©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the p21 Pathway of Growth Arrest and Apoptosis by the Integrin Cytoplasmic Domain (*)

(Received for publication, May 26, 1995; and in revised form, July 18, 1995)

Astrid S. Clarke Margaret M. Lotz Celia Chao Arthur M. Mercurio (§)

From the Laboratory of Cancer Biology, Deaconess Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The integrin alpha(6)beta(4), a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study alpha(6)beta(4)-mediated functions in more detail and to assess the contribution of the beta(4) cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta(4) integrin subunit. This cell line, termed RKO, expresses alpha(6)beta(1) but not alpha(6)beta(4), and it interacts with laminin-1 less avidly than similar cell lines that express alpha(6)beta(4). We expressed a full-length beta(4) cDNA, as well as a mutant cDNA that lacks the beta(4) cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full-length beta(4) cDNA in association with endogenous alpha6 exhibited partial G(1) arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha(6)beta(4) integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the beta(4) integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta(4) transfected RKO cells.


INTRODUCTION

The integrin alpha(6)beta(4) is a receptor for members of the laminin family of basement membrane components. Initial studies established that alpha(6)beta(4) is a receptor for laminin-1(1, 2, 3) , and subsequent work has shown that it also functions as a receptor for other laminin isoforms(4, 5) . In its capacity as a laminin receptor, alpha(6)beta(4) is involved in the formation and maintenance of hemidesmosomes (6, 7, 8) and in the dynamic adhesion and migration of carcinoma cells(2, 3) . Most likely, other functions of epithelial and carcinoma cells are dependent upon alpha(6)beta(4) because it plays such a pivotal role in mediating their interactions with laminin matrices. It is widely assumed that the unusually large and structurally unique cytoplasmic domain of the beta(4) integrin subunit associates with cytoskeletal and signaling molecules and that such associations provide the basis for the distinct functions associated with alpha(6)beta(4)(5, 8, 9) .

In our efforts to study alpha(6)beta(4)-mediated functions in more detail and to assess the contribution of the beta(4) cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta(4) integrin subunit. This cell line, termed RKO, expresses alpha(6)beta(1) but not alpha(6)beta(4)(2, 3) . In this study, we report that RKO transfectants, which expressed the full-length beta(4) cDNA in association with endogenous alpha(6) exhibited G(1) arrest and a basal rate of apoptosis, properties that were not evident in RKO cells transfected with either a beta(4) cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha(6)beta(4) integrin induced expression of the p21 (WAF1; Cip1) protein, an inhibitor of G(1) cyclin-dependent kinases(10, 11, 12) . These data suggest that the beta(4) integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta(4)-transfected RKO cells.


MATERIALS AND METHODS

Cloning of the beta(4)Integrin Subunit

A full-length beta(4) cDNA clone was isolated from the Clone A colon carcinoma cDNA library (Clontech). This library was screened using a polymerase chain reaction product that was obtained from a partial sequence of the beta(4) subunit that we previously published(13) . Multiple clones that encompassed the full-length beta(4) cDNA sequence were isolated and subcloned into pBluescript (Stratagene). The full-length beta(4) cDNA was ligated into the pRc/CMV expression vector (Invitrogen) using the beta(4) restriction sites BglII and BssHII and the unique EcoRI vector site. This cDNA lacked the 70- and 7-amino acid splice variants and the 5` upstream 49-base pair region(13, 14) . In addition, a beta(4) cDNA with a truncated cytoplasmic tail termed beta(4)-DeltaCYT was constructed by polymerase chain reaction of a 400-base pair region from base pairs 2010-2398(14) , which introduced a stop codon and an XbaI site after the first 4 amino acids of the tail. This product was digested with SmaI and XbaI and ligated together with the 5` end of beta(4) (digested with EcoRI and SmaI) into the mammalian expression pcDNA3 (Invitrogen) via the unique EcoRI and XbaI sites in this vector.

