Modulatory effects of PKC activity on increased 92-kDa gelatinase secretion by neonatal alveolar macrophages

Christophe Delacourt, Patricia Rouet-Benzineb, Christophe Delclaux, Jeannique L'Hour, Alain Harf, and Chantal Lafuma

Unité de Physiologie Respiratoire, Institut National de la Santé et de la Recherche Médicale Unité 296, and Unité de Physiopathologie Cellulaire et Fonctionnelle du Coeur et des Vaisseaux, Institut National de la Santé et de la Recherche Médicale Unité 400, Faculté de Médecine de Créteil, 94010 Créteil, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We previously demonstrated that alveolar macrophages (AMs) from neonatal rats can secrete more 92-kDa gelatinase than AMs from adult rats. In this study, we investigated the role of the protein kinase C (PKC) pathway in the transductional regulation of 92-kDa gelatinase secretion by rat AMs, and we also evaluated maturational changes in this role with increasing postnatal age. After AM stimulation by phorbol 12-myristate 13-acetate (PMA), we observed a dose-dependent increase in gelatinase secretion that was significantly more marked in AMs from 6-day-old rats than in AMs from adult rats and that was inhibited by the PKC inhibitor calphostin C. Adenosine 3',5'-cyclic monophosphate mimetics or concanavalin A failed to induce an increase in gelatinase secretion by AMs. Time-dependent variations in PKC activity after PMA stimulation differed significantly between 6-day-old rats and adult rats; PKC activity decreased in adult AMs (50%) but remained stable in 6-day-old AMs. We therefore investigated age-related differences in the intracellular proteolytic degradation of PKC, which is thought to be mediated by calpains. Leupeptin, used as a calpain inhibitor, inhibited the decrease in PKC activity after exposure of adult AMs to PMA and induced a greater than threefold increase in PMA-induced gelatinase secretion. Calpain activity was significantly lower in AM extracts from 6-day-old than from adult rats. The physiological implication of these developmental changes in 92-kDa gelatinase regulation was demonstrated by investigation of AMs from 1-day-old rats that showed a high level of spontaneous PKC-dependent gelatinase secretion coexisting with very low calpain activity. We conclude that sustained PKC activity is a key factor in the increased gelatinase secretion by AMs seen during the postnatal period and is due, at least in part, to reduced PKC degradation.

lung development; metalloproteinases; calpains; protein kinase C

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE NEONATAL LUNG is known to be at high risk for long-term sequelae after injurious processes (3, 5, 18). A major contributing factor to this increased susceptibility may be a specific functional pattern of neonatal alveolar macrophages (AMs), which may include reduced opsonic receptor function (16), defective ingestion or killing of bacteria (17), diminished free radical production (10), and impaired chemotaxis (17). We recently showed that AMs from newborn rats may also contribute to connective tissue degradation in the injured lung by secreting more 92-kDa gelatinase than AMs from adult rats (9). Among the different metalloproteinases synthesized by AMs, 92-kDa gelatinase is the most prominent (35). This enzyme cleaves basement membrane type IV collagen and efficiently degrades several denatured collagen types and gelatins (22). During normal postnatal lung growth, rapid turnover of type IV collagen has been demonstrated (2), as well as focal degradation of basement membranes, allowing transient intercellular contacts between pulmonary epithelial and interstitial cells (1). The increased spontaneous secretion of 92-kDa gelatinase that we demonstrated in neonatal AMs may contribute to these matrix changes. However, the balance between type IV collagen synthesis and degradation during normal lung development may be compromised during pulmonary insults, since we also found that stimulated neonatal AMs secreted four to five times more gelatinase than stimulated adult AMs (9). These differences in gelatinase secretion between neonatal and adult AMs may result from differences in intermediate signaling steps involved in the regulation of gelatinase production.

The aim of the present study was to gain insight into the maturation of signal transduction pathways involved in the stimulation of gelatinase secretion, with special attention to the modulatory role of the protein kinase C (PKC) pathway. PKC is a critical component of the signal transduction pathways used by cells to recognize and to respond to a variety of extracellular agents (25). There is evidence that PKC contributes to the regulation of 92-kDa gelatinase secretion in AMs. Phorbol esters, which bind directly to PKC (23), have been identified as potent stimulants of 92-kDa gelatinase secretion by AMs (35), and PKC inhibitors reduced lipopolysaccharide (LPS)-induced gelatinase secretion from macrophages (34, 36). Because phorbol esters also induced a higher level of 92-kDa gelatinase secretion from neonatal AMs than from adult AMs, we hypothesized that developmental changes in the state of activation and regulatory role of PKC may contribute to the observed functional differences between neonatal and adult AMs.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents. Gelatin, phorbol 12-myristate 13-acetate (PMA), concanavalin A (ConA), forskolin, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), EDTA, ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethylsulfonyl fluoride (PMSF), and calphostin C were obtained from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) was from GIBCO Life Technologies (Cergy Pontoise, France).

