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Identification of distinctive patterns of USP19-mediated growth regulation in normal and malignant cells.

Lu Y, Bedard N, Chevalier S, Wing SS - PLoS ONE (2011)

Bottom Line: Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels.This may occur through both KPC1 dependent and independent mechanisms.Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

View Article: PubMed Central - PubMed

Affiliation: Polypeptide Laboratory, Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27(Kip1). To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27(Kip1). However, in contrast to previous findings in fibroblasts, the stabilization of p27(Kip1) upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27(Kip1) and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

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Depletion of USP19 results in accumulation of p27Kip1 in DU145 cells.(A) p27Kip1 but not p21 or p16 accumulates in USP19-depleted DU145 cells. Equal amounts of protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. (B) Overexpression of USP19 in DU145 cells prevents USP19 siRNA induced increase of p27Kip1. Protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1B) were analyzed by immunoblotting with the indicated antibodies. Note that for the USP19 immunoblots, 300 µg of DU145-Vect protein and 20 µg of DU145-USP19 protein were used. (C) Silencing of USP19 does not increase p27Kip1 levels by increasing mRNA levels. Equal amounts of protein and RNA isolated from lysates of DU145 cells transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL) (as described in Fig. 1A) were analyzed by Western (top) and Northern (bottom) blots, respectively. (D) Silencing of USP19 stabilizes p27Kip1. DU145 cells were transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL). Forty-eight hours later, they were incubated with cycloheximide (CHX) to inhibit protein synthesis. At the indicated times, cells were lysed and proteins were analyzed by immunoblotting with the indicated antibodies. Shown are representative immunoblots and quantitation of p27Kip1 levels (means ± SE) from triplicate samples. *, rate of degradation of p27Kip1 was significantly inhibited in USP19 depleted DU145 cells (P<0.05) (two-way analysis of variance). (E) Levels of ubiquitin protein ligases, KPC1, Skp2 and Pirh2 are not affected upon USP19 depletion in DU145 cells. Equal amounts of protein from lysates (as in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. Quantification was performed on triplicate samples and did not reveal any significant differences in the levels of the ligases.
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pone-0015936-g003: Depletion of USP19 results in accumulation of p27Kip1 in DU145 cells.(A) p27Kip1 but not p21 or p16 accumulates in USP19-depleted DU145 cells. Equal amounts of protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. (B) Overexpression of USP19 in DU145 cells prevents USP19 siRNA induced increase of p27Kip1. Protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1B) were analyzed by immunoblotting with the indicated antibodies. Note that for the USP19 immunoblots, 300 µg of DU145-Vect protein and 20 µg of DU145-USP19 protein were used. (C) Silencing of USP19 does not increase p27Kip1 levels by increasing mRNA levels. Equal amounts of protein and RNA isolated from lysates of DU145 cells transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL) (as described in Fig. 1A) were analyzed by Western (top) and Northern (bottom) blots, respectively. (D) Silencing of USP19 stabilizes p27Kip1. DU145 cells were transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL). Forty-eight hours later, they were incubated with cycloheximide (CHX) to inhibit protein synthesis. At the indicated times, cells were lysed and proteins were analyzed by immunoblotting with the indicated antibodies. Shown are representative immunoblots and quantitation of p27Kip1 levels (means ± SE) from triplicate samples. *, rate of degradation of p27Kip1 was significantly inhibited in USP19 depleted DU145 cells (P<0.05) (two-way analysis of variance). (E) Levels of ubiquitin protein ligases, KPC1, Skp2 and Pirh2 are not affected upon USP19 depletion in DU145 cells. Equal amounts of protein from lysates (as in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. Quantification was performed on triplicate samples and did not reveal any significant differences in the levels of the ligases.

