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Cell Growth & Differentiation Vol. 10, 591-600, August 1999
© 1999 American Association for Cancer Research

Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Mediates the Growth-Inhibitory Effects of Retinoids1

Jing X. Kang2, Jennifer Bell, Richard L. Beard and Roshantha A. S. Chandraratna

Departments of Medicine, Massachusetts General Hospital, Charlestown, Massachusetts 02129, and Harvard Medical School, Boston, Massachusetts 02114 [J. X. K., J. B.]; and Retinoid Research, Allergan Pharmaceuticals, Irvine, California 92713 [R. L. B., R. A. S. C.]


    Abstract
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
Both retinoids and the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) have been shown to play an important role in controlling cell growth during embryonic development and oncogenesis. Our recent work (Kang et al., Proc. Natl. Acad. Sci. USA, 94:13671–13676, 1997; Kang et al., Proc. Natl. Acad. Sci. USA, 95: 13687–13691, 1998) revealed a direct biochemical interaction between retinoic acid (RA) and the M6P/IGF2R, thereby leading us to hypothesize that the M6P/IGF2R may mediate a growth-inhibiting effect of RA. To test this hypothesis, cell growth and apoptosis in response to RA and various receptor-selective retinoids were examined in cells that lack or overexpress the M6P/IGF2R. RA and those retinoids capable of binding to the M6P/IGF2R induced a remarkable morphological change with characteristics of round shape and reduced spreading, apoptosis, and growth inhibition in stably transfected mouse P388D1 cells overexpressing the M6P/IGF2R but not in the M6P/IGF2R-deficient P388D1 cells. These effects of RA were neither blocked by a potent RA nuclear receptor (RAR) antagonist (AGN193109), nor mimicked by a selective RAR agonist (TTNPB), suggesting that the observed effects of RA are independent of RARs. Similar effects of the retinoids were observed in cultured neonatal rat cardiac myocytes that have high levels of the M6P/IGF2R. Furthermore, overexpression of the M6P/IGF2R in a RA-resistant cancer cell line (HL-60R) that lacked functional RARs gave the cells a susceptibility to RA-induced apoptosis. These data suggest that the M6P/IGF2R may play an important role in mediating retinoid-induced apoptosis/growth-inhibition and provide insight into the similar biological effects of RA and the M6P/IGF2R on fetal development and carcinogenesis.


    INTRODUCTION
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
The M6P/IGF2R3 is a Mr 300,000 transmembrane glycoprotein known to have multiple important biological functions (reviewed in Refs. 1, 2, 3) , including: (a) binding and transporting M6P-bearing glycoproteins (e.g., lysosomal enzymes) from the TGN or the cell surface to lysosomes; (b) mediating the internalization and degradation of IGF2 ligand, a mitogen that normally acts through the IGF1 receptor; and (c) assisting in the activation of TGF-ß, a potent growth inhibitor for most cell types. These functions of the receptor give it a critical role in the suppression of cell growth in embryonic development and oncogenesis. This is evidenced by a growing number of animal as well as human studies (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . In mutant mice, loss of the M6P/IGF2R results in fetal overgrowth and perinatal lethality as a consequence of major cardiac abnormalities (4, 5, 6) . Human studies have also shown that loss or mutation of the M6P/IGF2R gene is associated with the etiology of several human cancers (7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .

RA and its analogues, the retinoids, exert diverse biological effects and control normal growth, fetal development, differentiation, morphogenesis, metabolism, and homeostasis (reviewed in Refs. 17 , 18) . Clinically, RA and some synthetic retinoids are known to be effective against a number of skin diseases and have shown promise for the treatment and prevention of several cancers (17 , 18) . Recently, retinoids have been linked to the induction of apoptosis in both in vivo and in vitro models of cell death (19, 20, 21, 22, 23, 24, 25, 26) . It is thought that the effects of retinoids are mediated through two classes of nuclear receptors, the RARs and the RXRs (17 , 18) . However, in some cases it has been shown that the retinoid-induced apoptosis or growth inhibition is independent of the action of the nuclear receptors (20, 21, 22, 23, 24, 25, 26, 27) , which suggests that other retinoid-response pathways may exist.

Recently, our work has demonstrated that the M6P/IGF2R is a novel receptor for RA (28) . RA binds to the M6P/IGF2R with high affinity (KD of 2–3 nM). The binding site for RA on the receptor protein is distinct from the binding sites for M6P and IGF2. Binding of RA enhances binding of the other ligands (e.g., M6P or M6P-containing proteins) to the receptor. In addition, binding of RA to the M6P/IGF2R seems to facilitate the endocytosis and sorting functions of the receptor (28) . Furthermore, our latest study shows that the interaction of RA with the M6P/IGF2R leads to an intracellular redistribution of the receptor and lysosomal enzymes (29) . These data suggest that RA may act as a functional promoter of the M6P/IGF2R by directly binding to its unique site on the receptor.

