulated proteins tested (c-Fos, c-Jun, cdk4, cyclin A, cyclin D3, and p27), cell lines transfected with vector alone behaved in a manner similar to the parental cells, Balb/c 3T3 (data not shown). Thus, these results further confirm biochemically that in the presence of T-KG, cells are unable to leave G 1 to enter the S phase of the cell cycle. Together, these results indicate that T-KG-expressing cell lines are capable of sensing the presence of growth factors, and respond by activating early gene expression. However, their progression through the cell cycle is arrested so that they are unable to reach the S phase of the cycle. DISCUSSION
FIG. 5. FACS analysis of the effect of T-KG on cell cycle progression. L TK- and L MK6 cells were seeded, synchronized and metal induced as indicated in Fig. 3. At 0, 16, or 24 h after serum stimulation, aliquots were withdrawn and nuclei were prepared for flow cytometric analysis in a FACScan. Individual nuclear DNA content, as reflected by fluorescence intensity (FL2-A parameter) of incorporated propidium iodide, is plotted in a histogram depicting relative cell numbers at each intensity. Each histogram is representative of data obtained from three similar experiments.
The data presented in this report strongly suggests that T-KG, a physiologically relevant cysteine proteinase inhibitor, can arrest cell proliferation. Expression of the protein in fibroblast cell lines leads to an alteration in growth-related parameters, including the ability to proliferate, both in logarithmically growing cultures and in G 0-arrested cells stimulated to proliferate in response to serum. Also, T-KG inhibits the ability of cells to enter the S phase of the cell cycle, as evidenced by a decreased rate of incorporation of radioactive precursors into DNA, as well as by FACS analysis, and by the observed impairment in the ability to properly reg-
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FIG. 6. T-KG does not affect the ability of cells to induce ERK activity in response to serum stimulation. Serum-starved B MCN-4 and B 2.3 cells, cultured in the presence of 1.25 M CdCl 2, were induced to proliferate by addition of 10% FBS. At different times, total cellular extracts were prepared and ERK activity (ERK1/ERK2) was measured using an “in gel” kinase assay. (Left) Only the first burst of ERK activity (up to 15 min) is represented. (Right) The full experiment (up to 10 h) is shown. Note that the first peak shown in the left panel appears in the right panel very close to the ordinate axis.
ulate both the degradation of p27 and cyclin D3 and the synthesis of cyclin A. On the other hand, both the unimpaired activation of the ERK pathway and the normal synthesis of early response proteins such as c-Fos and c-Jun suggest that cells in G 0 are still capable of sensing the presence of growth factors present in the serum and respond to their stimulation. It should be pointed out that the FACS analysis indicates that the effects we have observed are actually due to inhibition of cell proliferation and not to induction of apoptosis, as evidenced by the absence of an apoptotic peak in those experiments. This is in accordance with our unpublished observations indicating that, on the contrary, T-KG leads to a slight protection against etoposide-induced apoptotic death.
Cells that express T-KG are able to respond to mitogenic stimuli by activating the ERK pathway, and probably other signal transduction pathways as well, as demonstrated by the normal induction of several early response genes. Degradation of these molecules (c-Fos, c-Jun, and Jun B) also proceeds normally (data not shown). In the presence of T-KG, however, cells are arrested at a point in the cell cycle located before they start synthesizing cyclin A, normally at the beginning of the S phase [34]. The presence of T-KG also results in a failure to degrade both cyclin D3 and p27, which normally occurs in late G 1 in Balb/c 3T3 cells [34]. Since both cyclins [35] and p27 [36] have been shown to be degraded primarily by the ubiquitin/proteasome system, we favor the hypothesis that T-KG does not
FIG. 7. T-KG-expressing cells respond to serum stimulation by activating early gene expression. Cells (indicated under each panel) were serum starved for 3 days, and then were induced to proliferate by addition of 10% FBS as described in Fig. 3. Cellular extracts were prepared at different times after serum addition, and the abundance of c-fos (left), c-jun (middle), and cdk 4 (as a control for loading, right) was assessed by Western blotting as indicated under Materials and Methods.
