Early Life Polychlorinated Biphenyl 126 Exposure Disrupts Gut Microbiota and Metabolic Homeostasis in Mice Fed with High-Fat Diet in Adulthood.

Evidence supports the potential influence of persistent organic pollutants (POPs) on the pathogenesis and progression of obesity and diabetes. Diet-toxicant interactions appear to be important in diet-induced obesity/diabetes; however, the factors influencing this interaction, especially the early life environmental exposure, are unclear. Herein, we investigated the metabolic effects following early life five-day exposure (24 µg/kg body weight per day) to 3,3',4,4',5-pentacholorobiphenyl (PCB 126) at four months after exposure in mice fed with control (CTRL) or high-fat diet (HFD). Activation of aryl hydrocarbon receptor (AHR) signaling as well as higher levels of liver nucleotides were observed at 4 months after PCB 126 exposure in mice, independent of diet status. Inflammatory responses including higher levels of serum cytokines and adipose inflammatory gene expression caused by early life PCB 126 were observed only in HFD-fed mice in adulthood. Notably, early life PCB 126 exposure worsened HFD-induced impaired glucose homeostasis characterized by glucose intolerance and elevated gluconeogenesis and tricarboxylic acid (TCA) cycle flux without worsening the effects of HFD related to adiposity in adulthood. Furthermore, early life PCB 126 exposure resulted in diet-dependent changes in bacterial community structure and function later in life, as indicated by metagenomic and metabolomic analyses. These data contribute to a more comprehensive understanding of the interactions between diet and early life environmental chemical exposure.

Early Life Short-Term Exposure to Polychlorinated Biphenyl 126 in Mice Leads to Metabolic Dysfunction and Microbiota Changes in Adulthood.

Early life exposure to environmental pollutants may have long-term consequences and harmful impacts on health later in life. Here, we investigated the short- and long-term impact of early life 3,3',4,4',5-pentacholorobiphenyl (PCB 126) exposure (24 µg/kg body weight for five days) in mice on the host and gut microbiota using 16S rRNA gene sequencing, metagenomics, and 1H NMR- and mass spectrometry-based metabolomics. Induction of Cyp1a1, an aryl hydrocarbon receptor (AHR)-responsive gene, was observed at 6 days and 13 weeks after PCB 126 exposure consistent with the long half-life of PCB 126. Early life, Short-Term PCB 126 exposure resulted in metabolic abnormalities in adulthood including changes in liver amino acid and nucleotide metabolism as well as bile acid metabolism and increased hepatic lipogenesis. Interestingly, early life PCB 126 exposure had a greater impact on bacteria in adulthood at the community structure, metabolic, and functional levels. This study provides evidence for an association between early life environmental pollutant exposure and increased risk of metabolic disorders later in life and suggests the microbiome is a key target of environmental chemical exposure.

Emerging role of an immunomodulatory protein secretoglobin 3A2 in human diseases.

Secretoglobin (SCGB) 3A2 was first identified in 2001 as a protein exhibiting similarities in amino acid sequence and gene structure to SCGB1A1, a multi-functional cytokine-like molecule highly expressed in airway epithelial Club cells that was the first identified and extensively studied member of the SCGB gene superfamily. SCGB3A2 is a small secretory protein of ~10 kDa that forms a dimer and a tetramer. SCGB3A2 is predominantly expressed in airway epithelial Club cells, and has anti-inflammatory, growth factor, anti-fibrotic, and anti-cancer activities that influence various lung diseases. This review summarizes the current understanding of SCGB3A2 biological functions and its role in human diseases with emphasis on its mechanisms of actions and signaling pathway.

Secretoglobin 3A2 eliminates human cancer cells through pyroptosis.

Non-canonical inflammasome activation that recognizes intracellular lipopolysaccharide (LPS) causes pyroptosis, the inflammatory death of innate immune cells. The role of pyroptosis in innate immune cells is to rapidly eliminate pathogen-infected cells and limit the replication niche in the host body. Whether this rapid cell elimination process of pyroptosis plays a role in elimination of cancer cells is largely unknown. Our earlier study demonstrated that a multi-functional secreted protein, secretoglobin (SCGB) 3A2, chaperones LPS to cytosol, and activates caspase-11 and the non-canonical inflammasome pathway, leading to pyroptosis. Here we show that SCGB3A2 exhibits marked anti-cancer activity against 5 out of 11 of human non-small cell lung cancer cell lines in mouse xenographs, while no effect was observed in 6 of 6 small cell lung cancer cell lines examined. All SCGB3A2-LPS-sensitive cells express syndecan 1 (SDC1), a SCGB3A2 cell surface receptor, and caspase-4 (CASP4), a critical component of the non-canonical inflammasome pathway. Two epithelial-derived colon cancer cell lines expressing SDC1 and CASP4 were also susceptible to SCGB3A2-LPS treatment. TCGA analysis revealed that lung adenocarcinoma patients with higher SCGB3A2 mRNA levels exhibited better survival. These data suggest that SCGB3A2 uses the machinery of pyroptosis for the elimination of human cancer cells via the non-canonical inflammasome pathway, and that SCGB3A2 may serve as a novel therapeutic to treat cancer, perhaps in combination with immuno and/or targeted therapies.

Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates.

The body plan along the anteroposterior axis and regional identities are specified by the spatiotemporal expression of Hox genes. Multistep controls are required for their unique expression patterns; however, the molecular mechanisms behind the tight control of Hox genes are not fully understood. In this study, we demonstrated that the Lin28a/let-7 pathway is critical for axial elongation. Lin28a-/- mice exhibited axial shortening with mild skeletal transformations of vertebrae, which were consistent with results in mice with tail bud-specific mutants of Lin28a. The accumulation of let-7 in Lin28a-/- mice resulted in the reduction of PRC1 occupancy at the Hox cluster loci by targeting Cbx2. Consistently, Lin28a loss in embryonic stem-like cells led to aberrant induction of posterior Hox genes, which was rescued by the knockdown of let-7. These results suggest that the Lin28/let-7 pathway is involved in the modulation of the 'Hox code' via Polycomb regulation during axial patterning.

Spatiotemporal Expression of Three Secretoglobin Proteins, SCGB1A1, SCGB3A1, and SCGB3A2, in Mouse Airway Epithelia.

Secretoglobins (SCGBs) are cytokine-like small molecular weight secreted proteins with largely unknown biological functions. Three SCGB proteins, SCGB1A1, SCGB3A1, and SCGB3A2, are predominantly expressed in lung airways. To gain insight into the possible functional relationships among the SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice, using Scgb3a1-null and Scgb3a2-null mice as negative controls, by immunohistochemistry and by qRT-PCR analysis, respectively. The three SCGBs exhibited unique spatiotemporal expression patterns during embryogenesis. The lack of Scgb3a1 or Scgb3a2 did not affect expression of the other Scgb genes as determined by mRNA measurements. Moreover, the lack of Scgb3a1 or Scgb3a2 did not affect development of the pulmonary neuroepithelial bodies during embryogenesis, while the lack of Scgb3a2 may have resulted in slightly fewer ciliated cells than in the wild-type. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function.

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

Bhlha9 regulates apical ectodermal ridge formation during limb development.

Split hand/foot malformation (SHFM) and SHFM combined with long-bone deficiency (SHFLD) are congenital dysgeneses of the limb. Although six different loci/mutations (SHFM1-SHFM6) have been found from studies on families with SHFM, the causes and associated pathogenic mechanisms for a large number of patients remain unidentified. On the basis of the identification of a duplicated gene region involving BHLHA9 in some affected families, BHLHA9 was identified as a novel SHFM/SHFLD-related gene. Although Bhlha9 is predicted to participate in limb development as a transcription factor, its precise function is unclear. Therefore, to study its physiological function, we generated a Bhlha9-knockout mouse and investigated gene expression and the associated phenotype in the limb bud. Bhlha9-knockout mice showed syndactyly and poliosis in the limb. Moreover, some apical ectodermal ridge (AER) formation related genes, including Trp63, exhibited an aberrant expression pattern in the limb bud of Bhlha9-knockout mice; TP63 (Trp63) was regulated by Bhlha9 on the basis of in vitro analysis. These observations suggest that Bhlha9 regulates AER formation during limb/finger development by regulating the expression of some AER-formation-related genes and abnormal expression of Bhlha9 leads to SHFM and SHFLD via dysregulation of AER formation and associated gene expression.

Analysis of transcription factors expressed at the anterior mouse limb bud.

Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount in situ hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5-E11.5 limb bud. Among these genes, Irx3 showed a posteriorly expanded expression domain in Shh-/- limb buds and an anteriorly reduced expression domain in Gli3-/- limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated Irx3 transgenic mice in which Irx3 was expressed in the entire limb mesenchyme under the Prrx1 regulatory element. The Irx3 gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that Irx3 may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial-Mesenchymal Transition.

BACKGROUND: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. METHOD: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. RESULTS: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. CONCLUSION: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

Syndactyly in a novel Fras1(rdf) mutant results from interruption of signals for interdigital apoptosis.

