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Originally published In Press as doi:10.1074/jbc.M307857200 on October 29, 2003

J. Biol. Chem., Vol. 279, Issue 3, 1692-1702, January 16, 2004
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Regulation of {beta}1C and {beta}1A Integrin Expression in Prostate Carcinoma Cells*

Loredana Moro{ddagger}§, Elda Perlino{ddagger}, Ersilia Marra{ddagger}, Lucia R. Languino¶, and Margherita Greco{ddagger}

From the {ddagger}Institute of Biomembranes and Bioenergetics, National Research Council (C.N.R.), 70126 Bari, Italy and the Department of Cancer Biology and Cancer Center, University of Massachusetts, School of Medicine, Worcester, Massachusetts 01605

Received for publication, July 21, 2003 , and in revised form, October 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}1C and {beta}1A integrins are two splice variants of the human {beta}1 integrin subfamily that act as an inhibitor and a stimulator of cell proliferation, respectively. In neoplastic prostate epithelium, both these variants are down-regulated at the mRNA level, but only {beta}1C protein levels are reduced. We used an experimental model consisting of PNT1A, a normal immortalized prostate cell line, and LNCaP and PC-3, two prostate carcinoma cell lines, to investigate both the transcription/post-transcription and translation/post-translation processes of {beta}1C and {beta}1A. Transcriptional regulation played the key role for the reduction in {beta}1C and {beta}1A mRNA expression in cancer cells, as {beta}1C and {beta}1A mRNA half-lives were comparable in normal and cancer cells. {beta}1C translation rate decreased in cancer cells in agreement with the decrease in mRNA levels, whereas {beta}1A translation rate increased more than 2-fold, despite the reduction in mRNA levels. Both {beta}1C and {beta}1A proteins were degraded more rapidly in cancer than in normal cells, and pulse-chase experiments showed that intermediates and/or rates of {beta}1C and {beta}1A protein maturation differ in cancer versus normal cells. Inhibition of either calpain- or lysosomal-mediated proteolysis increased both {beta}1C and {beta}1A protein levels, the former in normal but not in cancer cells and the latter in both cell types, albeit at a higher extent in cancer than in normal cells. Interestingly, inhibition of the ubiquitin proteolytic pathway increased expression of ubiquitinated {beta}1C protein without affecting {beta}1A protein levels in cancer cells. These results show that transcriptional, translational, and post-translational processes, the last involving the ubiquitin proteolytic pathway, contribute to the selective loss of {beta}1C integrin, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The integrin family of transmembrane receptors consists of heterodimeric glycoproteins composed of an {alpha}- and a {beta}-subunit that, in addition to mediating cell adhesion to extracellular matrix proteins, play a pivotal role in regulating several cell functions, including proliferation, differentiation, migration, and intracellular signaling (1–5).

It is well established that the occurrence of alternatively spliced variants of both the {alpha}- and the {beta}-subunit contributes to the variety of biological functions of the integrin receptors (6, 7). To date five different cytoplasmic variants have been identified for the human {beta}1 subunit, namely {beta}1A, {beta}1B, {beta}1C, {beta}1C-2, {beta}1D, and they have been shown to differentially affect receptor localization, cell proliferation, adhesion and migration, interactions with intracellular proteins, as well as phosphorylation and activation of signaling proteins (6, 8–13).

The {beta}1C integrin is an alternatively spliced variant of the {beta}1 integrin subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain (14). This splice variant, at variance with its wild-type counterpart, i.e. the {beta}1A integrin, has been shown to inhibit cell proliferation in prostate cancer epithelial cells (12, 15), endothelial cells (12), fibroblasts (12, 16, 17), and CHO cells (13). In vivo, {beta}1C is mostly expressed in non-proliferative and differentiated epithelium (6, 18) whereas {beta}1A is expressed ubiquitously. At variance with {beta}1A, the {beta}1C protein is down-regulated in prostate adenocarcinoma (15, 18, 19), in some non-small-cell lung carcinomas (20), and inversely correlates with markers of cell proliferation in breast carcinoma (21). In neoplastic prostate specimens, it was shown that both {beta}1C and {beta}1A mRNA expression is down-regulated (15, 18, 19), whereas only {beta}1C protein levels are reduced or even lost (15, 18, 19). More recently, we have shown that a reduction in the transcriptional activity of the {beta}1 integrin gene plays a role in the mechanism of down-regulation of {beta}1C and {beta}1A mRNA levels in prostate adenocarcinoma tissue (22). Despite these preliminary findings, the molecular events that regulate the expression of {beta}1 integrin variants in different pathophysiological conditions remain obscure.

Studies concerning the regulation of {beta}1 integrin expression have been previously reported in which probes and antibodies used did not discriminate among different cytoplasmic splice variants. In this regard, {beta}1 integrin expression was reported to be regulated at the level of transcription by cell attachment to the extracellular matrix (23), by transforming growth factor (TGF)-{beta}1 treatment (24), and during differentiation (25) and cancer progression (26). On the other hand, a number of reports showed that expression of {beta}1 integrins is regulated both at the transcriptional and post-transcriptional/translational level (23, 27, 28). Moreover, post-translational mechanisms have been shown to play a role in regulating {beta}1 integrin expression levels: loss or reduction of {beta}1 integrins from the cell surface was associated with impairments in their glycosylation (i.e. maturation) process (25, 29, 30). Changes in protein stability have been involved in the regulation of integrin protein expression during carcinogenesis (31), but, to our knowledge, no report up to now has investigated the occurrence of changes in the rate of degradation of the {beta}1 integrins in different pathophysiological conditions.

