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Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus

Signaling pathways involved in LPS induced TNFalpha production in human adipocytes. LPS, with LBP and CD14 (coming from medium) bind to TLR4 on mature human adipose cells. Binding activates two main pathways in the cells. One pathway leads to the activation of NFkappaB (through TRAF6 and IKK). The other pathway passes through phosphorylated p38 MAP Kinase. Both pathways enable the activation of TNFalpha transcription, followed by cleavage of the protein via a membrane metalloprotease, ADAM17 or TACE, leading to the release of the soluble form of TNFalpha. The PI3K represents a third pathway, which activates NFkappaB and p38 MAPK. Another kinase, maybe the PI4K, plays an inhibitory role in the LPS activation of these 2 pathways. As far as the PKC is concerned, it probably activates IKK, or the p38 MAPKs, or both pathways. However, activation is only visible once the PI4K is inactive. It is therefore possible that PI4K constitutively inhibits PKC.
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Figure 7: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes. LPS, with LBP and CD14 (coming from medium) bind to TLR4 on mature human adipose cells. Binding activates two main pathways in the cells. One pathway leads to the activation of NFkappaB (through TRAF6 and IKK). The other pathway passes through phosphorylated p38 MAP Kinase. Both pathways enable the activation of TNFalpha transcription, followed by cleavage of the protein via a membrane metalloprotease, ADAM17 or TACE, leading to the release of the soluble form of TNFalpha. The PI3K represents a third pathway, which activates NFkappaB and p38 MAPK. Another kinase, maybe the PI4K, plays an inhibitory role in the LPS activation of these 2 pathways. As far as the PKC is concerned, it probably activates IKK, or the p38 MAPKs, or both pathways. However, activation is only visible once the PI4K is inactive. It is therefore possible that PI4K constitutively inhibits PKC.

Mentions: Furthermore, our results demonstrate that PI3K is partially implicated in LPS-activated adipocytes. However, there is a conflict in our results, as the use of 2 different PI3K inhibitors leads to opposite effects. Wortmannin brings about the activation of TNFalpha secretion. Indeed, this molecule is probably not specific to PI3K at the concentration that was used, and could perhaps inhibit other kinases, such as PI4K [29,30], which is probably implicated in limiting the LPS effect. Moreover, treatment with LY294002 at 100 and 500 nM leads to a decrease in TNFalpha secretion (Figure 4A2). As LY294002 is strictly specific to PI3K [31], it is highly plausible that PI3K is activated in the LPS-activated pathway. This functional outline appears to be different to the one identified in the monocyte/macrophage THP-1 cell line [27]. In THP1 cells, PI3K phosphorylates Akt, which in its active form is an inhibitor of the NFkappaB and p38 MAP Kinase pathways. Moreover, Akt2 is able to inactivate GSK3β, limiting the activation of NFkappaB. In mature human adipocytes, it seems that PI3K has no inhibitor effect upon NFkappaB and p38 MAP Kinase pathways. Thus, PI3K could be considered as being a third, minor, transduction pathway, as it accounts for 15% of the secretion. However, it would seem more realistic to consider PI3K as an upstream molecule of p38 MAPK and NFkappaB pathways (Figure 7).


Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

Signaling pathways involved in LPS induced TNFalpha production in human adipocytes. LPS, with LBP and CD14 (coming from medium) bind to TLR4 on mature human adipose cells. Binding activates two main pathways in the cells. One pathway leads to the activation of NFkappaB (through TRAF6 and IKK). The other pathway passes through phosphorylated p38 MAP Kinase. Both pathways enable the activation of TNFalpha transcription, followed by cleavage of the protein via a membrane metalloprotease, ADAM17 or TACE, leading to the release of the soluble form of TNFalpha. The PI3K represents a third pathway, which activates NFkappaB and p38 MAPK. Another kinase, maybe the PI4K, plays an inhibitory role in the LPS activation of these 2 pathways. As far as the PKC is concerned, it probably activates IKK, or the p38 MAPKs, or both pathways. However, activation is only visible once the PI4K is inactive. It is therefore possible that PI4K constitutively inhibits PKC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819999&req=5

Figure 7: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes. LPS, with LBP and CD14 (coming from medium) bind to TLR4 on mature human adipose cells. Binding activates two main pathways in the cells. One pathway leads to the activation of NFkappaB (through TRAF6 and IKK). The other pathway passes through phosphorylated p38 MAP Kinase. Both pathways enable the activation of TNFalpha transcription, followed by cleavage of the protein via a membrane metalloprotease, ADAM17 or TACE, leading to the release of the soluble form of TNFalpha. The PI3K represents a third pathway, which activates NFkappaB and p38 MAPK. Another kinase, maybe the PI4K, plays an inhibitory role in the LPS activation of these 2 pathways. As far as the PKC is concerned, it probably activates IKK, or the p38 MAPKs, or both pathways. However, activation is only visible once the PI4K is inactive. It is therefore possible that PI4K constitutively inhibits PKC.
Mentions: Furthermore, our results demonstrate that PI3K is partially implicated in LPS-activated adipocytes. However, there is a conflict in our results, as the use of 2 different PI3K inhibitors leads to opposite effects. Wortmannin brings about the activation of TNFalpha secretion. Indeed, this molecule is probably not specific to PI3K at the concentration that was used, and could perhaps inhibit other kinases, such as PI4K [29,30], which is probably implicated in limiting the LPS effect. Moreover, treatment with LY294002 at 100 and 500 nM leads to a decrease in TNFalpha secretion (Figure 4A2). As LY294002 is strictly specific to PI3K [31], it is highly plausible that PI3K is activated in the LPS-activated pathway. This functional outline appears to be different to the one identified in the monocyte/macrophage THP-1 cell line [27]. In THP1 cells, PI3K phosphorylates Akt, which in its active form is an inhibitor of the NFkappaB and p38 MAP Kinase pathways. Moreover, Akt2 is able to inactivate GSK3β, limiting the activation of NFkappaB. In mature human adipocytes, it seems that PI3K has no inhibitor effect upon NFkappaB and p38 MAP Kinase pathways. Thus, PI3K could be considered as being a third, minor, transduction pathway, as it accounts for 15% of the secretion. However, it would seem more realistic to consider PI3K as an upstream molecule of p38 MAPK and NFkappaB pathways (Figure 7).

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus