beta 1D Integrin Inhibits Cell Cycle Progression in Normal Myoblasts and Fibroblasts*

Alexey M. BelkinDagger § and S. Francesco Retta

From the Dagger  Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, the  Institute of Biology, University of Palermo, 90133 Palermo, and the Department of Biology, Genetics and Medical Chemistry, University of Torino, 10126 Torino, Italy

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Integrins are alpha beta heterodimeric transmembrane receptors involved in the regulation of cell growth and differentiation. The beta 1 integrin subunit is widely expressed in vivo and is represented by four alternatively spliced cytoplasmic domain isoforms. beta 1D is a muscle-specific variant of beta 1 integrin and a predominant beta 1 isoform in striated muscles. In the present study we showed that expression of the exogenous beta 1D integrin in C2C12 myoblasts and NIH 3T3 or REF 52 fibroblasts inhibited cell proliferation. Unlike the case of the common beta 1A isoform, adhesion of beta 1D-transfected C2C12 myoblasts specifically via the expressed integrin did not activate mitogen-activated protein kinases. The beta 1D-induced growth inhibitory signal was shown to occur late in the G1 phase of the cell cycle, before the G1-S transition. Ha-(12R)Ras, but not (Delta 22W)Raf-1 oncogene, was able to overcome completely the beta 1D-triggered cell growth arrest in NIH 3T3 fibroblasts. Since perturbation of the beta 1D amino acid sequence in beta 1A/beta 1D chimeric integrins decreased the growth inhibitory signal, the entire cytoplasmic domain of beta 1D appeared to be important for this function. However, an interleukin-2 receptor-beta 1D chimera containing the cytoplasmic domain of beta 1D still efficiently inhibited cell growth, showing that the ectodomain and the ligand-binding site in beta 1D were not required for the growth inhibitory signal. Together, our data showed a new specific function for the alternatively spliced beta 1D integrin isoform. Since the onset of beta 1D expression during myodifferentiation coincides with the timing of myoblast withdrawal from the cell cycle, the growth inhibitory properties of beta 1D demonstrated in this study might reflect the major function for this integrin in commitment of differentiating skeletal muscle cells in vivo.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Integrins are a large family of heterodimeric transmembrane cell adhesion receptors (1). They are involved in many aspects of cell behavior and are known to regulate a number of intracellular signaling pathways (2-4). One of the key integrin functions is an adhesion-mediated growth signaling (5). Integrin-mediated positive cell growth signaling cascade leads to activation of MAP1 kinases, which serves as a hallmark of cell proliferation (6-9). Integrins can synergize with growth factor receptors to relay the control of cell cycle progression in adhesion-dependent cell types (5, 10). Adhesion of different cell types to certain ECM proteins can either trigger proliferation or switch them to a differentiation program (11, 12). These opposite responses are possible due to redundant and overlapping expression of integrins and are mediated by ligation of different integrin alpha beta heterodimers on the cell surface (1). Differential coupling of the Shc adapter protein to integrin alpha  subunits causes selective activation of the MAP kinase cascade in adherent cells (13). This allows various integrin alpha beta heterodimers to transduce distinct signals from the ECM to the cell interior, including positive and negative growth signals in response to adhesion.

beta 1 integrin, the most ubiquitous beta  subunit, pairs with at least 10 different alpha  subunits to comprise receptors for a wide variety of ECM proteins. It is abundantly expressed in vivo on all proliferating as well as differentiated growth-arrested cell types, excluding red blood cells (1). beta 1 integrin is known to be involved in cell growth regulation in many cell types (14, 15). Four cytoplasmic domain variants generated by alternative splicing have been described for the beta 1 subunit (Refs. 16-21 and reviewed in Ref. 22). In most cell types beta 1 integrin is represented predominantly by the beta 1A isoform. The only noticeable exception is in differentiated striated muscles where it is displaced by the beta 1D integrin isoform (16). The other two minor beta 1 integrin variants with the alternatively spliced cytoplasmic domains, beta 1B and beta 1C, were identified several years ago (18-21). Unlike beta 1D integrin, these two isoforms are always coexpressed at low levels with the major beta 1A isoform (18, 19, 21). Recently, the beta 1C isoform has been shown to inhibit strongly cell growth upon transient expression in 10T1/2 fibroblasts (23). A short amino acid sequence Gln795-Gln802 within the cytoplasmic domain is essential for beta 1C-mediated growth arrest and is apparently unique for this integrin (24).