In light of the above findings, we investigated the regulation of the expression of {beta}1C and {beta}1A integrins at the transcriptional and post-transcriptional level, and as protein turnover and glycosylation in human prostate carcinoma cells. We show the following results. (i) A reduction in the transcriptional activity of the {beta}1 integrin gene can account for the down-regulation of {beta}1C and {beta}1A integrin mRNA levels in prostate cancer cells. (ii) Loss of {beta}1C protein in cancer cells depends not only on reduced transcription but also on reduced translation and increased protein degradation. (iii) Notwithstanding the reduction in the mRNA levels, the translation rate of the {beta}1A protein increases in cancer cells. (iv) The processing rates of {beta}1C and {beta}1A proteins are different in cancer versus normal cells. (v) The {beta}1C protein is a preferential target of the ubiquitin-proteasome proteolytic pathway in cancer cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—PNT1A cells, a human prostate normal cell line established by immortalization of normal adult prostate epithelial cells, LNCaP cells (clone FGC), a human prostate carcinoma cell line derived from a metastasis at the left supraclavicular lymph node, and PC-3 cells, a human prostate carcinoma cell line derived from a bone metastasis, were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). PNT1A cells were maintained in RPMI 1640 (Invitrogen Life Technologies, Milan, Italy) supplemented with 10% inactivated fetal bovine serum (FBS,1 BioSpa, Milan, Italy), 2 mM glutamine (Invitrogen Life Technologies), 100 units/ml of penicillin (Invitrogen Life Technologies), and 100 µg/ml of streptomycin (Invitrogen Life Technologies), at 37 °C in the presence of 5% CO2. PC-3 and LNCaP cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate (Invitrogen Life Technologies), 100 units/ml of penicillin and 100 µg/ml of streptomycin, at 37 °C in the presence of 5% CO2.

RNA Extraction and RT-PCR—Cells were grown to 60–70% confluence, washed twice with cold PBS and total cellular RNA was isolated using the TRIzol reagent (Invitrogen Life Technologies) following the manufacturer's instructions. Where indicated, cells were incubated with the transcription inhibitor actinomycin D (7.5 µg/ml; Sigma) before RNA extraction. Total RNA was reverse-transcribed, and the resulting cDNA amplified by PCR using the Titan One Tube RT-PCR System (Roche Applied Science), as described previously (22). Briefly, total RNA (1 µg) was reverse-transcribed for 30 min at 55 °C in 0.2 mM dNTPs (Roche Applied Science), 5 units of RNase Inhibitor (Promega, Milan, Italy), 1x RT-PCR buffer containing 3 mM MgCl2 (Roche Applied Science), 0.4 µM upstream primer (Invitrogen Life Technologies), 0.4 µM downstream primer (Invitrogen Life Technologies), 1 µl enzyme mix (AMV, TaqDNA polymerase and a proofreading polymerase; Roche Applied Science). After cDNA synthesis, a cycle of denaturation at 94 °C for 2 min followed by 60 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s and elongation at 68 °C for 45 s, were run. The primer pair for {beta}1A was 2259-2280 and 2476-2496, where underlining indicates the 5'-nucleotide position (predicted fragment size: 238 bp); the primer pair for {beta}1B was 2390-2410 and 2544-2564 (predicted fragment size: 272 bp); the primer pair for {beta}1C was 2441-2461 and 2592-2612 (predicted fragment size: 172 bp); the primer pair for {beta}1D was 2442-2465 and 2562-2582 (predicted fragment size: 142 bp). All PCR reactions were performed using a Perkin Elmer CETUS PCR System (PerkinElmer Life Sciences). The PCR products were analyzed on a 2% agarose gel, stained with ethidium bromide.

Northern Blotting Analysis—Northern blotting analysis was carried out as described previously (22). Briefly, RNA samples (10 µg/lane) were separated on formaldehyde-agarose gels and blotted onto nylon membranes (Hybond N+; Amersham Biosciences). The blots were hybridized with cDNA probes, previously labeled with [{alpha}-32P]dCTP (3,000 Ci/mmol; PerkinElmer Life Science Products, Boston, MA) by random primer extension (Megaprime DNA labeling kit; Amersham Biosciences), for 20 h at 42 °C. The filters were washed once with 2x SSPE, 0.1% SDS for 10 min at room temperature, then with 1x SSPE, 0.1% SDS at 42 °C, followed by several washes in 0.1x SSPE, 0.1% SDS, at 65 °C and finally exposed at –80 °C overnight or longer to Kodak X-Omat AR 5 film (Kodak, Rochester, NY). Radiolabeled probes were generated using either the 116-bp fragment specific for {beta}1C integrin or the full-length human {beta}1C cDNA (19). Quantitative analysis was performed by densitometric scanning of the autoradiographs using a GS-700 Imaging densitometer (Bio-Rad); multiple exposures of the same Northern blots in a linear range were performed. 28 S rRNA signals were used as controls to determine the integrity of RNA and equality of loading in each lane. The average of either {beta}1C or {beta}1 mRNA expression levels in PNT1A cells was set at 100 (arbitrary units). {beta}1C and {beta}1 mRNA levels in prostate carcinoma cells were calculated as percentage of PNT1A mRNA levels, hybridized on the same filter. The mean value (±S.E.) of results obtained from at least three experiments was calculated.

Immunoblotting Analysis and Immunoprecipitation—PNT1A, LNCaP and PC-3 cells were grown to 60–70% confluence, washed twice with cold PBS and lysed in 150 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µM calpain inhibitor. Where indicated, cells were incubated with either the protein synthesis inhibitor cycloheximide (12.5 µg/ml; Sigma) or the lysosomal inhibitors leupeptin (100 µM; Sigma) or pepstatin (100 µM; Sigma) or NH4Cl (50 mM), or the calpain inhibitor ALLN (10 µM; Calbiochem) or the ubiquitinproteasome inhibitor MG132 (10 µM; Calbiochem, La Jolla, CA) or the vehicle alone (Me2SO for leupeptin, pepstatin, ALLN, MG132, Sigma; ethanol for cycloheximide), before lysis. The protein content of each lysate was quantified using the Bio-Rad Dc protein assay reagent according to the manufacturer's protocol. Protein extracts (100 µg) were electrophoresed on 10% SDS-polyacrylamide gel under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Immunoblotting was performed as previously described (18), using either 5 µg/ml rabbit polyclonal affinity-purified antibody to {beta}1C integrin or a 1:500 dilution of rabbit polyclonal antibody to {beta}1A integrin (Chemicon Int., Temecula, CA) or 10 µg/ml of monoclonal antibody (mAb) to {beta}-tubulin (Sigma) for 16 h at 4 °C in Tris-buffered saline/Tween-20 (TBS-T) (20 mM Tris, pH 7.5, 150 mM NaCl, 0.3% Tween-20). The membrane was then washed three times in TBS-T and incubated with a 1:2000 horseradish peroxidase-conjugated goat affinity-purified antibody to either rabbit or mouse IgG (Amersham Biosciences), in TBS-T for 1 h at room temperature. After three washes in TBS-T, proteins were visualized using the enhanced chemiluminescent system (ECL, Amersham Biosciences) according to the manufacturer's instructions. Densitometric values for immunoreactive bands were quantified using a GS-700 Imaging densitometer (Bio-Rad). {beta}1C and {beta}1A protein levels in prostate cancer cells were calculated as percentage of the control (PNT1A cells), after normalization using {beta}-tubulin as control for protein loading.

