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Inhibition of E-selectin gene expression by transforming growth factor beta in endothelial cells involves coactivator integration of Smad and nuclear factor kappaB-mediated signals.

DiChiara MR, Kiely JM, Gimbrone MA, Lee ME, Perrella MA, Topper JN - J. Exp. Med. (2000)

Bottom Line: Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP).Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression.This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Transforming growth factor (TGF)-beta(1) is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-beta(1) can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-beta(1). Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-beta(1)-regulated Smad proteins and NFkappaB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

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(A) Induction of E-selectin mRNA in rat tissues by LPS can be inhibited by systemic pretreatment with TGF-β1. As described in Materials and Methods, rats received 4 mg/kg of LPS intraperitoneally, and 6 h later the indicated tissues were harvested and processed for RNA and protein extracts as described. (A) As shown by Northern analysis (left panel), E-selectin mRNA is not detected in control tissues but is detectable in the lung, liver, and hearts 6 h after LPS administration. In the right panels, tissues from LPS-treated animals that had received either vehicle or TGF-β1 pretreatment were similarly analyzed. Each pair of lanes represents two independently treated animals. Pretreatment with TGF-β1 markedly attenuated E-selectin mRNA induction (upper blots) but did not affect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels (lower blots). (B) NFκB-mediated DNA binding activity appears preserved after TGF-β1 pretreatment in vivo. The lungs and hearts of LPS-treated rats were processed to obtain nuclear extracts for DNA mobility shift analyses with the NFκB-specific oligo probes as described in the legend to Fig. 2. In the left panel, a specific shifted band is detected in lysates from LPS-treated rat lung that is not present in lysates from control lung and that is attenuated by immunodepletion with antip65/p50 antisera but not nonimmune IgG. The middle and right panels are lysates from lung and heart, respectively, from the animals that have received LPS with or without TGF-β1 pretreatment. Each pair of lanes represents two independently treated animals. TGF-β1 pretreatment in vivo does not appear to affect LPS-induced NFκB-mediated DNA binding in these tissues. (C) Western analysis for p65 and IκB levels in tissues from LPS and TGF-β1–pretreated rats. The levels of immunoreactive p65 and IκB in the rat tissues were assessed by immunoblot. Each pair of lanes represents two independently treated animals. Levels of p65 were not significantly altered in response to any of the treatment protocols. The levels of immunoreactive IκB appeared slightly depressed by LPS treatment in the absence of TGF-β1 pretreatment.
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Figure 3: (A) Induction of E-selectin mRNA in rat tissues by LPS can be inhibited by systemic pretreatment with TGF-β1. As described in Materials and Methods, rats received 4 mg/kg of LPS intraperitoneally, and 6 h later the indicated tissues were harvested and processed for RNA and protein extracts as described. (A) As shown by Northern analysis (left panel), E-selectin mRNA is not detected in control tissues but is detectable in the lung, liver, and hearts 6 h after LPS administration. In the right panels, tissues from LPS-treated animals that had received either vehicle or TGF-β1 pretreatment were similarly analyzed. Each pair of lanes represents two independently treated animals. Pretreatment with TGF-β1 markedly attenuated E-selectin mRNA induction (upper blots) but did not affect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels (lower blots). (B) NFκB-mediated DNA binding activity appears preserved after TGF-β1 pretreatment in vivo. The lungs and hearts of LPS-treated rats were processed to obtain nuclear extracts for DNA mobility shift analyses with the NFκB-specific oligo probes as described in the legend to Fig. 2. In the left panel, a specific shifted band is detected in lysates from LPS-treated rat lung that is not present in lysates from control lung and that is attenuated by immunodepletion with antip65/p50 antisera but not nonimmune IgG. The middle and right panels are lysates from lung and heart, respectively, from the animals that have received LPS with or without TGF-β1 pretreatment. Each pair of lanes represents two independently treated animals. TGF-β1 pretreatment in vivo does not appear to affect LPS-induced NFκB-mediated DNA binding in these tissues. (C) Western analysis for p65 and IκB levels in tissues from LPS and TGF-β1–pretreated rats. The levels of immunoreactive p65 and IκB in the rat tissues were assessed by immunoblot. Each pair of lanes represents two independently treated animals. Levels of p65 were not significantly altered in response to any of the treatment protocols. The levels of immunoreactive IκB appeared slightly depressed by LPS treatment in the absence of TGF-β1 pretreatment.

