©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Novel Structural Motif GlnGln in the Integrin Cytoplasmic Domain Regulates Cell Proliferation (*)

(Received for publication, July 5, 1995; and in revised form, August 8, 1995)

Mara Fornaro Duo-Qi Zheng Lucia R. Languino (§)

From the Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Alternative splicing of the integrin beta(1) subunit mRNA generates a variant form, beta, with a unique cytoplasmic domain that differs from beta(1) for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)^1 cells transiently transfected with cDNAs coding for human integrin beta or beta(1) subunits or mutants containing truncated forms of the beta cytoplasmic domain. A differential effect of beta and beta(1) on cell proliferation was observed. Expression of wild type beta(1) was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [^3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of beta. Cells expressing the beta mutant truncated at Leu and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta deletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln-Gln of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [^3H]thymidine uptake. The effect of beta expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta, full-length or deletion mutants, or beta(1) equally adhered to a functionally inhibitory monoclonal antibody against human beta(1) integrin. Expression of beta inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta was induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.


INTRODUCTION

The interactions of cells with extracellular matrix are predominantly regulated by the integrin family of cell surface receptors(1, 2, 3) .

Integrin functions, such as affinity for the ligand, subcellular localization, and signaling events, are regulated by the short cytoplasmic domain of both alpha and beta subunits(4, 5) . The cytoplasmic domain of beta(1) modulates migration(6, 7) , integrin localization(8, 9) , and focal adhesion kinase activation (6, 10, 11, 12) . Recent studies have also suggested that the beta(1) subfamily and its cytoplasmic domain participate in the control of cell differentiation (13) and proliferation. In the latter case, it has been shown that, first, beta(1)-ligation prevents apoptosis(14, 15) ; second, beta(1) integrins regulate cell growth and survival in normal human breast epithelium (16) and in other cells (17) ; and finally, domain-swapping of the integrin beta(1) subunit cytoplasmic domain differentially regulates the effect of fibronectin and of an integrin-activating antibody on cell proliferation(18) .

We have previously described beta (formerly beta), an alternatively spliced form of the ubiquitous integrin beta(1) (also designated beta) subunit which contains a unique 48-amino acid sequence in its cytoplasmic domain (19) and has no homology with beta cytoplasmic domain(20) . Based on the hypothesis that variant sequences in the beta(1) cytoplasmic domain might differentially modulate cell proliferation, we have investigated the ability of the integrin beta subunit and of beta deletion mutants, truncated in the cytoplasmic domain, to modulate cell growth.

We show here that a unique sequence Gln-Gln in the beta cytoplasmic domain negatively regulates cell growth and that beta expression correlates with a growth-arrested phenotype in cytokine-stimulated, vascular endothelial cells.


MATERIALS AND METHODS

Antibodies

The beta-specific rabbit sera were generated against keyhole limpet hemocyanin (Calbiochem)-coupled synthetic peptides (805-825, kkSCLSLPSTWDYRVKILFIRVP; 785-808, kkGVQWCDISSLQPLTSRFQQFSCLS) from the deduced sequence of beta(19, 21) . The anti-beta sera reacted specifically with the peptide used as antigen and with a recombinant bacterial protein containing the unique beta cytoplasmic sequence (not shown), but they did not recognize the 765-798 peptide or the recombinant protein generated from the deduced sequence of the wild type beta(1) cytoplasmic domain. The rabbit antiserum against the cytoplasmic domain of the human beta(1) subunit (22) was a generous gift of Dr. E. Ruoslahti. The following monoclonal antibodies were used: 13 (Becton Dickinson, San Jose, CA), P4C10 (Life Technologies, Inc.) and DF5 (Chemicon Inc., Temecula, CA) anti-human beta(1) integrin; 7E2, anti-hamster beta(1) integrin (a kind gift of Dr. R. L. Juliano) and 1C10, anti-human endothelial cells (Life Technologies, Inc.).