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FIG. 8. Cyclin levels confirm the inability of T-KG-expressing cells to reach the S phase of the cell cycle in response to serum stimulation. Cells (indicated in the middle) were serum starved for 3 days, and then were induced to proliferate by addition of 10% FBS as described in Fig. 3. Cellular extracts were prepared at different times after serum addition, and the abundance of cyclin A, cyclin D3, p27, and cdk 4 (as a control for loading) was assessed by Western blotting as indicated under Materials and Methods.
directly impinge on the ability of the cells to degrade these proteins, but rather, T-KG inhibits the progression of cells through the point in the cell cycle where these proteins should be normally degraded. It is interesting to consider the possible involvement of ERK in this phenomenon. The results shown in Fig. 6 (right panel) indicate that, compared to the controls, in T-KG-expressing cells there is a precipitous and very reproducible decrease in ERK activity approximately 5–9 hours into the G 1 phase. Thus, in these cells there is no detectable ERK activity at the time when the cells should be entering S phase. It has been shown that mitogen-activated protein kinases are required for the proliferative response of quiescent fibroblasts [37] and a sustained activation of p42/p44 MAPK is required for fibroblasts to pass the G 1 restriction point and enter into the S phase [38]. Therefore, we hypothesize that the sudden demise of ERK activity in T-KGexpressing cells could be the cause of the cell’s lack of progression through the cell cycle. Specifically, we propose that while in control cells, ERK activity remains elevated for several hours after serum stimulation (in fact, until the cells reach the G 1/S interphase), the
stabilization of MKP-1 in T-KG-expressing cell lines [33] leads to an early demise of ERK activity. This in turn could lead to the arrest of cells in late G 1 [39]. It should be emphasized that Western blot analysis has shown that expression of T-KG in the cell lines analyzed is actually below physiological range, as compared to the liver of old rats. Therefore our observations are not due to gross overexpression of T-KG to levels that are not physiologically relevant. This is true when comparing either cellular (in the cell lines vs the liver) or extracellular (in the conditioned medium vs serum) T-KG levels (data not shown). Thus, our results suggest that T-KG is a potent inhibitor of proliferation, able to arrest cell cycle progression even at very low doses. This raises several concerns, foremost among which is our ability to obtain stable cell lines by using a leaky promoter. While, as discussed, it is true that we were unable to obtain stable cell lines using the strong CMV promoter, our data indicates that inhibition of proliferation is already evident in the available cell lines even in the absence of inducer (data not shown). However, the inhibition of cell proliferation we have observed in logarithmically growing cells is only par-
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tial, and this is consistent with our observation that T-KG-expressing cell lines grow poorly (not shown). A different issue arises from reports in the literature indicating that kinins have a moderately proliferative effect on cells possessing kinin receptors (40, 41). We observe the opposite effect using a kinin precursor, T-KG. The difference could be due either to the difference in cell types, or to differences in the mechanisms. While it is known that kininogens cannot bind to kinin receptors [42], we cannot formally exclude the possibility that T-KG does act via these receptors, and the inhibitory effect we observe could be due to downregulation of the corresponding signal transduction pathways, including the ERK pathway, as a result of the long-term exposure to the ligand. On the other hand, a major difference between kinins and kininogens is the presence in the latter of cysteine proteinase inhibitory activity. While to date we have not shown that the effects we observe are due to the cysteine proteinase inhibitory function of T-KG, this is very likely to be so, since inhibition of cysteine proteinase activity is known to result in decreased cell proliferation, as well as impaired progression through the cell cycle [8, 12, 13, 43]. By using synthetic aldehyde thiol protease inhibitors, it has been demonstrated that progression of vascular smooth muscle cells requires cysteine protease activity in the transitions between G 0/G 1, G 1/S, and G 2/M [8]. In our experimental approach, we observe inhibition at the G 1/S, but we do not see an effect of T-KG at the G 0/G 1 interphase. This could reflect a difference either in the cell types used, or in specificity between chemical inhibitors and physiological ones, which are more likely to be compartmentalized within the cell. Together, these data indicate that T-KG expression leads to inhibition of cell proliferation, thus supporting our hypothesis that the age-related increase in both T-KG gene expression [20, 22, 23] and serum T-KG levels [21], could play a role in the well-known diminished proliferative capacity of tissues from old animals. We are gratefully indebted to Dr. P. Shaw (CHUV, Lausanne, Switzerland) for providing the vector pMT1-Cas. We also especially thank our colleagues Chris Sell and Mary Kay Francis for their critical reading of the manuscript. This work was supported by NIH Grant AG 13902, by a Minority Supplement to Grant AG 00378-24, and by FONDECYT Grant 1981064.
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T-Kininogen Inhibits Fibroblast Proliferation in the G 1 Phase of the Cell Cycle Claudio Torres, Min Li, Robin Walter, 1 and Felipe Sierra 1,2 MCP-Hahnemann University, 2900 Queen Lane, Philadelphia, Pennsylvania 19129; and Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile
By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblastderived cell lines that express biologically active Tkininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G 0 cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G 0 through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G 1 and S phases of the cell cycle. © 2001 Academic Press
Key Words: proliferation; cell cycle; T-kininogen; cysteine proteinases; MAP kinase.