AM isolation and culture. Sprague-Dawley rats were obtained from Charles River (Saint-Aubin les Elbeuf, France). Rat pups were tested on day 6 after birth and are called either 6-day-old rats or juvenile rats throughout this paper. Control adult rats were males weighing between 250 and 300 g.

AMs were obtained by in situ bronchoalveolar lavage (BAL) using a previously described technique (9). Briefly, rats were anesthetized with 5 mg/100 g body wt of intraperitoneally injected pentobarbital sodium and then were killed by exsanguination. The thorax was widely opened to expose the lungs and trachea. A small length of tubing was inserted into the trachea and ligated. BAL was carried out using 8-10 separate aliquots of warmed saline (37°C). Total volumes of BAL were 4 and 20 ml in 6-day-old and adult rats, respectively. Lavage fluids from several neonates (usually 4-6) were pooled to obtain a sufficient number of cells (at least 1.5-2 × 106 AMs). The lavage fluid was centrifuged at 300 g for 7 min, and the cell pellet was resuspended at 1 × 106 cells/ml in DMEM supplemented with 0.5% bovine serum albumin, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, and 2 mM glutamine. Cell smears were stained using the standard May-Grünwald-Giemsa procedure for each pool, and differential cell count always showed >98% AMs.

AMs were incubated in culture plates at 37°C for 2 h to allow attachment. Nonadherent cells were then removed by changing the culture medium. Cell cultures were maintained in a 95% air-5% CO2 atmosphere. After 24 h of culture, cell- conditioned media were collected, and cell viability was determined using trypan blue exclusion (viability was consistently >85%). The samples were frozen at -80°C until the assay. To determine the effects of various stimulants on 92-kDa gelatinase secretion, both neonatal and adult cell cultures were exposed, after removal of nonadherent cells, to PMA (10-6 to 10-8 M), ConA (10-20 µg/ml), forskolin (10-7 to 10-4 M), or DBcAMP (10-7 to 10-4 M) for 24 h.

Zymographic analysis of total gelatinase activity. Constant volumes of conditioned culture media (15 µl) were subjected to electrophoresis in 8% (wt/vol) polyacrylamide gels (PAGE) containing 1 mg/ml gelatin, in the presence of sodium dodecyl sulfate (SDS) under nonreducing conditions. After electrophoresis, gels were washed in 2.5% Triton X-100 for 1 h, rinsed briefly, and incubated at 37°C for 24 h in a buffer containing 100 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.40, and 10 mM CaCl2. The gels were then stained with Coomassie brilliant blue R250 and destained in a solution of 7.5% acetic acid and 5% methanol. Zones of proteolytic activity were seen as clear bands against a blue background.

Activities in the gel slabs were quantified using a computerized image-analysis program (National Institutes of Health Image 1.52, Macintosh) that quantifies both the surface area and the intensity of lysis bands. Results are expressed as arbitrary units (AU) per 24 h per 106 AMs. An internal standard was used in each gel to assess zymogram comparability. Linearity of this method for measuring enzymatic activity on zymograms over the range of activities found in unknown samples was previously evaluated (12).

The 92-kDa gelatinase activity was characterized by immunoblotting. Conditioned culture media from stimulated adult AMs were pooled and partially purified using gelatin-Sepharose chromatography, as previously described (9). Human purified 92-kDa gelatinase (Valbiotech, Paris, France) was used as a positive control. Samples were separated on SDS-PAGE and were electrophoretically transferred to nitrocellulose. After saturation of the excess protein binding sites with 5% bovine milk in 0.05 M Tris · HCl and 0.15 M NaCl, pH 7.6 (Tris-buffered saline), for 1 h at room temperature, the nitrocellulose was incubated with specific rabbit antibody to 92-kDa gelatinase at 1:200 dilution (Triple Point Biologics, Forest Grove, OR) in the above buffer for 1 h at room temperature. After thorough washing with Tris-buffered saline, the samples were incubated with peroxidase-labeled goat anti-rabbit antibody (1:1,000 dilution) in Tris-buffered saline containing 5% bovine milk for 1 h at room temperature. After washing, the immunoblots were visualized using enhanced chemiluminescence detection (Amersham, Buckinghamshire, UK).