Striated muscle myoblasts become irreversibly withdrawn from the cell cycle early in myodifferentiation before cell fusion occurs. The timing of cell cycle withdrawal (commitment to myodifferentiation) coincides with the appearance of muscle-specific beta 1D integrin in myocyte cultures and is in accordance with its increased expression in postmitotic growth-arrested myoblasts (16). We hypothesized that beta 1D might transmit a growth inhibitory signal in normal myoblasts in vivo. Here we demonstrate an inhibition of cell proliferation by beta 1D integrin when it is expressed transiently in normal myoblasts and fibroblasts.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Antibodies and Reagents-- Thymidine analog bromodeoxyuridine (BrdUrd) and mouse anti-BrdUrd mAb were from Sigma. Mouse mAb TS2/16 against human beta 1 integrin was provided by Dr. Martin Hemler (Dana Farber Cancer Institute, Boston) and used to detect the injected or transfected beta 1A and beta 1D integrins. Hamster anti-mouse beta 1 mAb HMbeta 1-1 for visualization of the endogenous beta 1 integrin was received from PharMingen (San Diego, CA). Anti-MAP kinase rabbit polyclonal antibody sc-93 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and was used for MAP kinase immunoprecipitation. Antibody against active (dually phosphorylated) MAP kinases was from Promega (Madison, WI). Rabbit polyclonal antibodies reacting with mouse cyclins A and E were obtained from Rockland (Gilbertsville, PA). Mouse mAb 7G7B6 against interleukin-2 receptor (IL2R) extracellular domain was from ATCC (Rockville, MD). Hamster mAbs reacting with mouse alpha 1, alpha 2, and alpha 5 integrins were obtained from PharMingen. Antibody against alpha 3 integrin cytoplasmic domain was kindly provided by Dr. Guido Tarone (University of Torino, Italy). Isoform-specific anti-beta 1A and anti-beta 1D antibodies were characterized previously (16).

Cell Cultures-- Mouse C2C12 myoblasts and NIH 3T3 fibroblasts were obtained from ATCC and used between 3 and 10 passages. Ha-(12R)Ras- and (Delta 22W)Raf-transfected NIH 3T3 fibroblasts were described previously (25-28). Briefly, activated Ha-(12R)Ras and activated (Delta 22W)Raf-1 (25, 26) were cloned into the expression vector pZIP-NeoSV(×)1 and transfected into NIH 3T3 cells using the calcium phosphate precipitation technique essentially as reported previously (25, 27). 24 h after transfection G418 was added to the growth medium at 1 mg/ml. The cells were selected for 10 days by which time all the cells in a control mock-transfected dish were dead. NIH 3T3 cells stably expressing Ha-(12R)Ras or (Delta 22W)Raf-1 displayed very similar highly transformed phenotype as detected by cell morphology, inhibited spreading, colony formation in soft agar, and profound cytoskeletal changes (26, 28). Rat embryo fibroblast line REF 52 was described previously (29). beta 1A-CHO and beta 1D-CHO stable transfectants were characterized previously (16, 17).

cDNA Constructs, Microinjection, and Transfection-- The cDNAs for human beta 1A and beta 1D cytoplasmic domain isoforms in pECE vector were described previously (16, 19). cDNAs encoding beta 1A/beta 1D chimeras were prepared using the beta 1A and beta 1D cDNA fragments and polymerase chain reaction-based mutagenesis. The sequences of the four mutant beta 1A/beta 1D integrin cDNAs were verified by dideoxy termination sequencing. IL2R (interleukin 2 receptor) cDNA and IL2R-beta 1A chimera (30) were kindly provided by Dr. Susan LaFlamme (Albany Medical College, Albany, NY). IL2R-beta 1D chimera was generated using polymerase chain reaction amplification of the 3' end of the beta 1D cDNA (16) with the forward primer 5'-TGTAGCTGGTGTGGTTGCTG-3' and the reverse primer 5'-TTCAAAGCTATTCTGGGCTG-3'. The polymerase chain reaction fragment was digested with HindIII endonuclease, and the resulting fragment encoding the beta 1D cytoplasmic domain was inserted in the HindIII site of the IL2R-beta 1A plasmid (30) to generate a plasmid encoding the IL2R-beta 1D chimera. The structure of the IL2R-beta 1D construct was confirmed by dideoxynucleotide sequence analysis using a primer upstream of the cloning site (5'-AGCGTCCTCCTCCTGAGTG-3').

For nuclear microinjections the beta 1A and beta 1D integrin cDNAs were used at 0.5 mg/ml to inject C2C12 myoblasts, REF 52 fibroblasts, or NIH 3T3 cells growing on fibronectin-coated glass coverslips (23). Transfection of C2C12 myoblasts, REF 52 fibroblasts, and NIH 3T3 cells, either wild-type or expressing Ha-(12R)Ras or (Delta 22W)Raf-1, was performed using SuperfectTM Transfection Reagent (Qiagen, Santa Clarita, CA). Cells at 40-60% confluency were incubated with the transfection complexes in regular serum-containing growth medium for 3-5 h. 5 µg of DNA and 30 µl of SuperfectTM was used for 60-mm dishes and 12 µg of DNA and 75 µl of SuperfectTM for 100-mm dishes. Four independent transient transfections were performed for each set of experiments. As judged by immunofluorescence with TS2/16 mAb, 28 ± 9% C2C12 myoblasts, 19 ± 6% REF fibroblasts, and 31 ± 14% NIH 3T3 cells (either wild-type or transfected with activated Ha-Ras or Raf-1) expressed the exogenous human beta 1A or beta 1D integrins 24 h after transfection.