INTRODUCTION
Cysteine proteinases provide a small percentage of the cell’s total proteolytic capabilities, and their virtually complete inhibition results in only a minor decline in total proteolytic rates. Nevertheless, several studies have indicated that they are implicated in the degra1
Current addresses: Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096, and Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile. 2 To whom reprint requests should be addressed. Fax: (562) 7355580. E-mail: [email protected].
dation of key regulatory factors including c-Fos [1], PKC [2], PKA [3], calmodulin [4], integrins [5], p53 [6], and pRb [7]. While some of these molecules are also degraded by the proteasome, alternative pathways, most likely involving calpain, appear to exist. Inhibition of their degradation could potentially lead to aberrant accumulation of these proteins and to a severe disturbance of important cellular functions, such as signal transduction and progression through the cell cycle. Indeed, several reports in the literature indicate that inhibition of cysteine proteinase activity results in inhibition of cell proliferation [8 –12]. Calpains appear to be the cysteine proteinases most likely involved in arresting cell cycle progression late in G 1 [13]. Furthermore, it has been shown that both addition of growth factors and malignant transformation of fibroblasts result in an induction of the cysteine proteinase, cathepsin L [14]. In many cancers, malignancy is closely paralleled by increases in the activity of cysteine proteinases such as cathepsin B [15, 16], and in some cases, a decrease in their inhibitors [17, 18]. Taken together, these data suggests that active cell proliferation requires cysteine proteinase activity. The precise role of cysteine proteinases in the control of cell proliferation is presently unclear. The activity of cysteine proteinases is primarily controlled by specific endogenous inhibitors, of which there are three major classes: stefins, cystatins, and kininogens [19]. Therefore, it is clear that any physiological or pathological circumstances that result in a change in the steady state level of these inhibitors could potentially result in a dysregulation of regulatory pathways involved in the control of cell proliferation. By differential screening of a cDNA library constructed from liver mRNA from old Sprague–Dawley rats, we have identified T-KG 3 as a major gene whose 3 Abbreviations used: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MKP-1, MAP kinase phosphatase-1; T-KG, T-kininogen; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; pRB, retinoblastoma protein; EGF, epidermal growth factor; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; TCA, trichloroacetic acid.
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expression increases with age in this tissue [20 –23]. Further studies have demonstrated that intrahepatic T-KG retains its biological activity as an inhibitor of cysteine proteinases and, in fact, is primarily found complexed with intracellular cysteine proteinases [24]. In both Sprague–Dawley and in (F344x Brown Norway) F1 rats, we have observed that the increase in serum T-KG consistently occurs 2.5 to 4 months before the death of the animal, irrespective of the age at which this event occurs [22, 23]. Therefore, T-KG represents a good candidate for a biomarker of the aging process in rats. The fact that serum levels of highmolecular-weight kininogen (HMW-KG) also increase with age in humans [25] as well as in rats [26] further supports the notion that overexpression of kininogens is a relevant age-related phenomenon. Because of these observations, we reasoned that inhibition of intracellular cysteine proteinases during aging could lead to a decreased proliferative capacity. While the liver is primarily a postmitotic tissue, an age-related decrease in the ability of the liver to regenerate after partial hepatectomy has been reported [27, 28], and we reasoned that this decreased proliferative capacity could result, at least in part, because of inhibition of cysteine proteinases by the increase in intracellular T-KG. As a model to test this idea, in the present study we used fibroblasts in culture to investigate the ability of T-KG to influence proliferative homeostasis. Our results indicate that expression of T-KG leads a diminished increase in cell number in logarithmically growing cultures. Furthermore, both FACS analysis and measurement of the ability of cells to incorporate radioactive precursors into DNA suggest that T-KG-producing cells are not capable of reaching the S phase of the cell cycle in response to addition of serum. However, biochemical analysis of cyclin levels, MAPK activation and expression of early response genes indicate that cells are capable of sensing growth stimulatory signals. We conclude that T-KG-expressing cell lines are arrested in their progression through the cell cycle, and this inhibition could have a significant impact in proliferative homeostasis in the aged. MATERIALS AND METHODS Materials. Media and serum were purchased from Life Technologies Inc. [ 3H]TdR and [ 3H]CdR were from NEN. Antibodies against cyclin D3, cyclin A, and c-Fos were purchased from Transduction Laboratories. Myelin basic protein was from Gibco-BRL. Aprotinin, pepstatin, leupeptin, and PMSF were from Boehringer, and most other chemicals were from Sigma, unless specified. Plasmid construction and stable transfection. The isolation and characterization of full-length T-KG cDNA clones has been described elsewhere [20]. The cDNA used in this study extends from ⫹57 (in between cap sites 2 and 3) to 14 bp past the polyadenylation site AAUAAA. This cDNA was cloned downstream of the mouse metallothionein I promoter (from ⫺151 to ⫹61) in the vector pMT1-Cas, a kind gift of Dr. Phillip Shaw (CHUV, Lausanne, Switzerland), re-
