BACH1 regulates erythrophagocytosis and iron-recycling in β-thalassemia

Macrophages play essential roles in erythrophagocytosis and iron recycling. β-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of β-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in β-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with β-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.     

“Wild type” GIST: Clinicopathological features and clinical practice

Gastrointestinal stromal tumor (GIST) is a mesenchymal tumor of the gastrointestinal tract. Mutation of KIT and PDGFRA genes is implicated in the tumorigenesis. Approximately 10% of GISTs do not harbor mutation of these genes, and they are designated as “wild type” GIST. They are classified into succinate dehydrogenase (SDH)‐deficient and non‐SDH‐deficient groups. SDH‐deficient group includes Carney triad and Carney Stratakis syndrome. The patients are young women. Tumors occur in the antrum of the stomach, and tumor cells are epithelioid. Lymph node metastasis is frequent. The non‐SDH‐deficient group includes neurofibromatosis (NF) type 1 and GISTs with mutations of BRAF, KRAS, and PIK3CA and with the ETV6‐NTRK3 fusion gene. GIST in NF occurs in the small intestine, and tumor cells are spindle shaped. GIST with BRAF mutation arises in the small intestine. Attention to the age, gender, family history and other neoplasms may raise the prediction of syndromic disease. Location of the tumor, morphology, and pleomorphism of the tumor cells are further informative. Lymphovascular invasion should be carefully evaluated. The determination of KIT expression is essential for the diagnosis. When wild type GIST is suspected, intensive genetic analysis is required. Further, a careful and long‐time observation is recommended.     

A Novel Regulatory Defect in the Branched‐Chain α‐Keto Acid Dehydrogenase Complex Due to a Mutation in the PPM1K Gene Causes a Mild Variant Phenotype of Maple Syrup Urine Disease

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched‐chain α‐keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease‐causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array‐based genotyping showed a copy‐neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild‐type PPM1K in PP2Cm‐deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm‐deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress‐signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype—and perhaps others.     

Histone methyltransferase Suv39h1 deficiency prevents Myc‐induced chromosomal instability in murine myeloid leukemias

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild‐type and histone methyltransferase Suv39h1‐deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc‐overexpressing wild‐type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end‐to‐end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc‐transduced Suv39h1‐deficient cells showed less critically short telomeres (P < 0.05) compared with Myc‐transduced wild‐type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc. © 2013 Wiley Periodicals, Inc.     

4-nitrophenol exposure in T24 human bladder cancer cells promotes proliferation,motilities, and epithelial-to-mesenchymal transition

Although health hazards of 4-nitrophenol (PNP) exposure have been reported, the adverse effects of PNP exposure on cancer biological features are still unknown. We investigated the effects of administration of PNP in T24 human bladder cancer cells. The results showed that PNP exposure promoted cellular proliferation, migration and invasion, inhibited adhesion and apoptosis in vitro. Using quantitative real-time PCR, we found that (1) the mRNA expression levels of cell-cycle regulators PCNA, cyclin D1 and COX-2 were increased in PNP-treated cells compared to controls, however, that of pro-apoptotic gene Bax was decreased; (2) the expression level of EMT-associated gene E-cadherin was decreased in PNP-treated cells, whereas those of N-cadherin, vimentin, snail, and slug were increased; (3) the expression levels of cancer-promoting genes HIF-1, IL-1β, VEGFα and K-Ras were enhanced, but those of tumor suppressors p53, PTEN and BRCA were decreased. There was a positive association between PNP exposure times and the promotion effects. Finally, we found that the expression level of PPARγ (γ1 isoform) was increased in PNP-treated T24 cells. GW9662, a specific PPARγ antagonist, attenuated PNP-induced cell migration and invasion. These findings indicate that PNP exposure may promote bladder cancer growth and progression involving PPARγ signaling. PPARγ is a potential target for development of novel intervention study on environment pollution. Environ. Mol. Mutagen. 61:316–328, 2020. © 2019 Wiley Periodicals, Inc.     

Macrophage PPARγ and impaired wound healing in type 2 diabetes

Macrophages undergo a transition from pro‐inflammatory to healing‐associated phenotypes that is critical for efficient wound healing. However, the regulation of this transition during normal and impaired healing remains to be elucidated. In our studies, the switch in macrophage phenotypes during skin wound healing was associated with up‐regulation of the peroxisome proliferator‐activated receptor (PPAR)γ and its downstream targets, along with increased mitochondrial content. In the setting of diabetes, up‐regulation of PPARγ activity was impaired by sustained expression of IL‐1β in both mouse and human wounds. In addition, experiments with myeloid‐specific PPARγ knockout mice indicated that loss of PPARγ in macrophages is sufficient to prolong wound inflammation and delay healing. Furthermore, PPARγ agonists promoted a healing‐associated macrophage phenotype both in vitro and in vivo, even in the diabetic wound environment. Importantly, topical administration of PPARγ agonists improved healing in diabetic mice, suggesting an appealing strategy for down‐regulating inflammation and improving the healing of chronic wounds. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression patterns common and unique to ulcerative colitis and celiac disease

