DCC is expressed in a CD166-positive subpopulation of chondrocytes in human osteoarthritic cartilage and modulates CRE activity

Abstract

Objective: In a recent study we determined a strong differential expression of DCC in OA compared to normal chondrocytes and a strong impact of the DCC receptor on cellular mobility triggered by its ligand Netrin-1. Migration of chondrocytes or their progenitor cells may play a role in remodeling of cartilage and pathological conditions. The purpose of this study is to identify subsets of chondrocytes expressing DCC and to understand signaling pathways used by DCC in chondrocytes.

Methods: Immunofluorescent histology of human cartilage was used to determine the expression pattern of CD166, DCC and p-CREB. Cell culture of chondrocytes and SW1353, transient transfection, siRNA transfection, EMSA, luciferase assay, quantitative RT-PCR, ELISA, and Western Blotting were used to study signaling down-stream of DCC.

Results: DCC expressing chondrocytes are mainly located in the surface layers of OA cartilage. These also express CD166 indicating that DCC expressing chondrocytes are progenitor cells. Interestingly, expression of DCC reduces cAMP levels, CREB DNA-binding activity and CRE activity in chondrocytes, whereas down-regulation of DCC results in induction of CRE signaling.

Conclusion: In summary, DCC is up-regulated in CD166-positive chondrogenic progenitor cells in OA and induces down-regulation of CREB. These findings indicate that migration of CD166 positive progenitor cells to sites of cartilage damage may be directed by regulation of DCC signaling.

Keywords: CRE activity; CREB; DCC; Repellent factors; migration; osteoarthritis.

Figure 1

DCC is increased in CD166 positive progenitor cells in OA cartilage. A. In immunofluorescent histology of normal cartilage CD166 positive cells are situated in the upper layers and do not express DCC (magnification × 20). B. Immunofluorescence stainings for DCC verified induction of DCC in OA cartilage preferentially in CD166 positive cells (magnification × 20).

Figure 2

Activity of NFkB, AP-1 and CREB signaling in normal chondrocytes after DCC or mock transfection. (A) DCC expression had no detectable effects on NFkB and AP-1 signaling by EMSA, whereas marked reduction of CREB DNA-binding activity was observed in normal chondrocytes. (B) The impact of DCC on CREB signaling was confirmed in chondrocytes of three additional donors (C) Specificity of the EMSA was confirmed by cold-competition of binding and by achieving a supershifted band using a specific anti-CREB antibody. (D) A luciferase reporter gene assay was performed in DCC-transfected chondrocytes versus controls and determined significant reduction of CREB activity after transfection of DCC, whereas NFkB activity stayed unchanged.

Figure 3

CREB activity is reduced in OA chondrocytes. CREB luciferase reporter gene assays were performed comparing normal chondrocytes to chondrocytes derived from OA patients. A significant difference in CREB activity was observed comparing normal and OA chondrocytes, whereas NFkB signaling was unchanged. (***: p < 0.001; ns: p > 0.05)

Figure 4

Down-regulation of DCC expression in OA chondrocytes and SW1353 chondrosarcoma cells results in induction of CRE signaling. OA chondrocytes were transfected with siRNA against DCC or control and CREB activity was determined by EMSA and CREB luciferase reporter gene assays. Down-regulation of DCC expression was confirmed on (A) mRNA level (OA chondrocytes and SW1353) and (B) protein level (SW1353). As SW1353 do not express DCC, expression was achieved by transient transfection of a DCC-expression plasmid prior to the experiment. DCC down-regulation resulted in higher CREB activity in (C) EMSAs and (D) luciferase reporter gene assays.

Figure 5

cAMP levels are down-regulated in chondrocytes after DCC transfection. After DCC transfection normal chondrocytes show a reduced cytoplasmic concentration of cAMP measured by ELISA (A). Expression of DCC after transfection was demonstrated by qRT-PCR (B).

Figure 6

DCC is increased and nuclear p-CREB is decreased in CD166 positive progenitor cells in OA cartilage. (A) In healthy cartilage chondrocytes of upper layers display a strong nuclear signal for p-CREB and a faint cytoplasmic signal for DCC (magnification x 40). (B) No nuclear staining for p-CREB was found in strongly DCC positive superficial chondrocytes of OA samples, whereas stationary chondrocytes in clusters showed nuclear staining for p-CREB (C) (magnification × 40).