FHL1C induces apoptosis in notch1-dependent T-ALL cells through an interaction with RBP-J
- Wei Fu†1,
- Kai Wang†1,
- Jun-Long Zhao†2,
- Heng-Chao Yu2,
- San-Zhong Li2,
- Yan Lin1,
- Liang Liang2,
- Si-Yong Huang1,
- Ying-Min Liang1Email author,
- Hua Han2Email author and
- Hong-Yan Qin2Email author
© Fu et al.; licensee BioMed Central Ltd. 2014
Received: 5 September 2013
Accepted: 17 June 2014
Published: 22 June 2014
Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSIs) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear.
Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software.
In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression.
Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL.
KeywordsT-cell acute lymphoblastic leukemia Notch signaling FHL1C RBP-J Apoptosis
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm that originates from immature T-cells. Although the currently used multi-agents chemotherapy results in 5-year relapse-free survival rates of over 75% in children and over 50% in adults, relapse usually is associated with resistances against chemotherapy and a very poor prognosis [1–3]. Therefore, it is essential to elucidate the molecular mechanisms underlying T-ALL progression to discover new therapeutic targets for the treatment of T-ALL.
Mutations in the Notch1 receptor have been demonstrated as the etiological cause of T-ALL [4, 5]. The first evidence of oncogenic Notch signaling was observed in T-ALL patients, involving translocation of a portion of the human Notch1 gene to the TCR locus . However, this event is rare in human T-ALL (less than 1%). In fact, more than 50% of T-ALL patients carry Notch1-activating mutations that are usually in the heterodimerization (HD) domain and proline/glutamic acid/serine/threonine-rich motifs (PEST) of the Notch1 receptor, which result in delayed degradation of Notch1 . Notch1 is one of the four mammalian Notch receptors that are single-pass transmembrane proteins consisting of functional extracellular, transmembrane, and intracellular domains. When the Notch receptor is triggered upon interaction with its ligands on neighboring cells, the Notch intracellular domain (NIC) is released from the membrane after proteolytic cleavages executed by γ-secretase-containing protease complexes. The NIC enters the nucleus and associates with the DNA-binding transcription factor RBP-J through its N-terminal RAM (RBP-J association molecule) domain, which transactivates promoters harboring RBP-J-binding sites by dissociating co-repressors, such as SMRT/N-CoR, HDAC, and MINT [1, 8], and recruiting co-activators including Mastermind-like (MAML) and p300/CBP . In T-ALL, activated Notch1 regulates cell proliferation and apoptosis by modulating the level and activities of the related molecules/pathways such as Hes1, c-Myc, PI3K/AKT, and NF-κB through canonical (RBP-J-dependent) and/or non-canonical (RBP-J-independent) signals [10, 11].
Considering the critical role of Notch activation in the progression of T-ALL, efforts have been made to cure T-ALL by blocking Notch signaling. Small molecule γ-secretase inhibitors (GSIs), which block the critical proteolytic steps required for Notch activation, can be applied for T-ALL treatment, but the clinical outcomes have been unsatisfactory. These outcomes might be attributed to the fact that γ-secretase is not specific for Notch receptors, and more importantly, GSIs only affect ligand-dependent Notch activation, not ligand-independent Notch activation resulting from chromosome translocation or point mutations. In addition, gastrointestinal toxicity and weak anti-leukemic effects on T-ALL also hinder the clinical application of GSIs [12, 13]. Another target for blocking Notch signaling in malignant T cell leukemia is RBP-J that mediates the effects of Notch1 mutants on downstream gene expression. Expression of a dominant-negative MAML1 (DN-MAML1) in T-ALL cell lines has been shown to antagonize Notch1 activation [14, 15]. Subsequently, Moellering et al. designed a stable α-helical peptide derived from MAML1 (SAHM1) based on the structure of DN-MAML1. They found that SAHM1 directly impedes assembly of the Notch1 transactivation complex in the nucleus and reduces malignant cell proliferation and promotes apoptosis. In contrast to GSIs, DN-MAML1 and SAHM1 inhibit Notch activation more efficiently because of their direct inhibition of Notch signals at the transcriptional factor level. However, as a multifunctional transcription activator, MAML1 is also not specific for Notch signaling . Thus, more effective Notch signal inhibitors are still required for the treatment of T-ALL.
Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) belongs to the four-and-a-half LIM domain protein family and is an alternatively spliced form of FHL1A/KyoT1. Selective use of exons results in a frame shift in translation, generating a WW-containing motif at the C-terminus of FHL1C, which can bind to RBP-J. Without a transcription activation domain, FHL1C/KyoT2 has been demonstrated to compete with NIC for RBP-J binding and suppress RBP-J-mediated Notch activation in vitro . These findings suggest that FHL1C may be another therapeutic target of T-ALL, but the role of FHL1C remains to be investigated in T-ALL cells. In the present study, we addressed this issue using T-ALL clinical samples and the T-ALL cell line Jurkat. We found that the expression level of FHL1C was lower in the peripheral blood mononuclear cells (PBMCs) of T-ALL patients than that in the controls. Overexpression of FHL1C or its various truncates containing the RBP-J-binding site or the minimal RBP-J-binding motif, all resulted in Jurkat cell apoptosis. Mechanistically, FHL1C-induced Jurkat cell apoptosis involved suppression of downstream target genes and key pathways of Notch signaling in T-ALL, including PI3K-AKT and NF-κB. These findings shed light on the design of new Notch inhibitors based on FHL1C to treat T-ALL.
Total RNA was extracted from a human skeletal muscle biopsy and then reverse transcribed using a commercially available kit from TAKARA (Dalian, China) with an oligo-dT primer. This patient had signed informed consent, and the protocol involving human samples was approved by the Ethics Committee of Tangdu Hospital, Fourth Military Medical University. FHL1C (GeneBank accession number: AF220153.1) was amplified by PCR with specific primers (Forward primer, 5′-ATGGCGGAGAAGTTTGACTGCCACTACT-3′; Reverse primer, 5′-TCACGGAGCATTTTTTGCAGTGGAAGCA-3′) (Additional file 1: Table S1). The 585 bp PCR product was cloned and confirmed by DNA sequencing. The full-length FHL1C cDNA was inserted into the expression vectors pEGFP-C1 (Clontech, Mountain View, CA) and pCMV-Myc (Clontech) to generate pEGFP-FHL1C and pCMV-Myc-FHL1C, respectively.
To construct EGFP-tagged truncates of FHL1C, LIM1, LIM2, and the C-terminal RBP-J-binding motif (RBPmotif) of FHL1C, various fragments were subcloned by PCR with the primers listed in Additional file 1: Table S1, and pEGFP-FHL1C expression vector was used as the template. The LIM1 and LIM2 domains were fused in frame at the 3′ terminus to the RBPmotif to generate LIM1R and LIM2R, respectively. LIM1R, LIM2R, and RBPmotif were then inserted in frame into pEGFP-C1 to generate pEGFP-LIM1R, pEGFP-LIM2R, and pEGFP-RBPmotif (Additional file 2: Figure S3A). To construct vectors for expression of EGFP fused to the minimal RBPmotif of FHL1C, double-stranded oligonucleotides encoding VWWPM, PVWWPMK, and APVWWPMKD peptides were synthesized and cloned in frame downstream of EGFP in pEGFP-C1. The plasmids were confirmed by DNA sequencing.
Patients, RNA extraction, RT-PCR, Sequencing
Blood samples were collected from T-ALL patients and normal healthy individuals (Additional file 3: Tables S3 and Additional file 4: Table S4). All patients and normal individuals involved in the study had signed informed consents for the use of their blood samples, except for children under the age of 18, who had their informed consents signed by their parents as their representatives. The protocols involving human samples were approved by the Ethics Committee of Tangdu Hospital, Fourth Military Medical University. Diagnoses had been made according to standard morphological, immunological, and molecular genetics criteria. PBMCs were separated by Ficoll-Hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and Jurkat cells using Trizol reagent (Invitrogen, Carlsbad, CA), and then reverse transcribed using the commercially available kit with random primers. cDNA was diluted appropriately and used for PCR, GAPDH was used as an internal control. DNA sequences corresponding to the HD and PEST domains were amplified using nested PCR according to previous report , and then sequencing was performed by Biotechnology Company.