To perform a coupled immunoprecipitation-immunoblotting assay, the whole cell extract (1 mg of protein) was first precleared and then incubated overnight at 4 °C with either a 1:20 dilution of rabbit polyclonal antibody to {beta}1C integrin (18) or a 1:100 dilution of polyclonal antibody to {beta}1A integrin (Chemicon). Immunocomplexes were recovered by binding to protein A-Sepharose (Sigma) and washed three times with 350 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100, 1 mM PMSF, and twice with 150 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton-X100, 1 mM PMSF. Immunocomplexes were analyzed by 10% SDS-PAGE under reducing conditions followed by transfer to PVDF membrane. Filters were immunoblotted using either a 1:1000 dilution of mAb to ubiquitin (Sigma) following the manufacturer's instruction, or 1 µg/ml mAb 13 to {beta}1 integrins (BD Biosciences-Transduction Laboratory, Temecula, CA), as previously described (19).

Nuclear Run-on Transcription Assay—Nuclear run-on assays were performed as described previously (22). Nuclei were isolated from 1 x 108 PNT1A, LNCaP, and PC-3 cells. In vitro run-on transcription was carried out by using 1 x 107 nuclei and 100 µCi of [{alpha}-32P]UTP (3,000 Ci/mmole; PerkinElmer Life Sciences)/assay for 30 min at 30 °C, with periodic mixing. Labeled transcripts were purified by phenol/chloroform extractions and ethanol precipitations. A total of 1.2 x 107 cpm (4.0 x 106 cpm/ml of prehybridization solution) of elongated nascent RNAs per assay was hybridized for 48 h at 42 °C to filter-immobilized cDNAs. The following cDNA fragments were used: the 116-bp specific {beta}1C fragment (nucleotides 2435–2550) isolated by EcoRI digestion from the pBluescript-{beta}1C plasmid (19), the 2.6 kb full-length {beta}1C fragment, isolated by EcoRI digestion from the pBluescript-full-length {beta}1C plasmid (14), and a 1.3-kb 28 S fragment isolated by BamH1 digestion from the p28S plasmid (32). After hybridization, the filters were washed once in 2x SSPE, 0.1% SDS for 10 min at 42 °C, twice in 1x SSPE, 0.1% SDS for 10 min at 42 °C, twice in 0.5x SSPE, 0.1% SDS for 10 min at 42 °C, once in 0.1x SSPE, 0.1% SDS for 10 min at 50 °C, and then exposed to Kodak X-OMAT AR 5 film (Kodak). Autoradiographs of the RNA-DNA hybrids were analyzed using a GS-700 Imaging densitometer (Bio-Rad). All values were normalized according to the signal of 28 S rRNA, used as an internal standard. The {beta}1 transcription rate in prostate carcinoma cells was calculated as percentage of the control (PNT1A cells).

35S-Metabolic Labeling—The rate of {beta}1 integrin protein synthesis and the stability of {beta}1 integrin proteins were determined in PNT1A, LNCaP and PC-3 cells maintained in culture on 100-cm dishes (~1 x 107 cells/dish). Cells were labeled for 3 h in 1 ml of methionine/cysteine-free RPMI 1640 medium (Sigma) containing 100 µCi/ml [35S]protein labeling mix ([35S]methionine/cysteine, Amersham Biosciences). Cells were washed twice with PBS and lysed with 100 µl/dish of lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µM calpain inhibitor) for 30 min at 4 °C. Cells were then scraped and insoluble material was removed by centrifugation at 10,000 x g for 30 min at 4 °C. In pulse-chase experiments, cells were incubated, before lysis, in fresh medium containing 10% FBS, supplemented with 10 mM methionine and 10 mM cysteine, for varying times (up to 96 h) after a 3-h pulse label. Cell lysates were precleared, and levels of trichloroacetic acid-precipitable radioactivity were determined (33). Trichloroacetic acid-precipitable counts per min (1 x 107) of precleared labeled cell extracts were immunoprecipitated overnight at 4 °C with either a 1:20 dilution of rabbit polyclonal antibody to {beta}1C integrin or a 1:100 dilution of polyclonal antibody to {beta}1A integrin or a 1:20 dilution of normal rabbit serum (NRS, Sigma), as negative control, and with either 50 µg/ml of mAb to {beta}-tubulin or 50 µg/ml of non-immune mouse IgG (Sigma), as negative control. Immunocomplexes were recovered by binding to protein A-Sepharose and washed as described above. Immunocomplexes were analyzed by 10% SDS-PAGE under reducing conditions followed by either fluorography (Amersham Biosciences) and drying in a gel dryer (Bio-Rad) or transfer to PVDF membrane. Dried gels and PVDF membranes were exposed to Hyperfilm films (Amersham Biosciences) at –80 °C for 1–4 days. After autoradiographic exposure, PVDF filters were immunoblotted with 1 µg/ml mAb 13 to {beta}1 integrins as control of the total amount of {beta}1 integrins that were immunoprecipitated. Densitometric values for immunoreactive and radioactive bands were quantified using a GS-700 Imaging densitometer (Bio-Rad).

Endoglycosidase Digestion—Immunoprecipitates were dissolved by boiling for 4 min in 10 mM sodium phosphate (pH 7.0) containing 2% SDS, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 µM calpain inhibitor. Samples (25 µl) were diluted 5 times with 100 mM sodium phosphate buffer (pH 7.0) containing protease inhibitors, divided into two aliquots, and 8 units/ml of endoglycosidase F (Endo-F; Roche Applied Science) were added to one of each pair. Following a 12-h incubation at 37 °C, an additional 6 units/ml of Endo-F was added, and samples were further incubated for 12 h at 37 °C. The incubations were terminated by adding SDS sample buffer. Samples were subjected to 10% SDS-PAGE, followed by fluorography and exposure to Hyperfilm films at –80 °C for 1–4 days.