Cells

The human umbilical vein endothelial cells (HUVEC) (Clonetics, San Diego, CA), were used at early passages, cultured, and stimulated with tumor necrosis factor alpha (TNF alpha, 100 units/ml; Genzyme Corp., Cambridge, MA) for 48 h as described(23) . The Chinese hamster ovary cell line was obtained from ATCC (Rockville, MD) and maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS).

cDNA Constructs

The human beta(1) cDNA was cloned into the pBJ-1 mammalian expression vector(24, 25) , and was provided by Dr. Y. Takada. The cDNA encoding the beta variant subunit (19) (provided by Dr. Y. Takada) was generated by subcloning the following cDNAs into XbaI/NotI sites in pBJ-1: the XbaI/HindIII cDNA fragment (2.3 kilobase pairs) that encodes the beta(1) extracellular and transmembrane (to nt 2357 of the beta(1) sequence) domains (26) and the HindIII (nt 2358)/EagI fragment (255 base pairs) isolated from PCR-1000 vector (Invitrogen, La Jolla, CA) (19) that encodes both the cytoplasmic region shared by beta(1) and beta and the beta-specific sequence (116 base pairs). To generate beta deletion mutants, site directed mutagenesis of the full-length human beta cDNA in pAlter-1 was carried out using the Altered Sites II system (Promega, Madison, WI). The following synthetic oligonucleotides were used as primers (the replaced nucleotide is underlined) to generate truncated forms of the beta cytoplasmic domain: beta Delta794 (nt 2476-2499) 5`-CAGCTCACTGCAACCTCTGACTTC-3`; beta Delta802 (nt 2501-2520) 5`-AGATTCCAGCAATTCTCCTG-3`; beta Delta812 (nt 2531-2554) 5`-CCGAGTACCTGGGATTACAGGGTG-3`; beta Delta817 (nt 2546-2571) 5`-TACAGGGTGAAAATCCTATTTATAAG-3`. These resulting deletion mutants lack, respectively, the last COOH-terminal 31, 23, 13, and 8 amino acids in the beta cytoplasmic domain (Table 1). The truncated forms of beta cDNA were sequenced by the dideoxy chain termination reaction using Sequenase 2.0 (U. S. Biochemical Corp.) and subcloned in the mammalian expression vector pBJ-1 for CHO cell transfection(27) . The deletion mutants beta Delta794 and Delta812 have been generated by Dr. Y. Takada.



In Vitro Translation and Immunoprecipitation

A cDNA encoding human beta was subcloned in Bluescript II SK+ (Stratagene, La Jolla, CA), in vitro transcribed and translated in the presence of [S]methionine (1,000 Ci/mmol; DuPont NEN) using the TNT(TM) coupled reticulocyte lysate system (Promega). Luciferase cDNA, provided by the manufacturer, was used as a control. The in vitro translated products were immunoprecipitated as described (22, 28) prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). HUVEC were surface-iodinated and immunoprecipitated as described(22) .

Cell Transfection

The following strategy was used to select cell transfectants to be used in proliferation assays. CHO cells were first transiently transfected by electroporation(29) , allowed to adhere to tissue culture dishes and after 20 h, serum-starved in DMEM for 48 h. Then, the transfected cells were analyzed by FACS (28) using P4C10 or DF5 (not shown) antibody to human beta(1) integrin, or 1C10, a control antibody (not shown). Positive cells expressing comparable levels of the full-length beta or beta(1) or beta truncated forms were sorted using FACStar or FACS IV (Becton Dickinson). After sorting, the transfectants (6-20 times 10^3) were resuspended in serum-free medium (30) , and added to 96-well microtiter plates (Flow Laboratories Inc., McLean, VA) coated with 10 µg/ml antibody 13 against the human beta(1) integrin to insure that only cells expressing the exogenous integrin subunit adhered. After 3 h and 30 min at 37 °C, the attached CHO cells were used for proliferation assays. In parallel, in each experiment, the number of attached cells was counted using a phase-contrast microscope after fixing and staining the cells with 0.5% Crystal Violet (Sigma)(28) .