Assay of PKC activity. To evaluate PKC activity, cell lysates were collected at baseline and at various times after stimulation. After removing the culture medium, each well containing 5 × 105 AMs was washed two times with phosphate-buffered saline (PBS) and was then resuspended in 200 µl of lysis buffer containing 20 mM Tris · HCl, pH 7.5, 0.25 M saccharose, 0.5% Triton X-100, 2 mM EDTA, 2 mM EGTA, 2 mM PMSF, and 0.1% leupeptin. Lysates were collected and further sonicated.

Ca2+- and phospholipid-dependent PKC was assayed on lysate samples by measuring 32P transferred from [gamma -32P]ATP to lysine-rich histone type III-S, as described by Thomas et al. (33). The reaction mixture (250 µl) contained 20 mM Tris · HCl, pH 7.4, 0.75 mM CaCl2, 100 µg/ml histone H1, 10 µM [gamma -32P]ATP (1,250 counts · min-1 · pmol-1), 31 µM bovine brain phosphatidylserine, and 0.5 µM 1,2-dioleyl-sn-glycerol. Twenty-five microliters of lysate samples were added to the incubation mixture. After 10 min at 30°C, the reaction was stopped by addition of 0.9 ml of 12% trichloroacetic acid (TCA). The acid-precipitable material was collected by centrifugation, dissolved in 1 N NaOH (100 µl), and precipitated again with 1 ml of 12% TCA. 32P incorporation was measured in the presence or in the absence of phospholipids. The linearity of the reaction over the 10-min period was verified in preliminary experiments. Results were expressed as picomoles per minute per 5 × 105 AMs.

In some experiments, we examined the subcellular distribution of PKC activity using the technique described by Pelech et al. (27), with the goal of assessing the PKC translocation between the cytosol and the membranes. Culture wells were placed on ice, and culture medium was removed. Each well was washed two times with ice-cold PBS. The soluble extract was produced when cells were incubated at 0°C for 10 min with 50 µM digitonin in 20 mM Tris · HCl, pH 7.4 (200 µl), with 2 mM EDTA, 0.5 mM EGTA, 2 mM PMSF, and 0.1% leupeptin. The cell remnants, after removal of the soluble extracts, were solubilized in 200 µl of the same buffer plus 0.5% Triton X-100 to obtain the particulate extract.

Assay of intracellular neutral cysteine proteinase activity. Activated PKC is thought to be degraded by Ca2+-activated neutral cysteine endopeptidases called calpains. To evaluate calpain activity in cell extracts, casein has been preferentially used as a test substrate because it is extensively hydrolyzed to acid-soluble peptides (8). Maximal in vitro proteinase activity is obtained at pH 7.5 in the presence of sulfhydryl reducing agents such as 2-mercaptoethanol (7). We therefore evaluated Ca2+-activated neutral protease activity in neonatal and adult AM extracts using [3H]casein as the substrate. Specific activity of radiolabeled casein was 1.4 µCi/50 µg. Cells harvested from BAL fluids were incubated in conditioned culture media as described above. Resting AMs were lysed in culture wells with 200 µl of 20 mM Tris · HCl buffer, pH 7.5, containing 1 mM EDTA, 1 mM EGTA, 2 mM PMSF, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100. The reaction mixture (500 µl) contained 20 mM Tris · HCl, pH 7.5, 5 mM 2-mercaptoethanol, 1 mM CaCl2, 50 µg [3H]casein, and 50 µl of lysate sample. Because 750 µM Ca2+ is required to obtain a full activation of both ubiquitous calpain isozymes, calpain I and calpain II (11), we determined the Ca2+ concentration in the reaction mixture using a specific Orion 9320 Ca2+ electrode connected to an SA 720 Orion ionometer (Boston, MA). Ca2+ concentration was found to be 920 µM. The reaction was allowed to proceed for 24 h at 37°C and was stopped by immersion in a ice bath immediately followed by addition of 170 µl of precipitating solution containing 12.5% TCA and 2.5% tannic acid. After 30 min, the mixture was centrifuged at 4°C for 10 min at 8,000 g, and the radioactivity of the supernatant was measured in a liquid scintillation counter (Minaxi Tri-Carb 4000 Series, United Technologies Packard). Each lysate sample was tested in the absence and in the presence of leupeptin to determine the caseinolytic activity due to calpains and other cysteine proteases. Samples were thus incubated in the reaction mixture for 30 min at 37°C with 1 mM leupeptin, and [3H]casein was then added. Results were expressed as micrograms degraded casein per 24 h per 5 × 105 AMs.

Statistical analysis. Results are expressed as means ± SE. Analysis of variance with repeated measures was used for the statistical analysis of differences in time-dependent responses between 6-day-old and adult rats. The nonparametric Mann-Whitney U-test was used to compare 6-day-old and adult rats. P values under 0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Total gelatinase activity. Total gelatinase activity in conditioned culture media was evaluated using zymography. As expected, AMs from 6-day-old and adult rats spontaneously released very low levels of 92-kDa gelatinase. Among the different stimuli used, forskolin, DBcAMP, and ConA failed to significantly increase gelatinase activity both in juvenile and in adult rats. On the contrary, PMA, which is known to directly activate PKC, induced significant increases in total gelatinase activity in both 6-day-old and adult rat AMs (Fig. 1). This increase was dose dependent and was significantly more marked on day 6 than in adulthood (P = 0.0001). The 92-kDa band was identified as a metalloproteinase based on the fact that it was completely inhibited by 10 mM EDTA but not by 2 mM PMSF or 2 mM N-ethylmaleimide (data not shown). Immunoblotting was performed under reducing conditions using anti-92-kDa gelatinase antibodies. Rat 92-kDa gelatinase was firmly recognized at the same molecular weight as human 92-kDa gelatinase used as positive control (Fig. 2). We selected 10-7 M PMA as the dose for further experiments. Despite higher levels of gelatinase activity after 24 h of PMA stimulation in neonates, the kinetics of the increase in gelatinase activity over time were similar in juvenile and adult rats (Table 1).


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Fig. 1.   Gelatinase activity released from rat alveolar macrophages (AMs). A: gelatin zymogram of AM-conditioned media from 6-day-old and adult rats after 24 h of culture under various conditions [resting cells (lanes a and e) and cells stimulated with 10-8 M phorbol 12-myristate 13-acetate (PMA; lanes b and f), 10-7 M PMA (lanes c and g), 10-6 M PMA (lanes d and h), forskolin (lane i), dibutyryl-cAMP (DBcAMP; lane j), and concanavalin A (lane k)]. B: quantification of PMA-induced 92-kDa gelatinase activity released from AMs of 6-day-old (bullet ) and adult (open circle ) rats. Gelatinase activity was measured by computerized image analysis of zymograms. Results are expressed as arbitrary units (AU) · 24 h-1 · 106 AMs-1. * Significant difference (P < 0.05) in the PMA-induced gelatinase secretion between newborns and adults; n = 4 pools of neonates and 4 adults.


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Fig. 2.   Immunoblotting of 92-kDa gelatinase. Lane 1: human purified 92-kDa gelatinase (100 ng); lane 2: adult rat 92-kDa gelatinase after partial purification (20 µl).

                              
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Table 1.   Kinetics of the 92-kDa gelatinase activity increase in rat AMs stimulated by 10-7 M PMA

To establish that PMA-induced secretion was mediated through PKC activity, AMs were preincubated with calphostin C (10-8 to 10-6 M), a specific and cell-permeable inhibitor of PKC (15), 15 min before 10-7 M PMA addition. Calphostin C induced a dose-dependent inhibition of the PMA-induced gelatinase activity (Fig. 3). Cell viability estimated by trypan blue exclusion was >90% in each test condition.


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Fig. 3.   Inhibition of PMA-induced 92-kDa gelatinase by a protein kinase C (PKC) inhibitor (calphostin C). AMs from 6-day-old (bullet ) and adult (open circle ) rats were preincubated with calphostin C (10-8 to 10-6 M) 15 min before exposure to 10-7 M PMA for 24 h. Gelatinase activity in conditioned culture media was evaluated using zymography and was quantified using computerized image analysis of zymograms. Results are expressed as percentages of PMA-induced gelatinase activity in the absence of inhibitor; n = 3 pools of neonates and 3 adults.

