Up-regulation of the alpha 2-Macroglobulin Signaling Receptor on Rheumatoid Synovial Fibroblasts*

(Received for publication, August 13, 1996, and in revised form, October 23, 1996)

Uma K. Misra , Mario Gonzalez-Gronow , Govind Gawdi and Salvatore V. Pizzo Dagger

From the Departments of Pathology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

In the present study, we demonstrate that the alpha 2-macroglobulin (alpha 2M) signaling receptor is up-regulated on rheumatoid synovial fibroblasts. In rheumatoid cells, 125I-alpha 2M-methylamine bound to two sites; namely, one of high affinity (Kd ~52 pM) and the second of lower affinity (Kd ~9.7 nM). In normal synovial fibroblasts only one site for 125I-alpha 2M-methylamine (Kd ~5.36 nM) was present. Receptor-associated protein did not inhibit the binding of alpha 2M-methylamine to the high affinity binding sites, but it caused a 70-80% reduction in its binding to low affinity binding sites establishing its identity as the low density lipoprotein receptor-related protein/alpha 2M receptor. Binding of alpha 2M-methylamine to rheumatoid but not normal synovial fibroblasts caused a rapid rise in inositol 1,4,5-trisphosphate synthesis with a peak reached within 10 s of ligand exposure. Concomitantly, rheumatoid but not normal cells showed a rise in intracellular Ca2+. Pretreatment of rheumatoid cells with Receptor-associated protein or pertussis toxin did not affect the alpha 2M-methylamine-induced increase in intracellular Ca2+. These are characteristic properties of ligation by alpha 2M-methylamine of the alpha 2M signaling receptor but not the lipoprotein receptor-related protein/alpha 2M receptor. Binding of alpha 2M-methylamine to rheumatoid synovial fibroblasts significantly increased the synthesis of DNA compared with normal synovial fibroblasts treated similarly.


INTRODUCTION

Human alpha 2-macroglobulin (alpha 2M)1 along with complement components C3 and C4 is a member of a large superfamily characterized by the presence of an internal beta -cysteinyl-gamma -glutamyl thiolester and a proteinase sensitive region (for review see Ref. 1). In the case of alpha 2M, proteinase cleavage results in "trapping" of the proteinase and inhibition of its ability to cleave most macromolecular substrates. The active site of the enzyme remains intact, and small proteinase substrates and even some proteins can access the entrapped proteinase and undergo cleavage (1). Attack of proteinases on alpha 2M results in a major conformational change of the inhibitor, which exposes receptor recognition sites on each of its four identical subunits (for review see Ref. 2). The presence of these receptors on various cells results in rapid removal of alpha 2M-proteinase complexes from the circulation so that these complexes do not remain a source of active proteinases. Direct attack on the inhibitor thiolester bonds by methylamine also causes exposure of these receptor recognition sites and rapid cellular uptake.

LRP/alpha 2MR has been identified as a receptor for alpha 2M activated by proteinase or methylamine (alpha 2M*) (3, 4). This receptor binds multiple ligands in addition to alpha 2M*, including Pseudomonas exotoxin A, plasminogen activators alone or in complex with plasminogen activator inhibitor type 1, lactoferrin, and lipoprotein lipase (for review see Ref. 5). Most of these ligands cannot cross-compete for binding to LRP/alpha 2MR, presumably because they interact with independent domains on this very large receptor. However, RAP, a 39-kDa protein that co-purifies with the receptor, blocks the binding of all known ligands to LRP/alpha 2MR (3, 5).

We previously demonstrated that binding of alpha 2M-methylamine to macrophages induces synthesis of IP3 and a rise in cytosolic calcium (6). Subsequently, we showed that a distinct alpha 2M receptor, but not LRP/alpha 2MR, is responsible for second messenger generation in macrophages exposed to alpha 2M-methylamine (7, 8, 9, 10). We have termed this receptor the alpha 2M signaling receptor (alpha 2MSR) (7, 8). The distribution, ligand binding, RAP inhibition and signaling characteristics of alpha 2MSR are quite different from LRP/alpha 2MR on murine macrophages (9, 10). Agonist-induced increases in IP3 cause a rise in cytosolic calcium by activating release from calcium-sequestering target organelles, and the increase in cytosolic calcium mediates a multitude of cellular responses (for review see Refs. 11 and 12). Elevated cytosolic calcium pools have recently been reported to modulate specific cell cycle events and DNA synthesis in 3T3 and DDT1MF-2 smooth muscle cells (13, 14).

We chose to study synovial fibroblasts for several reasons: 1) these cells, which play a critical role in the inflammatory response of rheumatoid arthritis, have never been examined for the presence of either alpha 2M receptor and 2) we have previously demonstrated that although both normal and rheumatoid synovial fibroblasts express plasminogen receptors, they are completely different structurally (15). Moreover, only the plasminogen receptor of rheumatoid synovial fibroblasts is coupled to a signaling cascade (16). In the present work, we examined normal and rheumatoid synovial fibroblasts for LRP/alpha 2MR and alpha 2MSR. These studies demonstrate that both cell types express LRP/alpha 2MR but only rheumatoid synovial fibroblasts express alpha 2MSR. Furthermore, we demonstrate that this receptor has ligand binding, signaling, and RAP inhibition characteristics similar to those reported for alpha 2MSR in murine macrophages (7, 8, 9). In addition, we also report that whereas alpha 2M-methylamine caused a 1.5-2-fold increase in DNA synthesis in rheumatoid synovial fibroblasts, the normal synovial fibroblasts remained unaffected, suggesting a growth factor-like activity of alpha 2M-methylamine in rheumatoid synovial fibroblasts and observations made with smooth muscle cells (17).


