Collagen synthesis and turnover following particle phagocytosis in dermal fibroblasts

161

Biochimica et Biophysica Acta, 607 (1980) 161--170 © Elsevier/North-Holland Biomedical Press

BBA 99620

COLLAGEN SYNTHESIS AND TURNOVER FOLLOWING PARTICLE PHAGOCYTOSIS IN DERMAL FIBROBLASTS

JOHN J. SAUK, Jr. Department of Oral Pathology, Medicine, and Genetics, School of Dentistry, University of Minnesota, Minneapolis, MN 55455 (U.S.A.) (Received July 17th, 1979) Key words: Collagen synthesis; Protein turnover; Phagocytosis; (Dermal fibrobIast)

Summary Dermal fibroblast collagens were isolated after cold pepsin/acetic acid extraction and characterized by differentiated salt precipitation, agarose molecu h r sieve chromatography, CM-cellulose chromatography, and identification of cyanogen bromide cleavage peptides. Subsequent to particle phagocytosis, collagens recovered as secretory products from latex-treated cells were quantitatively less in total collagen and deficient in type III collagen. Although the total levels of hydroxyproline synthesized were similar to control cell populations, hydroxyproline recovered as non
162

20%) collagens [5]. Although both collagens may have their origins in many cell types [6--10], both type I and type III collagens may be synthesized in vitro by dermal flbroblasts [11]. Gay et al. [6] have further demonstrated that both type I and type III collagens m a y be coincident gene products of a single fibroblast. In spite of this potential, the ratio of these collagen types synthesized and secreted in vitro may vary, depending upon the growth rate and nutritional requirements provided to the system [ 12]. The present investigation demonstrates that under conditions of undigestible particle phagocytosis, the gene product of collagens recovered as secretory products in vitro are deficient in type III collagen. Compartmental analysis, however, indicates that these alterations are the result of rapid intracellular protein turnover. Materials and Methods

Cell culture. Dermal flbroblast cell cultures were prepared from small pieces of embryonic mouse skins after the methods of Birkedal-Hansen et al. [13]. Cells were grown in the presence of minimal essential medium, 10% fetal calf serum, and 1% glutamine in an atmosphere of 95% air and 5% CO2 at 37°C. Upon reaching confluence the cells were freed from the supporting media using Ca2+ and Mg2+-free saline containing 0.1% EDTA and 0.125% trypsin and replated in 75-cm 2 Falcon flasks. Additional cell passages were carried out every three weeks by the same procedures with a dilution factor at each passage of 1 : 5. All experiments were performed on cells between the 3rd and 8th passages. Radioactive labelings were performed in confluent cell cultures for 24 h with complete medium containing [2-aH]glycine (50 ~Ci/ml (Amersham) and/or [14C]proline (5/~Ci/ml) (Amersham) in the presence of 100 /~g/ml /~-aminopropionitrile and 50 ~g/ml ascorbic acid. The purity of [14C]proline was determined on a Beckman amino acid analyzer and the level of contaminating hydroxy[~4C]proline was considered for each batch of isotope and appropriately corrected in the tabulation of results. In general, the level of contanimation was less than 450 cpm/5/~Ci of sample. Particle phagocytosis. In high-density cultures, just at confluence, some of the cells were incubated with 1.3S/ml 1 #m latex beads in complete media [14]. After 18 h the incubation media were removed and the cells were washed with two changes of fresh media. These conditions usually facilitated the phagocytosis of 50 -+ 7 latex beads/cell. Isolation of collagen from cell cultures. In most experiments, collagens of the medium and cell layer were analyzed independently. Usually, 1 mg/ml human type I collagen was utilized as a carrier and was dissolved in the medium prior to precipitation with (NH4)2SO4 (25% saturation) [15]. In some experiments, the resultant precipitates were treated with 100 ~g/ml pepsin in 0.05 M acetic acid (4°C for 3.5 h), dialyzed, and lyophilized [16]. Cell layers were extracted with 0.05 M acetic acid and 100 /~g/ml pepsin (4°C for 3.5 h), neutralized, and precipitated with (NH4)2SO4 as before. In select experiments the labeled collagens were isolated in the presence of type I, type II, and type III carrier collagens (1 mg/ml) and later fractionated from Tris-buffered neutral salt solutions (0.9M NaC1/0.05M Tris-HC1, pH 7.5) by differential salt precipitation [ 16,17 ].