Statistical Analysis—Data are reported as the mean ± S.E. Statistical analysis was performed by the Student's t test. All experiments were repeated at least twice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{beta}1C and {beta}1A Integrin Expression in Prostate Carcinoma Cells—To investigate the regulation of {beta}1C and {beta}1A integrin expression in prostate cancer cells, a normal human cell line (PNT1A), established by immortalization of normal adult prostate epithelial cells, non-tumorigenic in mice (34), and two prostate cancer epithelial cell lines, LNCaP (clone FGC) (35, 36) and PC-3 (37, 38), were used as the experimental model.

First control, it was investigated by RT-PCR analysis whether {beta}1C and {beta}1A were the only {beta}1 integrin cytoplasmic variants expressed in prostate cells (Fig. 1A). This was confirmed by observing that only the {beta}1A sequence was detected when primers common to {beta}1A and {beta}1C were used, suggesting that {beta}1C mRNA is expressed at lower levels than {beta}1A mRNA in human prostate cells, as previously reported in prostate tissue (22) and in other cell types (14), and that an amplification product of the expected molecular size was detected both in normal and in carcinoma cells when primers specific for {beta}1C were used. By using specific primers to amplify the {beta}1B and {beta}1D transcripts, no amplification product was detected (data not shown), as in Ref. 22. In order to check whether the steadystate levels of {beta}1C and {beta}1A mRNAs and proteins were regulated in cancer versus normal cell lines in a way comparable to the prostate tissue, Northern (Fig. 1B) and Western (Fig. 1C) blotting analysis were performed in PNT1A, LNCaP, and PC-3 cells. As shown in Fig. 1B, in LNCaP and PC-3 cells {beta}1C mRNA levels proved to decrease of 40.6% ± 3.3 (p < 0.02) and 55.7% ± 3.9 (p < 0.02), respectively, versus PNT1A cells. Decreases in {beta}1 mRNA, which reflects the {beta}1A mRNA, were also found for LNCaP and PC-3 versus PNT1A cells, 30.5% ± 2.9 (p < 0.02) and 35.5% ± 0.8 (p < 0.02), respectively. Fig. 1C shows that the {beta}1A protein was expressed in normal and carcinoma prostate cells at comparable levels, whereas, the {beta}1C protein was expressed in PNT1A cells, but was undetectable in either LNCaP or PC-3 cells.



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  		FIG. 1.
Expression of {beta}1C and {beta}1A integrins in carcinoma and normal prostate cells. A, RT-PCR analysis of {beta}1A (lanes 1–3) and {beta}1C (lanes 4–6) mRNAs in PNT1A (lanes 1 and 4), LNCaP (lanes 2 and 5), and PC-3 (lanes 3 and 6) cells. In lanes 1–3, no amplification of {beta}1C in combination with {beta}1A was observed. M, molecular size marker. B, Northern blotting analysis for {beta}1C and {beta}1 integrin mRNAs. 28 S rRNA signals of the ethidium bromide-stained gel were used as controls to determine equality of loading in each lane. Bottom, {beta}1C and {beta}1 mRNA expression levels in LNCaP and PC-3 cells were calculated as percentage of PNT1A mRNA levels (control), set at 100. Mean values ± S.E. from three different experiments are shown. C, immunoblotting analysis of {beta}1C and {beta}1A integrin proteins. PNT1A, LNCaP and PC-3 cell protein extracts (100 µg) were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, and immunostained using an antibody to either {beta}1C or {beta}1A integrin. {beta}-tubulin signals were used as loading controls. Bottom, {beta}1C and {beta}1A protein expression levels in LNCaP and PC-3 cells were calculated as percentage of PNT1A protein levels (control), set at 100. Mean values ± S.E. from three different experiments are shown.

 
These results show that {beta}1C and {beta}1A integrin mRNA and protein steady-state levels are regulated in LNCaP and PC-3 prostate cancer cell lines versus PNT1A normal cells in a way comparable to the prostate tissue.

Transcriptional Regulation of {beta}1 Integrins—In order to investigate whether, as previously reported in prostate tissue (22), a decreased transcription of the {beta}1 integrin gene could account for the down-regulation of {beta}1 integrin mRNA levels in prostate cancer cells, the {beta}1 integrin gene transcriptional activity was evaluated both in normal and carcinoma cell lines by nuclear run-on analysis, as described under "Experimental Procedures" (Fig. 2). The transcriptional rate of the {beta}1 integrin gene was found to be markedly reduced in LNCaP and PC-3 as compared with PNT1A cells: 40.3% ± 4.1 (p < 0.01) and 45.4% ± 4.7 (p < 0.01), respectively, when the {beta}1C autoradiographic signal was considered, and 32.4% ± 3.5 (p < 0.01) and 35.1% ± 3.7 (p < 0.01), respectively, when the {beta}1 signal was considered.



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  		FIG. 2.
Transcriptional regulation of {beta}1 integrins. Nuclear run-on transcription analysis of nuclei isolated from PNT1A, LNCaP, and PC-3 cells was performed. Equal amounts of 32P-labeled nuclear transcripts were hybridized to filters on which denatured cDNA (10 µg) for {beta}1C integrin, {beta}1 integrins, and 28 S rRNA had been immobilized. Densitometric values for {beta}1C and {beta}1 mRNAs were normalized to the internal standard 28 S rRNA. At the bottom, the {beta}1 integrin transcriptional activity in LNCaP and PC-3 cells was calculated as percentage of the transcriptional activity in PNT1A cells (control), set at 100, considering either {beta}1C or {beta}1 autoradiographic signals. Mean ± S.E. from three different experiments are shown.

 
In order to ascertain whether changes in mRNA stability could contribute to the down-regulation of {beta}1C and {beta}1A mRNA levels in prostate cancer cells, {beta}1C and {beta}1A mRNA half-life was estimated by their steady-state levels after cell treatment with actinomycin D, a transcription inhibitor. Cell viability in the presence of actinomycin D, as measured by Trypan Blue exclusion, was >97% in the three cell lines. After actinomycin D addition, a logarithmic decay was observed for {beta}1C mRNA levels, both in normal and neoplastic cells, but not for {beta}1 mRNA levels, that remained constant up to 48 h both in PNT1A and LNCaP and PC-3 cells (Fig. 3). No difference was detected in cancer versus normal cells with respect to {beta}1C mRNA half-life, which was ~12 h.



