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Activin B, a homodimer of the inhibin βB subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin βB gene and signalling pathways regulated by the canine inhibin βB. Using 5'- and 3'-rapid amplification of cDNA end (RACE) and RT-PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin βB gene. Immunoreactive inhibin βB molecules were detected at ~25 and ~14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin βB gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B-stimulated transcriptions of reporter genes, CAGA-luc and Hepcidin-luc, regulated by the canonical activin/transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) pathway, respectively. Activin B-induced CAGA-luc transcription was not detected in ALK7-deficient MDCK canine-derived cells; however, the forced expression of ALK7 resulted in the activin B-dependent expression in MDCK cells. Unexpectedly, the activin B-induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF-β receptor activity. The present study identified an experimentally isolated long form of the canine inhibin βB gene producing activin B that transactivates BMP- and activin/TGF-β-regulated gene expression.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-β (TGF-β)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-β/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. SIGNIFICANCE OF THE STUDY: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte-selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF-β activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte-selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.

Dietary vitamin A status affects energy metabolism. The present study explored the effect of all-trans retinoic acid (ATRA) on the expression levels of molecules and metabolites of brown adipocytes. Chronic ATRA treatment was initiated during the early stage (days 0-8) or late stage (days 8-12) of adipogenesis. Treatment with ATRA during the early and late stage of adipogenesis resulted in an increase in the expression level of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12, respectively, whereas expression of Pgc-1α, another gene expressed during brown adipogenesis, was unaffected by ATRA. Non-targeted metabolomic analyses indicated that the pathways related to the glucose metabolism were affected by ATRA, irrespective of the differentiation stage. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. ATRA decreased the cellular level of aconitic acid, fumaric acid, and malic acid on day 12 but not on day 8. Furthermore, ATRA increased the expression level of Hxk2 and downregulated the expressions of G6pdh and Pfkl/Pfkp on day 8 but not on day 12. Together, the results indicate that the chronic treatment with ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism. SIGNIFICANCE OF THE STUDY: Significance of the study treatment with all-trans retinoic acid (ATRA) during the early and late stage of adipogenesis increased the expression of Ucp1 and Cidea, genes highly expressed in brown adipocytes, on day 8 and day 12. Cellular levels of glucose 6-phosphate, fructose 6-phosphate, citric acid, and succinic acid decreased after ATRA treatment on days 8 and 12. In contrast, glucose level was higher in ATRA-treated cells on day 8, but it was lower on day 12. The present results indicate that ATRA stimulated the formation of activated brown adipocytes, eventually leading to alterations in the levels of cellular metabolites related to glucose metabolism.

Activity of brown/beige adipocytes is higher in women than in men. The expression level of uncoupling protein 1 (UCP1) is largely consistent with the thermogenic activity in brown/beige adipocytes. The present study examined the direct effects of sex hormones on Ucp1 expression in brown adipocytes and beige adipocytes, which were differentiated from HB2 brown preadipocytes and 3T3-L1 white preadipocytes, respectively; treatment with estradiol or testosterone was used during the early (days 0-8) or late stage (days 8-12) of brown adipogenesis and beige adipogenesis. On day 8 or day 12, cells were treated with or without isoproterenol (Iso), an agonist for the β-adrenergic receptor, for 4 hours. Furthermore, the sex of cells was examined; the sex-determining region y gene, which is located on the y chromosome, was present in HB2 cells, but not in 3T3-L1 cells, suggesting that HB2 cells and 3T3-L1 cells are male and female cells, respectively. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to Iso on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Treatment with testosterone during the early stage of brown adipogenesis did not affect Ucp1 expression but increased the responsiveness to Iso on Ucp1 induction by the treatment during the late stage of brown adipogenesis. The present results suggest that sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner. Direct effects of sex hormones in brown/beige adipogenesis were evaluated. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the β-adrenergic receptor, on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Testosterone during the late stage of brown adipogenesis increased the responsiveness to Iso on Ucp1 induction. Sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of β-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a β-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.

Activity of brown/beige adipocytes is higher in women than in men. The expression level of uncoupling protein 1 (UCP1) is largely consistent with the thermogenic activity in brown/beige adipocytes. The present study examined the direct effects of sex hormones on Ucp1 expression in brown adipocytes and beige adipocytes, which were differentiated from HB2 brown preadipocytes and 3T3-L1 white preadipocytes, respectively; treatment with estradiol or testosterone was used during the early (days 0-8) or late stage (days 8-12) of brown adipogenesis and beige adipogenesis. On day 8 or day 12, cells were treated with or without isoproterenol (Iso), an agonist for the β-adrenergic receptor, for 4 hours. Furthermore, the sex of cells was examined; the sex-determining region y gene, which is located on the y chromosome, was present in HB2 cells, but not in 3T3-L1 cells, suggesting that HB2 cells and 3T3-L1 cells are male and female cells, respectively. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to Iso on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Treatment with testosterone during the early stage of brown adipogenesis did not affect Ucp1 expression but increased the responsiveness to Iso on Ucp1 induction by the treatment during the late stage of brown adipogenesis. The present results suggest that sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner. Direct effects of sex hormones in brown/beige adipogenesis were evaluated. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the β-adrenergic receptor, on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Testosterone during the late stage of brown adipogenesis increased the responsiveness to Iso on Ucp1 induction. Sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner.

Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (−2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types. Copyright © 2013 John Wiley & Sons, Ltd.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of β-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a β-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

Magnesium (Mg) deficiency induces the production of free radicals, increases cytosolic ionized calcium concentration, and modulates the function of skeletal muscle in rats. The present study examined the effects of Mg deficiency on the gene expression of molecules related to myogenesis in the gastrocnemius muscle as well as in C2C12 myogenic cells. Ingestion of an Mg-deficient diet resulted in a lower weight of the gastrocnemius muscle and higher concentration of muscular TBARSs, an index of oxidative stress. Mg deficiency also enhanced the expression of Myod and myogenin. In vivo effects of Mg deficiency on myogenic gene expression were partially reproduced in in vitro C2C12 cells; expression of Myod was up-regulated by a mixed culture of myoblasts and myotubes with Mg-deficient medium, which related to the simultaneous up-regulation of Myhc IIb, a myotube-specific protein. The culture with Mg-deficient medium did not increase the gene transcript level of HO-1, another marker of oxidative stress, suggesting that Mg deficiency-induced Myod expression does not result from oxidative stress. Furthermore, oxidative stress induced by hydrogen peroxide did not increase Myod expression, whereas the expression of Myod, myogenin and Myhc IIb was decreased by oxidative stress from the initial phase of differentiation. The effects of Mg deficiency depended on the stages of myogenesis; myoblast culture in Mg-deficient differentiation medium did not affect the expression of Myod and Myhc IIb. The present study revealed stage-dependent effects of Mg deficiency on myogenesis.

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.

Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activator of NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-β potentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-specific transmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer of osteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment with TGF-β.

Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activator of NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-β potentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-specific transmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer of osteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment with TGF-β.

Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (-2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types.

Magnesium (Mg) deficiency induces the production of free radicals, increases cytosolic ionized calcium concentration, and modulates the function of skeletal muscle in rats. The present study examined the effects of Mg deficiency on the gene expression of molecules related to myogenesis in the gastrocnemius muscle as well as in C2C12 myogenic cells. Ingestion of an Mg-deficient diet resulted in a lower weight of the gastrocnemius muscle and higher concentration of muscular TBARSs, an index of oxidative stress. Mg deficiency also enhanced the expression of Myod and myogenin. In vivo effects of Mg deficiency on myogenic gene expression were partially reproduced in in vitro C2C12 cells; expression of Myod was up-regulated by a mixed culture of myoblasts and myotubes with Mg-deficient medium, which related to the simultaneous up-regulation of Myhc IIb, a myotube-specific protein. The culture with Mg-deficient medium did not increase the gene transcript level of HO-1, another marker of oxidative stress, suggesting that Mg deficiency-induced Myod expression does not result from oxidative stress. Furthermore, oxidative stress induced by hydrogen peroxide did not increase Myod expression, whereas the expression of Myod, myogenin and Myhc IIb was decreased by oxidative stress from the initial phase of differentiation. The effects of Mg deficiency depended on the stages of myogenesis; myoblast culture in Mg-deficient differentiation medium did not affect the expression of Myod and Myhc IIb. The present study revealed stage-dependent effects of Mg deficiency on myogenesis.

Changes in gene expression of TGF-beta family members and their receptors in response to treatment with H(2)O(2) and a calcium ionophore, A23187, were examined in C2C12 myoblasts and myotubes. The expression of Myf5, an initial regulator of myogenesis, was increased by A23187, and H(2)O(2) inhibited the up-regulation of Myf5. Treatment with H(2)O(2) decreased the expression of MHC IIb, a protein component of the myofibrils, irrespective of the presence of A23187, suggesting an inhibitory role of oxidative stress for myogenesis. Expression of ligands and receptors for the TGF-beta family was modulated in response to H(2)O(2) and A23187. Treatment with H(2)O(2) decreased expression of TGF-beta3, BMP-4, ALK4, ALK5, and ActRIIB, and increased expression of inhibin alpha and inhibin betaA in either the myoblast stage or the myotube stage, or both. A23187 potentiated down-regulation of BMP-4 and ALK4 expression, and up-regulation of TGF-beta1, TGF-beta2, inhibin alpha, inhibin betaA, ALK2, and ALK3 expression. These results indicate that oxidative stress and Ca(2+) influx affect expression of the TGF-beta family in C2C12 myoblasts and myotubes.

While analyzing the role of c-Jun NH2-terminal kinase (JNK) in neurogenesis in P19 embryonal carcinoma cells, we noticed that treatment with SP600125, a JNK inhibitor, increased the cell size markedly. SP600125-induced enlargement of P19 cells was time- and dose-dependent. The increased cell size in response to SP600125 was also detected in B6mt-1 embryonic stem cells. SP600125 treatment inhibited cell growth and increased DNA contents, indicating the inhibition of cell proliferation resulting from endoreduplication. Concurrently, the gene expression of p21, a regulator of G2/M arrest as well as G1 arrest, was increased in cells treated with SP600125. The increased cell size in response to SP600125 was detected even in P19 cells treated with colcemide, an inhibitor of cell cycle progression at the metaphase. The present study suggests that treatment with SP600125 progresses the cell cycle, skipping cytokinesis in P19 cells. Copyright © 2009 John Wiley & Sons, Ltd.