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In human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome.

Grabher D, Hofer S, Ortner D, Heufler C - PLoS ONE (2013)

Bottom Line: If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation.We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells.We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT
CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

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(A) FACS analysis of cell surface markers on untreated or 5 nM Bortezomib treated mature dendritic cells.Maturation marker expression (CD80, CD83 and CD86, MHC class I and MHC class II) on mature dendritic cells and mature dendritic cells treated with 5 nM Bortezomib were measured. Maturation markers show no difference when treated with 5 nM Bortezomib. Expression of MHC class I and MHC class II increase slightly in 5 nM Bortezomib treated dendritic cells. (B): Dendritic cells treated to inhibit the proteasome show stable CYTIP levels with SOCS-1 over expression. Mature dendritic cells treated with 5 nM Bortezomib and transfected with the indicated amounts of SOCS-1 encoding plasmid for 16 hours were harvested and protein lysates were prepared. 40 µg of lysates were applied to each lane and CYTIP levels (39 kDa) were detected by Western blot analysis. As a control, actin expression levels were determined. CYTIP expression levels correlated to actin expression in four independent experiments are shown. Dots represent CYTIP expression levels of the individual experiments in % of mock control. Lines are mean values. No significant decrease is obtained.
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pone-0057538-g005: (A) FACS analysis of cell surface markers on untreated or 5 nM Bortezomib treated mature dendritic cells.Maturation marker expression (CD80, CD83 and CD86, MHC class I and MHC class II) on mature dendritic cells and mature dendritic cells treated with 5 nM Bortezomib were measured. Maturation markers show no difference when treated with 5 nM Bortezomib. Expression of MHC class I and MHC class II increase slightly in 5 nM Bortezomib treated dendritic cells. (B): Dendritic cells treated to inhibit the proteasome show stable CYTIP levels with SOCS-1 over expression. Mature dendritic cells treated with 5 nM Bortezomib and transfected with the indicated amounts of SOCS-1 encoding plasmid for 16 hours were harvested and protein lysates were prepared. 40 µg of lysates were applied to each lane and CYTIP levels (39 kDa) were detected by Western blot analysis. As a control, actin expression levels were determined. CYTIP expression levels correlated to actin expression in four independent experiments are shown. Dots represent CYTIP expression levels of the individual experiments in % of mock control. Lines are mean values. No significant decrease is obtained.

Mentions: If SOCS-1 takes CYTIP to the proteasome for degradation treatment of monocyte dendritic cells with the proteasome inhibitor Bortezomib (Velcade®) should keep CYTIP levels constant when a SOCS-1 encoding plasmid is transfected in increasing concentrations. Treatment of dendritic cells with 7.5 nM Bortezomib has been shown to have an inhibitory effect on the maturation of dendritic cells while at 5 nM this effect was only seen as a tendency [16]. We therefore used 5 nM Bortezomib in our experiments to avoid inhibitory effects on dendritic cell maturation. Dendritic cells induced to mature and simultaneously treated with 5 nM Bortezomib indeed showed only marginal changes in the expression levels of the maturation markers CD80, CD83 and CD86. Expression of the MHC molecules (HLA-ABC and HLA-DR) was, however, increased, possibly because of an inhibition of their degradation by the proteasome (Fig. 5a). Dendritic cells were simultaneously matured and treated with 5 nM Bortezomib for 24 hours before transfection with the SOCS-1 encoding plasmid. CYTIP levels in Western blot analyses remained stable when dendritic cells were treated with Bortezomib (Fig. 5b). Statistical analysis using paired, two-tailed student’s t-test was performed on four independent experiments. CYTIP expression levels remained constant with the indicated amounts of SOCS-1 encoding plasmid used for transfection.


In human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome.

Grabher D, Hofer S, Ortner D, Heufler C - PLoS ONE (2013)

(A) FACS analysis of cell surface markers on untreated or 5 nM Bortezomib treated mature dendritic cells.Maturation marker expression (CD80, CD83 and CD86, MHC class I and MHC class II) on mature dendritic cells and mature dendritic cells treated with 5 nM Bortezomib were measured. Maturation markers show no difference when treated with 5 nM Bortezomib. Expression of MHC class I and MHC class II increase slightly in 5 nM Bortezomib treated dendritic cells. (B): Dendritic cells treated to inhibit the proteasome show stable CYTIP levels with SOCS-1 over expression. Mature dendritic cells treated with 5 nM Bortezomib and transfected with the indicated amounts of SOCS-1 encoding plasmid for 16 hours were harvested and protein lysates were prepared. 40 µg of lysates were applied to each lane and CYTIP levels (39 kDa) were detected by Western blot analysis. As a control, actin expression levels were determined. CYTIP expression levels correlated to actin expression in four independent experiments are shown. Dots represent CYTIP expression levels of the individual experiments in % of mock control. Lines are mean values. No significant decrease is obtained.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g005: (A) FACS analysis of cell surface markers on untreated or 5 nM Bortezomib treated mature dendritic cells.Maturation marker expression (CD80, CD83 and CD86, MHC class I and MHC class II) on mature dendritic cells and mature dendritic cells treated with 5 nM Bortezomib were measured. Maturation markers show no difference when treated with 5 nM Bortezomib. Expression of MHC class I and MHC class II increase slightly in 5 nM Bortezomib treated dendritic cells. (B): Dendritic cells treated to inhibit the proteasome show stable CYTIP levels with SOCS-1 over expression. Mature dendritic cells treated with 5 nM Bortezomib and transfected with the indicated amounts of SOCS-1 encoding plasmid for 16 hours were harvested and protein lysates were prepared. 40 µg of lysates were applied to each lane and CYTIP levels (39 kDa) were detected by Western blot analysis. As a control, actin expression levels were determined. CYTIP expression levels correlated to actin expression in four independent experiments are shown. Dots represent CYTIP expression levels of the individual experiments in % of mock control. Lines are mean values. No significant decrease is obtained.
Mentions: If SOCS-1 takes CYTIP to the proteasome for degradation treatment of monocyte dendritic cells with the proteasome inhibitor Bortezomib (Velcade®) should keep CYTIP levels constant when a SOCS-1 encoding plasmid is transfected in increasing concentrations. Treatment of dendritic cells with 7.5 nM Bortezomib has been shown to have an inhibitory effect on the maturation of dendritic cells while at 5 nM this effect was only seen as a tendency [16]. We therefore used 5 nM Bortezomib in our experiments to avoid inhibitory effects on dendritic cell maturation. Dendritic cells induced to mature and simultaneously treated with 5 nM Bortezomib indeed showed only marginal changes in the expression levels of the maturation markers CD80, CD83 and CD86. Expression of the MHC molecules (HLA-ABC and HLA-DR) was, however, increased, possibly because of an inhibition of their degradation by the proteasome (Fig. 5a). Dendritic cells were simultaneously matured and treated with 5 nM Bortezomib for 24 hours before transfection with the SOCS-1 encoding plasmid. CYTIP levels in Western blot analyses remained stable when dendritic cells were treated with Bortezomib (Fig. 5b). Statistical analysis using paired, two-tailed student’s t-test was performed on four independent experiments. CYTIP expression levels remained constant with the indicated amounts of SOCS-1 encoding plasmid used for transfection.

Bottom Line: If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation.We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells.We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT
CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

Show MeSH
Related in: MedlinePlus