Transfections and Flow Cytometry

The rectal carcinoma cell line RKO (15) was obtained from M. Brattain (Medical College of Ohio) and maintained in RPMI 1640 medium supplemented with 25 mM HEPES buffer, 10% FCS, (^1)1% penicillin/streptomycin, and 1% L-glutamine (Life Technologies, Inc.). RKO cells were transfected with either the full-length beta(4) construct, the beta(4)-DeltaCYT construct, or vector alone using 20 µl of Lipofectin reagent (Life Technologies, Inc.) and 10-15 µg of plasmid DNA. Geneticin (G418; Life Technologies, Inc.) was added to the growth medium at a concentration of 0.5 mg/ml for selection purposes. The transfected cells were sorted by FACS using the beta(4)-specific mAb UM-A9 (16) (provided by T. Carey, Michigan). FACS sorting was repeated twice to obtain a population of cells that exhibited significant surface expression of beta(4). These populations were then subcloned by single cell sorting into 96-well plates (Costar).

Growth Assays

The transfected cells were trypsinized, washed in divalent cation-free PBS (CMF-PBS), and resuspended in 5% FCS growth medium containing G418. Cells (1 times 10^4) were plated in triplicate 12-well plates (Costar) and allowed to grow for 3-6 days. Each well was harvested by trypsinization, and the number of cells was counted using a Coulter counter.

For propidium iodide staining, cells at approximately 50% confluency were plated overnight on either tissue culture plastic, EHS-laminin (4.2 µg/cm^2), or laminin-5 (0.2 µg/cm^2; provided by R. Burgeson) and then harvested by trypsinization. The cells were stained with propidium iodide (Sigma; 2 mg/ml in 4 mM sodium citrate containing 3% (w/v) Triton X-100 and RNase A (0.1 mg/ml)). The stained cells were analyzed by FACS.

DNA Fragmentation

Samples were prepared as described in Green and co-workers (17) for analysis of DNA fragmentation and analyzed by agarose gel electrophoresis (1.2%).

ApopTag Staining

Cells (1 times 10^6) were plated overnight on either plastic, EHS-laminin, or laminin-5 in normal culture medium (RPMI-H containing 10% FCS and G418), fixed in 1% paraformaldehyde in PBS, permeabilized in 70% ethanol, and then stained with the ApopTag reagent (apoptosis in situ detection kit, Oncor) and propidium iodide (as above). The stained cells were analyzed by FACS.

p21 Expression

For immunoblot analysis, cells (2 times 10^6) were plated for 18 h in 60-mm^2 dishes containing 5% FCS growth medium and G418. Some of the dishes were precoated with EHS-laminin (4.2 µg/cm^2) before the addition of cells. Cells were extracted for 10 min on ice in 10 mM Tris (pH 7.4) containing 10 mM NaCl, 3 mM MgCl(2), 1% (w/v) Tween 40, 0.5% sodium deoxycholate, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin and pepstatin (Boehringer Mannheim), and 50 µg/ml leupeptin (Boehringer Mannheim). Samples (10 µg of total protein) were resolved by SDS-polyacrylamide gel electrophoresis (12%), transferred to nitrocellulose, and probed for p21 using the mouse anti-human Sdi1 (p21) monoclonal antibody 6B6 (Pharmingen; 1 µg/ml) and a peroxidase-conjugated goat anti-mouse IgG (Kirkegaard & Perry; 0.5 µg/ml). Bound protein was detected by enhanced chemiluminescence (Amersham Corp.).

Cells for immunohistochemistry were plated for 18 h in complete growth medium on coverslips precoated with either poly-L-lysine (10 µg/cm^2) or EHS-laminin (5 µg/cm^2). Cells were fixed for 8 min in 4% paraformaldehyde, permeabilized for 2 min in 0.2% Triton X-100, and stained with the p21 (10 µg/ml) and a fluorescein-conjugated donkey anti-mouse IgG (Jackson Laboratories; 1:30). The cells were examined using a confocal microscope (Bio-Rad MRC 600, Bio-Rad Microsciences, Cambridge, MA) attached to a Zeiss Axiovert 35 equipped with a times63 Plan-Neofluar objective.