Integrin Analysis by Flow Cytometry-- The expression levels of the transfected human beta 1A and beta 1D integrins were determined using fluorescence-activated cell sorter analysis with TS2/16 mAb which reacts identically with these two beta 1 cytoplasmic domain variants (16, 17). The transfectants were incubated with 10 µg/ml of TS2/16 mAb for 1 h at 4 °C followed by fluorescein-labeled goat anti-mouse IgG. After the staining, cells were analyzed in a FACScan® flow cytometer (Becton Dickinson, Mountain View, CA).

Cell Proliferation Assay-- The cell proliferation assay based on BrdUrd incorporation into the nuclei was described earlier (23, 31). Cells transfected with beta 1A, beta 1D cDNAs, beta 1A/beta 1D chimeras, or IL2R-beta 1A and IL2R-beta 1D constructs were trypsinized on the day after transfection and were plated on fibronectin-coated glass coverslips in DMEM containing 10% FBS. The next day, the growth medium was replaced for DMEM with 0.5% FBS and cells were kept in low serum for 24 h. After that, cells were switched to the medium containing 10% FBS and 50 µM BrdUrd. 24 h later, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline and permeabilized with 0.5% Triton X-100. Before the immunostaining, cells were incubated first with DNase (0.1 units/µl, Promega) for 30 min at 37 °C.

To determine which phase of the cell cycle is affected by beta 1D integrin, NIH 3T3 cells on coverslips were starved in DMEM with 0.5% FBS for 2 days and then injected with beta 1A or beta 1D cDNA 0, 4, 8, 12, or 16 h after the addition of DMEM with 10% FBS. At least 250 cells were injected with each cDNA. More than 70% of the injected cells expressed the exogenous beta 1A or beta 1D proteins. In preliminary experiments, maximal protein expression was achieved 1-1.5 h after cDNA injection. 50 µM BrdUrd was added 18 h after the switch to 10% FBS, and the cells were incubated with BrdUrd for 6 h more. After BrdUrd treatment, cells were washed, fixed, and permeabilized as described above.

Immunostaining of the Transfected and Injected Cells-- To visualize simultaneously the transfected (injected) beta 1 integrins and nuclear incorporation of BrdUrd, fixed, permeabilized, and DNase-treated cells were coincubated with mAb TS2/16 against human beta 1 integrins and anti-BrdUrd mAb (1:100 dilution) for 1 h at 37 °C. Rhodamine-labeled donkey anti-mouse IgG (Chemicon, Temecula, CA) was used as secondary antibody to visualize the expressed beta 1 integrins at focal adhesions and accumulation of BrdUrd in the nuclei. Coverslips were viewed on a Zeiss Axiophot microscope equipped for epifluorescence. Micrographs were taken on T-max 400 film. 200 untransfected cells or cells expressing beta 1A, beta 1D, or beta 1A/beta 1D chimeras were counted and the nuclear staining for BrdUrd assessed.

BrdUrd-labeled C2C12 cells expressing IL2R-beta 1A and IL2R-beta 1D chimeric receptors were detected with mAb 7G7B6 against the extracellular domain of IL2 receptor (30) in combination with anti-BrdUrd mAb. 120 cells expressing IL2R construct, IL2R-beta 1A, or IL2R-beta 1D chimera were scored for the nuclear BrdUrd staining.

To detect nuclear localization of cyclins A and E in beta 1A- and beta 1D-transfected NIH 3T3 cells, they were initially treated as described above (see "Cell Proliferation Assay"). The cells were double-stained with mouse mAb TS2/16 and rabbit polyclonal antibodies against cyclins A and E, followed by a mixture of rhodamine-labeled donkey anti-mouse IgG and fluorescein-labeled goat anti-rabbit IgG (Chemicon). 120 beta 1A- and beta 1D-expressing NIH 3T3 cells were counted for both cyclin E and cyclin A expression.

In some experiments, intracellular localization of the endogenous beta 1A integrin was examined in NIH 3T3 cells expressing human beta 1D. In this case cells were incubated first with a mixture of mouse mAb TS2/16 against human beta 1 integrin, mouse mAb against BrdUrd, and hamster mAb HMbeta 1-1 specific for mouse beta 1 integrin (PharMingen). This was followed by coincubation with rhodamine-conjugated donkey anti-mouse IgG and fluorescein-labeled donkey anti-hamster IgG (Chemicon).