EXPERIMENTAL PROCEDURES

The methods employed in these studies have been described in detail previously (6, 7, 8, 9, 10, 15, 16). Hence they will be described only in brief.

Materials

Human alpha 2M was purified and reacted with methylamine as described previously (18). Fura-2/AM was obtained from Molecular Probes Inc. (Eugene, OR). Myo-[2-3H]inositol (specific activity, 10-20 Ci/mmol), sodium [3H]acetate (specific activity, 100 mCi/mmol), [3H]methylcholine chloride (specific activity, 60 Ci/mmol), and [3H]thymidine (specific activity, 70 Ci/mmol) were purchased from American Radiochemical (St. Louis, MO). The plasmid containing the LRP insert was a kind gift of Dr. Joachim Herz (University of Texas, Southwestern, Dallas, TX). All other reagents were of the highest grade available.

Cell Culture

Synovial fibroblasts were isolated by the explant method from synovial tissue removed from patients with a documented history of rheumatoid arthritis or from cadavers with no prior history of joint disease, cultured as described previously (15, 16), and used at passages 4-7. Greater than 98% of cells in these cultures are fibroblasts actively synthesizing collagen, as determined by reactivity with M38 monoclonal antibody specific for Type I procollagen (19). No significant reactivity was detected with CD14 monoclonal antibody LeuM3 specific for macrophages. Previous studies have demonstrated that primary synovial macrophages do not proliferate significantly under the culture conditions used for synovial fibroblasts and are not passaged using the gentle trypsinization procedures employed for synovial fibroblast cultures (20). Rheumatoid synovial tissue was obtained from the Division of Rheumatology, Department of Medicine. The studies described here were performed over a 2-year period with at least six different preparations of rheumatoid synovial fibroblasts. The results were not significantly different from preparation to preparation with respect to the parameters reported in the present study.

Detection of Receptor mRNAs by Slot Blot Hybridization

A partial human LRP/alpha 2MR cDNA fragment ranging from base pairs 188 to 6179 inserted into the plasmid pGEM 4 was used for hybridization with mRNAs isolated from normal and rheumatoid synovial cells. This plasmid was a kind gift of Dr. Joachim Herz. The probe was labeled with [alpha -32P]dCTP using a nick translation system (Life Technologies, Inc.) with DNA polymerase I/DNase I according to instructions provided by the manufacturer. mRNA from cells (1 × 106) grown to confluency was isolated and purified using the Micro-Fast Track mRNA isolation kit (Invitrogen, San Diego, CA) according to instructions provided by the manufacturer. Each mRNA sample in 100 µl of water was mixed with 300 µl of 6.15 M formaldehyde containing 1.3 M NaCl and 0.17 M sodium citrate, pH 7.0 (10 × SSC; 1 × SSC = 1.3 M NaCl and 0.17 M sodium citrate, pH 7.0), incubated for 15 min at 65 °C, loaded onto nitrocellulose paper using a Minifold II slot blotter (Schleicher & Schuell). The filter was washed with 400 µl of 10 × SSC and baked for 2 h at 80 °C under vacuum. The filter was immersed in 6 × SSC for 2 min and prehybridization buffer containing 0.5% SDS, 5 × Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA prewarmed to 68 °C was added (0.2 ml/cm2) and incubated for 2 h at 68 °C. The prehybridization buffer was removed, and hybridization buffer containing 6 × SSC, 10 mM EDTA, 2 × 106 CMP 32P-labeled denatured LRP/alpha 2MR cDNA probe, 5 × Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA was added (50 µl/cm2) and filter hybridized for 16 h at 68 °C. The filter was washed twice with 2 × SSC containing 0.5% SDS at 25 °C for 15 min followed by two washings with 2 × SSC containing 0.1% SDS and 0.1 × SSC containing 0.5% SDS, respectively. The filter was dried at 25 °C and subjected to autoradiography either by exposing it to x-ray film or using a PhosphorImager.

Binding of 125I-alpha 2M-Methylamine to Synovial Cells

The binding assay is essentially identical to that previously reported for binding of alpha 2M-methylamine to macrophages (21). In brief, human normal and rheumatoid synovial fibroblasts were cultured in 48-well tissue culture plates until the cell monolayers were confluent (70000-80000 cells/well). Prior to use in binding assays, monolayers were washed three times with HHBSS (Hanks' balanced salt solution containing 10 mM Hepes and 3.5 mM NaHCO3, pH 7.4). All binding assays were performed at 4 °C in RPMI 1640 (standard medium to which was added 10% iron supplemental calf serum, insulin (5 µg/ml), adenine (1.8 × 10-4 M), sodium pyruvate (2 µM), epidermal growth factor (20 ng/ml), penicillin (12.5 units/ml), and streptomycin (6 µg/ml)) containing 2% bovine serum albumin (16). 125I-alpha 2M-methylamine was incubated with the synovial fibroblasts for 60 min at 4 °C. Free ligand was separated from bound by rapidly aspirating the medium in the cold, and monolayers were washed extensively (7-8 times) with chilled RPMI 1640 medium containing 2% bovine serum albumin. The cells were lysed with 1 M NaOH, and bound radioactivity was determined in a gamma  counter. The specific binding of alpha 2M-methylamine to receptors was calculated by subtracting nonspecific binding determined in the presence of 5 mM EDTA from total binding (21).