sulting in the plasmid pMT-Kin. In some experiments, the cassette containing the T-KG cDNA under the control of the mouse metallothionein promoter was subcloned into pUC19, to give rise to plasmid pMT-Kin.pUC, which is devoid of Neo resistance and its associated SV40 early promoter region. No difference in expression or inducibility was observed when comparing these two constructs in transient assays. Stable transfections were performed by standard methods, and transfected cells were selected in 400 g/ml G418. As controls, Balb/c 3T3 cells were cotransfected with the empty vectors pCas-Neo and pMT1-Cas. No comparable control was prepared in the L TK- background, and in this case, the parental untransfected cell line was used as a control. Antibiotic selection gave rise to several clones, some of which have been further analyzed in this report. L MK6 cells are based on an L TK- background, while from Balb/c 3T3 cells we established B 2.3 cells, whose vector alone control are B MCN-4 cells, and B K.4 cells, whose control are B PUC-1 cells. Cell culture. Both L TK- and L MK-6 cells were maintained in minimum alpha medium (␣-MEM), while Balb/c 3T3, B 2.3, B MCN-4, B K.4, and B PUC-1 cells were maintained in Dulbecco’s MEM (DMEM). Both media were supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 g/ml). Cells were routinely plated at 1 ⫻ 10 4 cells/cm 2 and grown in a humidified 5% CO 2 atmosphere at 37°C. Unless otherwise noted, T-KG expression was induced 24 h after seeding, by addition of 75 M ZnSO 4, administered in two doses (25 and 50 M, respectively) 4 h apart. Expression of the protein was allowed to proceed for 24 h before harvesting the cells for analysis. Cultures in G 0 were obtained by incubation in medium containing 0.5% of FBS for 3 days, at which point most of the cells were quiescent as determined by loss of [ 3H]TdR incorporation. Expression of T-KG in G 0 cells was attained by incubation in the presence of 1.25 M CdCl 2 during the last 12 h of the serum starvation period, and serum stimulation was performed by refeeding in fresh medium containing 10% serum. It should be pointed out that the change of inducers (ZnSO 4 in logarithmically growing cells, and CdCl 2 in G 0 cells) is due to the high toxicity we observed when G 0 cells were treated with ZnSO 4. This effect was independent of the presence of T-KG. DNA synthesis. The level of DNA synthesis was measured by incorporation of [ 3H]TdR (Balb/c 3T3, B MCN-4, B 2.3, B PUC-1, and B K.4 cells) or [ 3H]CdR (L TK- and L MK-6 cells) into acid-insoluble material. For this, G 0 cells were stimulated to proliferate by addition of 10% FBS. Twelve hours later, 4 Ci/ml of [ 3H]TdR or [ 3H]CdR was added, and incubation was continued for 4 h. Cells were harvested (Packard, Harvester Filtermate 196, Meriden, CT), lysed, and fixed with 7% trichloroacetic acid. The acid-insoluble material was collected on glass filters (Skatron), dried, and counted in triplicate in a liquid scintillation counter (Packard, Matrix 9600, Meriden, CT). Flow cytometry. Quiescent cells induced with 1.25 M CdCl 2 for 12 h were stimulated for 0, 16, and 24 h with medium containing 10% FBS plus CdCl 2. Cells were prepared according to Vindelov et al. [29]. Briefly, washed cells were lysed in 250 mM sucrose, 40 mM trisodium citrate, pH 7.6, 0.1% NP-40, 1.5 mM spermine, 0.5 mM Tris, pH 7.6, 5% DMSO. The resulting nuclei were diluted 1:10 in the same buffer and incubated for 10 min at room temperature with 30 mg/ml trypsin, followed by 10 min in 0.5 mg/ml trypsin inhibitor, 100 g/ml RNAase A. DNA staining was accomplished by incubating the nuclei in 1 vol of 400 g/ml propidium iodide containing 1 g/ml spermine for 30 min, at 4°C in the dark. DNA fluorescence was measured with a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cells were excited at 488 nm, and detection was at 585 ⫾ 20 nm. Percentages of cells in G 0 plus G 1, S, and G 2 plus M phases of the cell cycle were determined using the FACScan CellFit program [30]. Western blot analysis. Total cellular extracts were prepared by washing the cell monolayers twice in ice-cold PBS, followed by lysis for 30 min at 4°C with rocking in a modified Hibi buffer [31] consist-
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FIG. 1. Basal and induced levels of T-KG expression in different transfected cell lines. Logarithmically growing cells were kept for 24 h in the absence (⫺) or presence (⫹) of 50 M ZnSO 4. Cells were then collected, lysed, and 25 g of total cellular proteins were analyzed by Western blot, using an anti-rat T-KG polyclonal antibody (a kind gift of Dr. J. Gauldie, University of Ontario, Canada), and developed by the ECL method (Amersham). L TK- are the parental cells from which the L MK-6 cell line was derived. All other cell lines are in a Balb/c 3T3 background. B PUC-2 and B MCN-4 were stably transfected with empty vector corresponding to the T-KG expression vectors used to produce B K.4 and B 2.3 cells, respectively.
