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Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

Poalas K, Hatchi EM, Cordeiro N, Dubois SM, Leclair HM, Leveau C, Alexia C, Gavard J, Vazquez A, Bidère N - Cell Commun. Signal (2013)

Bottom Line: We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD).From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMR_S 1014, Hôpital Paul Brousse, Villejuif 94800, France. nicolas.bidere@inserm.fr.

ABSTRACT

Background: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated.

Findings: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.

Conclusions: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

No MeSH data available.


Related in: MedlinePlus

Identification of USP34 as a negative regulator of TCR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 98 DUBs (2 siRNA/target) in Jurkat T lymphocytes stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I, top panel), or with 0.5 μg.ml-1 anti-CD3 and anti-CD28 (bottom panel). Fold activation compared to non-targeting (n.t.) siRNA-treated cells is shown. Green and red histograms indicate siRNA against USP34 and CYLD, respectively. (B) Nuclear (Nucl.) and cytosolic (Cyt.) fractions from Jurkat T cells stimulated with P/I as in (A) for 0 and 15 min were analyzed by immunoblot. PARP and tubulin served as loading and purity controls for nucleus and cytosol, respectively. Molecular weight markers (M.W.) are indicated. (C) Lysates from Jurkat cells transfected for four days with siRNA against CYLD, USP34 (three individual sequences), or with control n.t. siRNA were analyzed by immunoblot as indicated. (D and E) NF-κB reporter luciferase assay (mean ± S.D. of triplicate experiments) of n.t.-, CYLD- or USP34-silenced Jurkat cells stimulated as in (A). RLU, Relative Light Units; Unst, unstimulated cells. (F) Cells as in (C) were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 1 and 2 hours. mRNA levels of NFKBIA (IκBα), IL-2, TNFα, and ACTIN were measured by RT-PCR. (G) Enzyme-Linked ImmunoSorbent Assay (ELISA) of IL-2 secreted in the supernatant of Jurkat treated as in (C) and stimulated with P/I. (H) NF-κB reporter luciferase assay of Jurkat cells transfected with the catalytic domain of USP34 (V5-tagged USP34-CD) or with a control empty vector (EV) and stimulated as indicated. Histograms represent mean ± S.D. of triplicate experiments. Inset blot shows USP34-CD expression when overexpressed in HEK293T cells.
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Figure 1: Identification of USP34 as a negative regulator of TCR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 98 DUBs (2 siRNA/target) in Jurkat T lymphocytes stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I, top panel), or with 0.5 μg.ml-1 anti-CD3 and anti-CD28 (bottom panel). Fold activation compared to non-targeting (n.t.) siRNA-treated cells is shown. Green and red histograms indicate siRNA against USP34 and CYLD, respectively. (B) Nuclear (Nucl.) and cytosolic (Cyt.) fractions from Jurkat T cells stimulated with P/I as in (A) for 0 and 15 min were analyzed by immunoblot. PARP and tubulin served as loading and purity controls for nucleus and cytosol, respectively. Molecular weight markers (M.W.) are indicated. (C) Lysates from Jurkat cells transfected for four days with siRNA against CYLD, USP34 (three individual sequences), or with control n.t. siRNA were analyzed by immunoblot as indicated. (D and E) NF-κB reporter luciferase assay (mean ± S.D. of triplicate experiments) of n.t.-, CYLD- or USP34-silenced Jurkat cells stimulated as in (A). RLU, Relative Light Units; Unst, unstimulated cells. (F) Cells as in (C) were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 1 and 2 hours. mRNA levels of NFKBIA (IκBα), IL-2, TNFα, and ACTIN were measured by RT-PCR. (G) Enzyme-Linked ImmunoSorbent Assay (ELISA) of IL-2 secreted in the supernatant of Jurkat treated as in (C) and stimulated with P/I. (H) NF-κB reporter luciferase assay of Jurkat cells transfected with the catalytic domain of USP34 (V5-tagged USP34-CD) or with a control empty vector (EV) and stimulated as indicated. Histograms represent mean ± S.D. of triplicate experiments. Inset blot shows USP34-CD expression when overexpressed in HEK293T cells.