PKC activity. PKC activity was evaluated in cellular extracts from 6-day-old and adult rat AMs. Baseline levels of PKC activity did not differ significantly between neonatal and adult AMs (31.1 ± 4.1 and 33.3 ± 5.0 pmol · min-1 · 5 × 105 AMs-1, respectively). However, during stimulation with 10-7 M PMA, a time-dependent decrease in PKC activity was observed in adult AM extracts; surprisingly, no such decrease occurred with juvenile AM extracts (Fig. 4). PKC activity 1 h after PMA addition was only 50.4 ± 8.6% of basal values in adult AMs versus 93.5 ± 8.3% in juvenile AMs (P < 0.01).


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Fig. 4.   PKC activity in AM extracts from 6-day-old (bullet ) and adult (open circle ) rats. Adherent AMs were stimulated by 10-7 M PMA, and cellular extracts for evaluation of PKC activity were obtained after various stimulation times (5 min to 24 h). Results are expressed as percentages of resting PKC activity values. * Significant difference (P < 0.05) in PKC activity between newborns and adults; n = 5 pools of neonates and 7 adults.

In resting AMs, the membrane-bound fraction of PKC activity was high and similar in juvenile and adult rats (Table 2). After stimulation, this fraction tended to increase in juvenile AMs and to decrease in adult AMs, but the difference remained nonsignificant.

                              
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Table 2.   Membrane-bound fraction of protein kinase C activity in rat AMs

Relation between PKC activity and level of gelatinase secretion. We hypothesized that the sustained PKC activity seen in 6-day-old rats during the stimulation period contributed to the high level of PMA-induced gelatinase secretion in these animals. Assuming that the decrease in PKC activity during PMA stimulation seen in adult AMs was due to calpain-mediated PKC degradation (8), we preincubated adult AMs with leupeptin, a cell-permeable inhibitor of calpains (19).

As a first step, we verified the inhibitory effect of 1 mM leupeptin on PKC degradation in adult AMs. Adult cells were or were not preincubated with 1 mM leupeptin 30 min before addition of 10-7 M PMA. Cell lysates were investigated for measurement of PKC activity at baseline and after PMA stimulation for 1 and 24 h. Leupeptin (1 mM) had no effect on baseline PKC activity [22.1 ± 3.5 pmol · min-1 · 5 × 105 AMs-1 compared with 25.7 ± 1.9 pmol · min-1 · 5 × 105 AMs-1 in cells without leupeptin (n = 5)]. After 1 h of PMA stimulation, leupeptin completely inhibited the transient decrease in PKC activity (Fig. 5).


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Fig. 5.   Inhibitory effect of leupeptin on PKC activity downregulation in adult rat AMs. AMs were (open circle ) or were not (bullet ) preincubated with 10-3 M leupeptin 30 min before addition of 10-7 M PMA. Cell extracts for PKC activity measurements were collected at rest and after 1 and 24 h of stimulation. Results are expressed as percentages of resting PKC activity value. * Significant difference (P < 0.05) in the PKC activity between 2 conditions; n = 5 adults.

The effect of leupeptin on gelatinase activity was therefore evaluated. Leupeptin (10 µM to 1 mM) was added to culture media 30 min before stimulation of adult cells with 10-7 M PMA, and conditioned culture media collected after 24 h of stimulation were investigated for gelatinase activity. Preincubation with 1 mM leupeptin, but not 10 or 100 µM, induced a greater than threefold increase in gelatinase activity compared with PMA-stimulated cells without leupeptin (Fig. 6). Gelatinase activity after 1 mM leupeptin treatment was 83.3 ± 9.6 × 105 AU · 24 h-2 · 106 AMs-1, i.e., very similar to that observed with stimulated 6-day-old AMs in the absence of pretreatment (70.8 ± 7.5 × 105 AU · 24 h-1 · 106 AMs-1). Incubation with 1 mM leupeptin alone, without PMA, had no stimulating effect.


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Fig. 6.   Influence of leupeptin on PMA-induced 92-kDa gelatinase secretion in adult rat AMs. AMs from adult rats were incubated for 24 h in the presence (lanes b-e) or in the absence (lanes a and f) of 10-7 M PMA. Cells were preincubated or were not preincubated with leupeptin 30 min before stimulation (lane c, 10-5 M leupeptin; lane d, 10-4 M leupeptin; lanes e and f, 10-3 M leupeptin). A: gelatin zymogram; B: quantification by scanning densitometry. Filled bars, no PMA; open bars, 10-7 M PMA added. Results are expressed as AU · 24 h-1 · 106 AMs-1. * Significant difference in PMA-induced 92-kDa gelatinase secretion after preincubation with leupeptin compared with absence of leupeptin (lane b); n = 4 adults.

