Hiroshi Matsuzaki

Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux.

Regulation of intracellular Ca(2+) concentration is critical in numerous biological processes. Inositol 1,4,5-trisphosphate receptor (IP3R) functions as the Ca(2+) release channel on endoplasmic reticulum (ER) membranes. Much attention has been dedicated to mitochondrial Ca(2+) uptake via mitochondria-associated ER membranes (MAM) which is involved in intracellular Ca(2+) homeostasis; however, the molecular mechanisms that link the MAM to mitochondria still remain elusive. We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) expressed in lymphocytes physically interacts with IP3R. In this study, we first performed double-immunocytochemical staining of Tespa1 with a mitochondrial marker or an ER marker on an acute T lymphoblastic leukemia cell line, Jurkat cells, by using anti-ATP synthase or anti-calnexin antibody, respectively, and demonstrated that Tespa1 was localized very close to mitochondria and the Tespa1 localization was overlapped with restricted portion of ER. Next, we examined the effects of Tespa1 on the T cell receptor (TCR) stimulation-induced Ca(2+) flux by using Ca(2+) imaging in Jurkat cells. Reduction of Tespa1 protein by Tespa1-specific siRNA diminished TCR stimulation-induced Ca(2+) flux into both mitochondria and cytoplasm through the analyses of the mitochondrial Ca(2+) indicator (Rhod-2) and the cytoplasmic Ca(2+) indicator (Fluo-4), respectively. Furthermore, co-immunoprecipitation assay in HEK293 cells revealed that exogenous Tespa1 protein physically interacted with a MAM-associated protein, GRP75 (glucose-regulated protein 75), but not with an outer mitochondrial membrane protein, VDAC1 (voltage-dependent anion channel 1). All these results suggested that Tespa1 will participate in the molecular link between IP3R-mediated Ca(2+) release and mitochondrial Ca(2+) uptake in the MAM compartment.

Inhibition of Phosphodiesterase-4 (PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model

BackgroundWe previously established a three-dimensional (3-D) colonic crypt model using HKe3 cells which are human colorectal cancer (CRC) HCT116 cells with a disruption in oncogenic KRAS, and revealed the crucial roles of oncogenic KRAS both in inhibition of apoptosis and in disruption of cell polarity; however, the molecular mechanism of KRAS-induced these 3-D specific biological changes remains to be elucidated.ResultsAmong the genes that were upregulated by oncogenic KRAS in this model, we focused on the phosphodiesterase 4B (PDE4B) of which expression levels were found to be higher in clinical tumor samples from CRC patients in comparison to those from healthy control in the public datasets of gene expression analysis. PDE4B2 was specifically overexpressed among other PDE4 isoforms, and re-expression of oncogenic KRAS in HKe3 cells resulted in PDE4B overexpression. Furthermore, the inhibition of PDE4 catalytic activity using rolipram reverted the disorganization of HCT116 cells into the normal physiologic state of the epithelial cell polarity by inducing the apical assembly of ZO-1 (a tight junction marker) and E-cadherin (an adherens junction marker) and by increasing the activity of caspase-3 (an apoptosis marker) in luminal cavities. Notably, rolipram reduced the AKT phosphorylation, which is known to be associated with the disruption of luminal cavity formation and CRC development. Similar results were also obtained using PDE4B2-shRNAs. In addition, increased expression of PDE4B mRNA was found to be correlated with relapsed CRC in a public datasets of gene expression analysis.ConclusionsThese results collectively suggested that PDE4B is upregulated by oncogenic KRAS, and also that the inhibition of PDE4 catalytic activity can induce both epithelial cell polarity and luminal apoptosis in CRC, thus highlighting the utility of our 3-D culture (3 DC) model for the KRAS-induced development of CRC in 3-D microenvironment. Indeed, using this model, we found that PDE4B is a promising candidate for a therapeutic target as well as prognostic molecular marker in CRC. Further elucidation of the signaling network of PDE4B2 in 3 DC would provide a better understanding of CRC in vivo.

