Partial limitation of cellular functions and compensatory modulation of unfolded protein response pathways caused by double-knockout of ATF6α and ATF6ß.

Mammalian cells have three types of endoplasmic reticulum (ER) stress-sensing molecules: ATF6, IRE1, and PERK. Among these, ATF6 is unique in that it is processed in an ER-stress-specific manner and functions as a transcription factor for the activation of anti-ER stress genes (such as BiP). ATF6 is known to have two homologues, ATF6α and ATF6ß, and a greater understanding of their functions has been achieved through analyses using cultured cells. Physiological functions are also gradually being investigated in mice lacking ATF6α or ATF6ß. However, little is known about the effects on mouse organisms of the deletion of both the ATF6α and ATF6ß genes, since such double-knockout (DKO) mice suffer embryonic lethality at an early developmental stage. In this study, we generated and analyzed ATF6 DKO mice in which embryonic lethality was evaded by using Cre/loxP technology. Pancreatic ß cell-specific ATF6 DKO mice were born normally and lived without dysregulation of blood-glucose levels but had a reduced tolerance to glucose. Islets isolated from ATF6 DKO mice also showed low production and secretion of insulin and mild enhancement of IRE1 and PERK activity. We further examined the developmental abnormalities of systemic ATF6 DKO mice. The phenotypes of ATF6α-/-; ATF6ß-/- mice were similar to those previously reported, but ATF6α+/-; ATF6ß-/- and ATF6α-/-; ATF6ß+/- mice showed embryonic lethality at middle developmental stages, unlike those reported. Analysis of embryonic fibroblasts derived from these mice revealed that ATF6α and ATF6ß have a gene-dose-dependent functional redundancy and display distinct differences in their ability to induce BiP expression. (250 words).

Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate.

Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Neutrophil extracellular trap induction through peptidylarginine deiminase 4 activity is involved in 2,4,6-trinitrobenzenesulfonic acid-induced colitis.

Neutrophil extracellular traps (NETs) are induced in the innate immune response against infectious agents and are also implicated in the pathogenesis of various cancers and autoimmune diseases. Peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginine to citrulline, is also involved in NET formation. In this study, we investigated the pathogenic effect of PAD4 on NETs in inflammatory bowel disease using a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD4-deficient (PAD4KO) mice were generated by CRISPR-Cas9-mediated genomic editing. NETs were triggered in peritoneal neutrophils obtained from wild-type mice by A23187 (a calcium ionophore), but these responses were completely abolished in the PAD4KO mice. Experimental colitis was induced in wild-type and PAD4KO mice via an intrarectal injection of TNBS. TNBS injection resulted in body weight loss, extensive colonic erosion, and ulceration in wildtype mice. However, these responses were significantly attenuated following the administration of Cl-amidine (an inhibitor of pan-PADs) and DNase I (an inhibitor of NET formation), in combination with PAD4KO in mice. TNBS-induced increases in myeloperoxidase activity, inflammatory cytokine expression, and NET formation in the colon were significantly reduced following the administration of Cl-amidine, DNase I injection, and PAD4KO. These findings suggest that NET formation contributes to the pathogenesis of TNBS-induced colitis via PAD4. Thus, PAD4 is a promising target for the treatment of inflammatory bowel disease.

Venous volume and compliance in the calf and forearm does not change after acute endurance exercise performed at continuous or interval workloads.

Short-term endurance exercise training for 6-8 weeks leads to increases in venous volume and compliance in the limbs. However, it is not known whether these venous vascular properties are improved by acute endurance exercise. We examined the effects of acute endurance exercise involving continuous or interval workloads on venous volume and compliance in the exercising (calf) and non-exercising (forearm) limbs. Sixteen healthy young volunteers performed cycling exercise involving a continuous workload of 60% heart rate (HR) reserve or an interval workload of 40% HRreserve and 80% HRreserve, alternating every 2 min, for a total of 32 min each. Before and 60 min after acute cycling exercise, venous volume in the calf and forearm was measured by venous occlusion plethysmography during a cuff-deflation protocol with a venous collecting cuff wrapped to the thigh and upper arm and strain gauges attached to the calf and forearm. The cuff pressure was maintained at 60 mmHg for 8 min and was then deflated to 0 mmHg at a rate of 1 mmHg/s. Venous compliance was calculated as the numerical derivative of the cuff pressure-limb venous volume curve. In both the calf and forearm, the cuff pressure-venous volume curve and the cuff pressure-venous compliance relationship did not differ between before and 60 min after exercise involving continuous or interval workloads. These results suggest that acute exercise does not improve venous volume and compliance in both the exercising and non-exercising limbs.

Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice.