Cell Proliferation Assay

Transfected cells, attached to plates coated with the antibody 13 (as described above), were either incubated in serum-free DMEM or stimulated using 10% FCS for 18 h at 37 °C and then pulsed with 1 µCi of [^3H]thymidine/well (5.0 Ci/mmol; Amersham Life Sciences) during the last 3 h of the 18-h culture. The cells were washed and solubilized using 10% SDS in order to quantitate thymidine incorporation by liquid scintillation counting. Background trichloroacetic acid-soluble [^3H]thymidine, measured in ice-cold 5% trichloroacetic acid, was approximately 9.0% and was not subtracted from the values shown in this study. In each experiment, duplicate or triplicate observations were performed and the values are reported as mean ± standard error (S.E.). In the proliferation assay, the background was evaluated using mock-transfected cells. The number of mock-transfected cells that adhered to the antibody 13 was negligible and did not result in a detectable increase in [^3H]thymidine uptake (371 ± 109 cpm, in the absence of FCS and 512 ± 24 cpm, in the presence of 10% FCS). Group differences were compared using one-way analysis of variance, followed by Bonferroni post hoc contrast.


RESULTS AND DISCUSSION

Authenticity of the beta cDNA construct was first established by in vitro translation and immunoprecipitation. The translated polypeptide, which showed the expected molecular mass of 100 kDa, was specifically immunoprecipitated by an antiserum against the 785-808 beta cytoplasmic domain peptide or by P4C10, a monoclonal antibody that recognizes the extracellular portion of human beta(1) integrin (Fig. 1A), but not by an antiserum directed to the wild type beta cytoplasmic domain (Fig. 1A). While genetically engineered cell lines stably expressing the beta integrin could not be obtained, transient surface expression of beta was detected in CHO cells (Fig. 1B and not shown). In contrast, both transient (Fig. 1B) and stable (data not shown; (25) ) transfectants expressing the wild type beta(1) were obtained. Relatively low levels of beta and wild type beta(1) surface expression (Fig. 1B) were reproducibly observed under these experimental conditions. The inability of beta to be stably expressed in transfected cells prompted additional investigations on the possible regulatory role of this integrin variant in growth inhibition mechanisms. For these studies, CHO cells were transiently transfected with beta(1), beta, or beta deletion mutants lacking the COOH-terminal 31, 23, 13, or 8 amino acids (Table 1). Selection based on flow cytofluorometric sorting with P4C10 antibody against the human beta(1) extracellular domain allowed us to obtain transfected cells expressing comparable levels of all the integrin variants (Fig. 2, A and B). To further select cells expressing the exogenous integrins, the sorted CHO cells were panned on plates coated with 13, a functionally inhibitory monoclonal antibody to human beta(1)(10, 25) . The attached cells were tested in proliferation assays to compare the beta(1), beta, or beta deletion mutant functional activities on cell proliferation. Expression of beta in CHO cells caused a marked decrease of cell proliferation as measured by [^3H]thymidine uptake and as compared with the levels of proliferation of wild type beta(1) transfectants (Fig. 3A). FACS analysis performed using monoclonal antibody 7E2 (against hamster beta(1)) revealed comparable levels of expression of endogenous hamster beta(1) integrin in the beta and beta(1) transfectants and in mock-transfected CHO cells (data not shown), thus suggesting that the observed effect of beta on cell proliferation was not due to changes in the endogenous beta(1) integrin expression pattern. The increase of proliferation of the beta(1) transfectants varied between 6- and 10-fold (Fig. 3, A and C), and the results were not statistically different from the values of [^3H]thymidine uptake measured using mock-transfected CHO cells (1,248 ± 207 cpm, in the absence of FCS and 6,463 ± 245 cpm, in the presence of 10% FCS). In agreement with our observations, stable expression of beta(1) did not affect the increase of CHO cell proliferation triggered by 10% FCS as compared with mock-transfected cells(18) . Expression of beta Delta802, beta Delta812, and beta Delta817 mutants were equally effective in inhibiting cell proliferation (Fig. 3, A and B). In contrast, CHO cells transfected with beta Delta794 mutant proliferated in response to serum as efficiently as wild type beta(1) transfectants (12-fold increase in [^3H]thymidine uptake as compared with unstimulated cells; Fig. 3, C and D). The beta Delta802, beta Delta812, and the beta Delta817 transfectants responded to serum stimulation, with only a 2-fold proliferation increase (Fig. 3D).