On the basis of the direct biochemical link between RA and the M6P/IGF2R and the similarities of their roles in the control of embryonic development and oncogenesis, it is hypothesized that the M6P/IGF2R may mediate the effects of retinoids on cell growth. Specifically, interaction of retinoids with the M6P/IGF2R may lead to growth inhibition and/or apo-ptosis. To verify this hypothesis, several cell lines with different receptor profiles were tested for their responses to the treatments with various retinoids in terms of cell proliferation and death.


    RESULTS
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
To determine whether the M6P/IGF2R is involved in the growth-inhibitory effects of retinoids, several cell lines with different receptor profiles were tested for their responses to the treatments with various retinoids in terms of cell proliferation and death.


    P388D Cell Line.
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
Mouse macrophage P388D1 cells are the best characterized cells that lack the M6P/IGF2R (30, 31) . Transfer of the M6P/IGF2R gene into these cells has created a population of P388D1 cells that stably overexpress the receptor (31) . The responses of these two different populations of P388D1 cells (that lack or overexpress the M6P/IGF2R) to the treatment with RA are shown in Figs. 1Citation 2Citation 3Citation 4Citation .



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Fig. 1. Morphological changes induced by RA in P388D1 cells. Stably transformed P388D1 cells (provided by W. Sly) overexpressing the M6P/IGF2R (C and D) and control P388D1 cells (transfected with the control vector, receptor negative; A and B) were treated with (B and D) or without (A and C) 1 µM RA for 24 h. The photographs were taken directly from a 6-well culture plate using a phase microscope.

 


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Fig. 2. Effect of RA on actin cytoskeletal structure in P388D1 cells. Stably transformed P388D1 cells overexpressing the M6P/IGF2R (C and D) and control P388D1 cells (receptor negative; A and B) were treated with (B and D) or without (A and C) 1 µM RA for 24 h. Cells were fixed, permeabilized, and stained with phalloidin-FITC. Green, actin filament structure; red, cellular cytoplasm was stained with Even-blue.

 


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Fig. 3. Effect of RA on cell growth in P388D1 cells. Cells were grown in the presence or absence of 1 µM RA, and growth rate was measured by counting the viable cells in each treatment at specific time points. ({circ}), vector-transfected (receptor negative) cells without RA treatment (control); (•), vector-transfected cells with RA treatment; ({triangleup}), M6P/IGF2R cDNA-transfected (stably) cells without RA treatment; ({blacktriangleup}), M6P/IGF2R cDNA-transfected cells with RA treatment. Each point represents the mean of three separate experiments (n = 3; *, P < 0.05 versus control).

 


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Fig. 4. Effect of RA on apoptosis in P388D1 cells. Stably transformed P388D1 cells overexpressing the M6P/IGF2R (C and D) and control P388D1 cells (receptor negative; A and B) were treated with (B and D) or without (A and C) 1 µM RA for 48 h. Apoptotic cells were detected by TUNEL assay using the in situ apoptosis detection system, Fluorescein (Promega). Bright spots, (green fluorescence; D) fragmented DNA in apo-ptotic cells.

 
Treatment of the stably transfected cells with 1 µM RA for 24 h resulted in remarkable changes in cell morphology, characterized by round shape, smaller size, and loss of cell-cell connections or spreading attachments (plasma projections; Fig. 1Citation ), whereas the receptor-deficient cells (transfected with a control vector) showed no significant changes in cell morphology in response to the same treatment (Fig. 1)Citation . To determine whether the morphological changes observed in the P388D1 cells overexpressing M6P/IGF2R after RA treatment is associated with an alteration of cellular cytoskeletal structure or degradation of extracellular matrix, the cells were stained with FITC-phalloidin to observe changes in actin-filament organization. As shown in Fig. 2Citation , actin-fiber structure almost completely disappeared (was disrupted) in the receptor-expressing cells after RA treatment (1 µM for 24 h), whereas no significant change was observed in the M6P/IGF2R-deficient cells treated in the same manner. (It was noticed that the P388D1 cells stably expressing the M6P/IGF2R appeared to be more rounded and possessed less cytoskeletal structure than the M6P/IGF2R-negative P388D1 cells. This difference may reflect a response of the M6P/IGF2R-positive P388D1 cells to the low levels of retinoids that exist in the serum added to culture medium.) Accordingly, the stably transfected cells re-expressing the M6P/IGF2R grew much more slowly in the presence of RA when compared with the receptor-deficient cells (Fig. 3)Citation , which indicated an inhibitory effect on cell proliferation. In addition, fluorescence-staining of fragmented DNA indicated that a much higher number of apoptotic cells were present in the transfected cell cultures than in the control (receptor-deficient) cells after treatment with RA (Fig. 4)Citation . These data indicate that the effects of RA are related to the expression (or abundance) of the M6P/IGF2R in the cells.