Mentions: Since depletion of USP19 delayed G0/G1 to S progression, we explored possible mechanisms by assessing the steady-state levels of CKIs that inhibit G0/G1-S transition. USP19 siRNA, but not control siRNA led to an accumulation of p27Kip1, but not p16 or p21 as exemplified in the DU145 model (Fig. 3A). To confirm that this increased p27Kip1 was specific to USP19 depletion, USP19 siRNA was transfected in the stable DU145 cell line expressing rat USP19. In agreement with above, depletion of USP19 in DU145 cells expressing empty vector led to an induction of p27Kip1. However, in cells overexpressing rat USP19, the same siRNA treatment was unable to upregulate p27Kip1 (Fig. 3B), confirming that the upregulation of p27Kip1 is a specific response to USP19 depletion. This increased p27Kip1 induced by USP19 depletion did not appear to be due to increased gene transcription, as Northern blot analysis revealed that p27Kip1 mRNA levels did not change (Fig. 3C). These data suggest an involvement of USP19 in regulating the turnover of p27Kip1. To test this possibility, the rate of disappearance of p27Kip1 was measured in DU145 cells with or without USP19 depletion following inhibition of protein synthesis with cycloheximide. Indeed, in cells transfected with USP19 siRNA p27Kip1 levels remained relatively stable over time whereas the half-life of the protein was significantly lower in controls (Fig. 3D), implying that USP19 modulates the stability of p27Kip1. To determine whether the upregulation and stabilization of p27Kip1 in USP19 depleted DU145 cells was linked to changes in KPC1 expression as we previously reported [20] or the other E3s, Skp2 and Pirh2, also responsible for the ubiquitination of p27Kip1, we measured the levels of these proteins in response to USP19 depletion. Unexpectedly, we did not observe any changes in KPC1 (Fig. 3E), nor in Skp2 and Pirh2 levels (Fig. 3E). These observations indicate that USP19 can act through a KPC1 and also Skp2 and Pirh2 independent pathway(s) while still modulating cell growth and p27Kip1. Of interest the silencing of USP19 in PC-3 and 22RV1 cells, which also led to an inhibition of their proliferation resulted in a similar upregulation of p27Kip1 expression but also no changes in levels of the three ubiquitin ligases, KPC1, Skp2 or Pirh2 (Fig. S3). These results suggest that USP19 may lose its ability to regulate growth such as in the LNCaP cells or still exert this function through p27Kip1 as in a variety of other prostate cancer cell lines but this occurs through a pathway that bypasses the KPC1 ligase.


Identification of distinctive patterns of USP19-mediated growth regulation in normal and malignant cells.

Lu Y, Bedard N, Chevalier S, Wing SS - PLoS ONE (2011)