Real-time PCR was performed as triplicate using SYBR Premix EX Taq (TAKARA) with an ABI PRISM 7300 real-time PCR system (Applied Biosystems, Life Technologies, Carlsbad, CA) with β-actin as the reference control. Primers used for quantitative RT-PCR are listed in Additional file 5: Table S2.
Cell culture and transfection
Jurkat cells (ATCC, Rockville, MD) were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamate, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in saturated humidity with 5% CO2. HeLa and Cos7 cells (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) containing the supplements mentioned above.
HeLa and Cos7 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the recommended protocol. Jurkat cells (1 × 106) were transfected with a Nucleofector Kit V (Amaxa-Lonza, Cologne, Germany) using a Nucleofector I (program X-01) following the manufacturer’s optimized protocol.
HeLa or Cos7 cells were cultured in 24-well plates and transfected with 5 ng phRL-TK (Promega, Madison, WI), 80 ng pGa981-6 reporter plasmid , 200 ng pEF-BOS-Myc-NIC, and serial amounts (100, 300, and 500 ng) of plasmids carrying FHL1C or various truncates of FHL1C. The cells were harvested at 48 h post-transfection, and cell extracts were assayed for luciferase activity using a Gloma X™ 20/20 Luminometer (Promega). The luciferase activity was normalized to Renilla luciferase activity.
Flow cytometric analyses of cell cycle progression and apoptosis
Jurkat cells were resuspended in PBS and fixed in 70% ethanol on ice for 2 h. The cells were then stained with 20 mg/ml propidium iodide (PI) in PBS containing 0.1% Triton X-100 and 0.2 mg/ml RNase A for 30 min on ice. The cells were analyzed by a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA). Data were analyzed with CellQuest software.
Cell viability was routinely detected by trypan blue exclusion. Apoptosis was determined by staining with Annexin V-APC (eBiosciences, San Diego, CA) according to the manufacturer’s protocol, followed by flow cytometric analysis.
Co-immunoprecipitation and western blotting
pEGFP-FHL1C and pCMV-Myc-RBP-J were transfected into HeLa cells. Co-immunoprecipitation was performed as described previously  with an anti-Myc antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA). Western blotting was performed with anti-FHL1 (ProteinTech, Wuhan, China) or anti-Myc antibodies.
Western blotting analysis was performed routinely with primary antibodies including anti-AKT, anti-phospho-AKT (Signalway Antibody, Pearland, TX), anti-p50 (3354R-100; BioVision, Mountain View, CA), or anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Anti-rabbit IgG and anti-mouse IgG (Boster BioTec, Shanghai, China) were used as secondary antibodies. Anti-c-Rel, anti-IκBα antibodies were purchased from Eptiomics (Abcam, Burlingame, CA). An anti-caspase 3 antibody (H-277), anti-GFP antibody, normal goat IgG, and normal rabbit IgG were purchased from Santa Cruz Biotechnology.
Fractionation of subcellular components
Jurkat cells were washed twice with PBS at 4°C and then resuspended and incubated in buffer A (10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and a protease inhibitor cocktail) for 30 min on ice. After centrifugation at 4000 rpm for 20 min at 4°C, cytosolic fractions were collected, and the pellets were washed once in buffer A, resuspended in 1% NP-40 lysis buffer (10 mM Tris–HCl, pH 7.8, 0.5 mM EDTA, 250 mM NaCl, and the protease inhibitor cocktail), and then incubated for an additional 30 min on ice. After centrifugation at 10000 rpm for 15 min at 4°C, the nuclear factions were collected. Equal amounts of each fraction were analyzed by SDS-PAGE, followed by western blotting with the appropriate antibodies.
Cells were washed twice with PBS, fixed in 70% ethanol for 20 min, and then washed again with PBS. Hoechst diluted at 1:10,000 (final concentration: 0.12 μg/ml) was added to cells followed by incubation in the dark for 15 min. The cells were washed with PBS and visualized under a fluorescence microscope (BX51; Olympus, Tokyo, Japan).