To eliminate the possibility that the observed effects of RA in the P388D1 cells are mediated by its nuclear receptors (RARs; all-trans-RA does not bind to RXRs), receptor-selective retinoids, which selectively bind to either RARs or the M6P/IGF2R but not both, were tested for their effects on the growth of the cells. The compounds tested include the two RA analogues (retinol and retinol acetate) known not to bind to the RARs (32) and two synthetic retinoids [TTNPB (a potent RAR agonist) and AGN193109 (a potent RAR antagonist)] that can bind to the RARs with high affinity (33, 34, 35, 36, 37) . Their abilities to bind to the M6P/IGF2R were verified by a competitive photolabeling assay. As shown in Fig. 5ACitation , both retinol and retinol acetate seemed able to bind to the M6P/IGF2R with affinity similar to that of RA. However, the two synthetic retinoids, TTNPB and AGN193109 (which are structurally distinct from RA), failed to bind to the M6P/IGF2R (Fig. 5B)Citation . Treatment of the stably transfected P388D1 cells with retinol or retinol acetate induced significant effects on cell morphology (Fig. 6A)Citation and growth rate (Fig. 6B)Citation similar to those observed with RA. These two compounds had no significant effects on the control (M6P/IGF2R-deficient) cells (data not shown). In contrast, neither the RAR-agonist, TTNPB, nor the RAR-antagonist, AGN193109, alone induced similar effects after a 48-h treatment period (Fig. 7)Citation . Furthermore, the combined use of RA with the RAR-antagonist AGN193109 failed to block the effects of RA (Fig. 7)Citation , which suggested that the morphological and growth-inhibitory effects of RA in the cells were not RAR-dependent.



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Fig. 5. A, effects of various RA analogues on the binding of [3H]RA to the M6P/IGF2R. FBS proteins (100 µg) were incubated with [3H]RA (2 µM) in the absence (Cont.) or the presence of 25-fold molar excesses (50 µM) of unlabeled all-trans-RA (RA), retinol (ROH), or retinol acetate (ROHA). The samples were photolyzed and then processed for SDS/PAGE and fluorography. Both retinol and retinol acetate were able to compete with [3H]RA to bind to the IGF2R. B, competitive binding of RA and synthetic retinoids to the M6P/IGF2R. Partially purified M6P/IGF2R from neonatal rat serum was incubated with [3H]all-trans-RA (2 µM) in the absence (Cont.) or the presence of 25-fold molar excesses (50 µM) of unlabeled all-trans-RA (RA), a RAR agonist (TTNPB), or a RAR antagonist (AGN193109). The samples were photolyzed and then processed for SDS/PAGE and fluorography. Both TTNPB and AGN193109 were unable to compete with RA to bind to the IGF2R.

 


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Fig. 6. Effects of RA analogues on the morphology (A) and growth (B) of the stably transfected P388D1 cells overexpressing the M6P/IGF2R. Both the morphological data and the cell number shown were obtained at 48 h after treatment of the cells with 1 µM of all-trans-RA, retinol, or retinol acetete. Each point, the mean of three separate experiments (n = 3; *, P < 0.05 versus control). a, control (Cont.); b, RA (RA); c, retinol (ROH); d, retinol acetate (ROHA).

 


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Fig. 7. Effects of synthetic retinoids on the morphology (A) and growth (B) of the stably transfected P388D1 cells overexpressing the M6P/IGF2R. Both the morphological data and the cell number shown were obtained at 48 h after treatment of the cells with RA and/or synthetic retinoids. (a) control; (b) 0.25 µM RA; (c) 1 µM TTNPB (a potent RAR agonist); (d) 2 µM AGN193109 (a potent RAR antagonist); (e) 0.25 µM RA + 2 µM AGN193109. Each point, the mean of three separate experiments (n = 3; *, P < 0.05 versus control).