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

Tespa1 is a novel inositol 1,4,5-trisphosphate receptor binding protein in T and B lymphocytes

Tespa1 has been recently reported to be a critical molecule in T‐cell development, however, the precise molecular mechanisms of Tespa1 remain elusive. Here, we demonstrate that Tespa1 shows amino‐acid sequence homology to KRAS‐induced actin‐interacting protein (KRAP), an inositol 1,4,5‐trisphosphate receptor (IP3R) binding protein, and that Tespa1 physically associates with IP3R in T and B lymphocytes. Two‐consecutive phenylalanine residues (Phe185/Phe186) in Tespa1, which are conserved between Tespa1 and KRAP, are indispensable for the association between Tespa1 and IP3R. These findings suggest that Tespa1 plays critical roles in the immune system through the regulation of the IP3R.

Zfat-Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfat f/f-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfat f/f-LckCre mice resulted in a reduction in the number of CD4+CD8+double-positive (DP) cells, CD4+single positive cells and CD8+single positive cells. Indeed, in Zfat f/f-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.

Tespa1 protein is phosphorylated in response to store-operated calcium entry

We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) protein expressed in lymphocytes physically interacts with IP3R (Inositol 1,4,5-trisphosphate receptor), a Ca(2+) channel protein spanning endoplasmic reticulum (ER) membrane. However, the biochemical characterization of Tespa1 protein remains unknown. In this study, we have found that Tespa1 protein was posttranslationally modified upon intracellular Ca(2+) increase in thymocytes. Through the analyses using various inhibitors, store-operated Ca(2+) entry (SOCE) was found to be an essential factor for the Tespa1 protein modification induced by T cell receptor (TCR)-stimulation. Remarkably, the Ca(2+)-dependent Tespa1 protein modification was restored by in vitro protein phosphatase treatment, indicating that this modification was due to phosphorylation. Moreover, we examined whether Ca(2+)-dependent phosphorylation of Tespa1 protein would affect the physical association between Tespa1 and IP3R proteins, revealing that physical association of these proteins is maintained regardless of the presence or absence of phosphorylation of Tespa1. In addition, KRAP protein which represents substantial amino acid sequence homology to Tespa1 was also posttranslationally phosphorylated by intracellular Ca(2+) increase in HCT116 human colon cancer cells and HEK293 human embryonic kidney cells, suggesting that common signaling mechanism(s) may contribute to the molecular modification of Tespa1 and KRAP in different cellular processes. All these results suggested a novel molecular modification of Tespa1 and the existence of the regulatory pathway that SOCE affects the Tespa1-IP3R molecular complex.

Zfat‐Deficient CD4+CD8+ Double‐Positive Thymocytes Are Susceptible to Apoptosis With Deregulated Activation of p38 and JNK

Zfat, which is a nuclear protein harboring an AT‐hook domain and 18‐repeats of C2H2 zinc‐finger motif, is highly expressed in immune‐related tissues, including the thymus and spleen. T cell specific deletion of the Zfat gene by crossing Zfatf/f mice with LckCre mice yields a significant reduction in the number of CD4+CD8+ double‐positive (DP) thymocytes. However, physiological role for Zfat in T cell development in the thymus remains unknown. Here, we found that Zfat‐deficient DP thymocytes in Zfatf/f‐LckCre mice were susceptible to apoptosis both at an unstimulated state and in response to T cell receptor (TCR)‐stimulation. The phosphorylation levels of p38 and JNK were elevated in Zfat‐deficient thymocytes at an unstimulated state with an enhanced phosphorylation of ATF2 and with an over‐expression of Gadd45α⋅ On the other hand, the activation of JNK in the Zfat‐deficient thymocytes, but not p38, was strengthened and prolonged in response to TCR‐stimulation. All these results demonstrate that Zfat critically participates in the development of DP thymocytes through regulating the activities of p38 and JNK. J. Cell. Biochem. 116: 149–157, 2015.

