Blockade of OSMRß signaling ameliorates skin lesions in a mouse model of human atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe pruritus and eczematous skin lesions. Although IL-31, a type 2 helper T (Th2)-derived cytokine, is important to the development of pruritus and skin lesions in AD, the blockade of IL-31 signaling does not improve the skin lesions in AD. Oncostatin M (OSM), a member of IL-6 family of cytokines, plays important roles in the regulation of various inflammatory responses through OSM receptor ß subunit (OSMRß), a common receptor subunit for OSM and IL-31. However, the effects of OSM on the pathogenesis of AD remain to be elucidated. When AD model mice were treated with OSM, skin lesions were exacerbated and IL-4 production was increased in the lymph nodes. Next, we investigated the effects of the monoclonal antibody (mAb) against OSMRß on the pathogenesis of AD. Treatment with the anti-OSMRß mAb (7D2) reduced skin severity score in AD model mice. In addition to skin lesions, scratching behavior was decreased by 7D2 mAb with the reduction in the number of OSMRß-positive neurons in the dorsal root ganglia of AD model mice. 7D2 mAb also reduced the serum concentration of IL-4, IL-13, and IgE as well as the gene expressions of IL-4 and IL-13 in the lymph nodes of AD model mice. Blockade of both IL-31 and OSM signaling is suggested to suppress both pruritus and Th2 responses, resulting in the improvement of skin lesions in AD. The anti-OSMRß mAb may be a new therapeutic candidate for the treatment of AD.

Essential roles of the cytokine oncostatin M in crosstalk between muscle fibers and immune cells in skeletal muscle after aerobic exercise.

Crosstalk between muscle fibers and immune cells is well known in the processes of muscle repair after exercise, especially resistance exercise. In aerobic exercise, however, this crosstalk is not fully understood. In the present study, we found that macrophages, especially anti-inflammatory (M2) macrophages, and neutrophils accumulated in skeletal muscles of mice 24 h after a single bout of an aerobic exercise. The expression of oncostatin M (OSM), a member of the interleukin 6 family of cytokines, was also increased in muscle fibers immediately after the exercise. In addition, we determined that deficiency of OSM in mice inhibited the exercise-induced accumulation of M2 macrophages and neutrophils, whereas intramuscular injection of OSM increased these immune cells in skeletal muscles. Furthermore, the chemokines related to the recruitment of macrophages and neutrophils were induced in skeletal muscles after aerobic exercise, which were attenuated in OSM-deficient mice. Among them, CC chemokine ligand 2, CC chemokine ligand 7, and CXC chemokine ligand 1 were induced by OSM in skeletal muscles. Next, we analyzed the direct effects of OSM on the skeletal muscle macrophages, because the OSM receptor ß subunit was expressed predominantly in macrophages in the skeletal muscle. OSM directly induced the expression of these chemokines and anti-inflammatory markers in the skeletal muscle macrophages. From these findings, we conclude that OSM is essential for aerobic exercise-induced accumulation of M2 macrophages and neutrophils in the skeletal muscle partly through the regulation of chemokine expression in macrophages.

Comprehensive expression pattern of kin of irregular chiasm-like 3 in the adult mouse brain.

Kin of irregular chiasm-like 3 (Kirrel3), a member of the immunoglobulin superfamily, is expressed in the central nervous system during development and in adulthood. It has been reported that Kirrel3 is involved in the axonal fasciculation in the olfactory bulb, the neuronal migration in the pontine nucleus, and the synapse formation in the hippocampal neurons in mice. Although KIRREL3 mutations have been implicated in autism spectrum disorder and intellectual disability in humans, the comprehensive expression pattern of Kirrel3 in the adult brain is not fully understood. To better visualize Kirrel3 expression pattern and to gain insight into the role of Kirrel3 in the brain, we investigated the expression of Kirrel3 in the adult brain of Kirrel3-heterozygous (Kirrel3+/-) mice, in which Kirrel3-expressing cells could be identified by the expression of ß-galactosidase (ß-gal) in the nucleus of cells. The strong expression of ß-gal was observed in the hippocampus, cerebral cortex, olfactory bulb, amygdala, thalamus, and cerebellum. In the hippocampus, ß-gal was detected in the dentate gyrus and in the ventral parts of CA1 and CA3, which are known to be involved in the social recognition memory. Within the cerebral cortex, many cells with ß-gal expression were observed in the olfactory area and auditory area. In the striatum, neurons with ß-gal expression were mainly observed in the ventral striatum. Expression of ß-gal was observed in all layers in the cerebellum and olfactory bulb, except for the olfactory nerve layer. Double-immunofluorescence staining of ß-galactosidase with neuronal markers revealed that ß-gal expression was exclusively detected in neurons. These results suggest that Kirrel3 may be involved in the maintenance of neuronal networks, such as the maintenance of synaptic connectivity and plasticity in the motor, sensory, and cognitive circuits of adult brain.