 

    HL-60R Cell Line.
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
The human promyelocytic leukemia cell line HL-60R is normally resistant to RA treatment because the retinoid nuclear receptor function in this cell line has been voided because of a defect of RAR{alpha} (38, 39, 40) . In addition, HL-60 cells have also been shown to express relatively low levels of the M6P/IGF2R (30) . On this basis, we transfected HL-60R cells with a plasmid-encoding human M6P/IGF2R and then assessed their response to RA. Fig. 8Citation shows a set of representative data taken from one of three similar experiments (the other two experiments had similar results). Transient transfection with M6P/IGF2R cDNA resulted in overexpression of M6P/IGF2R in the cells, as measured by M6P-containing lysosomal enzyme (ß-glucuronidase) binding assay (Fig. 8A)Citation . However, transfection with the M6P/IGF2R cDNA did not alter the expression of the retinoid nuclear receptors (RAR{alpha} and RXRß) as measured by immunoblotting (not shown). (The transfection efficiency was 28% in this case as evaluated by transfection of a ß-gal vector with the same promotor). Accordingly, the cells transfected with the M6P/IGF2R cDNA showed a marked reduction in growth rate (Fig. 8B)Citation and a significant increase in apoptosis without antecedent differentiation (Fig. 8, C and D)Citation in response to RA (1 µM) when compared with the cells transfected with the parental vector. These data further suggest that the M6P/IGF2R mediates RA-induced apoptosis.



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Fig. 8. Effect of overexpression of M6P/IGF2R in HL-60R cells on their susceptibility to apoptosis in response to RA. HL-60R cells were transfected with a human M6P/IGF2R cDNA in plasmid pECE or the control pECE vector by the lipofection method. Cells were then incubated with or without 1 µM RA for 72 h, and the expression of M6P/IGF2R, cell growth rate, and cell death were assessed. A, overexpression of M6P/IGF2R was determined by measuring the M6P-inhibitable binding of ß-glucuronidase to saponin-permeabilized cells 48 h after transfection; Control, cells untransfected; Vector, cells transfected with pECE; M6P/IGF2R, cells transfected with M6P/IGF2R cDNA. B, cell growth rate, as measured by counting the viable cells of each treatments; {circ}, vector-transfected cells without RA treatment; •, vector-transfected cells with RA treatment; {square}, M6P/IGF2R cDNA-transfected cells without RA treatment; {blacksquare}, M6P/IGF2R cDNA-transfected cells with RA treatment. C, cytospin analysis of apo-ptosis. Cells transfected with the control vector (a and b) or with M6P/IGF2R cDNA (c and d) were treated with (b and d) or without (a and c) 1 µM RA for 3 days, centrifuged onto slides, and stained with LeukoStat stain. Arrows, typical apoptotic cells with pyknotic or fragmented nuclei.D, flow cytometry analysis of apoptosis. Cells transfected with control vector (a and b) or with M6P/IGF2R cDNA (c and d) were treated with (b and d) or without (a and c) 1 µM RA for 3 days and stained with FITC-labeled annexin V. The cells (10,000) were then analyzed by flow cytometry. The percentage of cells stained by FITC-labeled annexin V in each sample is indicated.

 

    Neonatal Rat Cardiac Myocytes.
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
Cultured neonatal rat cardiac myocytes, in which we first observed the binding of RA to the M6P/IGF2R, express very high levels of the M6P/IGF2R (28) . If the M6P/IGF2R indeed mediates the action of RA, it is expected that the response of these cells to RA should be highly sensitive. Thus, we examined the effects of RA and the receptor-selective retinoids on the growth of the cultured cardiac myocytes.

As shown in Fig. 9Citation , treatment of the cells with 1 µM RA for 48 h resulted in a marked alteration of cell morphology as well as inhibition of cell growth. The RA-treated cells rounded up, lost their spreading attachments, and exhibited much smaller cell colonies when compared with the control (untreated) cells (Fig. 9)Citation . Interestingly, retinol as well as retinol acetate exerted similar effects on cellular morphology and growth within the 48-h treatment. Staining of the nuclei with Hoechst 33258 revealed that there were many apoptotic cells in the cultures treated with RA, retinol, or retinol acetate (Fig. 10)Citation . [At an even higher concentration (5 µM for 72 h), retinol and retinol acetate were not effective in the induction of cell differentiation in HL-60 cells, which are sensitive to RA and RAR agonists (data not shown)]. However, the effects of RA on the myocytes could neither be mimicked by the RAR-agonist TTNPB (it had a much less significant effect within the 48-h treatment) nor be blocked by the RAR-antagonist AGN193109 (Fig. 11)Citation . The differential effects of retinoids on the growth of neonatal rat myocytes suggest that the retinoid-induced growth-inhibitory effects may be, at least in part, mediated by the M6P/IGF2R.



