Cytokine 72 (2015) 220–223
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short Communication
Leucine-rich glioma inactivated 3 and tumor necrosis factor-a regulate mutually through NF-jB Hyun A Kim, Nyoun Soo Kwon, Kwang Jin Baek, Dong-Seok Kim, Hye-Young Yun ⇑ Department of Biochemistry, Chung-Ang University, College of Medicine, 84 Heukseok-ro, Dongjak-gu, Seoul 156-861, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2014 Received in revised form 17 December 2014 Accepted 23 December 2014 Available online 31 January 2015 Keywords: LGI3 TNF-a NF-jB Adipokine Obesity
a b s t r a c t Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-a (TNF-a) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-a mRNA and protein. LGI3 treatment led to NF-jB activation and binding to an NF-jB binding site (523 to 514) in TNF-a promoter. TNF-a treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-a increased NF-jB binding to a predicted binding site (40 to 31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-a were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-a may play a role in adipose tissue inflammation in obesity. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family that is highly expressed in brain in a developmentally regulated manner [1]. Expression of LGI3 was shown to be regulated by neuronal restrictive silencer and AP-2 at transcription level [1]. We previously showed that LGI3 played regulatory roles in neuronal exocytosis and differentiation [2,3]. Besides the nervous system, LGI3 is expressed in diverse tissues including skin and adipose tissues [4,5]. We reported that ultraviolet B-irradiation-induced secretion of LGI3 promoted survival of human keratinocytes [5]. We also showed that LGI3 promoted keratinocyte migration and melanocyte pigmentation [6,7]. These findings led to the hypothesis that LGI3 may be a candidate cytokine secreted by and acting at multiple cell types. We found that LGI3 was expressed in adipose tissues and was downregulated during adipocyte differentiation and upregulated in adipose tissues of ob/ob mice and high fat diet (HFD)-fed mice [4,8]. We reported that LGI3 suppressed adipogenesis through its receptor, a disintegrin and metalloproteinase domain-containing protein 23 (ADAM23) and that LGI3 increased pro-inflammatory proteins including TNF-a in macrophage cells [4]. LGI3 was shown ⇑ Corresponding author at: Department of Biochemistry, Chung-Ang University, College of Medicine, 84 Heukseok-ro, Dongjak-gu, Seoul 156-861, Republic of Korea. Tel.: +82 2 820 5684; fax: +82 2 817 9866. E-mail address: hyyunoffi[email protected] (H.-Y. Yun). http://dx.doi.org/10.1016/j.cyto.2014.12.023 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
to downregulate adiponectin, an anti-inflammatory adipokine [8]. TNF-a and adiponectin are crucial adipokines that are perturbed in obesity and implicated in metabolic inflammation [9]. We proposed that LGI3 may be a pro-inflammatory adipokine that interplays with other adipokines in adipogenesis and metabolic inflammation [4]. In this study, we present the evidences that LGI3 and TNF-a upregulate each other through a key inflammatory transcription factor, NF-jB. 2. Materials and methods 2.1. Animals and cell cultures Animal protocols were approved by the Institutional Animal Care and Use Committee. For HFD-fed mice, female mice (4week-old C57BL/6, Central Lab Animal, Korea) were fed normal or high fat (60%kcal fat) diet for 16 weeks. White adipose tissues (WAT) were obtained from gonadal fat. 3T3-L1 preadipocytes (ATCC) were cultured, treated and harvested as previously described [4]. 2.2. Purification of recombinant LGI3 and protein analysis LGI3-His6 protein was purified as previously described [3]. TNF-
a was obtained from Invitrogen. The primary antibodies used were, LGI3-FL [2], LGI3, ADAM23, b-actin (Santa Cruz), TNF-a,
H.A Kim et al. / Cytokine 72 (2015) 220–223
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Fig. 1. Effect of LGI3 on TNF-a expression and NF-jB. (A, D) Western blot of the extract from 3T3-L1 cells treated with indicated concentrations of LGI3 for 24 h. (B) Western blot of the extract from the cells treated with 10 ng/ml LGI3 for varying time periods. (C) Quantitation of RNA from the cells treated with Dulbecco’s phosphate-buffered saline (DPBS)(Control) or 10 ng/ml LGI3 for 24 h. ⁄p