Transmission electron microscopy (TEM)
Sample preparation and observation under a transmission electron microscope were performed as described previously .
Data were analyzed with SPSS version 12.0 software. Results were expressed as the mean ± SD. Comparisons between groups were performed with the unpaired Student’s t-test. A P-value of less than 0.05 was considered statistically significant.
FHL1C is down-regulated in PBMCs from T-ALL patients
Overexpression of FHL1C induces apoptosis of T-ALL cells
FHL1C induces apoptosis of Jurkat cells through suppression of RBP-J-mediated transactivation
Similar to its murine homolog KyoT2, FHL1C also possesses a C-terminal RBPmotif, suggesting that FHL1C interacts with RBP-J and suppresses RBP-J-mediated transactivation. To confirm an interaction between FHL1C and RBP-J, we performed co-immunoprecipitation. HeLa cells were co-transfected with expression vectors for Myc-tagged RBP-J (pCMV-Myc-RBP-J) and EGFP-tagged FHL1C (pEGFP-FHL1C), and immunoprecipitation was performed with an anti-Myc antibody. Co-precipitated proteins were detected using an anti-FHL1 antibody by western blotting analysis. The results showed that GFP-FHL1C was well co-precipitated with RBP-J (Additional file 7: Figure S2A), suggesting that FHL1C interacts with RBP-J. Furthermore, we performed reporter assays using HeLa and Cos7 cells by transfection with pEGFP-FHL1C and a NIC expression vector. As a result, overexpression of FHL1C suppressed transactivation of the reporter harboring RBP-J-binding sites by NIC in a dose-dependent manner (Additional file 7: Figure S2B). This result demonstrated that FHL1C suppresses RBP-J-mediated transactivation by competing with NIC.
The C-terminal RBPmotifof FHL1C is sufficient to induce apoptosis of Jurkat cells
FHL1C/KyoT2 is composed of two N-terminal LIM domains and a 27 amino acid RBPmotif at the C-terminus . To determine which domain of FHL1C is critical for FHL1C-induced apoptosis of Jurkat cells, various EGFP fusion proteins in which EGFP was fused to full-length FHL1C, LIM1R, LIM2R, or RBPmotif were transfected into HeLa cells and then visualized under a confocal fluorescence microscope. As a result, these fusion proteins showed similar subcellular localization (Additional file 2: Figure S3A and S3B). Next, we examined the effect of these fusion proteins on RBP-J-mediated transactivation using a reporter assay. The results showed that all of the fusion proteins exhibited a transcription suppression effect on RBP-J-mediated transactivation of the reporter gene (Additional file 2: Figure S3C), although the full-length FHL1C fusion protein had the strongest activity.
These results raised the possibility of developing small peptides to disrupt Notch signaling in T-ALL cells. Therefore, as the first step, we determined which sequence in the RBPmotif of FHL1C could induce Jurkat cell apoptosis. Oligonucleotides encoding various lengths of the RBPmotif were synthesized, fused to the C-terminus of EGFP (Figure 5D), and then overexpressed in Jurkat cells by transfection. All constructs exhibited suppression of Notch-mediated transcriptional activation in reporter assays, but the construct carrying EGFP fused to the VWWPM motif showed suppression comparable with that of full-length FHL1C (Figure 5E). We next examined apoptosis by annexin-V staining. In the GFP+ cell population, overexpression of EGFP-VWWPM efficiently induced apoptosis of Jurkat cells, although the other two fusion proteins had similar effects (Figure 5F). Consistently, overexpression of EGFP fused to various lengths of the RBPmotif resulted in a reduction of the number of transfected GFP+ Jurkat cells (Figure 5G). These results suggest that a minimal RBP-J-binding sequence composed of five amino acids (VWWPM) is enough to induce apoptosis of T-ALL cells.
