(Received for publication, October 2, 1995)
From the
CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.
CD36 is a membrane glycoprotein expressed by erythrocyte precursors, mature monocytes, platelets, endothelial cells of the microvasculature, and mammary epithelial cells(1) . The CD36 cDNA predicts a polypeptide of 53-kDa with 10 potential N-linked glycosylation sites(2) . Depending on the cell type, CD36 displays different molecular masses (78, 88, or 94 KDa) corresponding to different glycoforms(1, 3) . Several functions, all depending on the molecule being expressed at the cell surface, have been ascribed to CD36. In platelets, the molecule acts as a receptor for the extracellular matrix proteins collagen (4) and thrombospondin 1(5, 6) . CD36 also mediates adherence of erythrocytes infected with Plasmodium falciparum to capillary endothelial cells, a phenomenon that contributes to the morbidity and mortality of malaria in humans(7) . Recently, CD36 has been reported to be involved in the phagocytosis of neutrophils and T lymphocytes undergoing apoptosis by macrophages(8, 9, 10) , as a receptor for oxidized low density lipoprotein on macrophages(11, 12) , and for fatty acid binding and transport in foam cells(13) . In many cell types including platelets, endothelial cells, and monocytes the role of CD36 as a cell surface receptor has been extended to that of a signal transduction molecule(14, 15, 16, 17) . An association with protein tyrosine kinases of the src gene family has been described in platelets and endothelial cells CD36(18, 19) .
The regulation of CD36 expression
during monocyte differentiation in terms of gene activation,
post-transcriptional and post-translational modifications and
intracellular transport is still poorly understood. In this study, we
have exploited U937 and THP-1 cells, two human lines that immortalize
different steps of monocytic maturation, to investigate the synthesis,
processing and intracellular transport of CD36 molecule. We find that
in both cell lines the expression of the CD36 antigen is induced by
phorbol esters (PMA) ()treatment, but with different
kinetics. Our results show that cells of the myelomonocytic lineage
have the capability of selectively modulating the rate of intracellular
transport of CD36, possibly by the transient association of the CD36
precursor with a glycoprotein of 33 kDa.
The monoclonal antibodies (mAbs) used in this study were NL07(3) , OKM5 (Ortho Diagnostics, Milan, Italy), Mo91(20) , all specific for CD36; the L31 anti-HLA class I(21) ; the OKMI anti-CD11b (Ortho Diagnostics), and the GAP 8.3 anti-CD45RA, B(22) . The IgG fraction was prepared from 1207 rabbit anti-CD36 antiserum (20) or from rabbit anti-mouse Ig µ-chain (23) by affinity chromatography on protein A-Sepharose. HRP-conjugated rabbit anti-mouse Ig and swine anti-rabbit Ig HRP were from Dako (Glostrup, Denmark). Controls were class-matched irrelevant monoclonal antibodies or a rabbit preimmune serum.
The molecular mass standards
included lysozyme (14 kDa), trypsin inhibitor (21 kDa), carbonic
anhydrase (30 kDa), ovalbumin (46 kDa), BSA (69 kDa), phosphorylase b (97 kDa), -galactosidase (116 kDa), and myosin (200
kDa), and were revealed by red Ponceau staining.
After washing in 0.1% Nonidet P-40 TBS, the immunocomplexes were eluted by treatment with SDS-PAGE sample buffer for 5 min at 100 °C and resolved by SDS-PAGE. Gels were either blotted to nitrocellulose or fixed and processed with Amplify (Amersham Corp., Milan, Italy) before exposure to Kodak X-Omat AR films or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Autoradiograms were analyzed by a computing densitometer (Molecular Dynamics).
Figure 1: Flow cytometry analysis of the surface expression of CD36 on resting and differentiated THP-1 and U937 cell lines. Indirect immunofluorescence tests were performed on untreated cells or cells treated with PMA for 48 h using the NL07 anti-CD36 mAb. The detection antibody was goat anti-mouse Ig-fluorescein isothiocyanate. Shadowed peak indicates control mouse Ig as fluorescence background. The ordinate shows cell numbers, and the abscissa shows fluorescence intensity.
Figure 2: Kinetics of CD36 expression in the PMA-differentiated THP-1 and U937 cell lines. Western blot analysis was performed using Mo91 anti-CD36 mAb and L31 anti-HLA class I mAb on whole extracts from U937 (a) or THP-1 (b) cells treated with 40 nM of PMA for times indicated in the figures and resolved on 7.5% SDS-PAGE. The arrowheads indicate the 74-kDa protein, the brackets indicate the broad band ranging between 90 and 105 kDa. Molecular mass markers are as indicated under ``Materials and Methods.''