Measurement of Inositol Phosphates in Synovial Fibroblasts

Normal and rheumatoid synovial fibroblasts were grown to confluency in six-well tissue culture plates (1-1.5 × 106 cells/well) in RPMI 1640 medium containing the additions as listed above. Prior to use for inositol phosphates measurements (6, 7, 8), medium from monolayers was aspirated, and a volume of inositol-free RPMI 1640 medium containing 0.25% bovine serum albumin was added followed by the addition of myo-[2-3H]inositol (8 µCi/ml) to each well. The plates were incubated at 37 °C for 16-18 h in a humidified CO2 (5%) incubator. The radiolabeled monolayers were washed five times with HHBSS containing 10 mM LiCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4, and a volume of HHBSS containing additions listed above was added to the monolayers. The monolayers were preincubated for 3 min at 37 °C prior to stimulation with alpha 2M-methylamine (100 nM) for varying periods of time. The reaction was terminated by aspirating the medium and adding 6.25% ice-cold perchloric acid. The cells were scrapped and transferred to tubes containing 5 mM EDTA and 1 ml of octylamine:freon (1:1, v/v), and the tubes centrifuged at 5600 × g for 20 min at 4 °C. The upper phase was applied to a 1-ml packed Dowex resin column (AG1-X8 formate; Bio-Rad) and sequentially eluted in a batch fashion with H2O, 50, 200, 400, 800, 1200, and 2000 mM ammonium formate containing 0.1 M formic acid, respectively (11). An aliquot was used for determining radioactivity in a liquid scintillation counter.

Measurement of [Ca2+]i in Synovial Cells

The intracellular calcium concentration ([Ca2+]i) in normal and rheumatoid synovial fibroblasts stimulated with alpha 2M-methylamine was measured according to methods published earlier (6, 7, 8). Briefly, the cells were plated on glass coverslips kept in 35-mm Petri dishes at a cell density of 1-1.5 × 105/cm2 and incubated in RPMI 1640 medium for 16-18 h at 37 °C in a humidified CO2 (5%) incubator. At the end of the incubation, 4 µM Fura-2/AM was added, and cells were incubated at 37 °C for 30 min in the dark and used for [Ca2+]i measurements in a digital imaging microscope as described (6, 7, 8). The effect of pertussis toxin (1 µg/ml) on alpha 2M-methylamine-induced changes in [Ca2+]i was studied as described previously (7, 8).

Synthesis of PAF in Synovial Cells from [3H]-Methylcholine Chloride

Rheumatoid and normal synovial fibroblasts were isolated and cultured to confluency as described above (16). Monolayers were washed with chilled HHBSS, a volume of quiescent medium was added followed by the addition of [3H] methylcholine chloride (2 µCi/ml), and cells were incubated as above for 16 h. Labeled monolayers were washed four times with HHBSS, a volume of quiescent medium was added, and monolayers were preincubated for 5 min at 37 °C before adding alpha 2M-methylamine (100 nM) and incubation continued. At specified time periods, the medium was quantiatively transferred to a clean glass tube to assess the secretion of newly synthesized PAF, and a volume of chilled, methanol containing 50 mM acetic acid to monolayers was added. The cells were scraped in to glass tubes, the wells were washed once with 1 ml of methanol, and the wash was combined with the cell suspension. Details of lipid extraction, PAF fractionation by thin layer chromatography, determination of radioactivity in cellular and secreted PAF, and identity of PAF are given in an earlier publication (22).

Synthesis of PAF in Synovial Cells from [3H] Sodium Acetate

Synovial fibroblasts were isolated and cultured to confluency as described above (16). The cells were washed twice with HHBSS and incubated overnight as above in quiescent medium added to monolayers followed by the addition of [3H]sodium acetate (20 µCi/ml), and cells were labeled for 30 min at 37 °C. The labeled cells were washed four times with HHBSS, a volume of quiescent medium was added, and cells were preincubated for 5 min before adding alpha 2M-methylamine (100 nM). Other details of quantitating radioactivity in cellular and secreted PAF were the same as described above.

Incorporation of [3H]Thymidine into Synovial Cells DNA

Rheumatoid and normal fibroblasts (70-80 × 103 cells/well) were plated in 24-well plates separately and grown to confluency as described (16). The monolayers were washed with HHBSS, a volume of quiescent medium was added along with [3H]thymidine (5 µCi/ml) and alpha 2M-methylamine) (100 nM), and cells were incubated for specific periods of time. In an additional set of studies, monolayers of rheumatoid synovial fibroblasts were treated with the cloned and expressed alpha 2-macroglobulin receptor-binding fragment (RBF) (9, 10) at concentrations of 50 pM or 1 nM. For some of these studies RAP (200 nM) was included in the incubation in addition to RBF. The reaction was terminated by aspirating the medium and washing monolayers six times with chilled HHBSS buffer. The cells were lysed in a volume of 1 N NaOH at 40 °C, and radioactivity was determined on an aliquot by scintillation counting.