MAP Kinase Assays-- Activation of MAP kinases in beta 1A- and beta 1D-transfected C2C12 cells was studied as reported earlier (6, 9, 10, 16, 32, 33), with some modifications. Briefly, cells transfected with human beta 1A and beta 1D cDNAs were trypsinized on the day after transfection. The remaining trypsin was inhibited with 0.5 mg/ml soybean trypsin inhibitor, and cells were washed several times in serum-free medium. Cells in DMEM, containing 2% bovine serum albumin, were plated on bacterial Petri dishes, precoated with purified TS2/16 mAb against the transfected beta 1 integrins, for 2 h at 37 °C. Unbound cells were washed out, and the adherent cells expressing human beta 1A or beta 1D integrins were lysed either in 1% SDS or in buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM HEPES, pH 7.5, with 1 mM sodium orthovanadate, 50 mM NaF, 1 mM p-nitrophenyl phosphate, 20 nM calyculin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Protein concentration in the samples was determined using Pierce BCA Protein Assay Reagent. Samples in 1% SDS (25 µg each) were used directly for SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting with polyclonal antibody against activated (dually phosphorylated) MAP kinases (Promega). 0.5 mg of cell lysates in 1% Nonidet P-40, 0.5% sodium deoxycholate buffer were used for immunoprecipitation with sc-93 anti-MAP kinase antibody (Santa Cruz Biotechnology) followed by immune kinase reaction with [gamma -32P]ATP and the exogenous substrate myelin basic protein (MBP). Phosphorylated MBP bands were analyzed by SDS electrophoresis and autoradiography (6, 16).

Metabolic Labeling, Immunoprecipitation, and Immunoblotting-- Cultured NIH 3T3 fibroblasts (wild-type or expressing exogenous Ha-(12R)Ras) were metabolically labeled with 0.1 mCi/ml Tran35S-label (ICN Biomedicals, Irvine, CA) in methionine- and cysteine-free medium for 12 h at 37 °C. After the labeling, cells were washed 3 times with phosphate-buffered saline and lysed on ice for 3 min with RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.5; containing 0.1% SDS, 1% Triton X-100 and 0.5% sodium deoxycholate). alpha 1, alpha 2, alpha 3, alpha 5, and the endogenous mouse beta 1 integrins were immunoprecipitated using the specific antibodies against these subunits. The resulting immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis on 10% gels.

Cultured Ha-(12R)Ras-NIH 3T3 cells expressing human either beta 1A or beta 1D integrins were lysed in RIPA buffer and immunoprecipitated with the antibodies against the endogenous mouse alpha  subunits or TS2/16 mAb against the transfected human beta 1 integrins. The immunoprecipitates were extensively washed and boiled in SDS sample buffer. After SDS-polyacrylamide gel electrophoresis, beta 1A and beta 1D isoforms were detected in the immunoprecipitates by immunoblotting with the isoform-specific antibodies (16, 17).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In our preliminary experiments we were not able to obtain stable expression of beta 1D in normal myoblasts, including mouse C2C12 cells or normal fibroblasts, such as REF 52 or NIH 3T3 cell lines, suggesting that this integrin might directly affect cell growth. To examine this possibility, we first microinjected either beta 1A or beta 1D cDNAs into the nuclei of these cells. The injected cells were serum-starved, then treated for 24 h with BrdUrd in serum-containing medium, and finally stained for both human beta 1 integrin and BrdUrd incorporation (Fig. 1). No nuclear staining was seen in beta 1A- and beta 1D-injected cells with anti-beta 1 integrin TS2/16 mAb alone (not shown). Whereas the majority of untransfected and beta 1A-transfected cells displayed bright nuclear staining with anti-BrdUrd mAb (Fig. 1, A and C, arrows), most beta 1D-transfected C2C12, REF 52, and NIH 3T3 cells did not incorporate BrdUrd into the nuclei (Fig. 1, B, D, and E, arrowheads). This indicated that beta 1D caused growth arrest in these cell types. In the injected cells, beta 1D colocalized with the endogenous beta 1A integrin at focal adhesions but did not cause a displacement or relocalization of beta 1A from these sites (Fig. 1, E and F). Therefore, we concluded that the observed inhibition of cell proliferation in beta 1D-transfected cells was caused by the expression of beta 1D integrin rather than the lack of the common beta 1A isoform at cell-matrix contacts.