Essential roles of oncostatin M receptor ß signaling in renal crystal formation in mice.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor ß (OSMRß)-deficient mice (OSMRß-/- mice). There were fewer renal crystal deposits in OSMRß-/- mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1ß), and fibrosis markers (TGF-ß, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRß-/- mice compared with those in WT mice. Immunofluorescence staining showed that OSMRß was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.

Abnormal behaviours relevant to neurodevelopmental disorders in Kirrel3-knockout mice.

In the nervous system, Kirrel3 is involved in neuronal migration, axonal fasciculation, and synapse formation. Recently, genetic links have been reported between mutations in the KIRREL3 gene and increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD) and intellectual disability. To elucidate the causal relationship between KIRREL3 deficiency and behavioural abnormalities relevant to neurodevelopmental disorders, we generated global Kirrel3-knockout (Kirrel3-/-) mice and investigated the detailed behavioural phenotypes. In the three-chambered social approach test, Kirrel3-/- mice displayed a significant preference for a mouse over a non-social object but no significant preference for a stranger mouse over a familiar mouse. Ultrasonic communications, including pup-to-mother calls, male-female courtship vocalisation and resident responses to intruder, were significantly impaired in Kirrel3-/- mice. Significant increases in locomotor activity and repetitive rearing were also observed in Kirrel3-/- mice. Furthermore, the performance of Kirrel3-/- mice in the rotarod test was significantly better than that of wild-type mice. In the acoustic startle test, Kirrel3-/- mice were significantly hypersensitive to acoustic stimuli. Anxiety-related behaviours and spatial or fear memory acquisition were normal in Kirrel3-/- mice. These findings suggest that Kirrel3-/- mice exhibit autistic-like behaviours, including social and communicative deficits, repetitive behaviours, and sensory abnormalities, as well as hyperactivity.

Oncostatin M in the development of metabolic syndrome and its potential as a novel therapeutic target.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, plays an important role in various biologic actions, including cell growth, neuronal development, and inflammatory responses. Recently, we demonstrated the unique relationship between OSM and metabolic syndrome in mice. Mice lacking OSM receptor ß subunit (OSMRß-/- mice) exhibited late-onset obesity. Before the onset of obesity, adipose tissue inflammation and insulin resistance were observed in OSMRß-/- mice. In addition, high-fat diet-induced metabolic disorders, including obesity, adipose tissue inflammation, insulin resistance, and hepatic steatosis, were aggravated in OSMRß-/- mice compared to those in wild-type mice. Consistent with these findings, OSM treatment dramatically improved these metabolic disorders in the mouse model of metabolic syndrome. Interestingly, OSM directly changed the phenotypes of adipose tissue macrophages toward anti-inflammatory M2 type. Furthermore, fatty acid content in the hepatocytes was decreased by OSM through expression regulation of several key enzymes of hepatic lipid metabolism. These findings suggest that OSM is a novel therapeutic target for metabolic syndrome.

Oncostatin M is a potential agent for the treatment of obesity and related metabolic disorders: a study in mice.