RESULTS

Representative RKO subclones from the full-length beta(4) cDNA and the beta(4)-DeltaCYT transfections that expressed varying levels of beta(4) surface expression were chosen for functional studies (Fig. 1A). No beta(4) expression was evident in subclones obtained from transfection of the expression vector alone (Neo). The association of the transfected beta(4) subunits with endogenous alpha(6) was confirmed by immunoprecipitation of surface-biotinylated cells with the A9 mAb (data not shown). Also, the full-length beta(4) transfectants exhibited increased adhesion, spreading, and migration on laminin-1 providing evidence that the expressed alpha(6)beta(4) integrin is functional (18) .


Figure 1: A, surface expression of the human beta(4) integrin subunit in RKO transfectants. Populations of transfected RKO cells expressing either a full-length beta(4) integrin (A7, B8, D4, F10) or a beta(4) cytoplasmic domain mutant (3C1, 5A3) on the cell surface were isolated by sequential FACS using UM-A9, a mAb specific for the beta(4) integrin subunit, subcloned by single cell sorting, and then analyzed by FACS. Mock transfectants (Neo 22 and Neo 26) were subcloned by limiting dilution analysis. For the mock transfectants, the scans obtained with a nonspecific IgG and UM-A9 are superimposed. For all of the other subclones, the UM-A9 scan (right-hand peak) is shown along with a mock transfectant scan (left-hand peak). B, analysis of DNA content in the bulk beta(4) transfectants. RKO cells transfected with the full-length beta(4) cDNA were enriched for beta(4) expression by sequential FACS using UM-A9, stained with propidium iodide, and analyzed by FACS to assess DNA content. The A(o), G(0)/G(1), S, and G(2)/M peaks are noted. C, analysis of DNA content in subclones of the beta(4) transfectants. Flow cytometry profiles obtained from propidium iodide staining of three subclones described in A are shown. For each profile, the percentage of cells in G(0)/G(1), S, and G(2) + M is noted in the upperright.



Initially, we observed that bulk sorts of the full-length beta(4) transfectants did not maintain high levels of beta(4) surface expression for more than 3-4 days after sorting. To gain insight into the behavior of these transfectants, we assessed their DNA content using propidium iodide. A significant A(o) peak, characteristic of cells undergoing apoptosis(19) , was evident in the bulk transfectants (Fig. 1B). Stable subclones that expressed full-length beta(4) cDNA maintained beta(4) surface expression, although the level of expression was less than that seen in the initial bulk sorts (Fig. 1A). These subclones grew noticeably more slowly than the mock transfectants. Propidium iodide staining revealed that the number of cells in G(1) was significantly greater in these transfectants than in the mock transfectants (Fig. 1C). The D4 subclone, for example, exhibited twice the percentage of total cells in G(1) compared with the mock transfectants. However, subclones that expressed the beta(4)-DeltaCYT subunit on the surface at levels comparable with that of full-length beta(4) exhibited no increase in the number of cells in G(1). To extend these observations, the doubling time for each of the subclones was determined (Table 1). The doubling time for RKO cells, as well as for the mock transfectants, is approximately 21 h. In contrast, the doubling times for subclones that expressed full-length beta(4) ranged from 25 to 39 h, and these times correlated with the level of beta(4) surface expression (cf. Table 1and Fig. 1). However, the beta(4)-DeltaCYT subclones exhibited doubling times similar to the Neo subclones, i.e. 20-21 h. Neither DNA content nor doubling times were affected significantly by growth of the subclones on EHS laminin-1 or laminin-5 compared with tissue culture plastic.