Mentions: To identify additional negative regulators of TCR-mediated NF-κB activation, we conducted a siRNA library screen against 98 DUBs through a gene reporter luciferase assay in Jurkat T cells stimulated with either anti-CD3 and anti-CD28 antibodies or PMA plus ionomycin to mimic TCR engagement (Figure 1A and Additional files 1 and 2). As expected, CYLD silencing led to an enhanced NF-κB activity upon TCR stimulation (Figure 1A). Furthermore, this screening also uncovered siRNA sequences specific for USP34 that potentiated NF-κB activation with a similar magnitude to CYLD siRNA (Figure 1A). USP34 encompasses a 404 kDa protein with a central catalytic domain [11]. However, little is known about this DUB, albeit it was previously linked to the Wnt developmental signaling pathway [12]. Subcellular fractionation experiments showed that USP34 was essentially distributed in the cytosol of cells regardless of TCR stimulation, and was notably absent from the nucleus and organelles (Figure 1B and Additional file 3A). We next verified by immunoblot that CYLD and USP34 endogenous levels were efficiently decreased by their respective siRNA sequences (Figure 1C). Of note, an additional siRNA duplex specific for USP34 was also included to reinforce our initial findings (named sequence 3). Consistent with the primary screening, NF-κB reporter activity was similarly boosted upon TCR stimulation in USP34- and CYLD-silenced Jurkat when compared to control non-targeting siRNA transfected cells (Figure 1D and E). As a consequence, the levels of the NF-κB targets NFKBIA (IκBα), interleukin-2 (IL-2) and TNFα, as measured by RT-PCR were increased in USP34-knocked down cells (Figure 1F). Accordingly, downstream IL-2 secretion was enhanced in supernatants of USP34-silenced cells (Figure 1G). Finally, ectopic expression of a plasmid encoding for the catalytic domain of USP34 (USP34-CD [13]) markedly dampened TCR-mediated NF-κB activity (Figure 1H). Because USP34-CD is a large segment (383 amino acids), it is possible that in addition to the catalytic domain, it also comprises a domain required for the binding to its partners to regulate NF-κB in lymphocytes. Collectively, our data suggest that USP34 is a cytosolic protein, which functions as a negative regulator of NF-κB upon TCR engagement.


Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

Poalas K, Hatchi EM, Cordeiro N, Dubois SM, Leclair HM, Leveau C, Alexia C, Gavard J, Vazquez A, Bidère N - Cell Commun. Signal (2013)