RESULTS

125I-alpha 2M-Methylamine Binding to Normal and Rheumatoid Synovial Fibroblasts

125I-alpha 2M-methylamine binds to both normal and rheumatoid cells, and the binding is saturable (Fig. 1A). Rheumatoid cells bound 2-3-fold more alpha 2M-methylamine than normal synovial cells (Fig. 1A). Rheumatoid cells showed two binding sites for alpha 2M-methylamine. One site had a high affinity (Kd ~52 pM) and the second site had a lower affinity (Kd ~9.7 nM) for alpha 2M-methylamine (Fig. 1B). In contrast normal synovial cells had only the lower affinity binding site (Kd ~ 5.6 nM) for alpha 2M-methylamine (Fig. 1B). Binding of alpha 2M-methylamine to the high affinity binding sites on rheumatoid cells was not inhibited by RAP (100-fold excess), whereas RAP greatly reduced binding of alpha 2M-methylamine to the lower affinity binding site in both normal and rheumatoid cells confirming its identity as LRP/alpha 2MR. The receptor number in rheumatoid cells was about 20% higher than normal synovial cells. We then employed slot blot hybridization analysis to detect mRNA for LRP/alpha 2MR in both types of synovial cells. As is evident from Fig. 2, both normal and rheumatoid synovial fibroblasts express LRP/alpha 2MR.


Fig. 1. 125I-alpha 2M-methylamine binding to synovial fibroblasts. A, binding of increasing concentrations of 125I-alpha 2M-methylamine to normal (bullet ) and rheumatoid (black-triangle) synovial cells in the absence of RAP. Binding in the presence of RAP (100-fold excess) to normal (open circle ) and rheumatoid (triangle ) cells is shown for comparison. Details of binding are given under "Experimental Procedures." Values are the means ± S.E. from three independent experiments performed in quadruplicate. The specific binding was calculated by subtracting nonspecific binding determined in presence of 5 mM EDTA from total binding as described previously (21). B, the Scatchard plot of the above binding data for normal (open circle ) and rheumatoid (bullet ) synovial cells. The slopes and Kd values were calculated using a Cricket program on a Macintosh IISi computer.
[View Larger Version of this Image (16K GIF file)]



Fig. 2. Northern blot of LRP/alpha 2MR mRNA in synovial fibroblasts. LRP/alpha 2MR mRNA was estimated by Northern blot analysis in human normal (lanes 2-4) and rheumatoid (lanes 6-8) synovial cells. The data are representative of two independent experiments. The details of extraction of mRNAs and hybridization with the cDNA probe for LRP/alpha 2MR mRNA are given under "Experimental Procedures." Different aliquots of mRNAs isolated from normal synovial cells (lane 2, 1.5 µl; lane 3, 3 µl; lane 4, 5 µl) and rheumatoid synovial cells (lane 6, 1.5 µl; lane 7, 3 µl; lane 8, 5 µl) were used for hybridization. The radioactivity of hybridized blots was detected by using a PhosphorImager (Molecular Diagnostics). Lane 1 is the blank, and lane 5 is the [alpha -32P]dCTP probe.
[View Larger Version of this Image (14K GIF file)]


The Effect of alpha 2M-Methylamine on Synovial Cell IP3 Synthesis and [Ca2+]i

We next studied the ability of alpha 2M-methylamine to induce IP3 synthesis in synovial fibroblasts. Fig. 3 demonstrates that there is a marked difference in the generation of IP3 upon binding of alpha 2M-methylamine to its receptors on normal and rheumatoid cells. With rheumatoid cells, ligation of alpha 2M-methylamine to its receptors rapidly but transiently increased IP3 levels by about 50-60%. Inclusion of RAP (100-fold excess) in the incubation did not significantly suppress the 50-60% induction of IP3 synthesis caused by alpha 2M-methylamine. In contrast, the binding of alpha 2M-methylamine to normal cells resulted in a very small increase in intracellular IP3 (<5%). Exposure of normal cells to higher ligand concentrations did not cause a greater rate of IP3 synthesis (data not shown). Concomitant with a rise in IP3, ligation of alpha 2M-methylamine to rheumatoid but not normal synovial fibroblasts resulted in a significant increase in [Ca2+]i (Fig. 4A). The cell shown in Fig. 4A demonstrated a 4-fold increase in [Ca2+]i from a resting value of 80-350 nM. For these studies multiple cells were analyzed by digital imaging microscopy using Fura-2/AM-loaded cells. For rheumatoid cells 90-95% of the cells responded to alpha 2M-methylamine treatment in a manner comparable with the cell shown in Fig. 4A. A somewhat lesser response rate was observed when peritoneal macrophages were exposed to alpha 2M-methylamine (6). Although 60-65% of normal cells showed some changes in [Ca2+]i, this response was minimal (<10% change). Table I summarizes the data from four separate studies of normal and five studies of rheumatoid synovial fibroblasts. alpha 2M-methylamine-induced increases in IP3 and [Ca2+]i in rheumatoid cells were not affected by RAP. Pertussis toxin had no effect on alpha 2M-methylamine-induced increase in [Ca2+]i (Fig. 4B), similar to macrophages where we have shown that a G protein coupled to the receptor is pertussis toxin-insensitive (7, 8).