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Fig. 1.   Expression of beta 1D integrin in C2C12 myoblasts, REF 52, and NIH 3T3 fibroblasts inhibits proliferation. A-F, effects of exogenous beta 1A and beta 1D expression on the nuclear incorporation of BrdUrd. A and B, C2C12 cells injected with human beta 1A integrin (A) or human beta 1D integrin (B). C and D, REF 52 cells injected with human beta 1A integrin (C) or human beta 1D integrin (D). A--D, cells were co-stained with anti-human beta 1 integrin mAb TS2/16 and anti-BrdUrd mAb. E and F, beta 1D-injected NIH 3T3 cells were double-stained for the expressed beta 1D integrin and BrdUrd (E) and the endogenous mouse beta 1A integrin (F). BrdUrd-positive nuclei in beta 1A-expressing cells are marked by arrows (A and C). BrdUrd-negative nuclei in beta 1D-expressing cells are indicated by arrowheads (B, D, and E).

Transient transfection of beta 1A and beta 1D cDNAs in C2C12, REF 52, and NIH 3T3 cells resulted in similar expression levels of these integrins on the cell surface as determined by flow cytometry with TS2/16 mAb (Table I). Quantitation of the growth inhibitory effect mediated by beta 1D in the transfectants showed that in the presence of this integrin only ~20% of C2C12 myoblasts (Fig. 2A) and ~14% of REF 52 fibroblasts (Fig. 2B) were entering the S phase, whereas more than 75% of beta 1A transfectants were progressing into the cell cycle under conditions of this assay. Because the efficiency of cell growth arrest depends on the level of the exogenous beta 1D, the few proliferating beta 1D-transfected cells might represent a subpopulation of the transfectants with the lower level of beta 1D expression. These results show that proliferation of normal myoblasts and fibroblasts is drastically inhibited by beta 1D integrin.

                              
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Table I
Surface expression levels of the exogenous human beta 1A and beta 1D integrins in the transfectants


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Fig. 2.   Quantitation of beta 1D integrin-mediated inhibition of DNA synthesis in C2C12 myoblasts and REF 52 fibroblasts. The percentages of C2C12 myoblasts (A) and REF 52 fibroblasts (B) showing nuclear staining with anti-BrdUrd mAb are depicted for untransfected cells, as well as for beta 1A- and beta 1D-transfected cells.

Analysis of MAP kinase activation in beta 1A- and beta 1D-transfected C2C12 cells was first performed in the absence of exogenous growth factors in cells adherent to the substrate specifically via the expressed integrin (Fig. 3, A and D). beta 1A-expressing cells displayed high levels of adhesion-dependent MAP kinase activation measured by both immunoblotting and immune complex kinase assays (Fig. 3, A and D; a and a'). In contrast, MAP kinase activity in the adherent beta 1D transfectants did not differ from the basal levels characteristic for these cells in suspension (Fig. 3, A and D; b and b'). Unlike C2C12 cells expressing beta 1D, beta 1D-CHO transfectants exhibited sharp activation of MAP kinases in response to beta 1D-mediated adhesion under the same experimental conditions (Fig. 3, B and E and Ref. 16). Therefore, the growth inhibitory effect of beta 1D strongly depends on cellular context and might be suppressed by enhanced activity of certain oncogenes.


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Fig. 3.   beta 1D inhibits adhesion-dependent activation of MAP kinases in C2C12 myoblasts. C2C12 myoblasts (A, C, D, and F) or CHO cells (B and E) expressing exogenous human beta 1A (a and a') or beta 1D (b and b') integrins were either kept in suspension (a and b) or plated for 2 h on plastic dishes coated with TS2/16 mAb against the expressed integrins (a' and b'). The experiments were performed either in serum-free medium (A, B, D, and E) or in 10% serum-containing medium (C and F). MAP kinase activation was measured by immunoblotting with antibody against dually phosphorylated MAP kinases (A-C) or by immune complex kinase assay with MBP as the exogenous substrate (D-F).

In the presence of growth factors both beta 1A- and beta 1D-transfected C2C12 cells displayed higher levels of MAP kinase activation compared with serum-free conditions (Fig. 3, C and F). However, whereas MAP kinase activation further increased upon adhesion in beta 1A-transfected cells (Fig. 3, C and F; a and a'), no such enhancement was seen in beta 1D-expressing cells in the presence of serum (Fig. 3, C and F; b and b'). This observation showed that beta 1D down-regulates MAP kinase activity in growth factor-stimulated C2C12 cells as well. Thus, the withdrawal of differentiating myoblasts from the cell cycle in vivo could be attributed at least partly to growth inhibitory effect of beta 1D integrin.