BACKGROUND: Fras1 encodes an extracellular matrix protein that is critical for the establishment of the epidermal basement membrane during gestation. In humans, mutations in FRAS1 cause Fraser Syndrome (FS), a pleiotropic condition with many clinical presentations such as limb, eye, kidney, and craniofacial deformations. Many of these defects are mimicked by loss of Fras1 in mice, and are preceded by the formation of epidermal blisters in utero. RESULTS: In this study, we identified a novel ENU-derived rounded foot (rdf) mouse mutant with highly penetrant hindlimb soft-tissue syndactyly, among other structural defects. Mapping and sequencing revealed that rdf is a novel loss-of-function nonsense allele of Fras1 (Fras1(rdf)). Focusing on the limb, we found that the Fras1(rdf) syndactyly phenotype originates from loss of interdigital cell death (ICD). Despite normal expression of bone morphogenetic protein (BMP) ligands and their receptors, the BMP downstream target gene Msx2, which is also necessary and sufficient to promote ICD, was down-regulated in the interdigital regions of Fras1(rdf) hindlimb buds. CONCLUSIONS: The close correlation between limb bud epidermal blistering, decreased Msx2 expression, and reduced ICD in the Fras1(rdf) hindlimb buds suggests that epithelium detachment from the mesenchyme may create a physical gap that interrupts the transmission of BMP, among other signals, resulting in soft tissue syndactyly.

Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex.

BACKGROUND: Limb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients. METHODS: This study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses. RESULTS: We identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome. CONCLUSIONS: These results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.

The AERO system: a 3D-like approach for recording gene expression patterns in the whole mouse embryo.

We have recently constructed a web-based database of gene expression in the mouse whole embryo, EMBRYS (http://embrys.jp/embrys/html/MainMenu.html). To allow examination of gene expression patterns to the fullest extent possible, this database provides both photo images and annotation data. However, since embryos develop via an intricate process of morphogenesis, it would be of great value to track embryonic gene expression from a three dimensional perspective. In fact, several methods have been developed to achieve this goal, but highly laborious procedures and specific operational skills are generally required. We utilized a novel microscopic technique that enables the easy capture of rotational, 3D-like images of the whole embryo. In this method, a rotary head equipped with two mirrors that are designed to obtain an image tilted at 45 degrees to the microscope stage captures serial images at 2-degree intervals. By a simple operation, 180 images are automatically collected. These 2D images obtained at multiple angles are then used to reconstruct 3D-like images, termed AERO images. By means of this system, over 800 AERO images of 191 gene expression patterns were captured. These images can be easily rotated on the computer screen using the EMBRYS database so that researchers can view an entire embryo by a virtual viewing on a computer screen in an unbiased or non-predetermined manner. The advantages afforded by this approach make it especially useful for generating data viewed in public databases.

Identification of novel transcription-regulating genes expressed during murine molar development.

BACKGROUND: The mechanism of tooth development is a complex process regulated by numerous genes including transcription factors, growth factors, and other intra- and extracellular molecules. Especially, transcription factors play a central role in gene expression, regulating a wide spectrum of biological processes including organogenesis. Substantial evidence has been demonstrated by a number of studies using genetically engineered animal models. However, detailed molecular mechanisms of tooth development have not been completely elucidated, partially because numerous genes that play essential roles in tooth development remain unidentified. RESULTS: In this study, we conducted an expression-based screening using gene expression database and in situ hybridization assays. Based on the gene expression database "EMBRYS," 207 out of 1,520 genes were expressed in the maxillary and/or mandibular processes and thus were selected for further analysis by section in situ hybridization. Among these candidates, 28 genes were newly identified as potential factors associated with tooth development by in situ hybridization assays with frontal sections of embryonic day 13.5 and 14.5 mouse embryos. The expression patterns were also examined at embryonic day 16.5 and 18.5. CONCLUSIONS: These results will contribute to elucidating the mechanisms of tooth development and to improving the technology for regeneration of tooth.

L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity.

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.

The myogenic transcriptional network.

Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program.

Developmental expression of sorting nexin 3 in the mouse central nervous system.

We previously reported that sorting nexin 3 (SNX3), a protein belonging to the sorting nexin family, regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The snx3 gene is disrupted in patients with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation, demonstrating that SNX3 plays an important role in the genesis of these organs during development. The present study was designed to determine the expression pattern of snx3 mRNA, particularly in the mouse central nervous system (CNS), from the embryonic stage to adulthood. Whole mount in situ hybridization of embryonic day (E) 9.5 and 10.5 mouse embryos revealed strong positive signals for snx3 mRNA in the forebrain, pharyngeal arches, eyes, and limb buds. In situ hybridization analyses of embryonic and neonatal brain sections revealed that snx3 mRNA is mainly expressed in the cerebral cortex, hippocampus, piriform cortex, cerebellum, and spinal cord. In adulthood, the expression of snx3 mRNA is observed in the cerebral cortex, hippocampus, piriform cortex, and cerebellar neurons. Thus, snx3 mRNA is expressed during neural development and in adult neural tissues, suggesting that SNX3 may play an important role in the development and function of the CNS.

MicroRNA-140 plays dual roles in both cartilage development and homeostasis.

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.

The Mohawk homeobox gene is a critical regulator of tendon differentiation.

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated Mkx(-/-) mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and Mkx(-/-) mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in Mkx(-/-) tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.