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  		FIG. 3.
Post-transcriptional regulation of {beta}1 integrin mRNAs. The half-lives of {beta}1C and {beta}1 mRNAs were evaluated by addition of actinomycin D (7.5 µg/ml) to PNT1A, LNCaP, and PC-3 cells. {beta}1C and {beta}1 steady-state mRNA levels were determined by Northern blotting up to 48 h after actinomycin D addition. The corresponding signal for 28 S rRNA of the ethidium bromide-stained gel was used to normalize the amount of total RNA loaded for each sample. Bottom, the degradation curves of {beta}1C and {beta}1 mRNAs are reported. {beta}1C and {beta}1 mRNA expression levels were calculated as percentage of the mRNA levels at time 0 (control), set at 100. Consistent results were obtained from two independent experiments.

 
Overall, these results show that transcriptional but not post-transcriptional processes account for the down-regulation of {beta}1C and {beta}1A mRNA steady-state levels in prostate cancer cells.

Translational Regulation of {beta}1C and {beta}1A Integrins—Notwithstanding both {beta}1C and {beta}1A mRNA levels were down-regulated, only the {beta}1C protein was found to be reduced or lost in prostate cancer cells (Fig. 1 and Refs. 18 and 19). In order to establish whether a different modulation of the rate of {beta}1C and {beta}1A protein synthesis was involved in the regulation of {beta}1C and {beta}1A expression in prostate cancer cells, we assayed the translation process by metabolic labeling of PNT1A, LNCaP, and PC-3 cells with [35S]methionine/cysteine, followed by immunoprecipitation studies (Fig. 4). Using a specific antibody to immunoprecipitate either {beta}1C or {beta}1A integrin, an 35S-labeled protein was detected, migrating with a similar molecular mass for the two integrin splice variants (indicated by the arrow). A larger amount of {beta}1A than {beta}1C protein could be immunoprecipitated from the PNT1A cell extracts, in agreement with the finding that {beta}1C mRNA levels are lower than those of {beta}1A (Fig. 1A). In LNCaP and PC-3 cells, [35S]methionine/cysteine incorporation into {beta}1C protein was reduced by 72.3% ± 5.4 (p < 0.001) and 74.9% ± 4.9 (p < 0.001), respectively, whereas [35S]methionine/cysteine incorporation into {beta}1A protein was increased to 316.5% ± 44.7 (p < 0.001) and 251.7% ± 42.1 (p < 0.001), respectively, as compared with PNT1A cells. The translational rate of the housekeeping protein {beta}-tubulin was also evaluated, as a control, and it was found comparable between normal and neoplastic prostate cells (data not shown). These results show that {beta}1C and {beta}1A integrin splice variants are translated with different efficiency in carcinoma cells.



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  		FIG. 4.
Translational regulation of {beta}1C and {beta}1A integrins. PNT1A, LNCaP, and PC-3 prostate cells were metabolically labeled with [35S]methionine/cysteine for 3 h, and proteins were immunoprecipitated with an antibody to either {beta}1C or {beta}1A integrin or with NRS, as negative control. Immunoprecipitated proteins were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis under reducing conditions. 35S-labeled proteins were visualized by fluorography. Positions of molecular mass standards (kDa) are indicated. Bottom, the relative amounts of the nascent {beta}1C and {beta}1A integrins in LNCaP and PC-3 cells were expressed as percentage of {beta}1C and {beta}1A content in PNT1A cells (control), set at 100. Mean values ± S.E. from four independent experiments are shown.

 
Stability and Glycosylation of {beta}1C and {beta}1A Proteins—In order to investigate whether changes in protein degradation rates could be involved in the differential regulation of {beta}1C and {beta}1A protein expression in prostate cancer cells, we measured the half-life of the two splice variant proteins. Firstly, translation was inhibited with cycloheximide and {beta}1C and {beta}1A steady-state protein levels were measured in normal and cancer prostate cells by immunoblotting analysis. Cell viability in the presence of cycloheximide was > 97% in the three cell lines, as measured by Trypan Blue exclusion. Fig. 5 shows that the {beta}1C protein levels declined logarithmically in normal cells, as well as the {beta}1A protein levels both in normal and neoplastic cells. The {beta}1C half-life was ~12 h in PNT1A cells whereas the {beta}1A half-life was ~32 h in PNT1A and ~18 h both in LNCaP and PC-3 cells (Fig. 5B). Unfortunately, the half-life of the {beta}1C protein could not be evaluated in LNCaP or PC-3 cells as it was not detectable by immunoblotting analysis. Controls were performed to ensure that the solvent itself caused no difference in {beta}1C or {beta}1A steady-state protein levels (Fig. 5A). In order to better investigate the post-translational regulation of {beta}1 integrin splice variants, pulse-chase experiments were performed and the stability of the newly-synthesized {beta}1 integrin proteins was analyzed as described under "Experimental Procedures." Fig. 6 shows the results of a typical pulse-chase experiment: the decay of {beta}1C and {beta}1A nascent proteins was logarithmic both in normal and carcinoma prostate cells, and {beta}1C protein half-life was ~13 h in normal cells and ~8 h in neoplastic cells, whereas {beta}1A protein half-life was ~55 h in normal cells and ~17 h in carcinoma cells. Thus, these results show that post-translational processes, at the level of protein degradation, are involved in the regulation of {beta}1C and {beta}1A protein expression in prostate carcinoma cells.



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  		FIG. 5.
Stability of {beta}1C and {beta}1A integrins in the presence of cycloheximide. The half-lives of {beta}1C and {beta}1A proteins were evaluated by addition of cycloheximide (12.5 µg/ml) to PNT1A, LNCaP, and PC-3 cells. A, {beta}1C and {beta}1A steady-state protein levels were determined by immunoblotting analysis up to 96 h after addition of either cycloheximide (+ cyx) or solvent alone (–cyx). Total protein extracts (100 µg) were separated on 10% SDS-polyacrylamide gels under reducing conditions, and immunostained using an antibody to either {beta}1C or {beta}1A integrin. {beta}-tubulin was used to normalize the amount of total proteins loaded for each sample. B, degradation curves of {beta}1C and {beta}1A proteins in the presence of cycloheximide are reported. {beta}1C and {beta}1A protein expression levels were calculated as percentage of the protein levels at time 0 (control), set at 100. Consistent results were obtained from two independent experiments.