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Fig. 9. Microphotographs showing the effects of various retinoids on cell morphology and growth of cultured neonatal rat cardiac myocytes. Cells were treated with ethanol (A, control) or 1 µM RA (B), retinol (C), or retinol acetate (D) for 48 h. (Normally, the cardiac myocytes grow in spreading colonies and exhibit spontaneous beating). The photographs were taken directly from a 6-well culture plate using a phase microscope.

 


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Fig. 10. Induction of apoptosis of neonatal rat cardiac myocytes by various retinoids. Neonatal rat heart cells were treated with ethanol (A, control), 1 µM RA (B), retinol (C), or retinol acetate (D) for 48 h. Cells were fixed and stained with Hoechst 33258 for nuclei. The brighter fluorescent spots are the nuclei of apoptotic cells, characterized by smaller-size, round-shape, and fragmentation.

 


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Fig. 11. Effects of RAR-selective retinoids on cell morphology and growth of cultured neonatal rat cardiac myocytes. Cells were treated with RA and/or synthetic retinoids for 48 h. The photographs were taken directly from a 6-well culture plate using a phase microscope. A, control; B, 0.25 µM RA; C, 0.25 µM RA + 2 µM AGN193109; D, 1 µM TTNPB.

 

    COS-7 Cells.
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
To further document the relationship between the M6P/IGF2R and cellular response to RA, we cotransfected COS-7 cells with a pECE/IGF2R and a pEGFP. The cotransfection allowed us to easily identify the transfected cells and detect their phenotypes in response to RA. The results showed (Fig. 12)Citation that the majority (50–75%) of cells transfected by M6P/IGF2R cDNA underwent apoptosis-like changes in morphology (rounding up, decreased size, and nuclear fragmentation) in response to RA treatment, whereas cells transfected with the parental (control) vector did not show such a significant response. These results also support a role for M6P/IGF2R in mediating the action of RA.



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Fig. 12. Response to RA of transfected COS-7 cells overexpressing the M6P/IGF2R. COS-7 cells were cotransfected with a GFP-vector (pEGFP) and a pECE/IGF2R or the parental pECE vector for 24 h by method of lipofection, followed by treatment with 1 µM RA for 48 h. Morphological changes of the transfected cells were viewed under a fluorescence microscope. A, pEGFP+pECE-transfected cells without RA treatment; B, pEGFP+pECE-transfected cells with RA treatment; C, pEGFP+pECE/IGF2R-transfected cells without RA treatment; D, pEGFP+pECE/IGF2R-transfected cells with RA treatment.

 

    DISCUSSION
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
The major goal of this study was to examine the role of the M6P/IGF2R in mediating the effect of retinoids on cell growth and death. Because the RARs, which exist in most cell types, are thought to mediate the most biological effects of RA, exclusion of the RARs’ involvement in a testing system is crucial for establishing a link between RA and the M6P/IGF2R. In this study, several unique cell lines with different receptor profiles (i.e., M6P/IGF2R and RARs) and receptor-selective retinoids were used to evaluate the relationship between the M6P/IGF2R and the retinoid-induced responses. Our results showed that RA and its analogues that are capable of binding to the M6P/IGF2R induced remarkable morphological changes, growth-inhibition, and apoptosis in cells overexpressing the M6P/IGF2R but not in M6P/IGF2R-deficient cells. These effects were also observed in the RAR-defective cells. Furthermore, the effects of RA were neither blocked by a RAR antagonist nor mimicked by the retinoids that selectively bind to the RARs. These data indicate a link between the RA-induced responses and the M6P/IGF2R. Therefore, the results of this study support the notion that the M6P/IGF2R may play a role in mediating the growth-inhibitory effects of retinoids.

There is increasing evidence that the M6P/IGF2R is involved in control of cell growth in carcinogenesis and fetal development. A growing number of studies (7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 41, 42) have suggested that the M6P/IGF2R is a tumor suppressor gene. It has been shown that expression of the M6P/IGF2R is significantly reduced in both rat and human hepatocellular carcinomas and that 70% of human hepatocellular tumors and 30% of human breast tumors have loss of heterozygosity at the M6P/IGF2R locus with mutations in the remaining allele (7, 8, 9, 10 , 14, 15, 16) . A major mutation was found in a region of the M6P/IGF2R gene coding for part of extracellular domain 10, a region closely associated with M6P-ligand binding (8 , 9) . Loss of heterozygosity at the M6P/IGF2R gene has also been shown to correlate with poor differentiation in early breast carcinomas (14) . In addition, mutation of the M6P/IGF2R gene was also found in genetically unstable cancers of the endometrium, stomach, and colorectum (12) . Furthermore, several studies (41, 42) show that, in mammary tumors regressing in response to d-limonene, the steady-state M6P/IGF2R mRNA level and apoptotic index increased 2- to 10-fold when compared with that of untreated tumors, whereas the M6P/IGF2R expression was unaltered in the nonresponsive tumors. Obviously, the M6P/IGF2R plays a critical role as a negative regulator of cell growth.

