Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2low Adult T-Cell Leukemia/Lymphoma.

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.

Peripheral T-Cell Lymphoma: From Biological Research to New Therapies.

This series of six articles (four reviews and two original articles) is presented by international leaders on peripheral T-cell lymphomas (PTCL) [...].

Identification and tracking of HTLV-1-infected T cell clones in virus-associated neurologic disease.

Human T lymphotropic virus type 1-assoicated (HTLV-1-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease caused by the persistent proliferation of HTLV-1-infected T cells. Here, we performed a T cell receptor (TCR) repertoire analysis focused on HTLV-1-infected cells to identify and track the infected T cell clones that are preserved in patients with HAM/TSP and migrate to the CNS. TCRß repertoire analysis revealed higher clonal expansion in HTLV-1-infected cells compared with noninfected cells from patients with HAM/TSP and asymptomatic carriers (ACs). TCR clonality in HTLV-1-infected cells was similar in patients with HAM/TSP and ACs. Longitudinal analysis showed that the TCR repertoire signature in HTLV-1-infected cells remained stable, and highly expanded infected clones were preserved within each patient with HAM/TSP over years. Expanded HTLV-1-infected clones revealed different distributions between cerebrospinal fluid (CSF) and peripheral blood and were enriched in the CSF of patients with HAM/TSP. Cluster analysis showed similarity in TCRß sequences in HTLV-1-infected cells, suggesting that they proliferate after common antigen stimulation. Our results indicate that exploring TCR repertoires of HTLV-1-infected cells can elucidate individual clonal dynamics and identify potential pathogenic clones expanded in the CNS.

Sodium ion influx regulates liquidity of biomolecular condensates in hyperosmotic stress response.

Biomolecular condensates are membraneless structures formed through phase separation. Recent studies have demonstrated that the material properties of biomolecular condensates are crucial for their biological functions and pathogenicity. However, the phase maintenance of biomolecular condensates in cells remains elusive. Here, we show that sodium ion (Na+) influx regulates the condensate liquidity under hyperosmotic stress. ASK3 condensates have higher fluidity at the high intracellular Na+ concentration derived from extracellular hyperosmotic solution. Moreover, we identified TRPM4 as a cation channel that allows Na+ influx under hyperosmotic stress. TRPM4 inhibition causes the liquid-to-solid phase transition of ASK3 condensates, leading to impairment of the ASK3 osmoresponse. In addition to ASK3 condensates, intracellular Na+ widely regulates the condensate liquidity and aggregate formation of biomolecules, including DCP1A, TAZ, and polyQ-protein, under hyperosmotic stress. Our findings demonstrate that changes in Na+ contribute to the cellular stress response via liquidity maintenance of biomolecular condensates.

Prognostic analysis of smoldering ATLL with skin eruptions based on genomic aberrations.

BACKGROUND: Patients with smoldering ATLL often present with a skin eruption due to skin infiltration of ATLL cells. Although skin eruption type is known to be associated with prognosis based on its pattern, it is unknown why different types of skin eruptions are associated with different prognoses. OBJECTIVE: Genomic analysis of patients with skin eruptions of smoldering ATLL will be performed to determine the mechanism of ATLL development and its association with prognosis. METHODS: DNA from skin biopsy specimens was used for targeted sequencing of 280 genes to examine the association between genomic variation and prognosis. RESULTS: Due to the small number of smoldering ATLL patients (27 cases), we could not find a clear relationship between skin eruption and prognosis in this study. Genomic analysis identified 247 genomic variants (108 genes), with an average of 9.2 variants and 3.2 variants as driver genes. Pathway analysis of the driver genes showed activation of the pathway associated with HTLV-1 infection, as well as activation of the signaling pathway observed throughout ATLL. Furthermore, multivariate analysis identified age>70 years and STAT3 mutation as prognostic risk factors and TBL1XR1 mutation as a risk factor for progression-free survival. CONCLUSION: Although the small number of patient samples did not allow us to determine a prognostic association with skin eruption, STAT3 mutation was identified as a prognostic risk factor for smoldering ATLL with skin eruption. Further studies are needed to increase the number of patients with this disease.

GPR56 C-terminal fragment mediates signal received by N-terminal fragment of another adhesion GPCR Latrophilin1 in neurons.