Fig. 3. Inositol 1,4,5-trisphosphate formation in alpha 2M-methylamine-stimulated synovial fibroblasts. myo-[2-3H]inositol-labeled normal (open circle ) and rheumatoid (bullet ) synovial cells were treated with alpha 2M-methylamine (100 nM) for varying periods of times and processed for quantitation of IP3 radioactivity as detailed under "Experimental Procedures." Each point is the average of two independent experiments performed in triplicate. The values are expressed as the percentage of changes in radioactivity over basal value ± the S.E. The asterisk indicates statistical significance (p < 0.05).
[View Larger Version of this Image (17K GIF file)]



Fig. 4. [Ca2+]i in alpha 2M-methylamine-stimulated single synovial cells. A, normal (open circle ) and rheumatoid (bullet ) synovial cells were preloaded with 4 µM of Fura-2/AM for 20 min at 37 °C, and changes in [Ca2+]i were measured as described previously (6, 7) after stimulation with alpha 2M-methylamine (100 nM) added at the arrow. B, rheumatoid synovial fibroblasts were treated with buffer (open circle ) or Pertussis toxin (1 µg/ml for 16 h) (triangle ) prior to stimulation with alpha 2M-methylamine (100 nM) added at arrow a. In a separate study, cells were treated with RAP (100-fold excess) (square ) added at arrow b 6 min prior to the addition of alpha 2M-methylamine added at arrow c. The changes in [Ca2+]i are representative of 4-5 independent experiments using 6-12 individual cells in each experiment. Table I shows the averaged data for all the cells studied.
[View Larger Version of this Image (19K GIF file)]


Table I.

Changes in [Ca2+]i in normal and rheumatoid synovial cells stimulated with alpha 2M-methylamine (100 nM)


Synovial cell type Number of studies Cells studied Response [Ca2+]i
Basal 5 min

% nM
Normal 4 34 60 -65 68.04  ± 7.72 89.74  ± 7.97
Rheumatoid 5 39 90 -95 58.62  ± 7.25 232.1  ± 15.11

DNA Synthesis in alpha 2M-Methylamine Synovial Cells

DNA synthesis from [3H]thymidine by normal and rheumatoid synovial fibroblasts stimulated with alpha 2M-methylamine was then compared (Fig. 5). DNA synthesis in both cell types was comparable with up to 4 h of treatment with alpha 2M-methylamine, but thereafter, alpha 2M-methylamine increased DNA synthesis in rheumatoid cells by about 2-fold compared with normal cells (Fig. 5). In unstimulated rheumatoid and normal cells the DNA synthesis was comparable (data not shown). The concentration of alpha 2M-methylamine chosen for this study, 100 nM, was based on previous studies with this ligand under a number of conditions. However, more recent studies and the present work suggest that alpha 2MSR binds ligands at very high affinity, Kd ~ 50 pM (9, 10). We therefore performed additional studies treating rheumatoid synovial fibroblasts with RBF at concentrations of 50 pM and 1 nM, respectively (Table II). At both ligand concentrations DNA synthesis was significantly increased. Moreover, inclusion of RAP (200 nM) caused little decrease in DNA synthesis despite being present at 4000- and 200-fold excess, respectively. We chose RBF for these studies in place of alpha 2M-methylamine to further demonstrate that the effects seen cannot be attributed to the presence of growth factors, which can bind to alpha 2M or alpha 2M-methylamine but not RBF (17). Based on the above studies, it is concluded that ligation of alpha 2MSR on rheumatoid synovial cells stimulates DNA synthesis.


Fig. 5. DNA synthesis in normal and rheumatoid synovial fibroblasts. Normal (open circle ) and rheumatoid (bullet ) synovial fibroblasts were stimulated with alpha 2M-methylamine (100 nM). Details are described under "Experimental Procedures." The values are the means ± S.E. from three individual experiments. The asterisk indicates statistical significance (p < 0.05).
[View Larger Version of this Image (15K GIF file)]


Table II.

Changes in DNA synthesis as measured by [3H]thymidine incorporation in rheumatoid synovial fibroblasts


Additionsa [3H] Thymidine uptake Change over basal levels

fmol/mg cell protein %
Basal (buffer) 1202.4  ± 41
RBF (50 pM) 1977.2  ± 76 64
RBF (50 pM) + RAP (200 nM) 2109.3  ± 92 75
RBF (1 nM) 2400.8  ± 71 99
RBF (1 nM) + RAP (200 nM) 1941.5  ± 58 61
RAP (200 nM) 1205.6  ± 54 0

a  The averages of eight separate determinates are shown ± S.E.