ing of 25 mM Hepes, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl 2, 0.2 mM EDTA, 0.1% Triton X-100, 2 mM Na pyrophosphate, 0.5 mM DTT, 20 mM -glycerophosphate, 0.1 mM Na orthovanadate, 10 g/ml leupeptin, 10 g/ml aprotinin, and 100 g/ml PMSF. Particulate matter was removed by centrifugation at 4°C, and protein concentration was determined by the Bradford method as commercialized by BioRad. A 30 to 50 g aliquot of each sample was separated by 10% SDS–PAGE and electrotransferred to nitrocellulose membranes (Schleicher and Schuell). Western blotting was performed under standard conditions using 5% nonfat milk as a blocking agent, horseradish peroxydaseconjugated goat anti-rabbit or anti-mouse IgG (Transduction Laboratories) as secondary antibodies, and the ECL system (Amersham) for detection. In all cases, data from Western blot analysis were normalized by stripping and re-probing the membranes with an anti- actin monoclonal antibody (ICN). MAP kinase “in gel” assay. ERK activity was determined by an ‘in-gel’ kinase assay, with myelin basic protein as substrate, according to the method described by Kameshita and Fujisawa [32] with the following modifications. Ten micrograms of protein was electrophoresed on a 12.5% polyacrylamide minigel containing 0.5 mg/ml of myelin basic protein. After SDS elimination, denaturation, and renaturation, the gel was equilibrated in kinase buffer, and the kinase reaction was initiated by placing the gel in fresh kinase buffer containing 30 M ATP and 100 Ci [␥- 32P]ATP for 1 h at room temperature with gentle agitation. After extensive washes in 5% (v/w) TCA, 1% sodium pyrophosphate, the gel was dried, and subjected to autoradiography: ERK activities (p44 ERK1 and p42 ERK2) were identified according to their molecular weight and the ability to phosphorylate myelin basic protein. Data analysis. Autoradiograms were scanned and quantified by using the ImageQuant image analysis software (Molecular Dynamics). All Western blot data were normalized against -actin as a control for gel loading (not shown). All experiments were performed at least three times, and statistical significance was determined by two-tailed Student t test.
RESULTS
T-KG inhibits cell proliferation. In order to examine the effect of T-KG expression on cell proliferation, we made constructs in which the full-length T-KG cDNA was under the control of either the strong constitutive CMV promoter, or under the control of the
weak inducible (though leaky) MT-1 promoter. While both constructs efficiently produce immunoreactive T-KG when transiently transfected into a variety of cell lines (data not shown), stable transfectants were obtained only with the weaker, inducible MT-1 promoter constructs. Our inability to obtain stable cell lines using the strong constitutive CMV promoter suggested the possibility that high levels of T-KG expression might be incompatible with cell growth. To test this hypothesis, we have made use of the cell lines that express T-KG under the control of the weaker MT-1 promoter. Figure 1 shows T-KG expression in three independent cell lines, both in the presence and in the absence of the inducer, ZnSO 4. Due to the leakiness of the promoter, all subsequent experiments were performed under induced conditions, and using untransfected and vector-alone transfected cell lines as controls. As a first approach to investigate the effect of T-KG on cell proliferation, we simply measured the increase in cell numbers during a 48 h-period in control and T-KG-producing cells. For this, cells were plated as described under Materials and Methods, and 24 h later, the cultures were induced for 24 h with 75 M ZnSO 4. Figure 2 shows that, while parental cells were able to increase their cell number to approximately 5 times the original seeding density, T-KG-expressing cells failed to grow significantly during the time of the assay. In order to confirm our observations made in logarithmically growing cells, we used serum deprivation to synchronize all the cell lines under study, and measured their ability to mount a proliferative response to the addition of 10% serum. Figure 3 shows that, while all control cells responded to the presence of serum by vigorous growth, T-KG-expressing cells have a negligible capacity to increase their numbers within the time of the experiment (24 h after serum stimulation).