The results described above suggested that expression of alpha(6)beta(4) could induce partial G(1) arrest and possibly apoptosis in RKO cells. To examine this possibility, we assayed ApopTag reactivity by FACS. ApopTag is a fluorescein-conjugated antibody that recognizes digoxigenin-tagged 3`-OH DNA ends generated by DNA fragmentation, and its use for the detection of apoptotic cells has been documented(20) . As shown in Table 2, approximately 8% of the D4 transfectants and 3% of the B8 transfectants were ApopTag positive. In comparison, fewer than 2% of the beta(4)-DeltaCYT or mock transfectants were ApopTag positive. Attachment to either EHS laminin-1 (data not shown) or laminin-5 (Table 2) did not alter the pattern of ApopTag staining. In addition, DNA fragmentation was evident in the B8 and D4 subclones but not in the beta(4)-DeltaCYT transfectants (Fig. 2). These results indicate that a low, but significant, rate of apoptosis occurs in the full-length beta(4) subclones.




Figure 2: DNA fragmentation in the RKO transfectants. DNA was extracted from the beta(4) transfectants as described under ``Materials and Methods'' and analyzed by agarose gel electrophoresis (1.2%). Lane1, 1-kilobase pair DNA ladder; lane2, 3C1, a beta(4)-DeltaCYT subclone; lane3, B8, a full-length beta(4) subclone; lane4, D4, a full-length beta(4) subclone.



A possible mechanism for the observed changes in growth that correlate with alpha(6)beta(4) expression was suggested by the report that RKO cells express relatively low levels of wild-type p53 (21) . Moreover, the growth-suppressive function of p53 can result from its ability to induce expression of p21 (WAF1; Cip1), an inhibitor of G(1) cyclin-dependent kinases(10, 11, 12) . Based on these observations, we hypothesized that expression of the alpha(6)beta(4) integrin in RKO cells increases p21 expression. This hypothesis was assessed initially by immunostaining using a p21-specific mAb. The results obtained revealed little p21 expression in either the Neo or beta(4)-DeltaCYT subclones (Fig. 3, A and B). In these subclones, fewer than 5% of the cells exhibited p21 staining. However, nuclear staining of p21 was much more evident in both the B8 and D4 subclones (Fig. 3, C and D). The frequency of nuclear staining was greater in the D4 subclone (30-40% cells stained) than it was in the B8 subclone (15-20% cells stained), an observation that correlates with the relative level of alpha(6)beta(4) expression in these two subclones. The expression of p21 in the subclones was also assessed by immunoblotting detergent extracts prepared from the beta(4) subclones. Relatively little p21 expression was evident in either the Neo or beta(4)-DeltaCYT subclones based on these immunoblots (Fig. 3E). The level of p21 expression in these subclones is consistent with other reports of cells that express low levels of wild-type p53(12) . In contrast, a substantial increase in p21 expression was seen in both the B8 and D4 beta(4) subclones. Attachment to EHS laminin-1 did not alter this pattern of p21 expression (Fig. 3E).


Figure 3: Expression of p21 in the RKO transfectants. A-D, detection of p21 by immunohistochemistry. A mock transfectant subclone (A), a beta(4)-DeltaCYT subclone (B), and two full-length beta(4) subclones B8 and D4 (C and D) were plated on poly-L-lysine and stained with a p21-specific mAb as described under ``Materials and Methods.'' Bar in A equals 50 µM. E, immunoblot detection of p21. RKO transfectants were plated on either tissue culture plastic (lanes 1, 3, 5, 7, and 9) or EHS laminin-1 (lanes 2, 4, 6, 8, and 10) for 18 h. After detergent extraction, the samples were normalized for protein content, resolved by SDS-polyacrylamide gel electrophoresis (12%), transferred to nitrocellulose, and blotted with the p21-specific mAb. Bound protein was detected by enhanced chemiluminescence. Lanes1 and 2, Neo-24, a mock transfectant subclone; lanes3 and 4, Neo-26, a mock transfectant subclone; lanes5 and 6, 3C1, a beta(4)-DeltaCyt subclone; lanes7 and 8, B8, a full-length beta(4) subclone; lanes9 and 10, D4, a full-length beta(4) subclone.




