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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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CYTIP and SOCS-1 expression levels at different time points during maturation in monocyte derived dendritic cells.(A) CYTIP expression levels during maturation of dendritic cells were measured at the indicated time points. Western blots were performed using rat anti human CYTIP antibody 1A3 and visualized with Alexa fluor 680 goat anti rat antibody. Three independent Western blot analyses were analyzed and show a peak expression for CYTIP at 72 hours after induction of maturation. One representative Western blot is shown. (B) SOCS-1 expression during maturation remains stable over time. Western blot analyses were performed with lysates harvested at the indicated time points using rabbit polyclonal anti SOCS-1 antibody and visualized with Alexa fluor 680 goat anti rabbit antibody. One representative Western blot is shown. Arbitrary units were set to show progression of CYTIP (A) and SOCS-1 (B) expression during maturation. Immature dendritic cells (iDC) were used as reference level at time point 0 of maturation.
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pone-0057538-g002: CYTIP and SOCS-1 expression levels at different time points during maturation in monocyte derived dendritic cells.(A) CYTIP expression levels during maturation of dendritic cells were measured at the indicated time points. Western blots were performed using rat anti human CYTIP antibody 1A3 and visualized with Alexa fluor 680 goat anti rat antibody. Three independent Western blot analyses were analyzed and show a peak expression for CYTIP at 72 hours after induction of maturation. One representative Western blot is shown. (B) SOCS-1 expression during maturation remains stable over time. Western blot analyses were performed with lysates harvested at the indicated time points using rabbit polyclonal anti SOCS-1 antibody and visualized with Alexa fluor 680 goat anti rabbit antibody. One representative Western blot is shown. Arbitrary units were set to show progression of CYTIP (A) and SOCS-1 (B) expression during maturation. Immature dendritic cells (iDC) were used as reference level at time point 0 of maturation.

Mentions: To evaluate expression levels over time, dendritic cells were matured at day 6 of culture and harvested 0, 24, 48, 72, 96 and 120 hours after maturation induction. Lysates of all time points were analyzed on Western blots for CYTIP and SOCS-1 levels. In three independent experiments we show that CYTIP expression levels increase during maturation up to 72 hours and decrease afterwards (Fig. 2A) while SOCS-1 expression levels remain constant (Fig. 2B). Statistical analysis was performed using paired, two-tailed student’s t-test. One representative Western blot each is shown with actin levels as loading control.


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)

CYTIP and SOCS-1 expression levels at different time points during maturation in monocyte derived dendritic cells.(A) CYTIP expression levels during maturation of dendritic cells were measured at the indicated time points. Western blots were performed using rat anti human CYTIP antibody 1A3 and visualized with Alexa fluor 680 goat anti rat antibody. Three independent Western blot analyses were analyzed and show a peak expression for CYTIP at 72 hours after induction of maturation. One representative Western blot is shown. (B) SOCS-1 expression during maturation remains stable over time. Western blot analyses were performed with lysates harvested at the indicated time points using rabbit polyclonal anti SOCS-1 antibody and visualized with Alexa fluor 680 goat anti rabbit antibody. One representative Western blot is shown. Arbitrary units were set to show progression of CYTIP (A) and SOCS-1 (B) expression during maturation. Immature dendritic cells (iDC) were used as reference level at time point 0 of maturation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g002: CYTIP and SOCS-1 expression levels at different time points during maturation in monocyte derived dendritic cells.(A) CYTIP expression levels during maturation of dendritic cells were measured at the indicated time points. Western blots were performed using rat anti human CYTIP antibody 1A3 and visualized with Alexa fluor 680 goat anti rat antibody. Three independent Western blot analyses were analyzed and show a peak expression for CYTIP at 72 hours after induction of maturation. One representative Western blot is shown. (B) SOCS-1 expression during maturation remains stable over time. Western blot analyses were performed with lysates harvested at the indicated time points using rabbit polyclonal anti SOCS-1 antibody and visualized with Alexa fluor 680 goat anti rabbit antibody. One representative Western blot is shown. Arbitrary units were set to show progression of CYTIP (A) and SOCS-1 (B) expression during maturation. Immature dendritic cells (iDC) were used as reference level at time point 0 of maturation.
Mentions: To evaluate expression levels over time, dendritic cells were matured at day 6 of culture and harvested 0, 24, 48, 72, 96 and 120 hours after maturation induction. Lysates of all time points were analyzed on Western blots for CYTIP and SOCS-1 levels. In three independent experiments we show that CYTIP expression levels increase during maturation up to 72 hours and decrease afterwards (Fig. 2A) while SOCS-1 expression levels remain constant (Fig. 2B). Statistical analysis was performed using paired, two-tailed student’s t-test. One representative Western blot each is shown with actin levels as loading control.

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