PAF Synthesis and Secretion in Synovial Cells Stimulated with alpha 2M-Methylamine

PAF is a naturally occurring biologically active phosphoglyceride that is produced by most cells involved in inflammatory responses including platelets, neutrophils, basophils, endothelial cells, monocytes, and tissue macophages (23, 24). We have recently demonstrated that ligation of alpha 2MSR causes an increase in PAF synthesis by macrophages (22). We therefore studied PAF synthesis in alpha 2M-methylamine-treated rheumatoid and normal synovial fibroblasts by the de novo pathway employing [3H]methylcholine as the substrate. The synthesis of PAF from [3H]methylcholine in both rheumatoid and normal cells were not significantly different (data not shown). PAF synthesis and secretion from [3H]acetate by the remodelling pathway, however, was stimulated by the addition of alpha 2M-methylamine to rheumatoid but not normal synovial cells (Fig. 6).


Fig. 6. Retention and secretion of PAF synthesized via the remodeling pathway. Normal (open circle ) and rheumatoid (bullet ) synovial fibroblasts exposed to alpha 2M-methylamine (100 nM) are compared. Experimental conditions are described under "Experimental Procedures." The values are an average of two experiments. A, retention of newly synthesized PAF by synovial fibroblasts. B, secretion of newly synthesized PAF by synovial fibroblasts. The values are expressed as the percentage of change over the basal value set at 100%. The basal values were obtained from buffer-treated cells under identical conditions except that alpha 2M-methylamine was absent.
[View Larger Version of this Image (15K GIF file)]



DISCUSSION

Many cell types express LRP/alpha 2MR, a receptor for multiple ligands including alpha 2M* (see 5). More recently, we have demonstrated that murine macrophages express a unique alpha 2M signaling receptor in addition to LRP/alpha 2MR (6, 7, 8, 9, 10). Ligation of alpha 2MSR by alpha 2M-methylamine or a cloned and expressed receptor binding fragment from the homologous rat alpha 1M causes a very rapid increase in IP3, which is followed rapidly by an increase in [Ca2+]i. Ligation of alpha 2MSR by alpha 2M-methylamine stimulates the activities of several phospholipases as well as protein kinase C (25). Rat vascular smooth muscle cells also express alpha 2MSR in addition to LRP/alpha 2MR (17).

The present study demonstrates that normal synovial fibroblasts express little, if any, of the very high affinity alpha 2M* receptor previously identified as the alpha 2M signaling receptor (7, 8, 9, 10). Exposure of normal synovial cells to alpha 2M-methylamine, moreover, caused almost no increase in IP3 and only a very small rise in [Ca2+]i. Whether this represents a very low level of expression of alpha 2MSR or is within experimental limits is difficult to determine. By contrast, rheumatoid synovial cells possess a very high affinity alpha 2M* receptor (Kd ~52 pM) and when exposed to alpha 2M-methylamine show a significant increase in IP3 synthesis and an increase in [Ca2+]i. The rise in [Ca2+]i was not blocked by RAP and was insensitive to pertussis toxin. These properties are identical to the characteristics of the macrophage alpha 2M signaling receptor, which we have previously reported (6, 7, 8). The increase in IP3 synthesis is consistent with that seen in murine macrophages exposed to alpha 2M-methylamine at a comparable ligand dose (6, 7, 8). The rise in [Ca2+]i is also comparable in magnitude when rheumatoid synovial fibroblasts and macrophages are compared. The pattern of response is, however, somewhat different. Almost all of the rheumatoid cells showed a very sustained response to alpha 2M-methylamine. By contrast, only 32% of the responding macrophages studied showed this very sustained response when exposed to similar concentrations of alpha 2M-methylamine (6). The remainder of the macrophages that responded showed several different forms of behavior including various oscillatory patterns (6). The significance of these patterns of [Ca2+]i regulation is unclear here as it is in most other ligand-responsive cells that bind various growth factors, hormones, or cytokines.

Binding of alpha 2M-methylamine to macrophage receptors is followed by a number of cellular events, including enhanced locomotion, chemotaxis, down-regulation of proteinase synthesis, suppression of respiratory burst, rapid secretion of prostaglandin E2, and prevention of interferon-gamma -induced cell rounding (for review see Ref. 2). In a recent report we have shown that ligation of alpha 2MSR with receptor recognized forms of alpha 2M causes tyrosine phosphorylation of PLCgamma and raises cytosolic pH in peritoneal macrophages (26). We also suggested that in macrophages, alpha 2M-methylamine may function as a growth factor (26). Weaver et al. (27) have recently shown that activated forms of alpha 2M increase expression of platelet-derived growth factor alpha -receptor in vascular smooth muscle cells as well as tyrosine phosphorylation of a 170-kDa protein. Moreover, alpha 2M* and RBF promote proliferation of these cells as a result of ligation of alpha 2MSR (17). The stimulation of DNA synthesis by alpha 2M-methylamine and RBF in rheumatoid synovial fibroblasts further confirms its role as a growth factor. Currently, we do not understand the mechanism(s) by which ligation of alpha 2MSR stimulates DNA synthesis in rheumatoid synovial fibroblasts. In recent years, a positive correlation between Ca2+ contents of IP3-sensitive and IP3-insensitive calcium pools and DNA synthesis has been demonstrated (28, 29, 30). In Swiss 3T3 cells, thapsigargin and di-tert-butylhydroxyquinone stimulated the reinitiation of DNA synthesis in synergy with either phorbol 12,13-dibutyrate or bombesin (13). It is also known that cell growth in DDT1MF-2 smooth muscle cells is linked to regulation of intracellular calcium pool contents (14, 31). Therefore, it is also possible that the alpha 2M-methylamine-induced increase in [Ca2+]i levels may result in a stimulation of DNA synthesis in rheumatoid synovial fibroblasts.