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FIG. 2. T-KG inhibits proliferation in logarithmically growing cells. Cell lines were seeded at 1 ⫻ 10 4 cells/cm 2. The following day, the cultures were treated with 75 M ZnSO 4 as indicated under Materials and Methods, and cells were allowed to grow for a further 24 h. At the end of this period, cells were trypsinized and counted in a Coulter counter. Results are expressed as cells/cm 2/10 ⫺3 and represent the mean of triplicate determinations ⫾ SE. Proliferation of T-KG-expressing cells is statistically diminished relative to either the parental cells and vector-alone transfected cells.
T-KG inhibits cell entry into the S phase of the cell cycle. In an effort to identify the stage(s) of the cell cycle where inhibition might occur, we measured the ability of serum stimulated cells to enter the S phase of the cell cycle. For this, cells in G 0, induced with CdCl 2 for 12 h, were induced to proliferate by addition of 10% FBS. After 12 h, 4 Ci/ml of [ 3H]TdR or [ 3H]CdR (for the thymidine kinase negative cell lines) were added, and incubation was continued for a further 4 h. The results shown in Fig. 4 indicate that T-KG inhibits cell entry into the S phase of the cell cycle. These results strongly suggest the existence of at least one point, located between G 0 and S, which is susceptible to inhibition by T-KG. These results were further confirmed by FACS analysis performed at 0, 16, and 24 h after serum stimulation. Figure 5 shows that, while the parental cell line (L TK-) responded to the addition of serum by a normal progression into S and G 2/M, the T-KG expressing cell line, L MK6, failed to leave the G 0/G 1 stage. Similar results have been obtained with B PUC-1/B K.4 cells (data not shown). In both of the T-KG-expressing cell lines (L MK6 and B K.4), we observe a rate of release from G 0/G 1 that reaches only less than 2% of the value observed in
control cell lines. We currently do not know why the inhibition of entry into S phase appears more pronounced when measured by FACS analysis than when measured by incorporation of radioactive precursors into DNA. Obviously, further experimentation will be required to solve this issue. T-KG-expressing cells are capable of sensing the stimulus given by serum. The data described thus far indicate that at least one point of inhibition of cell proliferation by T-KG occurs pre-S phase. We have previously observed that T-KG expression leads to a severe inhibition of the basal level of activity of the ERK pathway of signal transduction [33]. We hypothesized therefore that one possible explanation for our current observations could be an inability of T-KGexpressing cells to sense the presence of growth factors in the medium, due to a defect in signal transduction. Thus, we tested the ability of cells synchronized in G 0 to activate the ERK pathway in response to serum. Surprisingly, Fig. 6 shows that T-KG-expressing cells are capable of inducing ERK activity with kinetics and intensity comparable to that observed in control cell lines. The results were confirmed in a separate exper-
FIG. 3. T-KG inhibits cell proliferation in response to serum stimulation. Cells were seeded at 1 ⫻ 10 4 cells/cm 2, and the following day they were induced into G 0 by serum depletion. Two days later, the cultures were treated with 1.25 M CdCl 2 for 12 h. At that time, cells were either counted or induced to enter the cell cycle by addition of 10% FBS for a further 24 h. At the end of this period, cells were trypsinized and counted in a Coulter counter. Results are expressed as increase in cell number (cells/cm 2 24 h after serum stimulation minus cells/cm 2 before addition of serum). The results represent the mean of triplicate determinations ⫾ SEM. Proliferation of T-KG-expressing cells is statistically diminished relative to either the parental cells and vector-alone transfected cells.
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FIG. 4. T-KG inhibits entry into S phase. Cell lines were seeded and treated as in Fig. 3, except that 12 h after serum stimulation, they were given a 4-h pulse with 4 Ci/ml of either [ 3H]TdR or [ 3H]CdR (L TK- and L MK6 cells). Radioactivity incorporated into DNA was determined by binding to glass filters as described under Materials and Methods. Results are expressed as a fraction of the radioactivity incorporated in control cells (either transfected with vector alone or, in the case of L TK- cells, the parental cell line), in the presence of heavy metal induction.