AIMS/HYPOTHESIS: Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. METHODS: We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. RESULTS: Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12-15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. CONCLUSIONS/INTERPRETATION: Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. DATA AVAILABILITY: The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164 ).

Transgenic mouse model exhibiting weak red fluorescence before and strong green fluorescenceafter Cre/loxP-mediated recombination.

The Cre/loxP system is an indispensable tool for temporal and spatial control of gene function in mice. Many mice that express Cre and carry loxP sites in their genomes have been bred for functional analysis of various genes in vivo. Also, several reporter mice have been generated for monitoring of recombination by the Cre/loxP system. We have developed a Cre reporter gene with DsRed1 and Venus that exhibits a strong red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination in experiments using NIH3T3 cells. However, a transgenic mouse introduced with the same reporter gene exhibits a weak red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination. This property manifested ubiquitously in this mouse model and was maintained stably in mouse-derived fibroblasts. Use of the mouse model exhibiting the stronger red fluorescence might result in confusion of the Cre-dependent signal with false signals, because the Venus signal includes some fluorescence in the red region of the spectrum and the DsRed1 signal includes some fluorescence in the green region. However, we fortuitously obtained reporter mice that exhibit a weaker red fluorescence before Cre/loxP-mediated recombination. The use of this mouse model would decrease concern regarding errors in the identification of signals and should increase certainty in the detection of Cre activity in vivo.

PIN FORMED 2 Modulates the Transport of Arsenite in Arabidopsis thaliana.

Arsenic contamination is a major environmental issue, as it may lead to serious health hazard. The reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Root transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots compared with the wild-type. At the cellular level, arsenite specifically targets intracellular sorting of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 function results in arsenite hypersensitivity in roots. XFI coupled with XAS further revealed that loss of PIN2 function results in specific accumulation of arsenical species, but not the other metals such as iron, zinc, or calcium in the root tip. Collectively, these results suggest that PIN2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta.

Effect of short-term endurance training on venous compliance in the calf and forearm differs between continuous and interval exercise in humans.

We examined whether the effect of short-term endurance exercise training on venous compliance in the calf and forearm differed between continuous and interval workloads. Young healthy volunteers (10 women and 16 men) were randomly assigned to continuous (C-TRA; n = 8) and interval (I-TRA; n = 9) exercise training groups, and a control group (n = 9). Subjects in the C-TRA group performed a continuous cycling exercise at 60% of heart rate reserve (HRR), and subjects in the I-TRA group performed a cycling exercise consisting of alternating 2-min intervals at 40% HRR and 80% HRR. Training programs were performed for 40 min/day, 3 days/week for 8 weeks. Before and after training, limb volume in the calf and forearm was measured with subjects in the supine position by venous occlusion plethysmography using a venous collecting cuff placed around the thigh and upper arm. Cuff pressure was held at 60 mmHg for 8 min and then decreased to 0 mmHg at a rate of 1 mmHg/s. Venous compliance was calculated as the numerical derivative of the cuff pressure-limb volume curve. Calf venous compliance was increased after I-TRA, but not C-TRA. Forearm venous compliance was unchanged after C-TRA or I-TRA. These results suggest that the adaptation of venous compliance in response to endurance training for 8 week may occur in interval but not continuous exercise bouts and may be specific to the exercising limb.

Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member.

About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo, revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.

Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance.

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1-elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin-adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

IRE1-XBP1 pathway regulates oxidative proinsulin folding in pancreatic ß cells.

In mammalian pancreatic ß cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed ß cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic ß cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic ß cells.

Ectopic expression of the transcription factor MafB in basal keratinocytes induces hyperproliferation and perturbs epidermal homeostasis.

Mammalian epidermis is composed of four morphologically and functionally distinct layers of keratinocytes. The innermost basal layer consists of proliferating self-renewing keratinocytes, which also undergo asymmetric cell division to differentiate into postmitotic suprabasal cells throughout life. Control of the balance between growth and differentiation of basal cells is important for epidermal homeostasis to prevent skin disorders including malignancies; however, the underlying mechanism remains to be elucidated. Recently, MafB was identified as one of the transcription factors that regulate epidermal keratinocyte differentiation. MafB is expressed in postmitotic differentiating keratinocytes, and epidermal differentiation is partially impaired in MafB-deficient mice. To further establish the roles of MafB in the epidermis in vivo, we generated mice transgenic for MafB under the control of the basal cell-specific keratin (Krt) 14 promoter. In the epidermis of transgenic mice at embryonic day 18.5, the number of proliferating Krt14-positive basal-like cells was increased, and the granular and cornified layers were thickened. Furthermore, these MafB transgenic mice developed papillomas spontaneously with age. Therefore, MafB promotes differentiation in postmitotic keratinocytes and simultaneously has potential to promote growth when ectopically expressed in undifferentiated basal keratinocytes.