 



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  		FIG. 6.
Stability and processing of {beta}1C and {beta}1A nascent proteins. PNT1A, LNCaP, and PC-3 cells were pulse-labeled with [35S]methionine/cysteine (100 µCi/ml) for 3 h, then incubated in the absence of [35S]methionine/cysteine for different chasing periods before being lysed. {beta}1C and {beta}1A proteins were immunoprecipitated from protein extracts with an antibody to either {beta}1C or {beta}1A integrin, electrophoresed on 10% SDS-polyacrylamide gel under reducing conditions, and detected by fluorography. Positions of molecular mass standards (kDa) are indicated. Bottom, a quantification of {beta}1C and {beta}1A integrin turnover is reported. {beta}1C and {beta}1A protein levels were calculated as percentage of the protein levels at 0 h chasing period (control), set at 100. For each time point, the densitometric values of the autoradiographic signals of both the 126-kDa form and the forms with lower electrophoretic mobility were summed. Consistent results were obtained from two independent experiments.

 
The pulse-chase experiments were also used to investigate the maturation of {beta}1C and {beta}1A integrins in both normal and cancer cells. Fig. 6 shows, at 12 h of the chasing period in PNT1A cells, a decrease in the electrophoretic mobility of the nascent {beta}1C protein, with a 100% shift from the ~126-kDa form to one of ~130 kDa. Such a 130-kDa form was gradually degraded during the chasing period, as shown by the slight increase in mobility. In PC-3 and LNCaP cells, the rate of maturation of the {beta}1C protein was higher than in PNT1A cells: at 6 h of the chasing period, about half of the nascent {beta}1C protein showed a lower electrophoretic mobility, with a shift from ~126 to ~138 kDa. The 138-kDa form disappeared almost completely (more than 90%) at 12 h of the chasing period with a shift to the 126-kDa form that was then rapidly lost. As far as the {beta}1A protein is concerned, in PNT1A cells, at 6 h of the chasing period, approximately half of the nascent protein showed a lower electrophoretic mobility, with a shift from ~126 to ~138 kDa. The 138-kDa {beta}1A form represented ~85% of the total {beta}1A integrin at 12 h of the chasing period. Then, a gradual protein degradation, accompanied by a slight increase in the electrophoretic mobility, occurred. In PC-3 and LNCaP cells, a delay in the maturation of {beta}1A integrin was detected: at 6 h of the chasing period, only 10% of the nascent {beta}1A protein, versus 50% found in PNT1A cells, showed a lower electrophoretic mobility form, with an apparent molecular mass of ~138 kDa. The 138-kDa {beta}1A form represented about 65% of the total {beta}1A protein at 12 h of the chasing period. In PC-3 and LNCaP cells, the gradual {beta}1A protein degradation, which started at 12 h, was accompanied by an increase in electrophoretic mobility, occurring both the events with a rate higher than in PNT1A cells. Moreover, in prostate carcinoma cells maturation of the {beta}1A protein proceeded at a rate lower than {beta}1C maturation, as, at 6 h of the chasing period, ~ 10% of the {beta}1A nascent protein was fully processed versus ~50% of the {beta}1C nascent protein. In order to find out whether the changes in electrophoretic mobility during {beta}1C and {beta}1A integrin processing were due to N-glycosylation, {beta}1C and {beta}1A immunoprecipitates of PNT1A and PC-3 cells were digested with endoglycosidase F (Endo-F) before electrophoresis. Fig. 7 shows that, upon removal of the N-linked oligosaccharides by Endo-F digestion, the {beta}1 integrin core protein was similar during processing for {beta}1C and {beta}1A integrins both in normal and in neoplastic cells. Overall, these results show that changes in the maturation process of the {beta}1C and {beta}1A integrin proteins occur in carcinoma versus normal prostate cells.



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  		FIG. 7.
Sensitivity of {beta}1C and {beta}1A proteins to endoglycosidase F (Endo-F). PNT1A and PC-3 cells were pulse-labeled with [35S]methionine/cysteine (100 µCi/ml) for 3 h, then incubated in the absence of [35S]methionine/cysteine for different chasing periods up to 12 h before being lysed. {beta}1C and {beta}1A proteins were immunoprecipitated from protein extracts with an antibody to either {beta}1C or {beta}1A subunit, respectively. Immunoprecipitates were treated with (+) or without (–) Endo-F and then analyzed by 10% SDS-polyacrilamide gel under reducing conditions, and detected by fluorography. Positions of molecular mass standards (kDa) are indicated. Consistent results were obtained from two independent experiments.

 
Proteolysis Pathways Involved in the Degradation of {beta}1C and {beta}1A Proteins—In order to gain some insight into the post-translational mechanisms that regulate {beta}1C and {beta}1A protein levels, the occurrence of the lysosomal-, the calpain- and the ubiquitin-mediated proteolysis in the degradative processes of the two splice variants was investigated by incubating PNT1A and PC-3 cells for varying times (up to 12 h) with either leupeptin, a lysosomal inhibitor, or MG132, a proteasome inhibitor, or ALLN, a calpain inhibitor, and by measuring changes in {beta}1C and {beta}1A protein levels by immunoblotting (Fig. 8). Treatment of PC-3 cells with leupeptin resulted in induction of {beta}1C protein expression and in increased {beta}1A expression. After 12-h treatment, the {beta}1C protein levels resulted ~40–50% of those expressed in normal cells whereas the {beta}1A protein levels increased of ~90–100% versus non-treated PC-3 cells. Similar results were obtained when the lysosomal inhibitor pepstatin or the lysosomotropic agent NH4Cl were used (data not shown). Treatment of PC-3 cells with ALLN did not affect {beta}1C or {beta}1A protein expression but treatment with MG132 resulted in a rapid induction of {beta}1C protein expression without affecting {beta}1A expression. After 12-h treatment with MG132, the {beta}1C protein levels resulted ~70–80% of those expressed in normal cells. As far as normal cells are concerned, inhibition of either the lysosomal- or the calpain-mediated proteolysis increased both {beta}1C and {beta}1A protein levels: leupeptin, as well as pepstatin and NH4Cl (data not shown), caused an increase of ~ 45–50% both for {beta}1C and {beta}1A protein, whereas ALLN caused an increase of ~150% for {beta}1C and ~60–70% for {beta}1A protein after 12-h treatment. Neither {beta}1C nor {beta}1A protein levels were affected by MG132 treatment in normal prostate cells. Controls were performed to ensure that PNT1A and PC-3 cell viability, as judged by Trypan blue exclusion, was higher than 98% for all the treatments used (data not shown) and that incubation of normal and carcinoma cells with the solvent Me2SO did not affect {beta}1C or {beta}1A protein levels. These results show the following. (a) The lysosomal proteolytic pathway is involved in the degradation of both {beta}1C and {beta}1A protein in normal and cancer prostate cells, albeit at a higher extent in cancer than in normal cells. (b) A calpain-mediated proteolysis of both {beta}1C and {beta}1A protein occurs in normal but not in cancer cells. (c) The ubiquitin-proteasome pathway degrades selectively the {beta}1C protein in prostate cancer cells.