Intracellular neutral cysteine proteinase activity. Ca2+-activated neutral cysteine protease activity was evaluated using [3H]casein as the substrate. In resting AMs (Fig. 7), total caseinolytic activity was found to be significantly higher in cells from adult rats than from 6-day-old rats (P = 0.01). Under our reaction conditions, caseinolytic activity was almost entirely due to cysteine proteinases because 1 mM leupeptin inhibited because caseinolytic activity by >80% in both adult and juvenile AMs. Mean leupeptin-inhibitable proteinase activity, which represents intracellular neutral cysteine proteinase activity, was therefore 23.0 ± 1.5 and 15.1 ± 1.8 µg degraded casein · 24 h-1 · 5 × 105 AMs-1 in adult and 6-day-old rats, respectively (P < 0.03). When 5 mM EDTA was added to the reaction mixture instead of 1 mM CaCl2, caseinolytic activity was inhibited by 85%, attesting to the Ca2+ dependence of the measured proteinase activity.


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Fig. 7.   Ca2+-activated neutral protease activity in AM extracts from 6-day-old (open bars) and adult (filled bars) rats. [H3]casein was used as the substrate. Total caseinolytic activity was evaluated in the presence of 1 mM CaCl2. Fraction of total caseinolytic activity inhibited by 1 mM leupeptin was >80% in both adult and juvenile rats. Results are expressed as µg casein degraded · 24 h-1 · 5 × 105 AMs-1. * Significant difference (P < 0.05) between newborns and adults; n = 5 neonatal pools and 5 adults.

Role of PKC pathway in the spontaneously high level of gelatinase secretion immediately after birth. Because our data supported a large contribution of the PKC pathway to the increased 92-kDa gelatinase secretion by neonatal rat AMs, we investigated whether the PKC pathway was also involved in the spontaneous production of high levels of 92-kDa gelatinase activity by neonatal rat AMs immediately after birth (9). AMs were obtained by BAL from newborn rats during the first 24 postnatal hours, as previously described (9). AMs were isolated and cultured as described above in the presence or in the absence of 10-6 M calphostin C (>85% cell viability). After 24 h of incubation, gelatinase activity was evaluated in conditioned culture media using zymography. Calphostin C completely inhibited the high gelatinase activity released by control cells (Fig. 8). PKC activity in resting cells was 27.5 ± 6.7 pmol · min-1 · 5 × 105 AMs-1 (n = 3 pools), which was not significantly different from the resting values in 6-day-old and adult AMs. Intracellular neutral cysteine proteinase activity in neonatal AM extracts was 4.7 ± 2.5 µg degraded casein · 24 h-1 · 5 × 105 AMs-1 (n = 4 pools), which was significantly lower than in 6-day-old and adult rat AMs (15.1 and 23.0 µg degraded casein · 24 h-1 · 5 × 105 AMs-1, respectively; P = 0.0001).


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Fig. 8.   Gelatin zymogram of AM-conditioned media from <24-h newborn rats after 24 h of culture in the absence (control) or in the presence of 10-6 M calphostin C.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AMs play a key role in pulmonary defenses. After physiological or pathological stimulation, these cells are able to contribute to the degradation and remodeling of the extracellular matrix through the secretion of metalloproteinases and metalloproteinase inhibitors. The main metalloproteinase secreted by AMs is the 92-kDa gelatinase, which cleaves basement membrane type IV collagen as well as denatured collagens or gelatins. Uninhibited gelatinase activity can result in defective basement membrane organization, with increased permeability to inflammatory cells and dysregulation of cell attachment, growth, and differentiation (20). The neonatal period is characterized by continued pulmonary growth and by extensive lung remodeling (4). In particular, postnatal lung growth involves rapid basement membrane turnover as cells proliferate and alveoli form (2). Increased secretion of 92-kDa gelatinase by stimulated neonatal AMs may contribute to an imbalance between basement membrane collagen synthesis and degradation, leading to impaired lung development after lung injury. In the present study, we examined the intracellular signaling pathways that mediate the secretion of 92-kDa gelatinase in rat AMs. We found that gelatinase production was stimulated in a dose-dependent manner via the PKC pathway and that the increase in 92-kDa gelatinase secretion by neonatal AMs was due to sustained poststimulation PKC activity originating in decreased intracellular PKC degradation.