iment by Western blot using anti-active MAPK antibodies (NEBiolabs, data not shown). Furthermore, Fig. 7 indicates that the cells can respond to that stimulation, by activating the expression of the early response genes c-fos and c-jun. To establish more exactly the site of inhibition, we determined the effect of T-KG expression at the molecular level, by following the fate of some cell-cycle-regulated molecules as a function of time after release from serum starvation. In order to confirm that the T-KG-expressing cells are unable to enter the S phase, we probed for their ability to induce the S-phase-specific cyclin A. Figure 8 shows that cyclin A is induced by 16 h in the parental cell line as expected. In contrast,
no significant expression of cyclin A was observed in either of the T-KG cell lines tested. Moreover, we studied the effects of T-KG on the normal degradation of both cyclin D3 and p27 at the end of the G 1 phase. Figure 8 shows that in Balb/c 3T3 cells, both p27 and cyclin D3 levels decline at approximately the time cells enter the S phase (10 –12 h after serum stimulation), and the protein remains undetectable after that. In contrast, in both of the T-KG-expressing cell lines tested, p27 and cyclin D3 levels fail to decline. For all cell-cycle-regulated proteins tested (c-Fos, c-Jun, cdk4, cyclin A, cyclin D3, and p27), cell lines transfected with vector alone behaved in a manner similar to the parental cells, Balb/c 3T3 (data not shown). Thus, these results further confirm biochemically that in the presence of T-KG, cells are unable to leave G 1 to enter the S phase of the cell cycle. Together, these results indicate that T-KG-expressing cell lines are capable of sensing the presence of growth factors, and respond by activating early gene expression. However, their progression through the cell cycle is arrested so that they are unable to reach the S phase of the cycle. DISCUSSION
FIG. 5. FACS analysis of the effect of T-KG on cell cycle progression. L TK- and L MK6 cells were seeded, synchronized and metal induced as indicated in Fig. 3. At 0, 16, or 24 h after serum stimulation, aliquots were withdrawn and nuclei were prepared for flow cytometric analysis in a FACScan. Individual nuclear DNA content, as reflected by fluorescence intensity (FL2-A parameter) of incorporated propidium iodide, is plotted in a histogram depicting relative cell numbers at each intensity. Each histogram is representative of data obtained from three similar experiments.
The data presented in this report strongly suggests that T-KG, a physiologically relevant cysteine proteinase inhibitor, can arrest cell proliferation. Expression of the protein in fibroblast cell lines leads to an alteration in growth-related parameters, including the ability to proliferate, both in logarithmically growing cultures and in G 0-arrested cells stimulated to proliferate in response to serum. Also, T-KG inhibits the ability of cells to enter the S phase of the cell cycle, as evidenced by a decreased rate of incorporation of radioactive precursors into DNA, as well as by FACS analysis, and by the observed impairment in the ability to properly reg-
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FIG. 6. T-KG does not affect the ability of cells to induce ERK activity in response to serum stimulation. Serum-starved B MCN-4 and B 2.3 cells, cultured in the presence of 1.25 M CdCl 2, were induced to proliferate by addition of 10% FBS. At different times, total cellular extracts were prepared and ERK activity (ERK1/ERK2) was measured using an “in gel” kinase assay. (Left) Only the first burst of ERK activity (up to 15 min) is represented. (Right) The full experiment (up to 10 h) is shown. Note that the first peak shown in the left panel appears in the right panel very close to the ordinate axis.
ulate both the degradation of p27 and cyclin D3 and the synthesis of cyclin A. On the other hand, both the unimpaired activation of the ERK pathway and the normal synthesis of early response proteins such as c-Fos and c-Jun suggest that cells in G 0 are still capable of sensing the presence of growth factors present in the serum and respond to their stimulation. It should be pointed out that the FACS analysis indicates that the effects we have observed are actually due to inhibition of cell proliferation and not to induction of apoptosis, as evidenced by the absence of an apoptotic peak in those experiments. This is in accordance with our unpublished observations indicating that, on the contrary, T-KG leads to a slight protection against etoposide-induced apoptotic death.
Cells that express T-KG are able to respond to mitogenic stimuli by activating the ERK pathway, and probably other signal transduction pathways as well, as demonstrated by the normal induction of several early response genes. Degradation of these molecules (c-Fos, c-Jun, and Jun B) also proceeds normally (data not shown). In the presence of T-KG, however, cells are arrested at a point in the cell cycle located before they start synthesizing cyclin A, normally at the beginning of the S phase [34]. The presence of T-KG also results in a failure to degrade both cyclin D3 and p27, which normally occurs in late G 1 in Balb/c 3T3 cells [34]. Since both cyclins [35] and p27 [36] have been shown to be degraded primarily by the ubiquitin/proteasome system, we favor the hypothesis that T-KG does not
FIG. 7. T-KG-expressing cells respond to serum stimulation by activating early gene expression. Cells (indicated under each panel) were serum starved for 3 days, and then were induced to proliferate by addition of 10% FBS as described in Fig. 3. Cellular extracts were prepared at different times after serum addition, and the abundance of c-fos (left), c-jun (middle), and cdk 4 (as a control for loading, right) was assessed by Western blotting as indicated under Materials and Methods.