Autoimmune diseases like celiac disease (CeD) and ulcerative colitis (UC) show a common genetic background defined by the existence of shared susceptibility loci. We aimed to go deeper into this common genetic background through performing a cross‐disease study based on gene expression. We measured the expression of 21 genes located in 13 CeD‐UC susceptibility regions, and 10 genes in five CeD risk regions. Determinations were carried out in colon/rectum samples from 13 UC patients (inflamed and uninflamed tissue) and four colon samples from controls. Duodenal samples from 19 CeD patients and 12 controls were used for comparisons. Differences were analyzed using the Bayesian method. The shared chromosomal regions containing TNFAIP3, PTPN2, ICOSLG, C1orf106, and IL21 showed similar results in both diseases. FASLG, PLEK, CCR4, and TAGAP, all located in CeD risk loci, were up‐regulated in both CeD and UC patients. Finally, ZFP36L1, ZMIZ1, PUS10, UBE2L3, and BACH2 showed opposite results in CeD and UC. A high complexity underlies autoimmune common susceptibility loci, as the expression pattern of the studied genes does not always correlate with the one expected attending to the apparent genetic background. Differentially expressed genes such as ZFP36L1, ZMIZ1, PUS10, and BACH2 deserve further research in autoimmune diseases.     

Effect of Reduced c‐Kit Signaling on Bone Marrow Adiposity

c‐Kit (CD117) is required for normal differentiation of osteoblasts from bone marrow stromal cells and for normal bone formation. Osteoblasts and adipocytes originate from a common progenitor cell, and a reciprocal relationship in differentiation of the two lineages is often observed. Therefore, the effects of abnormal c‐kit signaling on bone marrow adiposity and adipocyte precursor pool size were evaluated in mouse strains with loss of function mutations in kit receptor or kit ligand. Additionally, to determine whether short‐duration pharmacological disruption of kit signaling influences bone marrow adiposity, we administered the kit receptor antagonist gleevec (imatinib mesilate) for 1 week to middle aged (13‐month‐old) male rats known to have high levels of bone marrow fat. Compared to wild‐type littermates, adipocytes were absent and adipocyte precursors greatly reduced in bone marrow from kit receptor‐deficient Kit^W/W‐ν mice. Administration of secreted kit ligand to membrane‐associated kit ligand‐deficient Kit^Sl/Sl‐d mice was ineffective in inducing bone marrow adipogenesis. These findings suggest that activation of kit receptor by the membrane‐associated form of kit ligand is required for kit signaling to promote bone marrow adipogenesis in mice. Rats treated with gleevec had lower adipocyte density compared to age‐matched controls, suggesting that kit signaling is required to maintain normal bone marrow adiposity. Taken together, our results indicate that c‐Kit signaling plays an important but previously unsuspected role in regulating bone marrow adiposity. Anat Rec, , 2011. © 2011 Wiley‐Liss, Inc.     

Novel oncogene and tumor suppressor mutations in KIT and PDGFRA wild type gastrointestinal stromal tumors revealed by next generation sequencing

Among gastrointestinal stromal tumors (GISTs) of 10–15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35G>T) mutation in both the primary gastric tumor and a post‐imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer‐related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors. © 2014 Wiley Periodicals, Inc.     

Over‐expression of BACH2 is related to ongoing somatic hypermutation of the immunoglobulin heavy chain gene variable region of de novo diffuse large B‐cell lymphoma

The basic region–leucine zipper (bZip) factor BTB, CNC homology 2 (BACH2) is known to have important roles in class switch recombination and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. In this study, we investigated the relationship between the expression of BACH2 and the status of SHM of the Ig heavy chain gene variable region (IgHV) for SHM in diffuse large B‐cell lymphoma (DLBCL). We examined 20 cases of DLBCL, 13 of which were germinal center B‐cell (GCB) DLBCL and 7 were non‐GCB DLBCL. Seven cases were negative, 6 were positive (cytoplasmic expression) and 7 were strongly positive (both nuclear and cytoplasmic expression) for BACH2. Confirmed mutation (CM) was identified in 8 cases and the CM index (number of confirmed mutations per 10 subclones) was distributed from 0 to 5. A CM index of 7 strongly positive (over‐expression) cases with BACH2 were distributed from 0 to 5, and that of 7 negative and 6 positive cases were distributed from 0 to 1. Over‐expression of BACH2 was statistically related to CM index (P = 0.008). In conclusion, over‐expression of BACH2 is critical for ongoing SHM of IgHV in DLBCL, and our data suggest that BACH2 may play an essential role for SHM of the Ig gene in B‐cell lymphoma.