To determine the phase of the cell cycle affected by beta 1D expression, we double-stained both populations of NIH 3T3 transfectants for the expressed human beta 1 integrin and either cyclin E or cyclin A (Fig. 4A). Scoring the beta 1A- and beta 1D-transfected cells for nuclear cyclin staining demonstrated that cyclin E expression did not significantly differ between the two populations. However, the percentage of beta 1A transfectants expressing cyclin A exceeded drastically that of beta 1D-expressing cells. Since the expression of cyclin A occurs specifically during the S phase and beta 1D blocks its appearance in the transfected NIH 3T3 cells, we concluded that beta 1D strongly interferes with the G1-S transition. To analyze further the timing of the beta 1D-triggered cell cycle block, we microinjected beta 1D cDNA at certain time points after serum stimulation of starved cells (Fig. 4B). DNA synthesis in NIH 3T3 cells became insensitive to inhibition by beta 1D integrin when its cDNA was injected ~12 h after serum stimulation. Previously, our estimations showed that the G1 phase lasts ~14-16 h in this cell type. Together, these two observations demonstrated that beta 1D-mediated growth arrest occurs late in the G1 phase before the beginning of the S phase.


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Fig. 4.   Determination of the cell cycle phase affected by beta 1D integrin. A, nuclear expression of cyclins E and A in beta 1A- and beta 1D-transfected NIH 3T3 cells. Cells were double-stained to detect the transfected beta 1 integrin and cyclins E and A. Shown are the percentages of beta 1A (dark bars) and beta 1D (hatched bars) transfectants with the nuclear expression of cyclins E and A. B, quiescent NIH 3T3 cells were switched to serum-containing medium and microinjected with human beta 1D cDNA at the indicated times thereafter. Cells were co-stained for beta 1D expression and BrdUrd incorporation. The percentages of beta 1D-NIH 3T3 cells with the nuclear BrdUrd expression are shown for each injection time point.

Previously we were able to select stable beta 1D integrin transfectants of CHO cells (16) and GD25 beta 1 integrin-deficient cells (17, 34), indicating that some cells were able to proliferate in the presence of beta 1D. Since both these cell lines display elevated activity of certain oncogenes, we rationalized that some of them can overcome the beta 1D-triggered cell cycle block. To test this suggestion directly, we transiently transfected beta 1D cDNA into NIH 3T3 cells expressing constitutively activated forms of Ha-Ras and Raf-1 oncogenes (25-28). Analysis of cell proliferation by the BrdUrd incorporation assay demonstrated that Ha-(12R)Ras completely abolished the inhibitory effect of beta 1D on cell growth (Fig. 5). However, (Delta 22W)Raf-1 was not able even to diminish this beta 1D-mediated effect. These data showed that certain oncogenes, such as Ha-Ras, overcome the beta 1D-mediated growth arrest, whereas some others, like Raf-1, cannot.


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Fig. 5.   Activated Ha-Ras, but not Raf-1 oncogene, overcomes the growth inhibitory signal triggered by beta 1D integrin expression. NIH 3T3 cells, either wild-type or stably expressing Ha-(12R)Ras or (Delta 22W)Raf-1 oncogenes, were transfected with human beta 1A (light bars) or beta 1D (dark bars) integrins. The percentages of beta 1A- and beta 1D-expressing cells incorporating BrdUrd in the nuclei were determined for each cell population.

Since integrin alpha  subunits are known to modulate cell growth through differential activation of MAP kinases (13), we asked whether activated Ha-Ras could alter the expression pattern of alpha  subunits in NIH 3T3 transfectants or their association with beta 1D integrin (Fig. 6). We did not see any detectable changes in the pattern of alpha  subunits associated with beta 1 integrin in NIH 3T3 cells transfected with Ha-(12R)Ras compared with the wild-type cells (Fig. 6, A and B). alpha 3 and alpha 5 integrins were the major beta 1-associated alpha  subunits in this cell line. The association of beta 1A and beta 1D integrins with the alpha  subunits was not altered in the corresponding transfectants (Fig. 6, C and D).


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Fig. 6.   Expression of activated Ha-Ras in NIH 3T3 cells does not alter expression of alpha  subunits or their association with the transfected beta 1A and beta 1D integrins. A and B, immunoprecipitation of 35S-labeled integrins from wild-type (A) or Ha-(12R)Ras-expressing (B) NIH 3T3 cells was performed with antibodies against mouse alpha 1 (a), alpha 2 (b), alpha 3 (c), beta 1 (d), or alpha 5 (e) subunits. C and D, immunoprecipitates from beta 1A-transfected (C) or beta 1D-transfected (D) Ha-(12R)Ras-NIH 3T3 cells with antibodies against mouse alpha 1 (a), alpha 2 (b), alpha 3 (c), alpha 5 (e), or human exogenous beta 1 (d) integrins were probed by immunoblotting with the isoform-specific antibodies against beta 1A (C) or beta 1D (D) integrins. Long and short arrows indicate the mature form and the precursor of the beta 1 integrin subunit, respectively.