PAF is a potent proinflammatory mediator (31, 32). Synthesis and secretion of PAF by alpha 2M-methylamine stimulated synovial fibroblasts offers a means by which proteinase generation and formation of alpha 2M-proteinase complexes can regulate activities by a variety of cells other than macrophages. Many cells such as endothelial cells, neutrophils, and T and B lymphocytes lack alpha 2M receptors (33). Therefore, these cells do not have a direct capability of responding to the generation of alpha 2M-proteinase complexes during tissue injury. However, by stimulating production and secretion of PAF, the binding of alpha 2M-proteinase complexes to an up-regulated alpha 2MSR in rheumatoid synovial fibroblasts offers the potential to regulate a variety of responses to tissue injury.

These studies may also be of significance in the disease rheumatoid arthritis. We have previously demonstrated that rheumatoid synovial fibroblasts have altered regulation of plasminogen activation as compared with normal synovial cells (16). Moreover, the structure of plasminogen receptors is very different on rheumatoid and normal cells (15). Binding of plasminogen to rheumatoid but not normal synovial fibroblasts activates a signaling mechanism that causes an increase in [Ca2+]i (15). Of significance, the increase in [Ca2+]i is triggered only after cell-bound plasminogen is activated by urinary-type plasminogen activator. Patients with rheumatoid arthritis demonstrate increased urinary-type plasminogen activator levels in synovial fluid relative to plasma levels (34, 35). Our own studies suggest a significantly higher capacity of rheumatoid compared with normal synovial cells with respect to plasminogen and urinary-type plasminogen activator binding and plasmin generation (16). Many studies have demonstrated that increased plasmin generation may directly increase cartilage proteoglycan degradation as well as lead to increased activation of collagenases and neutral proteoglycanases (for review see Ref. 16). Although alpha 2-antiplasmin is the major human plasmin inhibitor, it is present in very low concentrations in plasma and fluids, and it is rapidly consumed whenever large amounts of plasmin are generated. In these circumstances, alpha 2M becomes the major antiplasmin (for review see Ref. 33). alpha 2M is readily detected in rheumatoid synovial fluid (33) where it should rapidly react with collagenases and plasmin. alpha 2M-collagenase complexes have, in fact, been directly identified in rheumatoid synovial fluid (36). Thus significant amounts of receptor-recognized forms of alpha 2M will exist in the joint fluids of patients with rheumatoid arthritis. The presence of alpha 2MSR on rheumatoid synovial cells like the signaling plasminogen receptor is likely, therefore, to play a role in the pathogenesis of this disorder. The occurrence of macrophages in rheumatoid synovial tissue and fluids offers yet another means by which the alpha 2MSR may contribute to the evolution of the disease. Ligation of this receptor on macrophages results in the production of a number of proinflammatory prostaglandins, which will promote further tissue injury, releasing more proteinases, and therefore help to promote escalation of the process of joint destruction (37, 38).


FOOTNOTES

*   This work was supported by NHLBI, National Institutes of Health Grant HL-24066 and NCI, National Institutes of Health Grant CA-29589. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710.
1    The abbreviations used are: alpha 2M, alpha 2-macroglobulin; alpha 2M*, receptor-recognized forms of alpha 2M; LRP, low density lipoprotein-related protein; alpha 2MR, alpha 2M receptor; alpha 2MSR, the alpha 2M signaling receptor; RAP, receptor-associated protein; IP3, inositol 1,4,5-trisphosphate; AM, acetoxymethyl ester; [Ca2+]i, intracellular free Ca2+; RBF, receptor-binding fragment; PAF, platelet-activacting factor.