Mentions: To determine whether the results in cultured endothelial cells described above could be reproduced in an in vivo model, we examined the ability of systemic TGF-β1 to inhibit LPS-induced E-selectin expression in rats. Previous work has demonstrated the ability of systemically administered TGF-β1 to inhibit the expression of several inflammatory genes, such as inducible nitric oxide synthase II and heme oxygenase, and to ameliorate the course of LPS-induced shock, although the precise mechanism(s) of these effects have not been elucidated 222324. We initially examined the ability of LPS to induce E-selectin mRNA expression in whole organs in vivo. As demonstrated in Fig. 3 A, E-selectin mRNA was not detected in total RNA (15 μg) from the lung, liver, and heart of control rats but is readily detected after 6 h of LPS administration. Given that E-selectin is selectively expressed in vascular endothelium, the varying amounts of E-selectin mRNA detected (in a constant amount of total RNA) probably reflect both the degree to which endothelium contributes the overall RNA content of the individual tissues, and differences in the degree to which E-selectin expression is induced by LPS in these distinct tissue environments. To examine the effects of TGF-β1, a second set of rats received either vehicle or 20 μg/kg of active TGF-β1 intraperitoneally, followed 20 min later by 4 mg/kg of LPS. The lungs, heart, and liver of these animals were harvested 6 h later for RNA and protein analysis. As demonstrated in Fig. 3 A (right), TGF-β1 pretreatment markedly attenuated the level of E-selectin mRNA induced in all three tissues. To examine whether NFκB was being activated to similar extents, we next examined the DNA binding activity of protein lysates from rat tissues. As demonstrated in Fig. 3 B (left), lysates from LPS-treated rat lung tissue demonstrate a gel-shifted band that is not seen in lysates from control lungs and that can be abolished by immunodepletion with an αp65/p50 antisera. When these analyses were performed on lysates from tissues derived from the LPS-treated rats, no detectable difference in NFκB-mediated DNA binding was seen between the sham and TGF-β1–pretreated animals (Fig. 3 B). Fig. 3 C demonstrates that the levels of immunoreactive p65 appear to be equal among the groups and that the levels of IκBα are slightly diminished in the absence of TGF-β1 pretreatment, but not abolished. Taken together, these results indicate that LPS can effectively upregulate E-selectin expression in vivo, and that this effect can be significantly attenuated by systemic pretreatment with TGF-β1. Furthermore, this inhibition can occur in the absence of a detectable decrement in the levels of immunoreactive NFκB or the degree to which NFκB is activated as assessed by specific DNA binding assay.


Inhibition of E-selectin gene expression by transforming growth factor beta in endothelial cells involves coactivator integration of Smad and nuclear factor kappaB-mediated signals.

DiChiara MR, Kiely JM, Gimbrone MA, Lee ME, Perrella MA, Topper JN - J. Exp. Med. (2000)