Depletion of USP19 results in accumulation of p27Kip1 in DU145 cells.(A) p27Kip1 but not p21 or p16 accumulates in USP19-depleted DU145 cells. Equal amounts of protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. (B) Overexpression of USP19 in DU145 cells prevents USP19 siRNA induced increase of p27Kip1. Protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1B) were analyzed by immunoblotting with the indicated antibodies. Note that for the USP19 immunoblots, 300 µg of DU145-Vect protein and 20 µg of DU145-USP19 protein were used. (C) Silencing of USP19 does not increase p27Kip1 levels by increasing mRNA levels. Equal amounts of protein and RNA isolated from lysates of DU145 cells transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL) (as described in Fig. 1A) were analyzed by Western (top) and Northern (bottom) blots, respectively. (D) Silencing of USP19 stabilizes p27Kip1. DU145 cells were transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL). Forty-eight hours later, they were incubated with cycloheximide (CHX) to inhibit protein synthesis. At the indicated times, cells were lysed and proteins were analyzed by immunoblotting with the indicated antibodies. Shown are representative immunoblots and quantitation of p27Kip1 levels (means ± SE) from triplicate samples. *, rate of degradation of p27Kip1 was significantly inhibited in USP19 depleted DU145 cells (P<0.05) (two-way analysis of variance). (E) Levels of ubiquitin protein ligases, KPC1, Skp2 and Pirh2 are not affected upon USP19 depletion in DU145 cells. Equal amounts of protein from lysates (as in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. Quantification was performed on triplicate samples and did not reveal any significant differences in the levels of the ligases.
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pone-0015936-g003: Depletion of USP19 results in accumulation of p27Kip1 in DU145 cells.(A) p27Kip1 but not p21 or p16 accumulates in USP19-depleted DU145 cells. Equal amounts of protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. (B) Overexpression of USP19 in DU145 cells prevents USP19 siRNA induced increase of p27Kip1. Protein from lysates of cells transfected with control or USP19 siRNA oligonucleotides (as described in Fig. 1B) were analyzed by immunoblotting with the indicated antibodies. Note that for the USP19 immunoblots, 300 µg of DU145-Vect protein and 20 µg of DU145-USP19 protein were used. (C) Silencing of USP19 does not increase p27Kip1 levels by increasing mRNA levels. Equal amounts of protein and RNA isolated from lysates of DU145 cells transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL) (as described in Fig. 1A) were analyzed by Western (top) and Northern (bottom) blots, respectively. (D) Silencing of USP19 stabilizes p27Kip1. DU145 cells were transfected with USP19 siRNA oligonucleotide h1 or control oligonucleotide (CTL). Forty-eight hours later, they were incubated with cycloheximide (CHX) to inhibit protein synthesis. At the indicated times, cells were lysed and proteins were analyzed by immunoblotting with the indicated antibodies. Shown are representative immunoblots and quantitation of p27Kip1 levels (means ± SE) from triplicate samples. *, rate of degradation of p27Kip1 was significantly inhibited in USP19 depleted DU145 cells (P<0.05) (two-way analysis of variance). (E) Levels of ubiquitin protein ligases, KPC1, Skp2 and Pirh2 are not affected upon USP19 depletion in DU145 cells. Equal amounts of protein from lysates (as in Fig. 1A) were analyzed by immunoblotting with the indicated antibodies. Quantification was performed on triplicate samples and did not reveal any significant differences in the levels of the ligases.
Mentions: Since depletion of USP19 delayed G0/G1 to S progression, we explored possible mechanisms by assessing the steady-state levels of CKIs that inhibit G0/G1-S transition. USP19 siRNA, but not control siRNA led to an accumulation of p27Kip1, but not p16 or p21 as exemplified in the DU145 model (Fig. 3A). To confirm that this increased p27Kip1 was specific to USP19 depletion, USP19 siRNA was transfected in the stable DU145 cell line expressing rat USP19. In agreement with above, depletion of USP19 in DU145 cells expressing empty vector led to an induction of p27Kip1. However, in cells overexpressing rat USP19, the same siRNA treatment was unable to upregulate p27Kip1 (Fig. 3B), confirming that the upregulation of p27Kip1 is a specific response to USP19 depletion. This increased p27Kip1 induced by USP19 depletion did not appear to be due to increased gene transcription, as Northern blot analysis revealed that p27Kip1 mRNA levels did not change (Fig. 3C). These data suggest an involvement of USP19 in regulating the turnover of p27Kip1. To test this possibility, the rate of disappearance of p27Kip1 was measured in DU145 cells with or without USP19 depletion following inhibition of protein synthesis with cycloheximide. Indeed, in cells transfected with USP19 siRNA p27Kip1 levels remained relatively stable over time whereas the half-life of the protein was significantly lower in controls (Fig. 3D), implying that USP19 modulates the stability of p27Kip1. To determine whether the upregulation and stabilization of p27Kip1 in USP19 depleted DU145 cells was linked to changes in KPC1 expression as we previously reported [20] or the other E3s, Skp2 and Pirh2, also responsible for the ubiquitination of p27Kip1, we measured the levels of these proteins in response to USP19 depletion. Unexpectedly, we did not observe any changes in KPC1 (Fig. 3E), nor in Skp2 and Pirh2 levels (Fig. 3E). These observations indicate that USP19 can act through a KPC1 and also Skp2 and Pirh2 independent pathway(s) while still modulating cell growth and p27Kip1. Of interest the silencing of USP19 in PC-3 and 22RV1 cells, which also led to an inhibition of their proliferation resulted in a similar upregulation of p27Kip1 expression but also no changes in levels of the three ubiquitin ligases, KPC1, Skp2 or Pirh2 (Fig. S3). These results suggest that USP19 may lose its ability to regulate growth such as in the LNCaP cells or still exert this function through p27Kip1 as in a variety of other prostate cancer cell lines but this occurs through a pathway that bypasses the KPC1 ligase.

Bottom Line: Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels.This may occur through both KPC1 dependent and independent mechanisms.Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

View Article: PubMed Central - PubMed

Affiliation: Polypeptide Laboratory, Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27(Kip1). To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27(Kip1). However, in contrast to previous findings in fibroblasts, the stabilization of p27(Kip1) upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27(Kip1) and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

Show MeSH
Related in: MedlinePlus