We also wanted to examine which amino acid sequences in beta 1D integrin are required and sufficient for its growth inhibitory function. We therefore created beta 1 integrin chimeras by swapping beta 1A and beta 1D cytoplasmic domain sequences (Table II). All of these chimeric beta 1A/beta 1D integrins were expressed at similar levels on the cell surface and were targeted efficiently to focal adhesions of C2C12 cells, with the exception of chimera 4. None of the C2C12 transfectants expressing these chimeric integrins displayed abnormal adhesion or spreading (not shown). Interestingly, when examined by cell proliferation assay, none of the chimeras was as efficient as beta 1D with regard to growth inhibition (Fig. 7A). The only chimera that still caused a significant decrease of C2C12 cell proliferation was chimera 2, which lacked only 6 amino acids at the very C terminus of the beta 1D polypeptide (Fig. 7A, Table II). These data strongly suggest that there is no short amino acid motif within the beta 1D cytoplasmic tail, which is sufficient for this function. Instead, the whole C-terminal half of the cytodomain, encoded by exon D of the beta 1 integrin gene (16), appears to be essential for cell growth arrest.

                              
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Table II
Structure, surface expression levels, and focal adhesion localization of beta 1A/beta 1D integrin chimeras in C2C12 myoblasts


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Fig. 7.   Analysis of the amino acid sequences required for the growth inhibitory effect of beta 1D. A, effects of swapping the cytoplasmic domain sequences on the beta 1D integrin-mediated growth arrest. beta 1A (light bar), beta 1D (dark bar), and beta 1A/beta 1D chimeric integrins 1-4 (hatched bars; listed in Table II) were transfected in C2C12 myoblasts, and the percentages of proliferating cells in each population were determined by BrdUrd nuclear staining. B, the cytoplasmic domain of beta 1D integrin is sufficient for the growth inhibitory signal. IL2R as well as IL2R-beta 1A and IL2R-beta 1D chimeric receptors were transiently expressed in C2C12 cells. Their effects on cell cycle progression was determined using the BrdUrd incorporation assay. Shown are the percentages of cells re-entering the cell cycle in all three cell populations.

By trying to determine whether the extracellular domain and ligand binding have a role in the growth inhibitory function of beta 1D integrin, we took advantage of earlier engineered chimeric receptors, such as the IL2R-beta 1A chimera (30). This chimera contains the extracellular and transmembrane domains of interleukin 2 receptor and the cytoplasmic domain of beta 1A integrin. By replacing the beta 1A portion of this construct with the cDNA fragment encoding the beta 1D cytoplasmic domain (see "Materials and Methods"), we generated IL2R-beta 1D chimeric receptor. Both IL2R-beta 1D and IL2R-beta 1A chimeras were similarly expressed on the cell surface (Table III) and were targeted to focal adhesions upon transient expression in C2C12 cells (data not shown). The expression of these two chimeras as well as the wild-type IL2R in C2C12 myoblasts was followed by the analysis of cell proliferation using BrdUrd incorporation method (Fig. 7B). Our results showed that IL2R-beta 1A chimera only slightly decreased proliferation, most likely due to partial disruption of focal adhesions in some of the transfectants expressing high levels of this construct. In contrast, IL2R-beta 1D chimeric receptor strongly inhibited cell cycle progression in C2C12 myoblasts (Fig. 7B).

                              
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Table III
Surface expression levels of IL2R-beta 1A and IL2R-beta 1D chimeric receptors in C2C12 myoblasts

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this work we demonstrated that beta 1D, the muscle-specific variant of the beta 1 integrin subunit, inhibits cell cycle progression in normal myoblasts and fibroblasts. Our data reveal a novel function for the beta 1D cytoplasmic domain isoform, which is the predominant beta 1 integrin in striated muscles (16). This growth inhibitory function is specifically ascribable to the alternatively spliced cytoplasmic tail of beta 1D, since the entire cytodomain appears to be both necessary and sufficient for the beta 1D-mediated growth arrest.

beta 1D exhibits a dominant-positive effect over the beta 1A isoform due to enhanced interactions with both cytoskeletal and extracellular ligands and displaces the endogenous beta 1A from cell-matrix contacts when expressed in certain cell types (17). However, in C2C12, REF 52 or NIH 3T3 cells expressing high levels of beta 1D integrin, adhesion, and spreading on various ECM proteins was not visibly affected. Also, our recent data on transiently transfected beta 1D-C2C12 and beta 1D-NIH 3T3 cells indicated that even the cells expressing low amounts of beta 1D and still having the endogenous beta 1A at focal adhesions (Fig. 1, E and F) were unable to proliferate. These observations suggested that the beta 1D-triggered cell cycle block is not caused by the lack of beta 1A at cell-matrix adhesions and consequent loss of beta 1A-mediated signaling but is primarily due to active signaling by beta 1D. Meanwhile, further analysis is needed to elucidate whether this effect is based on the active inhibitory signal by beta 1D or blocking positive adhesion-mediated signaling caused by this integrin variant.

