Takuya Hiratsuka

ZFP521 contributes to pre-B-cell lymphomagenesis through modulation of the pre-B-cell receptor signaling pathway.

ZFP521 was previously identified as a putative gene involved in induction of B-cell lymphomagenesis. However, the contribution of ZFP521 to lymphomagenesis has not been confirmed. In this study, we sought to elucidate the role of ZFP521 in B-cell lymphomagenesis. To this end, we used a retroviral insertion method to show that ZFP521 was a target of mutagenesis in pre-B-lymphoblastic lymphoma cells. The pre-B-cell receptor (pre-BCR) signaling molecules BLNK, BTK and BANK1 were positively regulated by the ZFP521 gene, leading to enhancement of the pre-BCR signaling pathway. In addition, c-myc and c-jun were upregulated following activation of ZFP521. Stimulation of pre-BCR signaling using anti-Vpreb antibodies caused aberrant upregulation of c-myc and c-jun and of Ccnd3, which encodes cyclin D3, thereby inducing the growth of pre-B cells. Stimulation with Vpreb affected the growth of pre-B cells, and addition of interleukin (IL)-7 receptor exerted competitive effects on pre-B-cell growth. Knockdown of BTK and BANK1, targets of ZFP521, suppressed the effects of Vpreb stimulation on cell growth. Furthermore, in human lymphoblastic lymphoma, analogous to pre-B-cell lymphoma in mice, the expression of ZNF521, the homolog of ZFP521 in humans, was upregulated. In conclusion, our data showed that the ZFP521 gene comprehensively induced pre-B-cell lymphomagenesis by modulating the pre-B-cell receptor signaling pathway.

Dual retrovirus integration tagging: identification of new signaling molecules Fiz1 and Hipk2 that are involved in the IL-7 signaling pathway in B lymphoblastic lymphomas

IL‐7R, FLT3, and CD43 are surface antigens expressed during the transition from pro‐B to pre‐B cells in BM. To understand interactions between their signaling pathways, we analyzed spontaneous mouse B‐LBLs with dual MLV integration into Stat5a and Fiz1 or Stat5a and Hipk2. MLV integration resulted in up‐regulation of these genes in lymphoma cells compared with normal pro‐B cells from the BM. In lymphomas with both integrations into Stat5a and Fiz1, increases in phosphorylated STAT5A and expression of c‐Myc, a target gene of STAT5A, were observed following stimulation of the FLT3. Clones with the dual integrations grew faster in IL‐7 and FLT3L‐supplemented medium than clones with Stat5a integration alone. On the other hand, in lymphomas with integrations into Stat5a and Hipk2, increases in phosphorylated STAT5A and expression of c‐Myc were observed following cross‐linking of CD43. In conclusion, FLT3 and CD43 signaling pathways involve STAT5A via Fiz1 and Hipk2 in B‐LBLs. Identification of the dual MLV integration sites in B‐LBLs, therefore, will provide an excellent tool for identification of the signaling pathways in B‐LBLs.

Murine leukemia retrovirus integration induces the formation of transcription factor complexes on palindromic sequences in the signal transducer and activator of transcription factor 5a gene during the development of pre-B lymphomagenesis.

Murine leukemia retrovirus (MLV) vectors are highly effective tools for introducing a foreign gene into a target host genome. However, it remains unclear how integrated retroviral promoter activity is influenced by the upstream or downstream sequences and how the host cell phenotype is influenced by the integrated promoter activity. Herein, we analyzed a set of pre-B lymphoma clones in which the MLV genome was integrated into the signal transducer and activator of transcription factor 5a (Stat5a) gene. Among the clones, the lymphoma clones with a provirus integrating into the middle position of the palindromic target sequences showed significantly higher transcription of the Stat5a gene; and p300 and other transcriptional factors formed complexes, with binding to the proviral-host junctional DNA segment. By using a luciferase assay, the upstream and downstream sequences of the provirus contributed to the up-regulation of proviral promoter activity. In concomitance with the higher Stat5a transcription, the immunoglobulin gene recombination was arrested. Antiapoptotic activity was significantly higher, with an increase in Bcl-xL, one of the targets of STAT5A, when IL-7 was supplied. Thus, a minute difference between MLV integration sites can lead to large differences in the host phenotype through the formation of transcription factor complexes on the proviral-host junctional DNA segment, suggesting that caution is necessary in monitoring integration sites when working with MLV vectors.

Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser‐ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal‐regulated kinase was activated at the core of contracting platelet aggregates.

Intravital imaging of mouse urothelium reveals activation of extracellular signal‐regulated kinase by stretch‐induced intravesical release of ATP

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal‐regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two‐photon excitation microscope and a vacuum‐stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch‐induced ERK activation was reproduced in an hTERT‐immortalized human urothelial cell line (TRT‐HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch‐induced ERK activation in TRT‐HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP‐induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor‐expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks.

In Vitro HIV-1 Selective Integration into the Target Sequence and Decoy-Effect of the Modified Sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5′-(GTCCCTTCCCAGT )n(ACTG GGAAGGGAC)n-3′, and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.

STAT5A Modulates Chemokine Receptor CCR6 Expression and Enhances Pre-B Cell Growth in a CCL20-Dependent Manner.

Signal transducer and activator of transcription 5A (STAT5A) contributes to B‐cell responses to cytokines through suppressor of cytokine signaling (Socs) genes in innate immunity. However, its direct roles in B‐cell responses to chemokines are poorly understood. In this study, we examined the role of STAT5A in the innate immune response. We found that STAT5A upregulated the transcription of C‐C motif receptor 6 (Ccr6) to induce responses to its ligand, CCL20. STAT5A transcriptional activity proceeded through binding to the interferon‐γ activation site (GAS) element in the CCR6 promoter in the genome of pre‐B cells. High levels of STAT5A and CCR6 increased CCL20‐dependent colony growth of pre‐B cells. In human B‐lymphoblastic lymphoma with inflammation, STAT5A phosphorylation was correlated with CCR6 expression (P > 0.05 compared with that in cases without inflammation). In conclusion, our data supported our hypothesis that STAT5A enhanced the response of pre‐B cells to CCL20 to promote their growth.   J. Cell. Biochem. 117: 2630–2642, 2016.

A Quantitative trait locus responsible for inducing B-cell lymphoblastic lymphoma is a hotspot for microsatellite instability

(Cancer Sci 2010; 101: 800–805)

Recurrence of bilateral diffuse bronchiectasis after bilateral lung transplantation.

Abstract:  We report two cases of bilateral diffuse bronchiectasis in which early recurrence of the original lung disease occurred after bilateral lung transplantation (LT). Patient 1 underwent cadaveric LT. Recurrent bronchiectasis occurred 4 months later, and he died 6 years after LT. Patient 2 underwent living‐related lobar LT, bronchiectasis relapsed 4 months later, and he died 13 months after LT. Both cases were finally diagnosed as bilateral diffuse bronchiectasis by the pathological features of the explanted lungs: infiltration of inflammatory cells predominantly in the conducting airways with dilation of the bronchi of bilateral lungs and scarcity of foamy macrophages in the wall of the respiratory bronchioles. Similar pathological features were seen in autopsy specimens from patient 1 and a transbronchial biopsy specimen from patient 2. LT should be carried out with caution in patients with bilateral diffuse bronchiectasis. When performing LT in such patients, it is suggested that sinusitis should be controlled perioperatively.

Reassessment of H&E stained clot specimens and immunohistochemistry of phosphorylated Stat5 for histological diagnosis of MDS/MPN

Summary Few studies have comprehensively analysed histopathological findings of bone marrow clots for diagnosis of haematopoietic cell dysplasia. In particular, a limited number of studies have assessed the use of haematoxylin and eosin (H&E) staining, which is generally considered less informative than May-Giemsa staining. In the current study, the utility of bone marrow clot specimens for diagnosis was examined using H&E staining and immunohistochemistry. Patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN), including chronic myelomonocytic leukaemia (CMML), atypical chronic myeloid leukaemia (aCML) lacking Philadelphia chromosome, and juvenile myelomonocytic leukaemia (JMML), were selected for histological evaluation. H&E stained specimens were advantageous for observation of atypical basophilic staining of the cytoplasm and nucleus related to dysplasia. This finding was significantly supported for both MDS and MDS/MPN (p < 0.05 versus May-Giemsa staining); therefore, we concluded that H&E staining could be used for identification of dysplastic cells. In addition, despite the loss of tissue structure, phosphorylated Stat5 immunostaining was sufficiently useful for the observation of myelodysplastic blasts. Thus, clot specimens are useful for diagnosis of haematopoietic dysplasia by pathologists.