Noteworthy, PMA does not induce significant changes in the HLA class I molecule steady state levels (Fig. 2, bottom panels). Hence, induction of surface proteins does not appear to be a general phenomenon generated by PMA. The kinetics of appearance of p74 suggests that it might represent a precursor of CD36 whose synthesis is activated by PMA. Immunoprecipitation and pulse-chase experiments were thus designed to verify this possibility.
Figure 3:
Analysis of CD36 processing in U937 and
THP-1 cells by pulse-chase experiments. Cells treated for 12 h with PMA
were endogenously labeled with
[S]methionine/cysteine, then washed and
incubated in regular culture medium for various chase-time periods
before lysis and sequential immunoprecipitations with 1207 anti-CD36
rabbit serum, OKM1 mAb anti-CD11b, and GAP 8.3 mAb anti-CD45RA and B;
see ``Materials and Methods.'' The arrow indicates
the co-immunoprecipitated 33-kDa protein, the bracket indicates the mature CD36 protein, and the arrowhead indicates the gp74. Molecular mass markers are as indicated under
``Materials and Methods.''
Figure 4: Western blot analysis of immunoprecipitated CD36 from THP-1 differentiated cells. Immunoprecipitation was performed with 1207 anti-CD36 rabbit serum from extracts of the THP-1 cell line after 48 h of PMA differentiation or from THP-1 cells surface-labeled with succinimidobiotin undergoing the same treatment as described under ``Materials and Methods.'' Immunocomplexes were resolved on 7.5% SDS-PAGE and transferred to nitrocellulose membrane (lanes 1207). Control immunoprecipitations (lanes Ctrl) were performed with nonimmune rabbit serum. Western blots performed with anti-CD36 Mo91 mAb on immunocomplexes from unlabeled cells and on differentiated THP-1 whole extract fractionated on the same gel (lane Total Lys) were developed with rabbit anti-mouse Ig HRP (RaMIg-HRP). Immunocomplexes from cell surface-biotinylated proteins were detected on a nitrocellulose membrane by reaction with avidin-HRP. The arrowhead indicates the 74-kDa protein, the bracket indicates the broad band ranging between 90 and 105 kDa. Molecular mass markers are as indicated under ``Materials and Methods.''
When intact THP-1 cells are labeled with biotin prior to cell lysis, only the gp90-105 band is decorated by avidin (see the right lane), suggesting that p74 is not expressed on the cell surface. The faint band with an apparent molecular mass of 74 kDa detected by avidin in the immunoprecipitate(1207) might be due to the biotin labeling of the intracellular precursor from a few dead cells permeabilized during incubation. A weak band of 130-kDa molecular mass is present in the immunoprecipitated material(1207). This band, that was never detected in the total lysates, is likely to originate from artifactual cross-linking by succinimidobiotin.
If p74 were a precursor of mature CD36, treatment with N-glycosidase F, an enzyme that cleaves all types of N-linked glycosidic residues, should yield a single band of about 57 kDa(28) . As shown in Fig. 5(lanes 1 and 2) this is indeed the case. In PMA-differentiated U937 cells, both the gp90-105-kDa protein band and the one of 74 kDa yield a unique protein band of 57 kDa when N-linked sugars are removed. Treatment with endoglycosidase H allows one to distinguish high mannose (sensitive) from complex sugars. As evident from lanes 3 and 4, gp74 is sensitive to this glycosidase, while gp90-105 is largely resistant. The partial digestion of gp90-105 is probably due to incomplete processing of some of the CD36 glycans.
Figure 5: N-Glycosidase F and endoglycosidase H treatments. Cell extracts prepared from 48-h PMA-differentiated U937 were treated (+) with N-glycosidase F or with endoglycosidase H or incubated without enzyme(-) as described under ``Materials and Methods.'' Proteins were resolved on 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using Mo91 anti-CD36 mAb. Upper arrowhead indicates the 74-kDa protein, the bracket indicates the broad band ranging between 90 and 105 kDa, and the lower arrowhead indicates the 57-kDa protein. Molecular mass markers are as indicated under ``Materials and Methods.''
The PMA-induced accumulation of gp74 depends on de novo mRNA and protein synthesis. Consistent with gp74 being a precursor of CD36, both actinomycin D (a mRNA synthesis inhibitor) and cycloheximide (an inhibitor of protein synthesis) prevent the accumulation of the gp74 induced by PMA treatment (not shown).
Nonreducing gels (not shown) reveal that the p33 molecule is not disulfide-linked to CD36 precursor, while like gp74, p33 is endo-H-sensitive and is digested into a 22-24-kDa molecule (Fig. 6).
