Notch3 and pre-TCR interaction unveils distinct NF-kappaB pathways in T-cell development and leukemia - PubMed (original) (raw)

Notch3 and pre-TCR interaction unveils distinct NF-kappaB pathways in T-cell development and leukemia

Alessandra Vacca et al. EMBO J. 2006.

Abstract

Notch signaling plays a critical role in T-cell differentiation and leukemogenesis. We previously demonstrated that, while pre-TCR is required for thymocytes proliferation and leukemogenesis, it is dispensable for thymocyte differentiation in Notch3-transgenic mice. Notch3-transgenic premalignant thymocytes and T lymphoma cells overexpress pTalpha/pre-TCR and display constitutive activation of NF-kappaB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF-kappaB activation pathways. A pre-TCR-dependent pathway preferentially activates NF-kappaB via IKKbeta/IKKalpha/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2-A1 and IL7-receptor-alpha genes. In contrast, upon pTalpha deletion, Notch3 binds IKKalpha and maintains NF-kappaB activation through an alternative pathway, depending on an NIK-independent IKKalpha homodimer activity. The consequent NF-kappaB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2-A1 and IL7-receptor-alpha genes. Our data suggest that a finely tuned interplay between Notch3 and pre-TCR pathways converges on regulation of NF-kappaB activity, leading to differential NF-kappaB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/leukemogenesis.

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Figures

Figure 1

Figure 1

Notch3 constitutively activates NF-κB and the balance between different dimeric complexes depends on the presence of pre-TCR. (A) CD4+ and/or CD8+ subset distribution (detected by CD4 versus CD8 two-color FCA) of thymocytes from 4-week-old wt and Notch3-IC transgenic mice (N3-IC), and 8-week-old Notch3-IC/pTα−/− mice (N3-IC/pTα−/−); (B) EMSA of NF-κB complex in nuclear extracts from freshly isolated thymocytes of wt, N3-IC transgenic and N3-IC/pTα−/− mice. (C) The nuclear extracts were also incubated in the absence (−) or presence (+) of antibodies against individual NF-κB proteins (p50, p65, p52 and RelB, left panel; c-Rel, right panel) to characterize the complexes. An unrelated antibody was used as a negative control (not shown).

Figure 2

Figure 2

Nuclear translocation of different NF-κB complexes in Notch3 transgenic and Notch3/pTα−/− mice. (A) Western blot analysis of p52, p100, p50 and RelB in cytoplasmic and nuclear extracts from thymocytes of wt, N3-IC transgenic and N3-IC/pTα−/− double-mutant mice. In total, 50 μg of protein was loaded into each lane. (B) Immunofluorescence with anti-p52 antibody (red) and Hoechst staining (blue) of freshly isolated thymocytes from wt, Notch3 transgenic and N3-IC/pTα−/− mice. (C) Nuclear extracts from freshly isolated thymocytes from 4-week-old wt and Notch3 transgenic mice, and 8-week-old N3-IC/pTα−/− mice were revealed by Western blot with an anti-phospho-p65 (left panel). Whole-cell extracts (400 μg) of freshly isolated thymocytes was immunoprecipitated with mouse monoclonal anti-p65 or rabbit polyclonal anti-p50 antibodies and revealed by Western blot with anti-phospho-p65 (right panels). (D) p52/p100 mRNA expression was assayed by RT–PCR from freshly thymocytes derived from wt, Notch3 transgenic and N3-IC/pTα−/− double-mutant mice, β-actin was used as loading control. (E) p100 processing in thymocytes derived from wt, Notch3 transgenic and N3-IC/pTα−/−. Upper panel, graphic representation of p100 processing as determined by densitometry; lower panel, pulse-labeled cells chased for 3 h.