Adhesion GPCRs (aGPCRs) are a subfamily of GPCRs that are involved in cell adhesion, cell proliferation, and cell migration in various tissues. G protein-coupled receptor proteolytic site (GPS) of aGPCR is required to cleave the extracellular domain autocatalytically, generating two fragments; a N-terminal fragment (NTF) and a C-terminal fragment (CTF) containing seven transmembrane structure. NTF can interact with CTF non-covalently after cleavage, however the physiological significance of the cleavage of aGPCR at GPS, and also the interaction between NTF and CTF have not been fully clarified yet. In this study, we first investigated the expression profiles of two aGPCRs, GPR56/ADGRG1, and LPHN1/ADGRL1 in mouse brain, and found that the NTF and CTF of GPR56 independently expressed in different brain region at different developmental stages. Immunoprecipitation of GPR56CTF co-immunoprecipitated LPHN1NTF from mouse brain and HEK293T cells expressing both fragments. Stimulation with LPHN1 ligand, α-Latrotoxin N4C (αLTXN4C), to cells expressing LPHN1NTF and GPR56CTF increased intracellular Ca2+ concentration ([Ca2+ ]i). We also demonstrated that GPR56KO mouse neurons attenuated their Ca2+ response to αLTXN4C. These results suggest the possibility of functional and chimeric complex containing LPHN1NTF and GPR56CTF in neuronal signal transduction.

IMiD/CELMoD-induced growth suppression of adult T-cell leukemia/lymphoma cells via cereblon through downregulation of target proteins and their downstream effectors.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasia associated with human T-cell leukemia virus type 1 (HTLV-1) infection and has an extremely poor prognosis. Lenalidomide (LEN; a second-generation immunomodulatory drug [IMiD]) has been employed as an additional therapeutic option for ATL since 2017, but its mechanism of action has not been fully proven, and recent studies reported emerging concerns about the development of second primary malignancies in patients treated with long-term IMiD therapy. Our purpose in this study was to elucidate the IMiD-mediated anti-ATL mechanisms. Thirteen ATL-related cell lines were divided into LEN-sensitive or LEN-resistant groups. CRBN knockdown (KD) led to a loss of LEN efficacy and IKZF2-KD-induced LEN efficacy in resistant cells. DNA microarray analysis demonstrated distinct transcriptional alteration after LEN treatment between LEN-sensitive and LEN-resistant ATL cell lines. Oral treatment of LEN for ATL cell-transplanted severe combined immunodeficiency (SCID) mice also indicated clear suppressive effects on tumor growth. Finally, a novel cereblon modulator (CELMoD), iberdomide (IBE), exhibited a broader and deeper spectrum of growth suppression to ATL cells with efficient IKZF2 degradation, which was not observed in other IMiD treatments. Based on these findings, our study strongly supports the novel therapeutic advantages of IBE against aggressive and relapsed ATL.

The CGRP Receptor Antagonist MK0974 Induces EVI1high AML Cell Apoptosis by Disrupting ERK Signaling.

BACKGROUND/AIM: Acute myeloid leukemia (AML) with high expression of the oncogenic transcription factor ecotropic viral integration site-1 (EVI1) (EVI1high AML) is refractory, and there is an urgent need to develop treatment for EVI1high AML. We previously showed that calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein 1 (RAMP1) is highly expressed in EVI1high AML and participates in calcitonin gene-related peptide (CGRP)-induced stress hematopoiesis. This study examined whether MK0974 (a CGRP antagonist) acts as a therapeutic agent in CRLR/RAMP1high AML cell lines. MATERIALS AND METHODS: An in vitro experimental system was used to determine the effect of MK0974 on EVI1high AML cell lines. The expression of CRLR and RAMP1-3 in EVI1high and EVI1low AML lines was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). Next, MK0974 was added to the AML cell lines, and cell proliferation, cell cycle and apoptosis assays were carried out using flow cytometry (FCM). Proteins were evaluated using western blot analysis. We also generated AML cell lines with CRLR knockdown and evaluated whether the effect of MK0974 was reduced. RESULTS: Apoptosis was induced by adding MK0974 to the EVI1high AML cell line. In the EVI1high AML cell line, the addition of MK0974 attenuated the phosphorylation of ERK and p38. These effects were also attenuated by CRLR knockdown. CONCLUSION: MK0974, a CGRP receptor antagonist, inhibits the CRLR/RAMP1 complex and induces apoptosis, making it a potential therapeutic agent for CRLR/RAMP1high AML.

