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

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

Co-immunoprecipitation of SOCS-1 with CYTIP.

<p>Rat polyclonal anti CYTIP antibody (1A3) was used to co-immunoprecipitate CYTIP and its binding partners in 2 mg of mature dendritic cells lysate. Rabbit anti human SOCS-1 polyclonal antibody was used for detection and visualized with Alexa fluor 680 goat anti rabbit antibody. SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the immunoprecipitate (CO-IP) is shown. As a control, rat IgG1 Isotype control was used for co-immunoprecipitation (Isotype control). SM: size marker.</p

Co-immunoprecipitation of CYTIP with Ubiquitin.

<p>The immunocomplex obtained by incubating 2 mg of mature dendritic cells lysate with rat polyclonal anti CYTIP antibody (1A3) was analyzed by Western blot analyses with mouse anti ubiquitin and visualized with Alexa fluor 680 goat anti mouse antibody. Ubiquitination of the CYTIP precipitate is shown (Co-IP). As a control the precipitate obtained with rat isotype control is shown.</p

Transfection of mature dendritic cells (mDC) with increasing amounts of SOCS-1 encoding plasmid induces a decrease of CYTIP expression.

<p>1×10⁶ cells were transfected with the indicated amounts of SOCS-1 encoding plasmid. 16 hours later protein lysates were prepared and 40 µg of lysates were applied to each lane. CYTIP levels (39 kDa) were measured by Western blot analyses. As a control, actin expression levels were determined. Quantification of grey level pixel intensity was done with Odyssey software. One representative Western blot is shown. iDC: immature dendritic cells, mDC: mature dendritic cells, mock: transfection procedure without plasmid DNA, µg indicate the amount of plasmid used for transfection. CYTIP expression decreases significantly with SOCS-1 over expression 16 hours after transfection with 4 and 8 µg of SOCS-1 encoding plasmid.</p

CYTIP and SOCS-1 expression levels at different time points during maturation in monocyte derived dendritic cells.

<p>(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.</p

(A) FACS analysis of cell surface markers on untreated or 5 nM Bortezomib treated mature dendritic cells.

<p>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.</p