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  		FIG. 8.
Sensitivity of {beta}1C and {beta}1A proteins to lysosomal, calpain, and ubiquitin proteolytic pathways. PNT1A and PC-3 cells were treated with either leupeptin (100 µM) or ALLN (10 µM) or MG132 (10 µM) or the vehicle alone (Me2SO) for the indicated times. Cell lysates were analyzed for the levels of {beta}1C and {beta}1A proteins by immunoblotting, as described in the legend to Fig. 5. Similar results were obtained from two independent experiments.

 
If {beta}1C is a preferential target of the ubiquitin-proteasome pathway in cancer cells, inhibition of the proteasome activity with MG132 should accumulate ubiquitinated form/s of {beta}1C protein. To investigate this possibility, protein extracts of PC-3 cells treated with MG132 were immunoprecipitated with an antibody to either {beta}1C or {beta}1A, followed by immunoblotting analysis using an antibody to ubiquitin (Fig. 9). Upon treatment with MG132, ubiquitinated {beta}1C protein, migrating around 135 kDa, progressively accumulated as a function of time. As far as {beta}1A protein is concerned, two faint bands were observed 12 h after treatment but not in the earlier times. Of the two bands, one migrating around 135 kDa and the other around 105 kDa, the latter could represent an immature, non-glycosylated, {beta}1A protein form. As control of the total amount of {beta}1 integrins that were immunoprecipitated, immunoblotting was performed using an antibody that recognizes the extracellular domain of the {beta}1 integrins and, thus, all of the {beta}1 cytoplasmic variants. As shown in Fig. 9, the total amount of {beta}1A protein was significantly higher (at least 10-fold) than that of {beta}1C and did not change during MG132 treatment, at variance with {beta}1C protein levels that increased as a function of time (see also Fig. 8). Thus, in consideration of the much higher levels of {beta}1A versus {beta}1C protein, the amount of ubiquitinated {beta}1A protein, detected at 12 h, resulted extremely low as compared with that of {beta}1C. Overall, these results show that the ubiquitin-proteasome proteolytic pathway is involved in the selective down-regulation of {beta}1C protein in prostate carcinoma cells.



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  		FIG. 9.
Ubiquitination of {beta}1 integrins in prostate cancer cells. PC-3 cells were treated with MG-132 (10 µM) for the indicated times before being lysed. Cell lysates were first immunoprecipitated with an antibody to either {beta}1C or {beta}1A integrin and then immunoblotted with an antibody to ubiquitin. Immunoblotting with an antibody to {beta}1 integrins was performed as control of the total amount of {beta}1 integrins that were immunoprecipitated. Positions of molecular mass standards (kDa) are indicated. Consistent results were obtained from two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the molecular events involved in the regulation of {beta}1C and {beta}1A expression in normal and in cancer prostate cells. Indeed, the knowledge of the regulation of the expression of {beta}1 integrin variants in different pathophysiological conditions is rather poor; although it has been shown that {beta}1 integrin expression can be regulated both at the transcriptional (22–26, 30, 39) and post-transcriptional levels (23,25,27–31,40), these studies were performed on the {beta}1 integrin as a whole, with no discrimination among the various splice variants. We show here that, when comparing carcinoma versus normal prostate cells, {beta}1C and {beta}1A integrin variants share the same regulation at the transcriptional and post-transcriptional level, whereas they are differently regulated at the translational and post-translational level.

We show that, although in the investigated cancer cell lines LNCaP and PC-3 {beta}1C and {beta}1A mRNAs are expressed at lower levels than in the normal cell line PNT1A, the {beta}1A protein is expressed at comparable levels in LNCaP and PC-3 cells and in normal cells, but the {beta}1C protein is selectively lost in cancer cells. These results, together with the findings already reported in prostate tissue (18, 19), represented the starting background to further investigate the regulation of {beta}1C and {beta}1A expression in prostate cancer.

Changes in the transcriptional rate of the {beta}1 integrin gene have been previously shown to play a role in the regulation of {beta}1 integrin expression in cancer cells (26). A decrease in the transcriptional activity of the {beta}1 integrin gene has been recently demonstrated in human prostate cancer tissue (22). We show that a similar reduction also occurs in cancer cell lines, thus substantiating the validity of our model system. Moreover, we show that the stability of both {beta}1C and {beta}1A transcripts does not change during prostate malignant transformation, even though they differ from each other, the {beta}1C transcript half-life being much shorter than that of {beta}1A (~12 h versus > 48 h) both in normal and cancer cells. Since {beta}1C and {beta}1A mRNAs differ only for a 116-nucleotide encoding sequence, absent in the {beta}1A transcript (14), we suppose that such a region is responsible for the lower stability. The short {beta}1C mRNA half-life could be considered when discussing the lower mRNA expression levels of this variant compared with {beta}1A, as here reported in prostate cells as well as in other cell types (14, 41).

As the {beta}1A integrin supports cell proliferation (13, 15) and cancer cell invasion (42), it is conceivable that high levels of this protein are required in prostate cancer, even though the transcriptional activity of the {beta}1 integrin gene decreases. Thus, modifications in the translational/post-translational machinery in prostate cancer cells are expected to regulate {beta}1C and {beta}1A protein translation and/or degradation in an opposite manner, making it possible that low levels of {beta}1C protein, which strongly inhibits cell proliferation, but high levels of the {beta}1A protein, which favors cell proliferation and invasion, are expressed in prostate cancer.

In agreement with the above hypothesis, we show here that the {beta}1C translation rate decreases in prostate cancer cells, whereas the {beta}1A translation rate increases more than 2-fold versus the normal cells, notwithstanding the reduction in the mRNA levels. These results, in agreement with the general concept that deregulation of the protein synthesis machinery is a mechanism of neoplastic transformation, are consistent with several reports demonstrating constitutive high rates of protein synthesis for proteins having growth promoting properties (reviewed in Refs. 43 and 44).