DISCUSSION

We have shown that expression of the alpha(6)beta(4) integrin in RKO cells, a beta(4)-deficient carcinoma cell line, results in partial G(1) arrest as well as the apoptotic death of some transfectants. A possible mechanism for these observed changes in growth is provided by our finding that alpha(6)beta(4) also induces expression of the G(1) cyclin-dependent kinase inhibitor p21. The specificity of the observed induction of p21 expression is provided by the finding that the alpha(6)beta(4)-DeltaCYT integrin failed to affect p21 expression or growth even though it was expressed on the cell surface at levels comparable with the full-length integrin. In addition, we had previously observed that expression of either the E-cadherin (22) or galectin-3 cDNAs (^2)in RKO cells had no effect on their growth.

Our finding that alpha(6)beta(4) can trigger apoptosis in a population of transfectants is intriguing because our efforts to isolate transfectants with beta(4) surface expression greater than that observed in the D4 subclone, for example, were not successful. These transfectants did not survive for more than a few days after their selection by FACS. A reasonable interpretation for these data is that lower levels of alpha(6)beta(4) surface expression induce expression of p21 sufficient to induce partial G(1) arrest and some apoptosis and that higher levels of alpha(6)beta(4) expression result in more p21 expression and more widespread apoptotic death.

The beta(4)-dependent increase in growth arrest and p21 expression does not appear to depend on attachment to either a laminin-1 or laminin-5 matrix. For this reason, we suggest that the beta(4) cytoplasmic domain is linked constitutively to the p21 pathway of growth arrest and apoptosis in RKO cells. This possibility of constitutive activation of adhesion-dependent signaling pathways in transformed cells is supported, for example, by the recent report that the binding of focal adhesion kinase to SH2-containing proteins in v-src-transformed 3T3 cells is independent of integrin engagement and cell attachment(23) .

The results of this study raise the issue of the role of alpha(6)beta(4) in the regulation of normal cell growth and apoptosis. The intestinal epithelium may provide a superb model for examining such a relationship. In this epithelium, undifferentiated cells at the base of the crypts proliferate rapidly and give rise to differentiated enterocytes that migrate to the tips where they are sloughed off into the intestinal lumen. Apoptosis occurs at the tips and may provide the mechanism for cell loss in this structure(24) . Interestingly, p21 is expressed in mature enterocytes but not in undifferentiated crypt cells(25) , and there is evidence that alpha(6)beta(4) is not expressed in the undifferentiated crypt cells. (^3)For these reasons, the possibility of a functional relationship between alpha(6)beta(4) expression and p21 induction merits investigation.

Several reports have implicated cell adhesion as well as specific adhesion receptors in growth control and apoptosis(26, 27, 28, 29, 30) . The significance of our findings is that they establish a link between the cytoplasmic domain of a specific integrin subunit and the expression of a molecule known to be critical for growth suppression, apoptosis, and tumorigenesis. These findings should facilitate the elucidation of the signaling pathway(s) that results in the induction of p21 expression by cell surface receptors.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA44704. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient an American Cancer Society faculty research award. To whom correspondence should be addressed: Laboratory of Cancer Biology, Deaconess Hospital, Harvard Medical School, 50 Binney St., Boston, MA 02115. Tel.: 617-732-9874; Fax: 617-738-9188; mercurio{at}mbcrr.harvard.edu.

(^1)
The abbreviations used are: FCS, fetal calf serum; EHS, Englebreth-Holm-Swarm; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody.

(^2)
M. M. Lotz and A. M. Mercurio, unpublished observation.

(^3)
W. G. Carter, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. M. Brattain for providing the RKO cell line, Dr. T. Carey for the UM-A9 mAb, and Dr. R. Burgeson for purified laminin-5.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.