Zfat expression in ZsGreen reporter gene knock‑in mice: Implications for a novel function of Zfat in definitive erythropoiesis

Zinc finger and AT-hook domain containing (Zfat) is a transcriptional regulator harboring an AT-hook domain and 18 repeats of a C2H2 zinc-finger motif, which binds directly to the proximal region of transcription start sites in Zfat-target genes. It was previously reported that deletion of the Zfat gene in mice yields embryonic lethality by embryonic day 8.5 and impairs primitive hematopoiesis in yolk sac blood islands. In addition, Zfat has been reported to be involved in thymic T-cell development and peripheral T-cell homeostasis. In the present study, in order to obtain a precise understanding of the expression and function of Zfat, a knock-in mouse strain (ZfatZsG/+ mice), which expressed ZsGreen in the Zfat locus, was established. ZsGreen signals in tissues and cells of ZfatZsG/+ mice were examined by flow cytometric and histological analyses. Consistent with our previous studies, ZsGreen signals in ZfatZsG/+ mice were detected in the embryo and yolk sac blood islands, as well as in thymocytes, B and T cells. In the ZfatZsG/+ thymus, ZsGreen+ cells were identified not only in T-cell populations but also in thymic epithelial cells, suggesting the role of Zfat in antigen-presenting cells during thymic T-cell development. ZsGreen signals were observed in definitive erythroid progenitor cells in the fetal liver and adult bone marrow of ZfatZsG/+ mice. The proportion of ZsGreen+ cells in these tissues was highest at the early stage of erythroid differentiation, suggesting that Zfat serves particular roles in definitive erythropoiesis. Histological studies demonstrated that ZsGreen signals were detected in the pyramidal cells in the hippocampal CA1 region and the Purkinje cells in the cerebellum, suggesting novel functions of Zfat in nervous tissues. Taken together, these results indicated that the ZfatZsG/+ reporter mouse may be considered a useful tool for elucidating the expression and function of Zfat.

A CASE OF ADRENAL ADENOMATOID TUMOR DIAGNOSED BY IMMUNOHISTOCHEMICAL EVALUATION

An adenomatoid tumor is a benign tumor of mesothelial derivation, typically found in the genital track. However, though extremely rare, an adenomatoid tumor can be found in the adrenal gland, making it difficult to clinically and radiologically differentiate it from an adrenocortical tumor or a pheochromocytoma prior to surgery. We encountered a-52-year old man with an adenomatoid tumor in the adrenal gland, who presented with an incidentally discovered left adrenal mass revealed by PET-CT from his regular health examinations. He had been diagnosed with paroxysmal hypertension two years before and was being treated with a hypolipidemic agent. Abdominal computed tomography revealed a left adrenal mass measuring 25 × 15 mm, and the findings were different from the typical adrenocortical adenoma or pheochromocytoma. Although laboratory examinations of his blood samples indicated normal adrenal function, 24-hour urine specimens revealed high levels of 17-OHCS, 17-KS, and catecholamine. Both 131I-MIBG scintigraphy and phentolamine tests showed negative findings. The patient underwent a laparoscopic left adrenalectomy. The cut surface of the left adrenal gland weighing 21 g contained a white, solid mass measuring 25 × 15 × 20 mm within the adrenocortical tissue. Histologically, the tumor was composed of small tubules lined by eosinophilic tumor cells. The tumor cells were immunohistochemically positive for cytokeratins and calretinin, but negative for steroidogenic factor-1. Therefore, based on these findings, we diagnosed this tumor as an adrenal adenomatoid tumor. Histopathologically, the adrenal adenomatoid tumor may be difficult to distinguish from an adrenocortical adenoma, carcinoma, lymphangioma, hemangioma, angiosarcoma, or metastatic adenocarcinoma. Under these conditions, immunohistochemical examination is useful for definite diagnosis.