Figure 1: betain vitro translation and transient expression. A, human beta cDNA (lanes 1-5) or control luciferase cDNA (lanes 6 and 7) were in vitro transcribed and translated. Immunoprecipitation was performed using rabbit antisera raised against beta 785-808 peptide (a-beta; lanes 1 and 6), against beta 765-798 peptide (a-beta; lane 2), preimmune serum (lane 3), or monoclonal antibodies P4C10 (a-beta(1) mAb; lanes 4 and 7) or 1C10 (Control mAb; lane 5). The immunoprecipitates were separated on 7.5% SDS-PAGE in the presence of 2-mercaptoethanol and visualized by autoradiography. Prestained marker proteins in kilodaltons are shown. The arrow indicates the expected molecular mass of the beta translated product. B, CHO cells were transiently transfected with beta or beta(1) cDNAs or pBJ-1 and analyzed by FACS using P4C10, monoclonal antibody to human beta(1), followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The fluorescence intensity is expressed in arbitrary units. Dashed line, pBJ-1; thick line, beta; thin line, beta(1).




Figure 2: CHO cell surface expression of beta, beta(1), and beta deletion mutants. CHO cells were transiently transfected with beta, beta(1), beta Delta794, beta Delta802, beta Delta812, or beta Delta817 cDNAs. After transfection, the cells were starved for 48 h, stained using P4C10 monoclonal antibody to human beta(1), followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG, and sorted by FACS to isolate the cells expressing comparable surface levels of beta, beta(1), beta Delta794, beta Delta802, beta Delta812, or beta Delta817. A and B show the profiles of the sorted transfectants from two separate experiments.




Figure 3: DNA synthesis of CHO cell transfectants expressing beta, beta(1), and beta deletion mutants. CHO cell transfectants, expressing the full-length beta or beta(1) or the truncated beta forms, were isolated by flow cytometric sorting as in Fig. 2, and then 9,000 (panel A), 12,000 (panel B), or 6,000 (panel C) cells/well were added, in the absence of FCS, to 96-well plates coated with antibody 13 (10 µg/ml). Attached cells were incubated for 15 h at 37 °C in the absence or in the presence of 10% FCS and then for 3 h at 37 °C with 1 µCi/100 µl/well [^3H]thymidine to measure their proliferative response to FCS. A and B show the mean of duplicate observations ± S.E. values of thymidine incorporation in the absence (1, 3, and 5) or in the presence of 10% FCS (2, 4, and 6). C, data are expressed as percent of proliferation of the beta(1) transfectants. Values indicate [^3H]thymidine uptake measurements in the absence (1 and 3) or in the presence of 10% FCS (2 and 4). Results are mean ± S.E. values of triplicate determinations. D, data from two to three different experiments are expressed as -fold increase of proliferation, where the proliferation of serum-deprived cells was normalized to 1. Data are mean ± S.E. Group differences were compared using one-way analysis of variance followed by Bonferroni post hoc contrast. The difference in proliferation between beta Delta794-CHO cells and either beta Delta802-CHO, beta Delta812-CHO cells, or beta Delta817-CHO cells is statistically significant (p = 0.003; p = 0.004; p = 0.005, respectively). No significant differences were found comparing [^3H]thymidine uptake of beta Delta802-CHO with beta Delta812-CHO or with beta Delta817-CHO cells.



Despite the profound effect on cell proliferation mediated by the various integrin variants, cell adhesion of beta-CHO transfectants to the functionally inhibitory antibody 13 did not differ from that of the wild type beta(1)-CHO transfectants. In experiments parallel to those shown in Fig. 3A, the transfectants expressing wild type beta(1), beta, or beta deletion constructs were analyzed for their adhesive properties to antibody 13. The number of wild type beta(1)-CHO transfectants, attached to antibody 13-coated plates (3,200 ± 712 cells/well), did not differ from that observed using beta-CHO (3,920 ± 424 cells/well) or beta Delta802-CHO transfectants (3,200 ± 480 cells/well). In Fig. 3C, the attached cells were 3,100 ± 172 cells/well for beta(1)-CHO and 4,240 ± 320 cells/well for beta Delta794-CHO transfectants. Similarly, no differences were observed comparing the remaining mutants (not shown). These findings suggest that expression of the variant beta cytoplasmic domain does not affect ligand recognition and/or adhesive properties mediated by the intact heterodimeric receptor. It is unlikely that the association of beta with various alpha subunits plays a role in the observed effect since previous studies have demonstrated that cytoplasmic domains do not regulate the alphabeta pairing specificities, which appears to be controlled by extracellular determinants(31) .