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Molecular sieve chromatography. Collagen chains were resolved by chromatography on a column of agarose beads (Bio-Gel A-5m) utilizing 1 M CaC12, 0.05 M Tris-HCl, pH 6.5, after the procedures of Mayne et al. [16]. The molecular weights of CNBr peptides formed from isolated collagen chains obtained from CM-cellulose chromatography were determined as elution patterns from agarose columns of Bio-Gel A-1.Sm [15]. CM.cellulose chromatography. Radioactive peaks isolated from molecular sieve chromatography were desalted on Bio-Gel P-2 as described by Mayne et al. [16] and combined with 10 mg of a carrier type I or type III collagen (0.02 M (Na+) sodium acetate (pH 4.8); 1.0 M urea). The samples were denatured by heating to 45°C for 30 min and applied to a column (1.5 X 10 cm) of CM-cellulose (Whatman, CM-32). Chromatography was subsequently performed as previously described [15] utilizing a linear gradient from 0 to 0.14 M NaC1; however, the flow rates was 130 ml/h. Reduction and alkylation of type III collagen. Labeled v-chains obtained after agarose molecular sieve chromatography were reduced with 0.1 M 2-mercaptoethanol (5 M urea, pH 8.0) and alkylated with 0.2 M iodoacetic acid [15]. The samples were then directly applied and chromatographed on a calibrated column of agarose (Bio-Gel A-5m) for estimation of molecular weights. CMBr cleavage of isolated collagen chains. Collagen chains isolated from CM~ellulose on agarose chromatography were combined with 50 mg of a defined collagen chain, 5 ml of 70% formic acid, and 100 mg of CMBr. The reaction was then allowed to proceed under the conditions described by Miller et al. [18]. Chromatography of formed peptides was performed on CM-cellulose using the conditions specified by Mayne et al. [19]. Amino acid analysis. Samples were hydrolyzed under nitrogen at 110°C for 24 h in 6 N HC1 and amino acid analyses were performed utilizing an automatic amino acid analyzer [20]. For determination of radiolabeled amino acids, the effluent from the analyzer column was collected and radioactivity determinations made after the addition of scintillation fluid in a Beckman liquid scintillation counter. Turnover of radiolabeled collagen. Following continuous or°pulse-chase radiolabeling, media and cells were harvested and equally divided. One-half of each sample was hydrolyzed for amino acid analysis of radiolabeled hydroxyproline and proline. The remaining half of the sample was dialyzed against three changes of 1000 vols. of 0.05 M acetic acid at 4°C for 72 h. Samples were subsequently hydrolyzed as before and analyzed for radiolabeled amino acids. The amount of hydroxyproline after dialysis/radiolabeled hydroxyproline not subjected to dialysis (X100) was regarded as a measure of the percent of degradation of collagen during the radiolabeling period. Recovery of exogenous added labeled collagens. Guinea pig aorta and skin tissues were incubated in minimal essential media (100 ~g/ml ~-aminopropionitrile and 50 ~zg/ml ascorbic acid) containing [14C]proline at 37°C for 24 h. Collagens were isolated after the method of Mayne et al. [15] and redissolved in 1.0 M NaC1, 0.05 M Tris-HC1, pH 7.5. Type I and type III collagen were then selectively precipitated by differential salt precipitation [17]. Samples of the labeled collagens were added to control and latex-treated cells. T h e rate of degradation of each collagen type was determined either by dialysis of half of

164 each incubation mixture and amino acid analysis of radiolabeled hydroxyproline, or as V-components and a-chains isolated by molecular sieve chromatography. Results

Fig. 1 depicts comparative chromatograms on columns of agarose beads of [3H]glycine-labeled collagens isolated after limited pepsin treatment from media and cells of control and latex-treated cells. Separation by molecular weight as 7-components (Mr 285 000) and a-chains (Mr 95 000) are similar to those reported previously as a primary isolation of type III from type I collagens [15,16,21]. Initial fractionation of control cells by this method demonstrated a distribution of ~- and a ~ o m p o n e n t s typical for that reported for dermal fibroblasts (Fig. l a and b) [12,15,22]. Pretreatment of similar fibroblast populations at conditions facilitative for latex particle phagocytosis indicated a decrease in media levels of a-components (Fig. lc). In contrast, cellular extracted collagen chromatographic profiles from latex and control cells appeared to be nearly equal, demonstrating only a slight preponderance of a-chains to ~,-components in phagocytic cells (Fig. l b and d). Characterization of ~?-components In accordance with previous demonstrations that ~/-components depict type III collagens when isolated from ~-aminopropionitrile-treated cell populations after limited proteolysis at 4°C with pepsin [15,19,21], 7-components were subjected to (a) reduction, alkylation, and rechromatography on agarose beads; (b) rechromatography on CM-cellulose; (c) peptide mapping after cyanogen

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bromide cleavage of v~omponents, and (d) isolation by differential salt precipitation. In all instances, reduction and alkylation of V-components followed by rechromatography on Bio-Gel A-5m resulted in a quantitative resolution of radioactivity as a-chains (not shown). Chromatography of V-components on CM-cellulose were resolved as a single radioactivity peak eluting coincident with a carrier type III collagen and at a position similar to the elution pattern of ~12-components from type I collagen (Fig. 2). Cyanogen bromide cleavage peptides formed of [3H]glycine-labeled v~omponents mapped by CM-cellulose chromatography, and molecular sieve chromatography on Bio-Gel A-1.Sm (not shown), were in agood agreement with patterns and molecular weights obtained from carrier type III collagen. Isolation of collagen by differential salt precipitation after prior solubilization in 0.9 M NaC1/0.05 M Tris-HC1, pH 7.5, revealed quantitative recovery of v~omponent radioactivity with carrier type III collagen precipitating at 1.5 M NaC1. All a~omponents were further resolved by precipitation at 2.6 M NaC1 with carrier type I collagen. Insignificant radioactivity was further precipitated with carrier type II collagen at 4.4 M NaC1.