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FIG. 8. Cyclin levels confirm the inability of T-KG-expressing cells to reach the S phase of the cell cycle in response to serum stimulation. Cells (indicated in the middle) were serum starved for 3 days, and then were induced to proliferate by addition of 10% FBS as described in Fig. 3. Cellular extracts were prepared at different times after serum addition, and the abundance of cyclin A, cyclin D3, p27, and cdk 4 (as a control for loading) was assessed by Western blotting as indicated under Materials and Methods.
directly impinge on the ability of the cells to degrade these proteins, but rather, T-KG inhibits the progression of cells through the point in the cell cycle where these proteins should be normally degraded. It is interesting to consider the possible involvement of ERK in this phenomenon. The results shown in Fig. 6 (right panel) indicate that, compared to the controls, in T-KG-expressing cells there is a precipitous and very reproducible decrease in ERK activity approximately 5–9 hours into the G 1 phase. Thus, in these cells there is no detectable ERK activity at the time when the cells should be entering S phase. It has been shown that mitogen-activated protein kinases are required for the proliferative response of quiescent fibroblasts [37] and a sustained activation of p42/p44 MAPK is required for fibroblasts to pass the G 1 restriction point and enter into the S phase [38]. Therefore, we hypothesize that the sudden demise of ERK activity in T-KGexpressing cells could be the cause of the cell’s lack of progression through the cell cycle. Specifically, we propose that while in control cells, ERK activity remains elevated for several hours after serum stimulation (in fact, until the cells reach the G 1/S interphase), the
stabilization of MKP-1 in T-KG-expressing cell lines [33] leads to an early demise of ERK activity. This in turn could lead to the arrest of cells in late G 1 [39]. It should be emphasized that Western blot analysis has shown that expression of T-KG in the cell lines analyzed is actually below physiological range, as compared to the liver of old rats. Therefore our observations are not due to gross overexpression of T-KG to levels that are not physiologically relevant. This is true when comparing either cellular (in the cell lines vs the liver) or extracellular (in the conditioned medium vs serum) T-KG levels (data not shown). Thus, our results suggest that T-KG is a potent inhibitor of proliferation, able to arrest cell cycle progression even at very low doses. This raises several concerns, foremost among which is our ability to obtain stable cell lines by using a leaky promoter. While, as discussed, it is true that we were unable to obtain stable cell lines using the strong CMV promoter, our data indicates that inhibition of proliferation is already evident in the available cell lines even in the absence of inducer (data not shown). However, the inhibition of cell proliferation we have observed in logarithmically growing cells is only par-
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tial, and this is consistent with our observation that T-KG-expressing cell lines grow poorly (not shown). A different issue arises from reports in the literature indicating that kinins have a moderately proliferative effect on cells possessing kinin receptors (40, 41). We observe the opposite effect using a kinin precursor, T-KG. The difference could be due either to the difference in cell types, or to differences in the mechanisms. While it is known that kininogens cannot bind to kinin receptors [42], we cannot formally exclude the possibility that T-KG does act via these receptors, and the inhibitory effect we observe could be due to downregulation of the corresponding signal transduction pathways, including the ERK pathway, as a result of the long-term exposure to the ligand. On the other hand, a major difference between kinins and kininogens is the presence in the latter of cysteine proteinase inhibitory activity. While to date we have not shown that the effects we observe are due to the cysteine proteinase inhibitory function of T-KG, this is very likely to be so, since inhibition of cysteine proteinase activity is known to result in decreased cell proliferation, as well as impaired progression through the cell cycle [8, 12, 13, 43]. By using synthetic aldehyde thiol protease inhibitors, it has been demonstrated that progression of vascular smooth muscle cells requires cysteine protease activity in the transitions between G 0/G 1, G 1/S, and G 2/M [8]. In our experimental approach, we observe inhibition at the G 1/S, but we do not see an effect of T-KG at the G 0/G 1 interphase. This could reflect a difference either in the cell types used, or in specificity between chemical inhibitors and physiological ones, which are more likely to be compartmentalized within the cell. Together, these data indicate that T-KG expression leads to inhibition of cell proliferation, thus supporting our hypothesis that the age-related increase in both T-KG gene expression [20, 22, 23] and serum T-KG levels [21], could play a role in the well-known diminished proliferative capacity of tissues from old animals. We are gratefully indebted to Dr. P. Shaw (CHUV, Lausanne, Switzerland) for providing the vector pMT1-Cas. We also especially thank our colleagues Chris Sell and Mary Kay Francis for their critical reading of the manuscript. This work was supported by NIH Grant AG 13902, by a Minority Supplement to Grant AG 00378-24, and by FONDECYT Grant 1981064.
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