Overexpression of FHLIC inhibits downstream genes and key pathways of notch signaling in T-ALL progression
Furthermore, we examined the effects of FHL1C overexpression on the activation of PI3K/AKT and NF-κB by western blotting, which are critical pathways activated by Notch1 in T-ALL [29, 30]. We found that overexpression of FHL1C in Jurkat cells reduced the phosphorylation of AKT (Figure 6C and D). Activation of NF-κB is closely associated with Notch1-dependent T-ALL. Therefore, we examined the levels of p50, c-Rel, and IκB in the cytosolic and nuclear fractions of FHL1C-overexpressing Jurkat cells by western blotting. The results showed that the levels of p50 and c-Rel decreased significantly in the nuclear fraction. IκB was found primarily in the cytosolic fraction and was also decreased slightly upon FHL1C overexpression (Figure 6E–G). This data suggest that FHL1C might down-regulate NF-κB activity by inhibiting nuclear translocation of p50 and c-Rel.
The identification of activating point mutations in Notch1 in more than 50% of T-ALL cases has spurred the development of therapies targeting the Notch1 signaling pathway for the treatment of T-ALL. To date, most of these efforts have focused on inhibiting the activity of γ-secretase, an enzyme that is essential for Notch receptor activation. Small molecule GSIs that inhibit γ-secretase activity have been tested in clinical trials and shown down-regulation of Notch1 target genes in T-ALL cells [7, 31]. However, GSIs are not selective for Notch1 signaling and block other Notch receptors and physiological pathways requiring γ-secretase. Indeed, patients have developed marked fatigue and dose-limiting gastrointestinal toxicity in clinical trials of GSIs, because of the inhibition of Notch1 and Notch2 in intestinal crypt progenitors and/or stem cells, resulting in premature differentiation into goblet cells . However, Real et al. subsequently showed that the gut toxicity can be ameliorated by combinatorial therapy using GSIs and glucocorticoids . To avoid the side effects of GSIs, antibodies have been developed to specifically block the Notch1 receptor . However, it has been demonstrated that the hotspot region of Notch1 mutations in T-ALL is the PEST domain located in the C-terminus of Notch1, which leads to delayed NIC degradation and thus prolonged Notch signaling. Therefore, these mutations are less sensitive to anti-Notch antibodies [30, 34]. In addition, some tumor cells harboring chromosomal translocations or other genetic aberrations might not be suitable for antibody-mediated therapy . In addition to PEST domain mutations, another region of Notch1 mutations in T-ALL is the NRR region including the LNR and HD domains, in which mutations lead to ligand hypersensitivity and ligand-independent activation . Although anti-NRR antibodies have been developed, sustained treatment with these antibodies will likely cause vascular neoplasms . More recently, Roti et al. demonstrated that inhibition of SERCA (sacro/endoplasmic reticulum Ca2+-ATPase) calcium pumps preferentially affects the maturation and activity of mutant Notch1 receptors, leading to enhanced clearance of the mutant Notch protein. Even if SERCA can be specifically targeted, such inhibition does not effect on T-ALL cells with activated Myc mutations or lacking NRR region .
The transactivation complex NIC-RBP-J-MAML1 is critical for signaling from Notch receptors, and is thus becoming a promising therapeutic target for T-ALL at the transcription level. Recently, Moellering et al. showed that SAHM1 suppresses the transcriptional complexes of Notch signaling. Treatment of leukemic cells with SAHM1 inhibits cell proliferation in vitro and in a Notch1-driven T-ALL mouse model without prominent gut toxicity . In the current study, we found that overexpression of FHL1C induced apoptosis of the Jurkat T-ALL cell line in vitro. FHL1C overexpression down-regulated c-Myc expression and attenuated the PI3K/AKT pathway and NF-κB signaling. These mechanisms may be involved in the enhanced apoptosis of Jurkat cells overexpressing FHL1C (Additional file 8: Figure S4), and suggest that FHL1C may be another therapeutic target for T-ALL at the transcriptional level. Moreover, it has been shown that Pten plays an important role in negative regulation of PI3K/AKT signaling in T-ALL. However, because Jurkat cells lack active Pten protein expression, it is possible that FHL1C can suppress AKT by other mechanisms such as disruption of the NICD-P56Lck-PI3K complex [30, 38, 39]. Further studies are needed to investigate whether FHL1C can inhibit AKT activation through Pten in native T-ALL cells.