AIMS/HYPOTHESIS: Obesity and insulin resistance are closely associated with adipose tissue dysfunction caused by the abnormal recruitment of inflammatory cells, including macrophages. Oncostatin M (OSM), a member of the IL-6 family of cytokines, plays important roles in a variety of biological functions including the regulation of inflammatory responses. In previous reports, we have demonstrated that mice deficient in the OSM receptor ß subunit show obesity, adipose tissue inflammation, insulin resistance and hepatic steatosis, all of which are exacerbated by feeding the mice a high-fat diet. These results prompted us to test the therapeutic effects of OSM on obesity-induced metabolic disorders using mouse models of obesity. METHODS: In diet-induced obese and ob/ob mice, metabolic variables were assessed physiologically, histologically and biochemically after the intraperitoneal injection of recombinant mouse OSM twice a day for 1 week. RESULTS: Treatment with OSM improved obesity, adipose tissue inflammation, insulin resistance and hepatic steatosis in both mouse models. Although OSM reduced food intake, such therapeutic effects of OSM were observed even under pair-feeding conditions. Functionally, OSM directly changed the phenotype of adipose tissue macrophages from M1 type (inflammatory) to M2 type (anti-inflammatory). In the liver, OSM suppressed the expression of genes related to fatty acid synthesis and increased the expression of genes related to fatty acid oxidation. Furthermore, OSM decreased lipid absorption and increased the expression of active glucagon-like peptide-1 in the intestine. CONCLUSIONS/INTERPRETATION: We showed that OSM is a novel candidate to act as a powerful therapeutic agent for the treatment of obesity-induced metabolic disorders.

Deficiency of oncostatin M receptor ß (OSMRß) exacerbates high-fat diet-induced obesity and related metabolic disorders in mice.

Oncostatin M (OSM) belongs to the IL-6 family of cytokines and has diverse biological effects, including the modulation of inflammatory responses. In the present study we analyzed the roles of OSM signaling in obesity and related metabolic disorders. Under a high-fat diet condition, OSM receptor ß subunit-deficient (OSMRß(-/-)) mice exhibited increases in body weight and food intake compared with those observed in WT mice. In addition, adipose tissue inflammation, insulin resistance, and hepatic steatosis were more severe in OSMRß(-/-) mice than in wild-type (WT) mice. These metabolic phenotypes did not improve when OSMRß(-/-) mice were pair-fed with WT mice, suggesting that the effects of OSM signaling on these phenotypes are independent of the increases in the body weight and food intake. In the liver of OSMRß(-/-) mice, the insulin-induced phosphorylation of p70 S6 kinase remained intact, whereas insulin-induced FOXO1 phosphorylation was impaired. In addition, OSMRß(-/-) mice displayed a higher expression of genes related to de novo lipogenesis in the liver than WT mice. Furthermore, treatment of genetically obese ob/ob mice with OSM improved insulin resistance, adipose tissue inflammation, and hepatic steatosis. Intraportal administration of OSM into ob/ob mice activated STAT3 and increased the expression of long-chain acyl-CoA synthetase (ACSL) 3 and ACSL5 with decreased expression of fatty acid synthase in the liver, suggesting that OSM directly induces lipolysis and suppresses lipogenesis in the liver of obese mice. These findings suggest that defects in OSM signaling promote the deterioration of high-fat diet-induced obesity and related metabolic disorders.

Lack of oncostatin M receptor ß leads to adipose tissue inflammation and insulin resistance by switching macrophage phenotype.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, plays important roles in a variety of biological functions, including inflammatory responses. However, the roles of OSM in metabolic diseases are unknown. We herein analyzed the metabolic parameters of OSM receptor ß subunit-deficient (OSMRß(-/-)) mice under normal diet conditions. At 32 weeks of age, OSMRß(-/-) mice exhibited mature-onset obesity, severer hepatic steatosis, and insulin resistance. Surprisingly, insulin resistance without obesity was observed in OSMRß(-/-) mice at 16 weeks of age, suggesting that insulin resistance precedes obesity in OSMRß(-/-) mice. Both OSM and OSMRß were expressed strongly in the adipose tissue and little in some other metabolic organs, including the liver and skeletal muscle. In addition, OSMRß is mainly expressed in the adipose tissue macrophages (ATMs) but not in adipocytes. In OSMRß(-/-) mice, the ATMs were polarized to M1 phenotypes with the augmentation of adipose tissue inflammation. Treatment of OSMRß(-/-) mice with an anti-inflammatory agent, sodium salicylate, improved insulin resistance. In addition, the stimulation of a macrophage cell line, RAW264.7, and peritoneal exudate macrophages with OSM resulted in the increased expression of M2 markers, IL-10, arginase-1, and CD206. Furthermore, treatment of C57BL/6J mice with OSM increased insulin sensitivity and polarized the phenotypes of ATMs to M2. Thus, OSM suppresses the development of insulin resistance at least in part through the polarization of the macrophage phenotypes to M2, and OSMRß(-/-) mice provide a unique mouse model of metabolic diseases.