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167 T A B L E II RECOVERY OF DIALYZABLE HYDROXy[14C]PROLINE

AFTER PULSE-LABELING

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Characterization of a-components Fractionation of a-components on CM~ellulose in all instances resolved as two radioactivity peaks which coeluted with a l ( 1 ) and a2-chains of carrier type I collagen (Fig. 2). Mapping of cyanogen bromide radioactive peptides formed from these individual peaks on CM-cellulose and Bio-Gel A-1.5m (not shown) were confimatory of a l ( 1 ) and a2-chain compositions in each instance.

Dialyzable and non-dialyzable hydroxyproline analyses Analysis of the total hydroxyproline synthesized in control and latextreated cells indicated similar values (Table I). Compartmental analysis before and after dialysis, however, indicated that the levels of hydroxyproline retained as non
Recovery of labeled exogenous collagens after addition to cell populations [14C]Proline-labeled type I and type III collagens isolated from guinea pigs and added to both control and phagocytic cells were recovered at similar levels as non
The present report demonstrates that under conditions of undigestible particle phagocytosis, dermal fibroblasts will, in vitro, secrete into the media a collagen phenotype characterized by CM-cellulose chromatography and cyanogen bromide mapping only as type I collagen. In addition, the amount of collagen isolated as molecular weight components from Bio-Gel A-5m molecular sieve chromatography is less than that for control cells. These data would seem to either imply a decreased rate of collagen synthesis in general, and an absence of type III collagen synthesis or a decreased rate of collagen secretion.

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169 However, if total hydroxyproline is used as a measure of collagen synthesis, values for phagocytic and control cells are almost equal (Table I). Compartmental analysis of collagen chains further indicates that cellular levels of type III collagen are present in both cell populations and that the a m o u n t of collagen isolated by molecular sieve chromatography as V-components and as a-chains are similar for phagocytic and control cells. Turnover of collagen in media and cells, measured as a fraction of nondialyzable to total hydroxyproline for both latex-treated and control cells, indicates that 33% of control and 68% of phagocytic cell synthesized [14C]proline-labeled collagen is degraded within a short period of time to dialyzable peptides. The greatest amount of protein containing hydroxyproline turnover being associated with phagocytic cells (Table I). The reduced recovery of type III labeled guinea pig collagen from phagocytic cells as ~/-components or a-chains on molecular sieve chromatography implies an ensuing extracellular proteolysis as a result of increased neutral proteinase secretion [1--3], whereby type III collagen is probably degraded near the site susceptible to collagenase [23]. However, the inability to demonstrate progressive degradation of collagen in pulse-chase experiments and the recovery of near equal quantities of labeled guinea pig exogenous added type I and type III collagen as non-dialyzable hydroxyproline proteins would appear to dispel extracellular proteolysis as a major mechanism of collagen turnover. Although Burke et al. [24] have demonstrated that pepsin extraction of type III collagen results in a quantiative underestimation of this collagen, this p h e n o m e n o n is in great part dependent upon the temperature (15°C) and time of extraction (75 h). In contrast, we have observed that if the temperature of limited proteolysis with purified pepsin is maintained at no greater that 4°C and for periods not exceeding 5 h, the quantitative loss of type III collagen is n o t significant. Miller (Miller, E.J., personal communication) has observed similar results, and the data reported by Mayne et al. [15] in a variety of cell types demonstrates a similar experience with limited pepsin extraction of type III conagen. These present data thus indicate the existence of a mechanism of rapid collagen degradation as elucidated by Bienkowski et al. [25] and Steinberg [26] by which degradation of newly synthesized collagen is a rapid procedure n o t due to an extracellular collagenase, or to phagocytosis and digestion within phagosomes. The suggestion that this is a normal homeostatic mechanism by which cells modulate the quantity or quality of collagen being secreted is supported by the demonstration of this phenomenon in a variety of cells and tissues [25], by the demonstration of increased degradation in young cultures in which underhydroxylated collagen is made [26], and an enhanced breakdown in ascorbate deficiency [27]. Although the present report does not specifically identify those features of collagen synthesis in phagocytic cells which lead to an increased rate of degradation of newly synthesized collagen, the mechanism appears to function at some posttranslational stage. Thus, the net effects of latex ingestion are the transformation of a primarily collagensecreting cell to that of a phagocyte, with the qualitative and quantitative alterations observed in collagen secreted being attributes to rapid protein turnover.

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Acknowledgement This study was supported in part by U.S.P.H.S. Grants GM 22167 and GM 24558. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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