4µ8C Inhibits Insulin Secretion Independent of IRE1α RNase Activity.

IRE1α plays an important role in the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. 4µ8C, a well-known inhibitor of IRE1α RNase activity, is commonly used to analyze IRE1α function during ER stress in cultured mammalian cells. However, the off-target effects of 4µ8C remain elusive. Pancreatic ß-cells synthesize a large amount of insulin in response to high glucose stimulation, and IRE1α plays an important role in insulin secretion from pancreatic ß-cells. Here, to analyze the role of IRE1α in pancreatic ß-cells, we examined insulin secretion after 4µ8C treatment. Although 4µ8C inhibited insulin secretion within 2 hr, neither insulin synthesis nor maturation was inhibited by 4µ8C under the same conditions. This result prompted us to examine the precise effects of 4µ8C on insulin secretion in pancreatic ß-cells. Unexpectedly, with just 5 min of treatment, 4µ8C blocked insulin secretion in cultured pancreatic ß-cells as well as in pancreatic islets. Furthermore, insulin secretion was prevented by 4µ8C, even in pancreatic ß-cells lacking the IRE1α RNase domain, suggesting that 4µ8C blocked the late stage of the insulin secretory process, independent of the IRE1α-XBP1 pathway. Our results indicate that 4µ8C has an off-target effect on insulin secretion in pancreatic ß-cells. These findings inform the researchers in the field that the use of 4µ8C requires the special consideration for the future studies.Key words: 4µ8C, XBP1, insulin, IRE1α, pancreatic ß-cells.

Pathogenic Mechanism of Diabetes Development Due to Dysfunction of Unfolded Protein Response.

The endoplasmic reticulum (ER) is an organelle in which newly synthesized secretory and membrane proteins are folded and assembled. Various stresses cause the accumulation of unfolded or misfolded proteins in the ER, resulting in ER dysfunction. This condition is termed ER stress. To cope with ER stress, cells activate a signaling pathway termed the unfolded protein response (UPR). Recently, accumulating evidence suggests that the UPR plays a pivotal role in pancreatic ß cells. Pancreatic ß cells producing a large amount of insulin are highly sensitive when the UPR is impaired. In mammalian cells, three principal ER stress sensors, PERK, IRE1, and ATF6, initiate the UPR. Activated PERK attenuates protein translation through eIF2α phosphorylation to cope with the ER stress. PERK KO mice develop diabetes by 2-4 weeks of age due to progressive ß-cell loss. IRE1α noncanonically splices the XBP1 mRNA, leading to the upregulation of the ERAD components and ER molecular chaperones. This pathway is constitutively activated in pancreatic ß cells. To clarify the physiological role of the IRE1α pathway in ß cells, we generated pancreatic-ß-cell-specific IRE1α-conditional KO (cKO) mice and IRE1α-cKO insulinoma cell lines. Here, we show that IRE1α is required for the upregulation of insulin-folding enzymes in pancreatic ß cells to balance insulin-folding enzymes with insulin.

Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway.

Mammalian inositol-requiring enzyme 1α (IRE1α) is the most conserved of all endoplasmic reticulum (ER) stress sensors, which includes activating transcription factor (ATF) 6 and double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK). IRE1α has been known to splice X-box binding protein 1 (XBP1) mRNA, which is induced by ATF6 under ER stress. This spliced XBP1 mRNA is translated into the active transcription factor that promotes the expression of specific genes to alleviate ER stress. Herein, we report that in addition to the induction of XBP1 expression by ATF6, IRE1α expression is induced by ATF4, which is downstream of PERK, under ER stress. Increased IRE1α expression results in a higher splicing ratio of XBP1 mRNA. This effect was not transient and affected not only the intensity but also the duration of the activated state of this pathway. These multiple regulatory mechanisms may modulate the response to various levels or types of ER stress.

Hes7 3'UTR is required for somite segmentation function.

A set of genes in the posterior end of developing mouse embryos shows oscillatory expression, thereby regulating periodic somite segmentation. Although the mechanism for generating oscillation has extensively been clarified, what regulates the oscillation period is still unclear. We attempted to elongate the oscillation period by increasing the time to transcribe Hes7 in this research. We generated knock-in mice, in which a large intron was inserted into Hes7 3'UTR. The exogenous intron was unexpectedly not properly spliced out and the transcripts were prematurely terminated. Consequently, Hes7 mRNA lost its 3'UTR, thereby reducing the amount of Hes7 protein. Oscillation was damped in the knock-in embryos and periodic somite segmentation does not occur properly. Thus, we demonstrated that Hes7 3'UTR is essential to accumulate adequate amounts of Hes7 protein for the somite segmentation clock that orchestrates periodic somite formation.

A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads.

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).