Dynamic expression pattern of leucine-rich repeat neuronal protein 4 in the mouse dorsal root ganglia during development.

A member of leucine-rich repeat neuronal protein (Lrrn) family, Lrrn4, is a type I transmembrane protein and functions as a cell adhesion molecule. In our previous report, Lrrn4 is expressed in a subset of small-sized dorsal root ganglion (DRG) neurons of the adult mice. In the present study, we investigated the expression pattern of Lrrn4 in the developing DRGs. The expression of Lrrn4 was first observed in 7% of total DRG neurons at embryonic day (E) 13.5, gradually increasing to 44% at E17.5, reached the maximum level between E17.5 and postnatal day (P) 7, decreased drastically after P7, and became the adult level by P14. Interestingly, the expression of Lrrn4 was mainly observed in TrkC-positive neurons at E13.5, and the predominant expression was shifted from TrkC-positive neurons to TrkA-positive neurons between E15.5 and E17.5. As the central afferents of TrkC-positive and TrkA-positive neurons begin to penetrate into the spinal cord to form synapse with secondary neurons at E13.5 and E15.5, respectively, the time course of Lrrn4 expression may suggest the contribution of Lrrn4 to synaptic formation. In addition, some cell adhesion molecules containing leucine-rich repeat are identified as synaptic adhesion molecules, suggesting that the spatiotemporal expression pattern of Lrrn4 contributes to the development of synaptic function in the DRG neurons.

Expression pattern of leucine-rich repeat neuronal protein 4 in adult mouse dorsal root ganglia.

A member of leucine-rich repeat neuronal protein family, leucine-rich repeat neuronal protein 4 (Lrrn4), is a type I transmembrane protein. Previously, we have reported that Lrrn4 is expressed in various regions of the central nervous system (CNS) and involved in the memory retention. However, little is known about the role of Lrrn4 in the peripheral nervous system (PNS). Northern blot analysis revealed that Lrrn4 mRNA was expressed predominantly in the dorsal root ganglia (DRGs) with low levels in some regions of the CNS. To identify Lrrn4-expressing cells in the DRGs, we performed in situ hybridization histochemistry and LacZ staining in Lrrn4-heterozygous (Lrrn4+/-) mice generated by the replacement of Lrrn4 gene with ß-galactosidase gene. In the adult DRGs, 8% of total DRG neurons contained Lrrn4 mRNA, which was exclusively expressed in the small-sized neurons. LacZ staining combined with immunohistochemistry revealed that approximately 42% and 58% of Lrrn4-positive neurons contained receptor tyrosine kinase A (TrkA)- and Ret-immunoreactivity, respectively. After sciatic nerve axotomy, the expression of Lrrn4 mRNA was reduced in injured side of the DRGs. Thus, Lrrn4 is expressed in a subset of nociceptive neurons and might contribute to the maintenance of nociceptive circuits.

Regulation of AMP-activated protein kinase signaling by AFF4 protein, member of AF4 (ALL1-fused gene from chromosome 4) family of transcription factors, in hypothalamic neurons.