Figure 3

Figure 3

Notch3 regulates the assembling and activation of different IKK complexes, depending on the presence of pre-TCR. (A) Western blot analysis of whole-cell extracts (50 μg) from freshly isolated thymocytes probed with anti-IκBα, IKKβ and anti-IKKα. The β-tubulin expression was used as loading control. (B) In vitro kinase assay using both GST-IκBα (left panel) and GST-p100 (right panel) as exogenous substrates performed on IKKα immunoprecipitation from cell lysates of thymocytes from 4-week-old wt and Notch3 transgenic mice, and 8-week-old N3-IC/pTα−/− mice. The upper panels show a graphic representation of both IκBα and p100 phospho-substrates amount as determined by densitometry. (C) Total cell extracts (400 μg) derived from wt, N3-IC and N3-IC/pTα−/− thymocytes were immunoprecipitated with mouse monoclonal anti-IKKα antibody and revealed in Western Blot with both anti-IKKα and anti- IKKβ antibodies. The upper panel shows a graphic representation of the IKKα/IKKβ complex amount as determined by densitometry. (D) Upper panel, Western blot analysis of whole-cell extracts (50 μg) from freshly isolated thymocytes probed with an anti-NIK antibody. Lower panel, total cell extracts (400 μg) derived from wt, N3-IC and N3-IC/pTα−/− thymocytes, immunoprecipitated with anti-NIK and revealed in Western blot with anti-Notch3 antibody. (E) Total cell extracts (400 μg) derived from wt, Notch3 and N3-IC/pTα−/− thymocytes were immunoprecipitated with mouse monoclonal anti-IKKα antibody and revealed in Western blot with both anti-NIK antibody (upper panel) and anti-Notch3 antibody (lower panel). (F) Western blot analysis of whole-cell extracts (50 μg) from freshly isolated thymocytes probed with anti-phospho-IKKα. The β-tubulin expression was used as loading control. (G) IκBα stability analysis in thymocytes derived from wt, Notch3 transgenic and N3-IC/pTα−/− by cycloheximide (CHX) treatment (right panel). The β-tubulin expression was used as loading control. Graphic representation of IκBα protein half-lives as determined by densitometry (left panel).

Figure 4

Figure 4

NF-κB target genes are differentially regulated by Notch3 depending on the presence or absence of pre-TCR. (A) Cyclin D1, (B) Bcl2 A1 and (C) IL7 receptor alpha expression was analyzed by RT–PCR from primary thymocytes derived from wt, Notch3 transgenic and N3-IC/pTα−/− double-mutant mice. β-Actin was used as loading control.

Figure 5

Figure 5

Notch3 induces the recruitment of different NF-κB complexes on gene promoters, depending on the presence of pre-TCR. Schematic representation of (A) cyclin D1, (B) Bcl2 A1 and (C) IL7 receptor α promoter regions assessed by chromatin immunoprecipitation are indicated. Primary T cells derived from Notch3 and N3-IC/pTα−/− mice were processed by using antibodies directed against p65, RelB, p52, and RNA polymerase II (pol II) or no antibody as control. Immunoprecipitated DNA was analyzed by PCR with (A) cyclin D1, (B) Bcl2 A1 and (C) IL-7 receptor α promoter-specific primers. The amount of input promoter DNA is also shown. The results are representative of three similar experiments.

Figure 6

Figure 6

Speculative model of the mechanisms by which Notch3 induces the activation of different NF-κB pathways. (A) Outline of the molecular events triggered by Notch3 in the presence of a functional pre-TCR. Signals arising from Notch3 and pTα/pre-TCR cooperation mainly involve the β and γ subunits of the IKK complex, via PKCθ, resulting in degradation of IκBα, nuclear translocation and DNA binding of p50/RelA heterodimers. These molecular events are strictly associated to canonical NF-κB activation pathway and result in prosurvival/proliferative gene activation. In the presence of pre-TCR, Notch3 also increases the association between IKKα and NIK, possibly leading to a further increase of the overall NF-κB activation, via the non-canonical pathway. (B) In the absence of pre-TCR Notch3 switches on another pathway, directly interacting with and upregulating IKKα function, which enhances the phosphorylation level of p52 precursor, p100, and results in a higher p52/RelB heterodimer nuclear translocation. As a consequence, p52/RelB heterodimers accumulate in the nucleus and regulate genes that are crucial for the survival and differentiation of thymocytes.

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