Identification of USP34 as a negative regulator of TCR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 98 DUBs (2 siRNA/target) in Jurkat T lymphocytes stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I, top panel), or with 0.5 μg.ml-1 anti-CD3 and anti-CD28 (bottom panel). Fold activation compared to non-targeting (n.t.) siRNA-treated cells is shown. Green and red histograms indicate siRNA against USP34 and CYLD, respectively. (B) Nuclear (Nucl.) and cytosolic (Cyt.) fractions from Jurkat T cells stimulated with P/I as in (A) for 0 and 15 min were analyzed by immunoblot. PARP and tubulin served as loading and purity controls for nucleus and cytosol, respectively. Molecular weight markers (M.W.) are indicated. (C) Lysates from Jurkat cells transfected for four days with siRNA against CYLD, USP34 (three individual sequences), or with control n.t. siRNA were analyzed by immunoblot as indicated. (D and E) NF-κB reporter luciferase assay (mean ± S.D. of triplicate experiments) of n.t.-, CYLD- or USP34-silenced Jurkat cells stimulated as in (A). RLU, Relative Light Units; Unst, unstimulated cells. (F) Cells as in (C) were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 1 and 2 hours. mRNA levels of NFKBIA (IκBα), IL-2, TNFα, and ACTIN were measured by RT-PCR. (G) Enzyme-Linked ImmunoSorbent Assay (ELISA) of IL-2 secreted in the supernatant of Jurkat treated as in (C) and stimulated with P/I. (H) NF-κB reporter luciferase assay of Jurkat cells transfected with the catalytic domain of USP34 (V5-tagged USP34-CD) or with a control empty vector (EV) and stimulated as indicated. Histograms represent mean ± S.D. of triplicate experiments. Inset blot shows USP34-CD expression when overexpressed in HEK293T cells.
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Figure 1: Identification of USP34 as a negative regulator of TCR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 98 DUBs (2 siRNA/target) in Jurkat T lymphocytes stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I, top panel), or with 0.5 μg.ml-1 anti-CD3 and anti-CD28 (bottom panel). Fold activation compared to non-targeting (n.t.) siRNA-treated cells is shown. Green and red histograms indicate siRNA against USP34 and CYLD, respectively. (B) Nuclear (Nucl.) and cytosolic (Cyt.) fractions from Jurkat T cells stimulated with P/I as in (A) for 0 and 15 min were analyzed by immunoblot. PARP and tubulin served as loading and purity controls for nucleus and cytosol, respectively. Molecular weight markers (M.W.) are indicated. (C) Lysates from Jurkat cells transfected for four days with siRNA against CYLD, USP34 (three individual sequences), or with control n.t. siRNA were analyzed by immunoblot as indicated. (D and E) NF-κB reporter luciferase assay (mean ± S.D. of triplicate experiments) of n.t.-, CYLD- or USP34-silenced Jurkat cells stimulated as in (A). RLU, Relative Light Units; Unst, unstimulated cells. (F) Cells as in (C) were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 1 and 2 hours. mRNA levels of NFKBIA (IκBα), IL-2, TNFα, and ACTIN were measured by RT-PCR. (G) Enzyme-Linked ImmunoSorbent Assay (ELISA) of IL-2 secreted in the supernatant of Jurkat treated as in (C) and stimulated with P/I. (H) NF-κB reporter luciferase assay of Jurkat cells transfected with the catalytic domain of USP34 (V5-tagged USP34-CD) or with a control empty vector (EV) and stimulated as indicated. Histograms represent mean ± S.D. of triplicate experiments. Inset blot shows USP34-CD expression when overexpressed in HEK293T cells.
Mentions: To identify additional negative regulators of TCR-mediated NF-κB activation, we conducted a siRNA library screen against 98 DUBs through a gene reporter luciferase assay in Jurkat T cells stimulated with either anti-CD3 and anti-CD28 antibodies or PMA plus ionomycin to mimic TCR engagement (Figure 1A and Additional files 1 and 2). As expected, CYLD silencing led to an enhanced NF-κB activity upon TCR stimulation (Figure 1A). Furthermore, this screening also uncovered siRNA sequences specific for USP34 that potentiated NF-κB activation with a similar magnitude to CYLD siRNA (Figure 1A). USP34 encompasses a 404 kDa protein with a central catalytic domain [11]. However, little is known about this DUB, albeit it was previously linked to the Wnt developmental signaling pathway [12]. Subcellular fractionation experiments showed that USP34 was essentially distributed in the cytosol of cells regardless of TCR stimulation, and was notably absent from the nucleus and organelles (Figure 1B and Additional file 3A). We next verified by immunoblot that CYLD and USP34 endogenous levels were efficiently decreased by their respective siRNA sequences (Figure 1C). Of note, an additional siRNA duplex specific for USP34 was also included to reinforce our initial findings (named sequence 3). Consistent with the primary screening, NF-κB reporter activity was similarly boosted upon TCR stimulation in USP34- and CYLD-silenced Jurkat when compared to control non-targeting siRNA transfected cells (Figure 1D and E). As a consequence, the levels of the NF-κB targets NFKBIA (IκBα), interleukin-2 (IL-2) and TNFα, as measured by RT-PCR were increased in USP34-knocked down cells (Figure 1F). Accordingly, downstream IL-2 secretion was enhanced in supernatants of USP34-silenced cells (Figure 1G). Finally, ectopic expression of a plasmid encoding for the catalytic domain of USP34 (USP34-CD [13]) markedly dampened TCR-mediated NF-κB activity (Figure 1H). Because USP34-CD is a large segment (383 amino acids), it is possible that in addition to the catalytic domain, it also comprises a domain required for the binding to its partners to regulate NF-κB in lymphocytes. Collectively, our data suggest that USP34 is a cytosolic protein, which functions as a negative regulator of NF-κB upon TCR engagement.

Bottom Line: We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD).From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM UMR_S 1014, Hôpital Paul Brousse, Villejuif 94800, France. nicolas.bidere@inserm.fr.

ABSTRACT

Background: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated.

Findings: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.

Conclusions: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

No MeSH data available.


Related in: MedlinePlus