Figure 6: Endoglycosidase treatment of the 33-kDa co-immunoprecipitated protein. 1207 anti-CD36 rabbit serum immunoprecipitated samples from endogenously labeled U937 cells after PMA treatment were incubated with (+) or without(-) 10 milliunits of endoglycosidase H for 12-18 h at 37 °C, in appropriate digestion buffer; see ``Material and Methods.'' Treated samples were run on 5-15% gradient SDS-PAGE in reducing conditions, and dried gels were analyzed on a PhosphorImager (Molecular Dynamics). Black arrows indicate undigested gp74 and gp33, and white arrows indicate shifted proteins after endoglycosidase treatment. Molecular mass markers are as indicated under ``Materials and Methods.''
The first possibility is excluded by
adsorption experiments. Adsorption of the 1207 anti-CD36 rabbit serum
with human platelets, which express abundant mature CD36 but no
gp74gp33 complexes, (
)results not only in a
significant reduction of gp74 present in the immunoprecipitates, but
also in a similar reduction of gp33 (Table 1). As a control,
adsorption on platelets did not affect the efficiency of a rabbit serum
anti-IgM to immunoprecipitate radiolabeled mouse Ig µ-chain (Table 1).
The second hypothesis was excluded by two lines of evidence. First, neither polyclonal rabbit serum anti-CD36 nor the Mo91 mAb detected gp33 in Western blots of whole lysates or on anti-CD36 immunoprecipitates (not shown). Second, different peptides are generated by trypsin digestion from gel-purified gp74 and gp33. While the gp74 yields four peptides of 27, 21, 18, and 14 kDa, respectively, three peptides of 30, 25, and 23 kDa are generated by limited proteolysis of gp33 (Fig. 7). Hence, these results suggest that gp33 is a protein that associates with the 74-kDa CD36 precursor.
Figure 7: Peptide mapping by limited proteolysis and SDS-PAGE analysis of gp74 and gp33. Immunocomplexes obtained from endogenously labeled U937 cells after 12 h of PMA treatment were separated by 10% SDS-PAGE. The gp74 and gp33 protein bands (arrowheads) were eluted from the gel, acetone-precipitated, and treated with 100 µg/ml trypsin protease as indicated under ``Materials and Methods.'' They were then loaded onto a 12% acrylamide gel. Dried gels were exposed to a PhosphorImager or film. Molecular mass markers (MW) are as indicated under ``Materials and Methods.''
In the present study, the U937 promonocytic and the THP-1 monocytic cell lines have been exploited to analyze the synthesis, processing, and surface expression of CD36 during monocyte/macrophage differentiation. Some of the events that characterize this developmental program, including changes in the growth rate, adherence, and expression of surface markers can be mimicked in these cell lines by stimulation with PMA(26, 27) . Compared with blood monocytes, in vitro differentiated macrophages and alveolar macrophages showed a down-regulation of CD36 surface expression (29) that is consistent with the different degree of differentiation and CD36 expression observed in the the two cell lines used in the study.
The most intriguing finding that emerges from our experiments is that the intracellular processing of CD36 is faster in U937 than in THP-1. Crucial to this conclusion is the identification of different molecular glycoforms of CD36 that represent discrete stages in the biogenesis of this membrane protein. That the 74-kDa protein is a precursor of the mature CD36 (gp90-105) is demonstrated by the following lines of evidence: (i) antibodies raised against the mature CD36 recognize the gp74 both in Western blot and immunoprecipitation assays, (ii) treatment with N-glycosidase-F reduces both the mature CD36 and the 74-kDa form to a single protein band of 57 kDa, and (iii) pulse-chase experiments clearly define a precursor product relationship between gp74 and gp90-105.
As in other
glycoproteins, these size differences are likely to reflect different
intracellular locations. The selective accessibility to biotin
indicates that only the mature CD36 (gp90-105) is expressed on
the plasma membrane. On the other hand, the sensitivity of gp74 to the
endoglycosidase H, an enzyme that cleaves the high mannose glycans
characteristic of protein that did not reach the medial Golgi apparatus (30) , suggests that the low molecular weight precursor is
localized in an early compartment of the secretory pathway. Hence, the
steady state ratio between the two bands can be taken as an indication
of the efficiency of intracellular processing and transport. With this
in mind, it appears that the transport of CD36 is slower in THP-1 than
in U937 at all time points after PMA admininstration and that the rate
of synthesis of the precursor has little influence on the subsequent
processing events. As reported by others (31) we confirmed that
PMA induces an increase in the rate of transcription and accumulation
of specific transcripts, ()but does not alter the
posttranslational events that modulate the expression of CD36 on the
cell membrane in the two cell lines. At least two features seem to
retard the transport of CD36 to the surface of THP-1. First, transport
of the protein to the Golgi apparatus is severely impaired in these
cells, as indicated by the abundance of the endo-H-sensitive gp74 and
its slow conversion into the more mature forms. Second, as evident from
the pulse-chase experiments (Fig. 3) and confirmed by detergent
insolubility analyses (not shown), gp90-105 is degraded at a
faster rate in THP-1 cells.