A number of investigations have shown that cell glycosylation plays a significant role in malignant transformation (reviewed in Refs. 45 and 46) and can participate in integrin protein degradation (47, 48). {beta}1 integrin subunits contain at least twelve potential N-glycosylation sites (Asn-X-(Ser/Thr)) in the extracellular domain (49) and their processing, involving addition of N-linked carbohydrate side chains within the Golgi, occurs with concurrent shifts in the electrophoretic mobility (50). We show here that the {beta}1C, but not the {beta}1A neo-synthesized protein is glycosylated in cancer cells at a higher rate than in normal cells. In addition, since in prostate cancer cells, at 6 h of the chasing period, the {beta}1C nascent protein is present (even if partially) in a form of molecular mass (~138 kDa) higher than that observed at 12 h in normal cells (~130 kDa), and since Endo-F treatment of immunoprecipitates removes the shifts in electrophoretic mobility of the {beta}1 integrin nascent proteins, we suggest that intermediates of the {beta}1C protein during processing may be differently glycosylated in cancer versus normal cells. We also show that the glycosylation rate of the {beta}1C protein is different from that of {beta}1A both in normal and in cancer cells, as in PNT1A cells it is lower and in PC-3 and LNCaP cells it is higher than that of {beta}1A. As aberrant N-glycosylation of {beta}1 integrins has been shown to affect cell adhesion (51) and migration (52), ascertaining whether the changes both in the processing rates and in the processing intermediates of the {beta}1 integrin variants depend on certain features of the glycosylation could be a good goal to pursue.

We show here that the {beta}1C protein is degraded more rapidly in cancer versus normal cells. However, we have found that the {beta}1A protein half-life in cancer cells is also lower than in normal cells, suggesting that a general, non-splice variant-specific, higher turnover of the {beta}1 proteins occurs in cancer cells. Such a conclusion is not unique: in fact, it has been already shown that rapid degradation of normal proteins is a powerful, rapid and specific means whereby key regulatory proteins, involved in the control of cell division, are irreversibly inactivated in cancer cells (reviewed in Refs. 53 and 54). In addition, we find that the {beta}1C protein half-life is significantly shorter than that of {beta}1A both in cancer and in normal cells, suggesting that the specific 48-amino acid sequence in the cytoplasmic domain of the {beta}1C protein (14) could be a selected target for proteolysis to occur.

Two major proteolytic systems exist in mammalian cells, the lysosomal and the non-lysosomal systems (55). Degradation of plasma membrane proteins has been shown to take place in lysosomes (56–58). In agreement with this finding, we show that lysosomal degradation of both {beta}1C and {beta}1A proteins occurs in normal and in cancer prostate cells, but that it is more active in the neoplastic cells, thus contributing to the loss of {beta}1C in these cells. Among the non-lysosomal pathways, the calpain and the ubiquitin-mediated proteolysis have been shown to be involved in the degradation of many regulatory proteins (53, 54, 59, 60). We show here that in normal, but not in cancer prostate cells, the calpain-mediated proteolysis is involved in the degradation of both {beta}1C and {beta}1A protein. It should be noted that many integrin subunits (including {beta}1A, {beta}1D, {beta}2, {beta}3, {beta}4, and {beta}7) have been shown to be calpain-sensitive (61–63). In particular, cleavage of the {beta}1A cytoplasmic domain had been previously demonstrated in normal cells (63). Our results show that also the {beta}1C protein is a target of the calpain-mediated proteolysis in normal cells. On the other hand, we show that in cancer cells, expression of {beta}1C protein, normally absent in these cells, is induced by inhibition of the ubiquitin proteolytic pathway, and reaches levels close to those expressed in normal cells, without any effect on {beta}1A protein levels, thus providing evidence that the {beta}1C protein is a preferential target of the ubiquitin-proteasome pathway in cancer cells. This is further confirmed by the preferential accumulation of ubiquitinated {beta}1C and not {beta}1A protein when cancer cells are treated with a proteasome inhibitor. We find that neither {beta}1C nor {beta}1A integrin is degraded by the ubiquitin-proteasome pathway in normal cells, indicating that the activity of the ubiquitin proteolysis, as well as that of calpain, as discussed above, may vary between normal and carcinoma cells. These findings add another member, i.e. the {beta}1C protein, to the list of growth inhibitory molecules that are preferentially degraded by the ubiquitin-proteasome pathway in cancer cells (reviewed in Refs. 64 and 65) and that might be targeted by proteasome inhibitors in anticancer therapy (65, 66).

In conclusion, our data demonstrate that the differential expression of {beta}1C and {beta}1A integrins in prostate cancer cells is regulated at the transcriptional, post-transcriptional, translational and post-translational levels via both common (transcriptional) and variant-specific (post-transcriptional/translational/post-translational) mechanisms. Interestingly, we highlight a role for the ubiquitin-proteasome proteolytic pathway in the selective loss of {beta}1C protein expression in prostate cancer cells. Since in vivo down-regulation of {beta}1C seems to occur at an early stage in the pathogenesis of prostate cancer (18, 19), the identification of the specific molecular events that contribute to the loss of {beta}1C, but not {beta}1A protein in prostate cancer cells could help in giving some insights into the molecular basis of prostate malignant transformation.


   FOOTNOTES
 
* This work was supported by the MURST-PPRST Cluster 03 grant (to E. M.), by the ARMY, Prostate Cancer Research Program Grant DAMD17-98-1-8506 (to L. R. L.), and by a grant from the Associazione Italiana Ricerca sul Cancro (AIRC) (to E. P.). 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. Back

§ To whom correspondence should be addressed: Institute of Biomembranes and Bioenergetics, National Research Council (C.N.R.), Via Amendola 165/A, 70126 Bari, Italy. Tel.: 39-080-544-2412; Fax: 39-080-544-3317; E-mail: csmmlm22{at}area.ba.cnr.it.

1 The abbreviations used are: FBS, fetal bovine serum; ALLN, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; AMV, avian myeloblastosis virus; cyx, cycloheximide; Me2SO, dimethyl sulfoxide; dNTPs, deoxynucleotides; Endo-F, endoglycosidase F; mAb, monoclonal antibody; NRS, normal rabbit serum; PBS, phosphate-buffered saline solution; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SSPE, saline sodium phosphate ethylenediaminetetraacetic acid buffer; CHO, chinese hamster ovary. Back


   ACKNOWLEDGMENTS
 
We thank R. Lusardi for linguistic consultation.



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 ABSTRACT
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 DISCUSSION
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