Maturational differences in gelatinase production from rat AMs. Among the different stimuli used in our study, only PMA significantly increased 92-kDa gelatinase activity from rat AMs. Both 6-day-old and adult rat AMs responded to PMA in a dose-dependent manner, but secreted 92-kDa gelatinase activity rose 17-fold in juvenile AMs versus only 7-fold in adult cells, whereas basal activities were comparable. These results confirm our previous report of an increased response of neonatal cells to PMA (9) and suggest that the PKC pathway may play a pivotal role in the transductional regulation of 92-kDa gelatinase production. As is the case with other phorbol esters, PMA can directly activate PKC, thus bypassing the endogenous pathway involving production of diacylglycerol (DAG) from inositol phospholipids after stimulation of cell surface receptors (23). Also, the age-related differences in PMA-induced gelatinase secretion suggest maturational differences in the intracellular signaling mechanisms involved in 92-kDa gelatinase secretion by rat AMs and particularly in the modulatory role of the PKC pathway.

Role of PKC pathway in transductional regulation of 92-kDa gelatinase production. The intracellular signal transduction pathways that mediate the secretion of 92-kDa gelatinase in macrophages are incompletely understood. LPS-stimulated 92-kDa gelatinase production has been shown to involve both PKC (34, 36) and protein tyrosine kinase (36) activities. Regulation via a prostaglandin E2-mediated adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway has also been proposed (28). Our results argue for a predominant role of the PKC pathway in the modulation of 92-kDa gelatinase secretion by rat AMs.

First, gelatinase secretion increased after exposure to phorbol ester, which directly stimulates PKC, but not to agents in which stimulatory effects are not thought to be primarily mediated through the PKC pathway. Thus cAMP mimetics such as DBcAMP and the adenylate cyclase activator forskolin failed to increase 92-kDa gelatinase in stimulated AMs. In keeping with our findings, Xie et al. (36) found that an inhibitor of a cAMP-dependent protein kinase (protein kinase A) failed to modify LPS-induced 92-kDa gelatinase secretion by macrophages (36). Similarly, ConA failed to increase gelatinase activity in rat AMs, although previous studies have demonstrated that this agent can induce matrix metalloproteinase expression in cell types other than AMs via different mechanisms than phorbol esters (6, 26).

Second, changes in intracellular PKC activities in adult rat AMs were associated with modifications in gelatinase secretion. Phorbol esters activate PKC by mimicking the effect of the natural PKC activator DAG. DAG production is short lived, and thus levels of DAG rapidly return to basal values, leading to a reversible activation of PKC (25). In contrast, phorbol esters may cause sustained PKC activation, which may lead in turn to downregulation of the PKC proteins themselves. Calpain-mediated proteolysis of membrane-bound PKC may be responsible for this downregulation (8). This phenomenon was observed in our study in PMA-stimulated adult rat AMs. PKC activity diminished by 50% within 1 h of addition of PMA to the culture media. Leupeptin, used as a calpain inhibitor, prevented this downregulation, leading to sustained PKC activity in adult rat AMs and to increased 92-kDa gelatinase secretion. These effects demonstrate the key role of the PKC signaling pathway in the regulation of gelatinase secretion.

Finally, the inhibitory effect of calphostin C on 92-kDa gelatinase secretion by stimulated or resting AMs is further evidence of an effect of PKC on the regulation of 92-kDa gelatinase secretion. Calphostin C is a potent and selective PKC inhibitor that acts at the phorbol ester binding site. In particular, calphostin C has been found to induce 1,000-fold greater inhibition of PKC than other protein kinases, such as cAMP-dependent protein kinase and tyrosine-specific kinase (15). The complete inhibition of PMA-induced gelatinase secretion obtained in our study with 1 µM calphostin C excludes an inhibitory effect due to protein kinases other than PKC (15). We also used calphostin C on resting cells from newborn rats aged <24 h. The spontaneously high level of gelatinase activity seen in these cells was completely inhibited by calphostin C. This suggests that sustained activation of PKC is present in these neonatal cells and is essential to the persistence of a high level of gelatinase activity.

Maturational differences in the modulatory role of the PKC pathway. Our results indicate that the increased 92-kDa gelatinase secretion in response to PMA in 6-day-old rat AMs was related to sustained activation of PKC. Maintenance of PKC activation may be due to an unusual interaction of this enzyme with membranes or to a slow rate of PKC degradation. Our results argue for a low capacity of PKC degradation in 6-day-old AMs.

