Regulation of β1C and β1A Integrin Expression in Prostate Carcinoma Cells

Loredana Moro,Elda Perlino,Ersilia Marra,Lucia R Languino,Margherita Greco

doi:10.1074/jbc.m307857200

Abstract

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.

Highlights

␤1C and ␤1A integrins are two splice variants of the human ␤1 integrin subfamily that act as an inhibitor and a stimulator of cell proliferation, respectively
We show the following results. (i) A reduction in the transcriptional activity of the ␤1 integrin gene can account for the down-regulation of ␤1C and ␤1A integrin mRNA levels in prostate cancer cells. (ii) Loss of ␤1C protein in cancer cells depends on reduced transcription and on reduced translation and increased protein degradation. (iii) Notwithstanding the reduction in the mRNA levels, the translation rate of the ␤1A protein increases in cancer cells. (iv) The processing rates of ␤1C and ␤1A proteins are different in cancer versus normal cells. (v) The ␤1C protein is a preferential target of the ubiquitin-proteasome proteolytic pathway in cancer cells
These results show that ␤1C and ␤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

Summary

EXPERIMENTAL PROCEDURES

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). Cells were washed twice with PBS and lysed with 100 ␮l/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 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin, 1 ␮M calpain inhibitor) for 30 min at 4 °C. Trichloroacetic acid-precipitable counts per min (1 ϫ 107) of precleared labeled cell extracts were immunoprecipitated overnight at 4 °C with either a 1:20 dilution of rabbit polyclonal antibody to ␤1C integrin or a 1:100 dilution of polyclonal antibody to ␤1A integrin or a 1:20 dilution of normal rabbit serum (NRS, Sigma), as negative control, and with either 50 ␮g/ml of mAb to ␤-tubulin or 50 ␮g/ml of non-immune mouse IgG (Sigma), as negative control. 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.

RESULTS

Findings

DISCUSSION