Recently, another beta 1 integrin isoform, beta 1C, was shown to generate a strong growth inhibitory signal upon its transient expression in 10T1/2 fibroblasts (23) or CHO cells (24). Expression of this integrin in vivo is detectable in some blood cells (21) and is up-regulated in tumor necrosis factor-alpha -stimulated endothelial cells (24). No information is available yet concerning the mechanisms of growth arrest mediated by beta 1D and beta 1C isoforms. In both these cases, the cytoplasmic domains alone are sufficient to generate the inhibitory signal, whereas the transmembrane and extracellular domains, and therefore ligand binding, are not required. However, in the case of beta 1C integrin, a short specific motif Gln795-Gln802 is responsible for this function (24), whereas the entire cytodomain appears to be important for the growth arrest triggered by beta 1D. Another distinction between the mechanisms of growth inhibition by these integrins relates to the fact that beta 1C displays its antiproliferative potential in all the cell types tested so far (23, 24), although beta 1D growth inhibitory effect is clearly cell type-specific.

Certain oncogenes, as shown here for Ha-(12R)Ras, are able to completely suppress the beta 1D-mediated growth arrest. This explains why we previously were able to obtain stable expression of beta 1D integrin in some cell types that had elevated activity of some oncogenes (CHO and GD25 beta 1-minus cells) but failed to select stable transfectants with other cells displaying a more normal phenotype. This property of beta 1D is also consistent with the fact that some transformed cells of muscle origin, e.g. human rhabdomyosarcoma (RD) cells, proliferate even though expressing significant amounts of beta 1D integrin.2 Yet, others have shown that expression of c-myc and v-myc oncogenes in C2C12 myocytes does not significantly alter either their differentiation patterns or commitment (irreversible withdrawal from the cell cycle), (35). Taken together, these observations show that the growth inhibitory effect of beta 1D can be suppressed by some but not all oncogenes.

At present it remains unclear why activated Ha-Ras is able to overcome the growth inhibitory signal from beta 1D whereas activated Raf-1 can not. Most existing models of anchorage-dependent cell growth imply that Ras is positioned upstream from Raf-1 in the integrin-mediated MAP kinase cascade. Also, activated Raf-1 and Ha-Ras have similar transforming activity in NIH 3T3 fibroblasts as judged by cell morphology, growth in soft agar and cytoskeletal changes (26, 28). However, one recent report showed that integrin-mediated activation of MAP kinases, MEK (MAP kinase kinase) and Raf-1 in NIH 3T3 cells appeared to be independent of Ras (33). Also, activation of the Raf-1/MAP kinase cascade was shown to be insufficient for Ras transformation of RIE-1 epithelial cells (26). These recent observations imply that oncogenic Ras triggers certain Raf-independent signals essential for cellular transformation and also reevaluate the roles of Ras and Raf in propagation of adhesion-mediated growth signals from integrins to MAP kinases.

Previous analysis of beta 1D expression in mouse myogenic cultures (16) and during mouse embryogenesis3 showed that the onset of its biosynthesis coincides with the time point of irreversible growth arrest during myocyte differentiation. This indicates that the growth inhibitory properties of beta 1D, demonstrated by transient transfection assays in this study, might be related to the major function of this muscle-specific integrin in vivo. Further work is apparently needed to elucidate the role of specific signaling events in the cell growth arrest mediated by beta 1D integrin.

    ACKNOWLEDGEMENTS

We thank Dr. Keith Burridge (University of North Carolina, Chapel Hill), in whose lab a part of this work was performed, for support and encouragement. We are grateful to Drs. Guido Tarone and Fiorella Balzac (University of Torino, Italy) for providing us with cDNAs encoding beta 1A/beta 1D integrin chimeras. Dr. Susan LaFlamme (Albany Medical College, Albany, NY) kindly supplied IL2R and IL2R-beta 1A constructs used in this study. We are indebted to Drs. Geoffrey Clark and Channing Der (University of North Carolina, Chapel Hill, NC) for providing us with NIH 3T3 cells expressing Ha-(12R)Ras and (Delta 22W)Raf-1 oncogenes. We also thank Dr. Kenneth Ingham (Biochemistry Department, American Red Cross) for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health R29 Grant CA77697 (to A. M. B.) and National Institutes of Health Grant GM29860 (to Dr. Keith Burridge).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0725; Fax: 301-738-0794; E-mail: belkina{at}usa.redcross.org.

1 The abbreviations used are: MAP, mitogen-activated protein; ECM, extracellular matrix; BrdUrd, bromodeoxyuridine; MBP, myelin basic protein; mAb, monoclonal antibody; IL2R, interleukin-2 receptor; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; FBS, fetal bovine serum.

3 M. Brancaccio, S. Cabodi, A. M. Belkin, G. Collo, V. E. Koteliansky, D. Tomatis, F. Altruda, L. Silengo, and G. Tarone, manuscript in preparation.

2 A. M. Belkin, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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