FHL1 is a member of the FHL protein family that contains four-and-a-half LIM domains. FHL1 family members interact with many proteins through their LIM domains, including transcription factors, enzymes, and cytoskeleton proteins. These proteins play important roles in cell differentiation and cytoskeleton formation. Recent studies have shown that FHL1 also has important functions in tumorigenesis and cancer progression. FHL1 expression is suppressed in a variety of tumors including lung cancer, breast cancer, brain tumors, and gastric cancer [40, 41]. In contrast, some reports show that FHL1 is expressed at a high level in a squamous cell carcinoma cell line . FHL1 is aberrantly expressed in most T-ALL cell lines, particularly those exhibiting deregulated TLX1/HOX11 expression after specific chromosome translocation . In our study using PBMCs from T-ALL patients, we detected FHL1A expression in two cases, but the significance and underlying mechanism are unclear. We also detected significant down-regulation of FHL1C expression in PBMCs of T-ALL patient, accompanied by up-regulation of Hes1, a Notch target gene involved in T-ALL progression. These results suggest that FHL1C may be involved in T-ALL progression and can be used as a therapeutic target of the disease. However, the mechanism regulating FHL1C expression in T-ALL cells remains unknown, and whether FHL1C is involved in other cancers is unclear. In addition, although FHL1B (KyoT3) is another isoform of FHL1, which encodes a 34 kDa polypeptide containing the same RBPmotif found in FHL1C [44, 45], we did not detect FHL1B expression in T-ALL patients or normal healthy individuals.
FHL1C/KyoT2 encodes a 22 kDa protein sharing the two N-terminal LIM domains with FHL1A, and a 27 amino acid RBP-J-binding region at the C-terminus generated by alternative splicing. FHL1C/KyoT2 may participate in suppression of RBP-J-mediated Notch signaling by two mechanisms: competing with NIC for binding to RBP-J [17, 46] or recruitment of co-repressors. The LIM domain is a protein interaction interface that is involved in linking proteins with the actin cytoskeleton and/or transcriptional machinery [47, 48]. Our previous studies have shown that KyoT2 might suppress RBP-J-mediated Notch transactivation by recruiting the Polycomb suppression complex including RING1 and HPC2 through the LIM domains. Furthermore, KyoT2-mediated repression of Notch transactivation may be regulated by sumoylation involving PIAS1 [17, 46, 49]. In this study, we showed that overexpression of FHL1C induced apoptosis of Jurkat cells. Through a series of structure-function analyses, we found that such apoptosis was mainly mediated through the C-terminal RBPmotif of FHL1C, suggesting that competitive binding to RBP-J might be the major mechanism. Nevertheless, we cannot exclude the involvement of other interacting molecules. More importantly, we found that a minimal pentapeptide motif, VWWPM, suppressed RBP-J-mediated Notch activation and induced apoptosis of T-ALL cells at a relatively high efficiency. We expect that this peptide sequence will benefit future Notch-targeted therapies of T-ALL.
Taken together, our study revealed that overexpression of FHL1C induces Jurkat cell apoptosis. This finding may provide new insights into the design of new Notch inhibitors based on FHL1C to treat T-ALL in the future.
Four-and-half LIM domain protein1C
Hairy and enhancer of split
Notch intracellular domain
Peripheral blood mononuclear cells
RBP-J association molecule
Recombination signal binding protein-J
Stapled α-helical peptides derived from MAML1
Sacro/endoplasmic reticulum Ca2+-ATPase.
We are grateful to Prof. Tasuku Honjo for Plasmids. We thank Ms. Lei Feng and Ms. Xiu-Li Cao for technical assistance and Prof. Chun-Mei Wang for TEM experiment analysis. This study was carried out in the Graduates Innovation Center of Fourth Military Medical University. This work was support by grants from National Natural Science Foundation of China (81071874, 31101054, 81100354, 81300397).
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