In the hypothalamus, fasting induces a member of the AF4 family of transcription factors, AFF4, which was originally identified as a fusion partner of the mixed-lineage leukemia gene in infant acute lymphoblastic leukemia. However, the roles of AFF4 in the hypothalamus remain unclear. We show herein that expression of AFF4 increased upon addition of ghrelin and fasting in the growth hormone secretagogue receptor-expressing neurons of the hypothalamus. In the growth hormone secretagogue receptor-expressing hypothalamic neuronal cell line GT1-7, ghrelin markedly induced expression of AFF4 in a time- and dose-dependent manner. Overexpression of AFF4 in GT1-7 cells specifically induced expression of the AMP-activated protein kinase (AMPK) α2 subunit but failed to induce other AMPK subunits and AMPK upstream kinases. The promoter activity of the AMPKα2 gene increased upon addition of AFF4, suggesting that AFF4 regulates transcription of the AMPKα2 gene. Additionally, AFF4 also increased the phosphorylation of acetyl-CoA carboxylase α (ACCα), a downstream target of AMPK. In GT1-7 cells, ghrelin phosphorylated ACCα through AMPKα phosphorylation in the early phase (15 min) of the activation. However, ghrelin-induced expression of AMPKα2 and phosphorylation of ACCα in the late phase (2 h) of the activation were independent of AMPKα phosphorylation. Attenuation of expression of AFF4 by its siRNA in GT1-7 cells decreased ghrelin-induced AMPKα2 expression and ACCα phosphorylation in the late phase of the activation. AFF4 may therefore help to maintain activation of AMPK downstream signaling under conditions of prolonged stimulation with ghrelin, such as during fasting.

Site-specific subtypes of macrophages recruited after peripheral nerve injury.

After partial ligation of mouse sciatic nerve, the subtypes of macrophages were examined in the injured nerve and dorsal root ganglia (DRGs). Many M1 macrophages, which were inducible nitric oxide synthase (iNOS)-positive and arginase-1 (Arg-1)-negative, and neutrophils infiltrated the injured nerve. In contrast, almost all macrophages infiltrating the ipsilateral side of DRGs after the nerve injury were iNOS⁻/Arg-1⁺, M2 type. The infiltration of M1 and M2 macrophages was first observed in the injured nerve and ipsilateral DRGs on days 1 and 2, respectively. In addition, the macrophage infiltration preceded the activation of microglia in the ipsilateral dorsal horn of spinal cord. Thus, infiltrating macrophages after peripheral nerve injury may play unique roles dependent on the location in the development of neuropathic pain.

Oncostatin M regulates neural precursor activity in the adult brain.

The regulation of neural precursor cell (NPC) activity is the major determinant of the rate of neuronal production in neurogenic regions of the adult brain. Here, we show that Oncostatin M (Osm) and its receptor, OsmRß, are both expressed in the subventricular zone (SVZ) and that in contradistinction to leukemia inhibitory factor and ciliary neutrophic factor, Osm directly inhibits the proliferation of adult NPCs as measured by a decreased level of neurosphere formation in vitro. Similarly, intraventricular infusion of Osm dramatically decreases the pool of NPCs in both the SVZ and the hippocampus. In keeping with the inhibitory action of Osm, we reveal that mice lacking OsmRß have substantially more NPCs in the SVZ, the hippocampus and the olfactory bulb, demonstrating that endogenous Osm signaling is important for NPC homeostasis. Finally, we show that Osm can also inhibit clonal growth of glioblastoma-derived neurospheres, further supporting the close link between NPCs and tumor stem cells.

Regulation of ghrelin signaling by a leptin-induced gene, negative regulatory element-binding protein, in the hypothalamic neurons.

Leptin, the product of the ob gene, plays important roles in the regulation of food intake and body weight through its receptor in the hypothalamus. To identify novel transcripts induced by leptin, we performed cDNA subtraction based on selective suppression of the polymerase chain reaction by using mRNA prepared from the forebrain of leptin-injected ob/ob mice. One of the genes isolated was a mouse homolog of human negative regulatory element-binding protein (NREBP). Its expression was markedly increased by leptin in the growth hormone secretagogue-receptor (GHS-R)-positive neurons of the arcuate nucleus and ventromedial hypothalamic nucleus. The promoter region of GHS-R contains one NREBP binding sequence, suggesting that NREBP regulates GHS-R transcription. Luciferase reporter assays showed that NREBP repressed GHS-R promoter activity in a hypothalamic neuronal cell line, GT1-7, and its repressive activity was abolished by the replacement of negative regulatory element in GHS-R promoter. Overexpression of NREBP reduced the protein expression of endogenous GHS-R without affecting the expression of ob-Rb in GT1-7 cells. To determine the functional importance of NREBP in the hypothalamus, we assessed the effects of NREBP on ghrelin action. Although phosphorylation of AMP-activated protein kinase α (AMPKα) was induced by ghrelin in GT1-7 cells, NREBP repressed ghrelin-induced AMPKα phosphorylation. These results suggest that leptin-induced NREBP is an important regulator of GHS-R expression in the hypothalamus and provides a novel molecular link between leptin and ghrelin signaling.