Activating Mutation in the Receptor Tyrosine Kinase FLT3 with Clinicopathological Relevance in Canine Mast Cell Tumors.

Recent research has focused on the receptor tyrosine kinase (RTK) KIT which is involved in the pathogenesis of canine mast cell tumors (MCT). However, the role of other RTKs in this neoplasm remains unclear. The present study aimed to determine the frequency of FLT3 mutations and to evaluate the mutational status and clinicopathological relevance of canine MCT patients. There were a total of 20 cases that were cytologically and histopathological diagnosed as canine MCTs; genomic polymerase chain reaction (PCR) and Sanger sequencing were used to identify mutations. For the juxtamembrane (JM) domain, the FLT3 14/15 primer pair was used to investigate exon 14/15 loci. Based on genomic PCR amplification of exon 14/15 and 20 of the FLT3 gene and Sanger sequencing of 20 cases of canine MCTs, the overall frequency of FLT3 mutation in canine MCTs was 75%. The majority of FLT3 mutations (70%) were internal tandem duplications (ITD) of the JM domain, while one case arose from deletion mutations of the tyrosine kinase domain (TKD). However, double mutations were not observed in this study. Furthermore, there is also clinicopathological relevance to MCT dogs carrying FLT3-ITD mutations, showing a tendency toward leukocytosis due to neutrophilia, and resembling human acute myeloid leukemia (AML) with FLT3-ITD mutations. A subset of MCTs with FLT3-ITD mutations, showing an enhanced signal of phosphorylated ERK1/2 identified by immunoblotting, suggests that an activating mutation may be driven by a distinct signal of the ERK pathway. Our results indicate that FLT3-ITD mutation is an oncogenic driver of canine MCTs, and that it shares some clinicopathologic features with human AML. These findings may offer new opportunities for further studies on canine mast cell tumorigenesis and a novel therapeutic target for canine MCT cases harboring FLT3-ITD mutations.

RAISING is a high-performance method for identifying random transgene integration sites.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes.

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection.

Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Oncogenic isoform switch of tumor suppressor BCL11B in adult T-cell leukemia/lymphoma.

B-Cell leukemia/lymphoma 11B (BCL11B) is a transcription factor important for T-cell development and acts as a tumor suppressor gene in T-cell acute lymphoblastic leukemia. Here, we identified BCL11B as a candidate leukemia-associated gene in human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma (ATLL). Interestingly, the short form lacking exon 3 (BCL11B/S) protein was more highly expressed than the full-length BCL11B (BCL11B/L) in leukemic cells from most of the ATLL patients, although expression ratios of BCL11B/L to BCL11B/S were almost equal in control CD4+ T cells. BCL11B/S and BCL11B/L exhibited distinct subcellular localization and differential effects on cellular growth; BCL11B/L expression exhibited nuclear localization and inhibited cell growth in ATLL cells, whereas BCL11B/S exhibited nucleocytoplasmic distribution and accelerated cell growth. Furthermore, BCL11B/S expression accelerated the development of T-cell leukemia/lymphomas in transgenic mice carrying HTLV-1/HBZ, a critical viral factor in leukemogenesis, whereas these phenotypes did not occur in the double transgenic mice carrying BCL11B/L and HTLV-1/HBZ. In HTLV-1-infected T-cell lines, BCL11B expression is downregulated by HTLV-1/Tax, a viral factor necessary at the early stage of leukemogenesis. These results suggest that downregulation of BCL11B/L expression and upregulation of BCL11B/S may contribute to the development and progression of ATLL.