Further evidence supporting a role for the M6P/IGF2R in the suppression of cell growth comes from recent studies (4, 5, 6) using mutant mice containing a homologous deletion of the M6P/IGF2R gene. These studies show that loss of the M6P/IGF2R results in fetal overgrowth and perinatal lethality as a consequence of major cardiac abnormalities (ventricular hyperplasia, marked enlargement of the heart due to an increase in the number of cells; Refs. 5 , 6 ). In addition, the expression of this receptor is developmentally regulated, with the receptor being high in fetal and neonatal tissues and declining postnatally (43) , which suggests an essential role for the receptor in control of normal fetal growth and development.

The effects of the M6P/IGF2R on cell growth may be attributable to its capability to bind multiple ligands (in distinct sites). With our recent discovery (28 , 29) , the receptor is now known to bind at least three different classes of ligands: (a) M6P (carbohydrate); (b) IGF2 (protein); and (c) RA (lipid). Many cellular proteins, such as lysosomal enzymes (1, 2, 3) , TGF-ß (44) , human leukemia inhibitory factor (45) , DNase (46) , and so forth, contain an M6P group and, thereby, are the ligands for the M6P/IGF2R. Binding of the M6P-containing proteins (through their M6P group) to the M6P/IGF2R is required for their trafficking, maturation/activation, or functional activities. Particularly, the targeting or sorting of newly synthesized lysosomal enzymes from the TGN to the endosomes/lysosomes—an essential process for the maturation and cellular retention of lysosomal enzymes including certain proteases (e.g., cathepsin B and cathepsin D)—relies on binding to and trafficking with the M6P/IGF2R. Thus, the M6P/IGF2R is a key factor in the regulation of lysosomal functions. Defect or loss of the interaction between newly synthesized lysosomal enzymes and the M6P/IGF2R, which leads to increased levels of both intracellular and extracellular pro-lysosomal enzymes (e.g., pro-cathepsin D and B) with a decrease in the mature form of the enzymes, has been reported in tumor cells (47, 48, 49) . This, together with the fact that some lysosomal proteases (e.g., cathepsins) are involved in apoptosis (50, 51, 52) , suggests a potential link between the M6P/IGF2R-mediated lysosomal functions and cell growth regulation (carcinogenesis). More importantly, the binding of latent TGF-ß to the M6P/IGF2R is known to be essential for its activation (44) . Because TGF-ß is a potent growth inhibitor for most cell types, this role of the M6P/IGF2R may have a profound impact on cell growth. Furthermore, the binding of IGF2 (a mitogen, normally acting through the IGF-I receptor) to the M6P/IGF2R at the cell surface leads to the internalization and lysosomal degradation of the mitogen. In this manner, the receptor serves as a suppressor of IGF2 proliferative actions. Therefore, the M6P/IGF2R plays an important role in the suppression of cell growth.

Because of the multiple growth-inhibiting effects of the M6P/IGF2R, it is conceivable that any agent capable of up-regulating the functional activities of this receptor could have a profound impact on cell growth and death. We propose that RA is such an agent that acts as a functional promoter of the M6P/IGF2R. Specifically, the binding of RA to the M6P/IGF2R may lead to increased binding affinity for other ligands (i.e., M6P, IGF2), rapid endocytosis, and efficient sorting/trafficking of enzymes. As a result, one or more of the growth-inhibiting actions (increased activation and cellular retention of apoptosis-related enzymes, increased degradation of IGF2, and increased activation of TGF-ß) may occur. This is supported by previous studies (28 , 29 , 53, 54, 55) . Our early studies show that (a) RA binds to the M6P/IGF2R with high affinity (28) ; (b) its binding increases the binding of M6P-ligands to the receptor (28) ; (c) RA increases the endocytosis of exogenous M6P-bearing glycoproteins (e.g., lysosomal enzymes; Ref. 28 ); (d) RA enhances the intracellular lysosomal enzyme activity (28) ; (e) RA facilitates the trafficking of M6P/IGF2R and lysosomal enzymes from the TGN to the lysosomes (29) ; and (f) RA also enhances the internalization of IGF2 (28) . In addition, it has been shown that RA can increase the activation of TGF-ß (53, 54, 55) . Although more elaborate studies are needed to elucidate the interaction pathway, the data available thus far have provided insight into a new mechanism by which RA induces growth-inhibition through interaction with the M6P/IGF2R and also a possible explanation for the recent observations that RA could induce apoptosis in some cells independent of the retinoid nuclear receptor (20, 21, 22, 23, 24, 25, 26, 27) .