An intracellular localization of CD36
has already been reported in platelets, where some mature CD36 is
stored within the -granules and can be rapidly transported to the
plasma membrane upon activation(32) . In uninduced monocytic
cell lines, however, the intracellular pool of CD36 is small (Fig. 2). Moreover, the PMA-induced expression of CD36 is
dependent on RNA and protein synthesis, suggesting that the molecule is
neither recycled from intracytoplasmic stores, as observed for
platelets, nor does it represent a degradation product.
Although the rate of bulk flow membrane traffic can be regulated(33) , faster transport per se is not sufficient to explain the more efficient processing of CD36 in U937. Other membrane molecules, either PMA-inducible (CD11b) or constitutively expressed (CD45R), are indeed processed and transported intracellularly at similar rates in U937 and THP-1.
It is well established that individual proteins are secreted
at different rates by the same cell(34) . An important limiting
step of intracellular transport occurs at the endoplasmic
reticulum-Golgi apparatus boundary and generally reflects the rates at
which the folding of the cargo protein takes place. Also integral
membrane proteins are subject to the same quality control events that
restrain transport to structurally mature molecules, providing an
additional step for regulating gene expression during development.
Perhaps the best examples of such posttranslational regulatory
mechanisms come from the stage-specific expression of antigen receptors
on T and B lymphocytes (see (35) and (36) for
reviews). As for these multimeric proteins, subunit assembly is
essential for negotiating transport to the plasma membrane; the absence
of a single subunit is generally sufficient to cause retention and
degradation of the other components (37) . However, there are
also cases in which the assembly of existing subunits is dynamically
regulated. For instance, for unknown reasons B lymphocytes are unable
to polymerize and secrete IgM(23) . Similarly, immature
thymocytes synthesize but fail to assemble T cell antigen receptor
- and
-chains, degrading them in the endoplasmic
reticulum(38) . If multimeric proteins hence can be regulated
at the level of assembly, only the rate of folding would modulate the
transport of monomeric molecules. As most of the chaperones identified
so far that catalyze the folding of nascent proteins are ubiquitous,
abundant proteins, it is not easy to envisage a model that explains the
different rate of transport of CD36. The latter is thought to be a
monomeric receptor (see (1) ). However, in platelets the mature
CD36 transiently associates with proteins involved in transducing
signals (18) , while in endothelial cells, it is present as a
detergent-insoluble complex(39) . These interactions may be
responsible for the detergent ``insolubility'' of
gp90-105 observed in U937 and THP-1, but it is less likely that
they mediate the different processing of gp74.
An appealing
hypothesis is that the observed association with gp33 may regulate the
transport and processing of the CD36 precursor, determining its
intracellular retention or the expression on cell surface. Like the
CD36 precursor to which it is noncovalently bound, gp33, or at least
the fraction that associates with gp74, also is retained in a pre-Golgi
compartment, as demonstrated by sensitivity to endoglycosidase H. The
association between gp33 and gp74 inversely correlates with processing
of the latter. Hence, only in THP-1, where processing of CD36 is slow,
does gp33 coprecipitate at extended chase times. Lastly, in U937
dissociation of gp33 correlates with the acquisition of endo-H
resistance. The disappearance of radioactive gp33 upon chase indicates
that it is mostly newly synthesized gp33 that associates with the CD36
precursors. These results suggest that gp33 interact only once with the
CD36 precursor. This behavior differs from other known chaperone
molecules, such as BiP, that have been shown to associate sequentially
with more than one newly synthesized proteins (40) . It is of
note that a molecule that displays similar biochemical properties to
gp33 is coprecipitated from COS cells transfected with CD36. A conservation among species and cell types would suggest a
general role for this protein.
A CD36 deficiency has been described as the absence of surface expression of CD36 molecule(41, 42) . Recently, it has been shown that the substitution of proline-90 to serine directly leads to CD36 deficiency, impairing the maturation of the CD36 precursor and addressing its intracellular degradation(43) . It will be of interest to investigate whether gp33 associates with these mutants.
In conclusion, it appears that, during their differentiation, monocytic cell lines exploit many levels, including alterations in intracellular transport, to regulate the expression of CD36. It remains to be seen whether the regulatory mechanisms demonstrated here for cultured neoplastic cells are exploited, as postulated by others(44) , also in the normal myelomonocytic maturating pathways.