Integrin α6A (ITGA6A)-type Splice Variant in Extracellular Vesicles Has a Potential as a Novel Marker of the Early Recurrence of Pancreatic Cancer.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide, with a poor prognosis. Owing to the difficulty of early diagnosis, the aim of this study was to isolate biomarkers from extracellular vesicles (EVs) that can lead to early diagnosis. MATERIALS AND METHODS: EVs in the culture supernatant were isolated from a pancreatic cancer cell line (PK-1) and expanded by using two-dimensional gel electrophoresis, and protein identification from each spot was performed by using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were classified and compared with previously reported results for EVs from murine pancreatic cancer PAN02 cells, and their expression specificity was examined using PDAC cell lines and patient-derived PDAC tissues. In addition, the significance of selected biomarker(s) was examined based on the changes in biomarkers in the blood EVs of PDAC patients after surgery. RESULTS: We found that the ITGA6A splice variant was predominantly expressed in several pancreatic cancer cell lines and blood EVs from patients with PDAC, whereas the ITGA6B splice variant was predominantly expressed in EVs from the blood of normal volunteers. In the expression pattern of ITGA6 in EVs from blood samples of two PDAC patients before and after resection surgery, the expression of ITGA6A in EVs significantly decreased after surgery and increased several months before clinical recurrence. Furthermore, the increased expression of ITGA6A in EVs occurred much earlier than that of CA19-9. CONCLUSION: Determination of ITGA6A expression in blood EVs in PDAC patients could be a useful blood marker for the early diagnosis of PDAC recurrence.

Effective Utilization of Vaccinium virgatum Aiton Stems as Functional Materials: Major Constituent Analysis and Bioactivity Evaluation.

We previously reported that rabbit-eye blueberry (Vaccinium virgatum Aiton) leaves exhibit multiple functions. In this study, we evaluated whether V. virgatum stems can also be used as functional materials similar to leaves and clarified the major constituents and their biological activity (antioxidant activity and anti-adult T cell leukemia (ATL) activity). Water extracts of V. virgatum stems were separated into 19 fractions using a Diaion HP-20 open column. Sugars and organic acids were detected in the highly water-soluble fractions. Polyphenols and proanthocyanidin were detected in the hydrous methanol-soluble fractions. In biological activity evaluations, a difference in antioxidant activity was observed in the water-containing methanol-eluted fractions, and fractions exhibiting anti-ATL activity differed depending on cell type. These results suggest that blueberry stems, like leaves, are rich in polyphenols and exhibit antioxidant activity and inhibit ATL cell growth. In the future, aerial parts of blueberries, including stems and leaves, could be used as functional materials and/or medicinal resources.

Inhibition of adult T-cell leukemia cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB-PAC) inhibits the proliferation of HTLV-1-infected adult T-cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB-PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV-1-infected and ATL cells. After hot water extraction with fractionation with methanol-acetone, BB-PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB-PAC polymerization degree of 14. The basic structure of BB-PAC is mainly B-type bonds, with A-type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome-dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome-dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB-PAC treatment. Therefore, BB-PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV-1-infected and ATL cells.

TAS-116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T-cell leukemia.

Adult T-cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T-cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor-κB (NF-κB)-mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti-ATL effects of a novel oral HSP90 inhibitor, TAS-116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS-116 achieved IC50 values of less than 0.5 µmol/L in 10 ATL-related cell lines and less than 1 µmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS-116 also showed significant inhibitory effects against tumor cell growth in ATL cell-xenografted mice. Furthermore, gene expression profiling of TAS-116-treated Tax-positive or -negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF-κB pathway in Tax-positive cells and cell-cycle arrest in Tax-negative cells and primary ATL cells. TAS-116 suppressed the activator protein-1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS-116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti-ATL therapeutic agent.

Role of Mel1/Prdm16 in bone differentiation and morphology.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16-/-) was embryonic lethal at E 11.5. Heterozygous mice showed abnormal cartilage and bone formation in the postnatal skull and long bones, suggesting that Mel1/Prdm16 expression plays an important role in bone development. In osteoblast and chondrocyte cell lines, Mel1/Prdm16 promotes the differentiation of chondrocytes and regulates the differentiation of osteoblasts. Transient repression of the master regulator Runx2 is required for chondrocyte differentiation at an early stage of differentiation. However, in Mel1/Prdm16-suppressed ATDC5 cells, the initial suppression of Runx2 was lacking and its expression was upregulated at the beginning of differentiation, suggesting that chondrogenic differentiation is suppressed in Mel1/Prdm16+/- mesenchymal progenitor cells because Runx2 expression is upregulated during the early stage of differentiation. Thus, the Mel1/Prdm16 gene may be involved in the early repression of Runx2 expression during osteochondral differentiation and promote chondrogenic differentiation.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

Expression of cell adhesion molecule 1 in human and murine endometrial glandular cells and its increase during the proliferative phase by estrogen and cell density.

AIMS: Cell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. MAIN METHODS: CADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. KEY FINDINGS: Immunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from -223 to -84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. SIGNIFICANCE: Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.