The link between the retinoid-M6P/IGF2R interaction and cell death or growth-inhibition can help us understand many physiological and pathological phenomena related to cell growth. For example, during pattern-formation or tissue remodeling in embryogenesis, both the level of M6P/IGF2R and the concentration of retinoids are remarkably high in cells that undergo apoptosis or in tissues to be removed (20 , 43 , 56) . In this context, the excessive use or overdose of retinoids during embryogenesis can cause an increase in embryo resorption, a reduction in fetal body weight, and fetal deformities, perhaps because of excessive cell death (20 , 57) . Conversely, the loss or mutation of the M6P/IGF2R results in fetal overgrowth (4, 5, 6) or cancer formation (7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .

It has been well established that retinoids act via a nuclear receptor RARs/RXRs system. Our findings now add a new mode of action for retinoids. It is possible that both of these systems coexist in most cell types. However, their roles in regulation of cellular functions may be distinct and may vary with cell types and physiological states. In the RARs/RXRs system, RA functions as a transcriptional factor to up- or down-regulate gene expression, a process in which many cofactors may also be involved. This can be viewed as "team-work/network" and "off or on" mode. In contrast, in the M6P/IGF2R system, RA acts as a functional accelerator of the multiple functional protein with no need of protein expression. The overall cellular responsiveness induced by the interaction depends on the total number of the ligand-receptor complex formed, which is determined by the density of the receptor within a cell and/or the concentration of RA. (Effect can be observed within 24 h). This system can be relatively regarded as "Short-cut/localtalk" and "less or more" mode. Nevertheless, the relationship between these two systems in terms of the regulation of cell growth and death needs to be further studied.

In summary, our data demonstrate that the M6P/IGF2R plays an important role in mediating the growth-inhibitory effects of retinoids. This discovery adds a new regulatory pathway of apoptosis and thereby provides new insight into the etiology, prevention, and treatment of cancer and developmental defects.


    MATERIALS AND METHODS
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 
Materials.
All-trans-RA, retinol, and retinol acetate were obtained from Sigma. The synthetic retinoids used (AGN193109, TTNPB) were produced by Allergan Inc. (Irvine, CA). Stably transfected P388D1 cells re-expressing the M6P/IGF2R were kindly provided by Dr. William S. Sly (St. Louis University). HL-60R cells were kindly provided by Dr. S. J. Collins (Fred Hutchinson Cancer Research Center).

Cell Culture.
P388D1 cells were routinely maintained in MEM-{alpha} supplemented with 10% FBS. (The stably transfected P388D1 cells were regularly challenged with G418). HL-60R cells were cultured in suspension in RPMI 1640 supplemented with 10% FBS. Cardiac myocytes were isolated from 1-day-old neonatal Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation system (Worthington). The isolated cells were cultured on Petri dishes with culture medium (F-10 nutrient mixture, 10% horse serum, 5% FBS, 50 µg/ml streptomycin, and 50 units/ml penicillin G). Cells were used for experiments after 2–6 days of culture.

Cell Transfections.
For transient transfection of HL-60R cells, 5 x 105 cells were washed with serum-free medium and transfected with 2.5 µg of M6P/IGF2R cDNA or the control vector pECE using Dosper Liposomal Transfection Reagent (Boehringer Mannheim) following the manufacturer’s protocol. Twenty-four h after transfection, cells were washed and resuspended in fresh culture medium (5% FBS) with or without 1 µM RA. The transfection efficiency was evaluated by transfection of a ß-galactosidase vector with the same promoter. COS-7 cells were cotransfected with a GFP-vector (pEGFP) and a pECE/IGF2R or the parental pECE vector for 24 h by the method of lipofection, followed by treatment with 1 µM RA for 48 h.

ß-Glucuronidase Binding Assay.
Cells were permeabilized with 0.25% saponin in 50 mM Hepes (pH 7.0), 150 mM NaCl, 5 mM ß-glycerophosphate. 0.5% BSA, 10 mM M6P for 30 min on ice. The cells were then washed three times with PBS (pH 7.2) containing 0.05% saponin. They were incubated with 20,000 units/ml ß-glucuronidase in 50 mM Hepes (pH 7.5), 150 mM NaCl, 5 mM ß-glycerophosphate, 0.5% BSA, and 0.5% saponin with or without 10 mM M6P for 3 h on ice. The cells were then washed five times with PBS containing 0.05% saponin and were solubilized in 1% sodium deoxycholate. The solubilized cells were assayed for ß-glucuronidase (58) and protein. Specific enzyme binding was calculated by subtracting nonspecific binding (in the presence of 10 mM M6P) from total binding.