It has been often observed that, upon cell stimulation, primarily cytosolic PKC is translocated to membranes, where it exhibits catalytic activity. However, the physiological significance of this translocation is still unclear, and the association of PKC with membranes does not always reflect the state of activation of the enzyme (25). In the present study, we found a high fraction (40%) of membrane-associated PKC in resting AMs, with no difference between neonates and adults. A similar subcellular distribution of PKC has already been reported in rat AMs, with an even higher fraction (60%) of membrane-bound PKC (29). Even after PMA stimulation, no age-related difference in the fraction of membrane-associated PKC was observed, but, as a consequence of PMA activation, PKC became more susceptible to downregulation in adult AMs.

Available data suggest that PKC is normally cleaved by Ca2+-activated neutral proteases, also called calpains. Two ubiquitous calpain species have been characterized. Both share similar biochemical characteristics, except for the Ca2+ concentration required for activation in vitro (31). Insights into the physiological effects of calpains have been provided by studies on neutrophil activation (30), platelet activation (32), phorbol ester-induced alterations in lung epithelial cell morphology (13), and impaired NK cell activity in the beige mouse (14). In our study, we found a clear link between leupeptin-inhibitable degradation of PKC and 92-kDa gelatinase secretion in rat AMs, and we demonstrated that the absence of PKC degradation during the first hour after AM stimulation was responsible for the increased secretion of gelatinase by juvenile AMs. We further demonstrated that the amount of Ca2+-activated neutral proteases was significantly less in 6-day-old rat AMs than in adult cells, suggesting that the persistence of PKC activity after PMA stimulation of juvenile AMs may have been at least partly due to a lower capacity of PKC degradation. To our knowledge, this is the first study reporting evidence of postnatal PKC signaling pathway maturation and of its potential physiological implications.

Other PKC regulatory systems than calpains may be involved, according to the time of stimulation, as suggested by the partial recovery of PKC activity after the first hour of stimulation in adult AMs. Further studies are needed to describe more precisely these late regulatory systems for PKC activity. Nevertheless, these late phenomena appeared to have no influence on gelatinase secretion compared with the regulation of PKC degradation within 1 h of stimulation. Indeed, the inhibition of the initial PKC degradation in adult AMs was sufficient to increase gelatinase secretion to levels similar to those measured in juvenile AMs.

Physiological implications. Sustained PKC activation can be experimentally induced by exposure to phorbol esters and also occurs in response to several physiological mechanisms. These include high charge density in the membrane (25), sustained production of DAG by phospholipases, especially by phospholipase D (24), and possibly other proteins that may stabilize the association of active PKC to a specific locus on the membrane (21). Our results suggest that the persistence of PKC activation may also involve the capacity of the cell to degrade activated PKC. Indeed, inhibition of calpain activity in adult AMs or physiologically low calpain activity in cells from 6-day-old rats resulted in an absence of PKC downregulation after cell stimulation.

Sustained activation of PKC may be essential for the persistence of cellular responses over a long period of time, and our results suggest such a physiological sustained PKC activity in neonatal AMs immediately after birth. The high levels of spontaneously released 92-kDa gelatinase that we reported previously (9) were PKC dependent, but the PKC activity measured in neonatal cells was comparable to that measured in resting cells from older rats. A major finding of this study was the very low intracellular neutral cysteine proteinase activity in neonatal AMs, even compared with 6-day-old rat AMs. This is consistent with the hypothesis that neonatal cells immediately after birth may be "programmed" to have low calpain activity and that this may result in sustained PKC activity responsible for release of high levels of gelatinase activity. This pattern may be useful in neonates to ensure intense physiological remodeling of basement membranes (1, 2) or migration of AMs into the alveoli because 92-kDa gelatinase has been shown to permit migration of inflammatory cells across basement membranes (11). Developmental changes consist of a gradual increase in calpain activity from birth to adulthood, with an intermediary state in 6-day-old rat AMs characterized by a low level of gelatinase secretion at rest and by a potential for sustained PKC activation with secretion of large amounts of gelatinase during cell stimulation. Adult AMs are characterized by a high level of calpain activity responsible for rapid PKC degradation after cell stimulation and, consequently, for a low level of gelatinase secretion. Whether these in vitro findings can be extrapolated to in vivo responses to lung injury remains to be established, but increased 92-kDa gelatinase secretion from AMs could contribute to the increased frequency of sequelae after lung injury during the neonatal period.

    FOOTNOTES

Address for reprint requests: C. Delacourt, Unité de Physiologie Respiratoire, INSERM U 296, Faculté de Médecine, 8, rue du Général Sarrail, 94010 Creteil, France.

Received 3 December 1996; accepted in final form 1 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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