The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that S100A11 and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-alpha induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-alpha-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA.

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1β in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1β responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.

A multi-antigen multi-peptide vaccine, targeting proteins expressed in pre-invasive breast lesions, can stimulate Type I CD4+ T-cells which have been shown to be deficient in both breast cancer patients and mice that develop mammary tumors. Transgenic mice (TgMMTV-neu) were immunized with a multi-antigen peptide vaccine specific for neu, IGFBP-2 and IGF-IR at a time when some of the animals already had pre-invasive lesions (18 weeks of age). While immunization with each individual antigen was partially effective in inhibiting tumor growth, immunization with the multi-antigen vaccine was highly effective, blocking development of palpable lesions in 65% of mice and slowing tumor growth in the infrequent palpable tumors which did arise. Protection was mediated by CD4+ T-cells and the few slow-growing tumors that did develop demonstrated a significant increase in intratumoral CD8+ T-cells as compared to controls (p=0.0007). We also combined the vaccine with agents that were, by themselves, partially effective inhibitors of tumor progression in this model; lapatinib and the RXR agonist bexarotene. While the combination of lapatinib and vaccination performed similarly to vaccination alone (p=0.735), bexarotene and vaccination significantly enhanced disease free survival (p<0.0001) and approximately 90% of mice showed no pathologic evidence of carcinomas at 1 year. The vaccine also demonstrated significant clinical efficacy in an additional transgenic model of breast cancer (TgC3(I)-Tag). Chemo-immunoprevention combinations may be an effective approach to breast cancer prevention even when the vaccine is administered in the presence of subclinical disease.