REFERENCES

  1. Sottrup-Jensen, L. (1987) in Plasma Proteins (Putnam, F. W., ed), pp. 191-291, Academic Press, New York
  2. Chu, C. T., and Pizzo, S. V. (1994) Lab. Invest. 71, 792-812 [Medline] [Order article via Infotrieve]
  3. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990) J. Biol. Chem. 265, 17401-17404 [Abstract/Free Full Text]
  4. Kristensen, T., Moestrup, S. K., Gliemann, J., Bendtsen, L., Sand, O., and Sottrup-Jensen, L (1990) FEBS Lett. 276, 151-155 [CrossRef][Medline] [Order article via Infotrieve]
  5. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601-638 [CrossRef][Medline] [Order article via Infotrieve]
  6. Misra, U. K., Chu, C. T., Rubenstein, D. S., Gawdi, G., and Pizzo, S. V. (1993) Biochem. J. 290, 885-891 [Medline] [Order article via Infotrieve]
  7. Misra, U. K., Chu, C. T.-C., Gawdi, G., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 12541-12547 [Abstract/Free Full Text]
  8. Misra, U. K., Chu, C. T.-C., Gawdi, G., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 18303-18306 [Abstract/Free Full Text]
  9. Howard, G. C., Misra, U. K., DeCamp, D. L., and Pizzo, S. V. (1996) J. Clin. Invest. 97, 1193-1203 [Abstract/Free Full Text]
  10. Howard, G. C., Yamaguchi, Y., Misra, U. K., Gawdi, G., Nelsen, A., DeCamp, D. L., and Pizzo, S. V. (1996) J. Biol. Chem. 271, 14105-14111 [Abstract/Free Full Text]
  11. Berridge, M. D. (1983) Biochem. J. 212, 849-858 [Medline] [Order article via Infotrieve]
  12. Putney, J. W., and Bird, G. S. (1993) Endocrinol. Rev. 14, 610-631 [Medline] [Order article via Infotrieve]
  13. Charlesworth, A., and Rozengurt, E. (1994) J. Biol. Chem. 269, 32528-32538 [Abstract/Free Full Text]
  14. Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961 [Abstract/Free Full Text]
  15. Gonzalez-Gronow, M., Gawdi, G., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 4360-4366 [Abstract/Free Full Text]
  16. Gonzalez-Gronow, M., Gawdi, G., and Pizzo, S. V. (1993) J. Biol. Chem.. 268, 20791-20795 [Abstract/Free Full Text]
  17. Webb, D. J., Hussaini, J. M., Weaver, A. M., Atkins, T. L., Chu, C. T., Pizzo, S. V., Owens, G. K., and Gonias, S. L. (1995) Eur. J. Biochem. 234, 714-722 [Abstract]
  18. Imber, M. J., and Pizzo, S. V. (1981) J. Biol. Chem. 256, 8134-8139 [Abstract/Free Full Text]
  19. McDonald, J. A., Broekelman, T. J., Matheke, M. J., Crouch, E., Koo, M., and Kuhn, C. (1986) J. Clin. Invest. 78, 1237-1244 [Medline] [Order article via Infotrieve]
  20. Weinberg, J. B., and Athens, J. B. (1993) Wintrobe's Clinical Hematology, 9th ed., pp. 267-298, Lea and Febiger, Philadelphia
  21. Enghild, J. J., Thøgersen, I. B., Roche, P. A., and Pizzo, S. V. (1989) Biochemistry 28, 1400-1412
  22. Misra, U. K., and Pizzo, S. V. (1996) J. Cell. Biochem. 61, 39-47 [CrossRef][Medline] [Order article via Infotrieve]
  23. Venable, M. E., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Lipid Res. 34, 691-702 [Medline] [Order article via Infotrieve]
  24. Snyder, J. (1995) Biochem. J. 305, 689-705 [Medline] [Order article via Infotrieve]
  25. Misra, U. K., and Pizzo, S. V. (1994) Ann. N. Y. Acad. Sci. 737, 486-489 [Medline] [Order article via Infotrieve]
  26. Misra, U. K., Gawdi, G., and Pizzo, S. V. (1995) Biochem. J. 309, 151-158 [Medline] [Order article via Infotrieve]
  27. Weaver, A. M., Owens, G. K., and Gonias, S. L. (1995) J. Biol. Chem. 270, 30741-30748 [Abstract/Free Full Text]
  28. Zweifach, A., and Lewis, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6295-6299 [Abstract]
  29. Lee, K.-M., Toscas, K., and Villereal, M. L. (1993) J. Biol. Chem. 268, 9945-9948 [Abstract/Free Full Text]
  30. Newcomb, T. G., Mullins, R. D., and Sisken, J. E. (1993) Cell Calcium 14, 539-549 [Medline] [Order article via Infotrieve]
  31. Short, A. D., Klein, M. G., Schneider, M. F., and Gill, D. L. (1993) J. Biol. Chem. 268, 25887-25893 [Abstract/Free Full Text]
  32. Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1990) J. Biol. Chem. 265, 17381-17384 [Free Full Text]
  33. Salvesen, G., and Pizzo, S. V. (1993) in Hemostasis and Thromboses. Basic Principles and Clinical Practice (Colman, R. W., Hirsh, J., Marder, V. J., and Salzman, E. W., eds), pp. 241-258, J. B. Lippincott Co., Philadelphia
  34. Kummer, J. A., Abbink, J. J., DeBoer, J. P., Roem, D., Nieuwenhuys, E. J., Kamp, A. M., Swaak, T. J. G., and Hack, C. E. (1992) Arthritis Rheum. 35, 884-893 [Medline] [Order article via Infotrieve]
  35. Brommer, E. J. P., Doojewaard, G., Dijkmans, B. A. C., and Breedveld, F. C. (1992) Ann. Rheum. Dis. 51, 965-968 [Abstract]
  36. Abe, S., and Nagai, Y. (1973) J. Biochem. (Tokyo) 73, 897-900 [Medline] [Order article via Infotrieve]
  37. Hoffman, M. R., Pizzo, S. V., and Weinberg, J. B. (1988) Agents Actions 25, 360-368 [Medline] [Order article via Infotrieve]
  38. Uhing, R. J., Martensen, C. H., Rubenstein, D. S., Hollenbach, P. W., and Pizzo, S. V. (1991) Biochim. Biophys. Acta 1093, 115-120 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.