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Short Communication
Leucine-rich glioma inactivated 3 and tumor necrosis factor-a regulate mutually through NF-jB Hyun A Kim, Nyoun Soo Kwon, Kwang Jin Baek, Dong-Seok Kim, Hye-Young Yun ⇑ Department of Biochemistry, Chung-Ang University, College of Medicine, 84 Heukseok-ro, Dongjak-gu, Seoul 156-861, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2014 Received in revised form 17 December 2014 Accepted 23 December 2014 Available online 31 January 2015 Keywords: LGI3 TNF-a NF-jB Adipokine Obesity
a b s t r a c t Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-a (TNF-a) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-a mRNA and protein. LGI3 treatment led to NF-jB activation and binding to an NF-jB binding site (523 to 514) in TNF-a promoter. TNF-a treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-a increased NF-jB binding to a predicted binding site (40 to 31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-a were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-a may play a role in adipose tissue inflammation in obesity. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family that is highly expressed in brain in a developmentally regulated manner [1]. Expression of LGI3 was shown to be regulated by neuronal restrictive silencer and AP-2 at transcription level [1]. We previously showed that LGI3 played regulatory roles in neuronal exocytosis and differentiation [2,3]. Besides the nervous system, LGI3 is expressed in diverse tissues including skin and adipose tissues [4,5]. We reported that ultraviolet B-irradiation-induced secretion of LGI3 promoted survival of human keratinocytes [5]. We also showed that LGI3 promoted keratinocyte migration and melanocyte pigmentation [6,7]. These findings led to the hypothesis that LGI3 may be a candidate cytokine secreted by and acting at multiple cell types. We found that LGI3 was expressed in adipose tissues and was downregulated during adipocyte differentiation and upregulated in adipose tissues of ob/ob mice and high fat diet (HFD)-fed mice [4,8]. We reported that LGI3 suppressed adipogenesis through its receptor, a disintegrin and metalloproteinase domain-containing protein 23 (ADAM23) and that LGI3 increased pro-inflammatory proteins including TNF-a in macrophage cells [4]. LGI3 was shown ⇑ Corresponding author at: Department of Biochemistry, Chung-Ang University, College of Medicine, 84 Heukseok-ro, Dongjak-gu, Seoul 156-861, Republic of Korea. Tel.: +82 2 820 5684; fax: +82 2 817 9866. E-mail address: hyyunoffi[email protected] (H.-Y. Yun). http://dx.doi.org/10.1016/j.cyto.2014.12.023 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
to downregulate adiponectin, an anti-inflammatory adipokine [8]. TNF-a and adiponectin are crucial adipokines that are perturbed in obesity and implicated in metabolic inflammation [9]. We proposed that LGI3 may be a pro-inflammatory adipokine that interplays with other adipokines in adipogenesis and metabolic inflammation [4]. In this study, we present the evidences that LGI3 and TNF-a upregulate each other through a key inflammatory transcription factor, NF-jB. 2. Materials and methods 2.1. Animals and cell cultures Animal protocols were approved by the Institutional Animal Care and Use Committee. For HFD-fed mice, female mice (4week-old C57BL/6, Central Lab Animal, Korea) were fed normal or high fat (60%kcal fat) diet for 16 weeks. White adipose tissues (WAT) were obtained from gonadal fat. 3T3-L1 preadipocytes (ATCC) were cultured, treated and harvested as previously described [4]. 2.2. Purification of recombinant LGI3 and protein analysis LGI3-His6 protein was purified as previously described [3]. TNF-
a was obtained from Invitrogen. The primary antibodies used were, LGI3-FL [2], LGI3, ADAM23, b-actin (Santa Cruz), TNF-a,
H.A Kim et al. / Cytokine 72 (2015) 220–223
221
Fig. 1. Effect of LGI3 on TNF-a expression and NF-jB. (A, D) Western blot of the extract from 3T3-L1 cells treated with indicated concentrations of LGI3 for 24 h. (B) Western blot of the extract from the cells treated with 10 ng/ml LGI3 for varying time periods. (C) Quantitation of RNA from the cells treated with Dulbecco’s phosphate-buffered saline (DPBS)(Control) or 10 ng/ml LGI3 for 24 h. ⁄p