TIMP-3 recruits quiescent hematopoietic stem cells into active cell cycle and expands multipotent progenitor pool.

Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3-deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs.

TIM1 is an endogenous ligand for LMIR5/CD300b: LMIR5 deficiency ameliorates mouse kidney ischemia/reperfusion injury.

Leukocyte mono-immunoglobulin (Ig)-like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc-induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1-LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.

The absence of interleukin-6 enhanced arsenite-induced renal injury by promoting autophagy of tubular epithelial cells with aberrant extracellular signal-regulated kinase activation.

Sodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6(-/-)) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6(-/-) mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction.

Detailed expression pattern of Foxp1 and its possible roles in neurons of the spinal cord during embryogenesis.

A member of winged-helix/forkhead transcription factors, Foxp1, is expressed in the developing spinal cord during mouse embryogenesis. To shed light on the potential role of Foxp1 in neurons of the developing spinal cord, we investigated the detailed expression pattern of Foxp1 between embryonic day (E) 9.5 and E17.5. At E10.25, some postmitotic neurons with strong expression of Foxp1 (Foxp1(high)) were first detected in the ventral half of the brachial spinal cord. By E11.5, Foxp1(high) neurons increased in the ventral spinal cord at the limb levels. All of Foxp1(high) neurons at the limb levels were Islet2(+)/Lhx3(-) motor neurons (MNs) of the lateral motor column and some neurons that expressed Foxp1 weakly (Foxp1(low)) at the thoracic level were MNs of the preganglionic motor column. Between E12.5 and E17.5, Foxp1(low) neurons were also observed in the intermediate zone throughout the ventral spinal cord, all of which were Pax2(+), En1(+), Evx1(-), Chx10(-), Gata3(-), and Lhx3(-) V1 interneurons. Interestingly, no colocalization of Foxp1 with Lhx3 was observed in the developing spinal cord. In addition, overexpression of Foxp1 markedly attenuated the endogenous expression of Lhx3 in a neuroendocrine cell line. Chromatin immunoprecipitation assays in a neuronal cell line and E13.5 spinal cords revealed an interaction between Foxp1 and the consensus motif in the Lhx3 promoter. These results suggest that Foxp1 may play some important roles in the determination of neuronal fates of the ventral spinal cord, possibly through the suppression of Lhx3 expression.

Wnt modulators, SFRP-1, and SFRP-2 are expressed in osteoblasts and differentially regulate hematopoietic stem cells.

Wnt signaling has been implicated in the self-renewal of hematopoietic stem cells (HSCs). Secreted frizzled-related proteins (SFRPs) are a family of soluble proteins containing a region homologous to a receptor for Wnt, Frizzled, and are thought to act as endogenous modulators for Wnt signaling. This study examined the role of SFRPs in HSC regulation. Among the four family members, SFRP-1 and SFRP-2 are specifically induced in the bone marrow in response to myelosuppression, and immunostaining revealed that both proteins were expressed in osteoblasts. Interestingly, SFRP-1 reduced the number of multipotent progenitors in in vitro culture of CD34(-)KSL cells, while SFRP-2 did not. Furthermore, SFRP-1 compromised the long-term repopulating activity of HSCs, whereas SFRP-2 did not affect or even enhanced it in the same setting. These results indicate that although both SFRP-1 and SFRP-2 act as inhibitors for Wnt signaling in vitro, they differentially affect the homeostasis of HSCs.