Human S100A9 protein is stabilized by inflammatory stimuli via the formation of proteolytically-resistant homodimers

Abstract

S100A8 and S100A9 are Ca(2+)-binding proteins that are associated with acute and chronic inflammation and cancer. They form predominantly heterodimers even if there are data supporting homodimer formation. We investigated the stability of the heterodimer in myeloid and S100A8/S100A9 over-expressing COS cells. In both cases, S100A8 and S100A9 proteins were not completely degraded even 48 hrs after blocking protein synthesis. In contrast, in single transfected cells, S100A8 protein was completely degraded after 24 h, while S100A9 was completely unstable. However, S100A9 protein expression was rescued upon S100A8 co-expression or inhibition of proteasomal activity. Furthermore, S100A9, but not S100A8, could be stabilized by LPS, IL-1β and TNFα treatment. Interestingly, stimulation of S100A9-transfected COS cells with proteasomal inhibitor or IL-1β lead to the formation of protease resistant S100A9 homodimers. In summary, our data indicated that S100A9 protein is extremely unstable but can be rescued upon co-expression with S100A8 protein or inflammatory stimuli, via proteolytically resistant homodimer formation. The formation of S100A9 homodimers by this mechanism may constitute an amplification step during an inflammatory reaction.

Figure 1. hS100A8 and hS100A9 form a stable heterodimer in THP-1.

THP-1 were treated with DSS for 30 min in ice. Subsequently samples were used for Western blot and stained for hS100A9 and hS100A8 (A). THP-1 cells were treated with 10 µg/ml cycloheximide for 4 h, 24 h and 48 h. Samples were, then, collected and Western blot for hS100A9 and hS100A8 was performed (B).

Figure 2. The hS100A8/hS100A9 heterodimers were more stable than hS100A8 and hS100A9 homodimers.

COS cells were transfected with hS100A8 and hS100A9 separately or together. After 24 h, COS cells were treated with 100 µg/ml cycloheximide for 4 h, 24 h and 48 h. Samples were collected and analyzed by Western blot. Filters were stained with (A) anti-hS100A9 or (B) anti-hS100A8. In the lower charts, the relative band intensity compared to non-stimulated cells are indicated.

Figure 3. hS100A9 protein was unstable in COS cells but could be stabilized by MG132 or co-transfection with hS100A8.

COS cells were transfected with hS100A8 and hS100A9 constructs either separately or together. 24 h after transfection, the cells were stimulated for 8 h with 1 or 10 µM MG132 and subsequently analyzed by Western blot using (A) anti-hS100A9 or (B) anti-hS100A8. The first three lanes (NT) of each panel represented non-transfected cells. From lane 4 to 6, COS cells were transfected with (A; A9 Transfected) hS100A9 or (B; A8 Transfected) hS100A8 separately, while from lane 7 to 9 (A8/A9 Transfected) cells were co-transfected.

Figure 4. hS100A9 protein was unstable in LEP cells but could be rescued by MG132 and hS100A8.

hS100A8 and hS100A9 expression vectors were transfected into human fibroblasts (LEP cells) as described before for COS cells. After SDS-PAGE and Western blots were performed either with (A) anti-hS100A9 or (B) anti-hS100A8. Lane 1 (NT) represented non-transfected cells; from lane 2 to lane 4 cells were transfected with hS100A9 (panel A; A9 T) or with hS100A8 (panel B; A8 T); in lane 5 (A8/A9 T) cells were co-transfected with both constructs.

Figure 5. hS100A8 associates with hS100A9 in co-transfected COS cells.

COS cells were co-transfected with expression vectors for both hS100A8 and hS100A9. 24 h later, hS100A9 was immunoprecipitated and analyzed for hS100A8 expression by Western blot (Panel 2, lane 1). The same experiment was repeated immunoprecipitating hS100A8 and staining for hS100A9 (Panel 1, lane 1). Lane 2 and 3 represented the controls. In brief, in control samples, COS cells were co-transfected and a full Co-IP experiment was performed but without the cell extract, or the antibody, respectively. In panel 1, lane 4, COS cells were transfected only with hS100A9-carrying vector. The hS100A9 protein was immunoprecipitated and Western blot performed with hS100A8 staining.

Figure 6. LPS and IL1β induced hS100A9 protein stabilization.

COS cells were transfected either with hS100A8 or hS100A9 constructs, as described above. 24 h after transfection, COS cells were stimulated with either 1 or 10 ng/ml IL1β or alternatively with either 10 or 100 ng/ml LPS. Samples were collected and analyzed by Western blot. Filters were stained with (A) anti-hS100A9 and (B) anti-hS100A8. Lane 1 (NT) represented non-transfected cells; from lane 2 to 6, COS cells were transfected with (A; A9 T) hS100A9 or (B; A8 T) hS100A8 separately; in lane 7 (A8/A9 T) COS cells were co-transfected with both plasmids.

Figure 7. IL1β promotes the formation of protease-resistant hS100A9 homodimers.

COS cells were transfected with hS100A8 and hS100A9 expression vectors, either separately or together. 24 h after transfection, transfected cells were treated either with MG132 or IL1β. Then, COS cells were incubated with 1 mM DSS on ice for 30 minutes and analyzed by Western blot (hS100A9 (A) and hS100A8 (B)).