Morphological Evaluation.
For HL-60R cells, after transfection and treatment with RA, 0.2 ml of cells was removed for a cytospin preparation. The cells on slides were then fixed and stained with LeukoStat stain (Fisher) according to the manufacturer’s instruction. Cells were scored as apoptotic if there was evidence of nuclear pyknosis and fragmentation, cytoplasmic condensation, and basophilia. For the adherent cells (P388D1 cells, cardiac myocytes, and COS cells) grown on coverslips, morphological changes were observed and photographed directly under a phase microscope.

Flow Cytometry.
Flow cytometry analysis of apoptosis in HL-60R cells was carried out using an ApoAlert Annexin V Apoptosis kit (Clontech). Cells (1 x 106 were incubated for 10 min with 1.0 µg/ml Annexin V-FITC in binding buffer, and 10,000 cells were analyzed by a FACScan (Coulter).

Stain for Cytoskeletal Structure.
Cells grown on coverslips were fixed with 4% paraformaldehydate for 30 min followed by a wash in PBS. The cells were then permeabilized with 0.1% Triton X-100 for 5 min. After they were washed with PBS, the coverslips were incubated with phalloidin-FITC (Sigma) at 1:100 at room temperature for 30 min and washed and mounted on microscope-slides with Vectashied.

Photoaffinity Labeling of the M6P/IGF2R with [3H]RA.
Photoaffinity labeling using [3H]RA as a photoaffinity reagent was performed as we described previously (28) . Briefly, [3H]RA (usually 2 µM) with or without 100 µM unlabeled RA or other retinoids tested was incubated with partially purified M6P/IGF2R proteins (100 µg) from FBS or neonatal rat serum at room temperature with agitation for 1 h in the dark. The samples were placed on ice and exposed to an intense 365-nm UV light source (Model B 100AP, Upland, CA) for 7 min. The protein samples were concentrated by a vacuum drier or by filtration with a Biomax-100 centrifugal Filter (Millipore). The resultant protein pellets were used for SDS-PAGE and autoradiography.

Assessment of Cell Proliferation.
Cell proliferation was assessed 24, 48, 72, and 96 h after exposure to retinoids. The number of viable cells in each treatment was determined by the trypan blue exclusion method using a hemocytometer. To avoid bias, counting was done blindly for each sample by two individuals (counters; J. X. K. and J. B.), and each sample was counted twice.

Nuclear Stain for Apoptosis.
Hoechst staining was performed to detect changes in nuclei. Briefly, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 1 µg/ml Hoechst 33258 (Sigma). Apoptotic changes (fragmented nucleus, chromatin condensation, and so forth) were examined under a fluorescent microscope. Apoptotic cells were also detected by TUNEL assay using an in situ apoptosis detection system, Fluorescein (Promega).

Statistical Analysis.
Data are presented as mean ± SD. Comparison between individual treatment and control (treament versus control) was made by using paired Student’s t test, and a P < 0.05 was considered statistically significant.


    Acknowledgments
 
We thank Dr. W. Sly for the M6P/IGF2R cDNA and the stably transfected P388D1 cells and Dr. S. J. Collins for HL-60R cells. We are grateful to Dr. Alexander Leaf for his support and encouragement.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Cancer Institute Grant R01 CA-79553 (to J. X. K.). Back

2 To whom requests for reprints should be addressed, at Massachusetts General Hospital, Room 4433, 149 13th Street, Charlestown, MA 02129-2000. Phone: (617) 726-8509; Fax: (617) 726-6144; E-mail: kang.jing{at}mgh.harvard.edu Back

3 The abbreviations used are: M6P/IGF2R, M6P/IGF2 receptor; M6P, mannose 6-phosphate; IGF1, insulin-like growth factor I; IGF2, insulin-like growth factor II; RA, retinoic acid; RAR, retinoic acid nuclear receptor; RXR, retinoid X receptor; TGF, transforming growth factor; TGN, trans-Golgi network; GFP, green fluorescence protein; pEGFP, plasmid encoding GFP; pECE/IGF2R, plasmid encoding the M6P/IGF2R; FBS, fetal bovine serum; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. Back

Received for publication 5/11/99. Revision received 6/25/99. Accepted for publication 6/28/99.


    References
 TOP
 Abstract
 INTRODUCTION
 RESULTS
 P388D Cell Line.
 HL-60R Cell Line.
 Neonatal Rat Cardiac Myocytes.
 COS-7 Cells.
 DISCUSSION
 MATERIALS AND METHODS
 References
 

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