These results demonstrate that expression of the integrin beta subunit exerts a negative regulatory function on CHO cell proliferation, and that this effect appears to be restricted to residues 795-802 in the variant cytoplasmic domain. Although we might speculate that the Gln-Gln region may play a direct role in the interaction with as yet unidentified intracellular ligand(s), other mechanisms, including secondary structure and conformational remodeling, cannot be presently excluded.

We have here described for the first time a role for the sequence Gln-Gln in mediating beta effect on cell proliferation. Recent studies have highlighted a role for beta in negatively regulating cell cycle progression in late G(1), when beta cDNA was microinjected in model 10T1/2 fibroblasts(32) . In that study, using a different cell type and experimental system, a role for only the extreme 13 carboxyl-terminal amino acids was shown in the inhibitory effect of cell cycle progression. This may be a consequence of the normal (33) as opposed to the tumorigenic CHO (34) cell phenotypes used in the two studies. Furthermore, in the present study, direct ligand engagement of beta(1) integrin by the functionally blocking antibody 13 in the absence of serum and/or matrix deposited by the cells might have unmasked a highly selective role for the Gln-Gln sequence in regulating cell growth.

In order to explore a potential pathophysiological role for beta in regulation of cell growth, endothelial cells were stimulated with inflammatory cytokines to induce a growth-arrested phenotype(35) . Consistent with previous observations demonstrating absence of beta transcript in growing endothelium(19) , an antiserum specific for beta did not immunoprecipitate any labeled polypeptide from surface-iodinated HUVEC (Fig. 4). In contrast, immunoprecipitation studies from TNF alpha-stimulated HUVEC revealed detectable surface levels of beta integrin (Fig. 4). In parallel experiments, immunohistochemical staining with the antiserum to beta characterized in Fig. 1, demonstrated prominent expression in severely injured and growth-impaired liver tissue.^2 Combined with the data presented above, we speculate that during inflammatory responses associated with prominent cytokine release, preferential expression of beta by the injured cells may contribute to a low or non-proliferative phenotype that may impair tissue repair and compromise its regenerative response(s).


Figure 4: TNF alpha-stimulated endothelial cells express beta. HUVEC were incubated for 48 h at 37 °C in the presence of TNF alpha (lanes 1, 3, and 5) or in its absence (lanes 2 and 4) and then surface-iodinated and immunoprecipitated using antibodies to beta (a-beta, lanes 1 and 2); beta (a-beta, lanes 3 and 4), and normal rabbit serum (Control serum; lane 5). The immunoprecipitates were separated using 7.5% SDS-PAGE in the presence of 2-mercaptoethanol and visualized by autoradiography. Prestained marker proteins in kilodaltons are shown.




FOOTNOTES

*
This work was supported in part by American Cancer Society Grant IRG-IN 31-36 (to L. R. L.). 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.

§
To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, P. O. Box 208023, 310 Cedar St., New Haven, CT 06520. Tel.: 203-737-1454; languino@biomed.med.yale.edu.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; HUVEC, human umbilical vein endothelial cell(s); DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; TNF, tumor necrosis factor; nt, nucleotide(s); FACS, fluorescence-activated cell sorting; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Fornaro, G. Tallini, and L. R. Languino, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. J. A. Madri, E. Ruoslahti, and D. Wright for continuous critical discussion; the Protein and DNA facilities of the R. W. Johnson Pharmaceutical Research Institute for technical assistance, Dr. P. L. Perrotta for assisting with statistical analysis, Rocco Carbone for support in performing flow cytometric analysis, and L. Iodice for help with the preparation of the manuscript.


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