(A) Induction of E-selectin mRNA in rat tissues by LPS can be inhibited by systemic pretreatment with TGF-β1. As described in Materials and Methods, rats received 4 mg/kg of LPS intraperitoneally, and 6 h later the indicated tissues were harvested and processed for RNA and protein extracts as described. (A) As shown by Northern analysis (left panel), E-selectin mRNA is not detected in control tissues but is detectable in the lung, liver, and hearts 6 h after LPS administration. In the right panels, tissues from LPS-treated animals that had received either vehicle or TGF-β1 pretreatment were similarly analyzed. Each pair of lanes represents two independently treated animals. Pretreatment with TGF-β1 markedly attenuated E-selectin mRNA induction (upper blots) but did not affect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels (lower blots). (B) NFκB-mediated DNA binding activity appears preserved after TGF-β1 pretreatment in vivo. The lungs and hearts of LPS-treated rats were processed to obtain nuclear extracts for DNA mobility shift analyses with the NFκB-specific oligo probes as described in the legend to Fig. 2. In the left panel, a specific shifted band is detected in lysates from LPS-treated rat lung that is not present in lysates from control lung and that is attenuated by immunodepletion with antip65/p50 antisera but not nonimmune IgG. The middle and right panels are lysates from lung and heart, respectively, from the animals that have received LPS with or without TGF-β1 pretreatment. Each pair of lanes represents two independently treated animals. TGF-β1 pretreatment in vivo does not appear to affect LPS-induced NFκB-mediated DNA binding in these tissues. (C) Western analysis for p65 and IκB levels in tissues from LPS and TGF-β1–pretreated rats. The levels of immunoreactive p65 and IκB in the rat tissues were assessed by immunoblot. Each pair of lanes represents two independently treated animals. Levels of p65 were not significantly altered in response to any of the treatment protocols. The levels of immunoreactive IκB appeared slightly depressed by LPS treatment in the absence of TGF-β1 pretreatment.
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Figure 3: (A) Induction of E-selectin mRNA in rat tissues by LPS can be inhibited by systemic pretreatment with TGF-β1. As described in Materials and Methods, rats received 4 mg/kg of LPS intraperitoneally, and 6 h later the indicated tissues were harvested and processed for RNA and protein extracts as described. (A) As shown by Northern analysis (left panel), E-selectin mRNA is not detected in control tissues but is detectable in the lung, liver, and hearts 6 h after LPS administration. In the right panels, tissues from LPS-treated animals that had received either vehicle or TGF-β1 pretreatment were similarly analyzed. Each pair of lanes represents two independently treated animals. Pretreatment with TGF-β1 markedly attenuated E-selectin mRNA induction (upper blots) but did not affect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels (lower blots). (B) NFκB-mediated DNA binding activity appears preserved after TGF-β1 pretreatment in vivo. The lungs and hearts of LPS-treated rats were processed to obtain nuclear extracts for DNA mobility shift analyses with the NFκB-specific oligo probes as described in the legend to Fig. 2. In the left panel, a specific shifted band is detected in lysates from LPS-treated rat lung that is not present in lysates from control lung and that is attenuated by immunodepletion with antip65/p50 antisera but not nonimmune IgG. The middle and right panels are lysates from lung and heart, respectively, from the animals that have received LPS with or without TGF-β1 pretreatment. Each pair of lanes represents two independently treated animals. TGF-β1 pretreatment in vivo does not appear to affect LPS-induced NFκB-mediated DNA binding in these tissues. (C) Western analysis for p65 and IκB levels in tissues from LPS and TGF-β1–pretreated rats. The levels of immunoreactive p65 and IκB in the rat tissues were assessed by immunoblot. Each pair of lanes represents two independently treated animals. Levels of p65 were not significantly altered in response to any of the treatment protocols. The levels of immunoreactive IκB appeared slightly depressed by LPS treatment in the absence of TGF-β1 pretreatment.
Mentions: To determine whether the results in cultured endothelial cells described above could be reproduced in an in vivo model, we examined the ability of systemic TGF-β1 to inhibit LPS-induced E-selectin expression in rats. Previous work has demonstrated the ability of systemically administered TGF-β1 to inhibit the expression of several inflammatory genes, such as inducible nitric oxide synthase II and heme oxygenase, and to ameliorate the course of LPS-induced shock, although the precise mechanism(s) of these effects have not been elucidated 222324. We initially examined the ability of LPS to induce E-selectin mRNA expression in whole organs in vivo. As demonstrated in Fig. 3 A, E-selectin mRNA was not detected in total RNA (15 μg) from the lung, liver, and heart of control rats but is readily detected after 6 h of LPS administration. Given that E-selectin is selectively expressed in vascular endothelium, the varying amounts of E-selectin mRNA detected (in a constant amount of total RNA) probably reflect both the degree to which endothelium contributes the overall RNA content of the individual tissues, and differences in the degree to which E-selectin expression is induced by LPS in these distinct tissue environments. To examine the effects of TGF-β1, a second set of rats received either vehicle or 20 μg/kg of active TGF-β1 intraperitoneally, followed 20 min later by 4 mg/kg of LPS. The lungs, heart, and liver of these animals were harvested 6 h later for RNA and protein analysis. As demonstrated in Fig. 3 A (right), TGF-β1 pretreatment markedly attenuated the level of E-selectin mRNA induced in all three tissues. To examine whether NFκB was being activated to similar extents, we next examined the DNA binding activity of protein lysates from rat tissues. As demonstrated in Fig. 3 B (left), lysates from LPS-treated rat lung tissue demonstrate a gel-shifted band that is not seen in lysates from control lungs and that can be abolished by immunodepletion with an αp65/p50 antisera. When these analyses were performed on lysates from tissues derived from the LPS-treated rats, no detectable difference in NFκB-mediated DNA binding was seen between the sham and TGF-β1–pretreated animals (Fig. 3 B). Fig. 3 C demonstrates that the levels of immunoreactive p65 appear to be equal among the groups and that the levels of IκBα are slightly diminished in the absence of TGF-β1 pretreatment, but not abolished. Taken together, these results indicate that LPS can effectively upregulate E-selectin expression in vivo, and that this effect can be significantly attenuated by systemic pretreatment with TGF-β1. Furthermore, this inhibition can occur in the absence of a detectable decrement in the levels of immunoreactive NFκB or the degree to which NFκB is activated as assessed by specific DNA binding assay.

Bottom Line: Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP).Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression.This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
Transforming growth factor (TGF)-beta(1) is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-beta(1) can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-beta(1). Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-beta(1)-regulated Smad proteins and NFkappaB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

Show MeSH
Related in: MedlinePlus