In the search for putative “natural” ligands of RAGE, radiolabelled soluble RAGE was used to probe bovine lung extract, the material from which labeled AGE-BSA retrieved RAGE as a binding partner (1
). The discovery that S100A12 and amphoterin (HMGB1) bound RAGE initially presented a quandary for several reasons: first, S100s and amphoterin were largely recognized as intracellular molecules (1
). S100s were located in the intracellular space wherein at least in part through their “EF-hand” domains, these molecules mediated calcium signaling, phagocytosis and cellular migration (28
). Amphoterin was described initially as a non-histone DNA binding protein located in the nucleus (29
). Second, the key signatures of S100s and amphoterin struck the cords of inflammation, cellular migration and invasive potential. How might such molecules engage RAGE and, specifically, were such mechanisms related to diabetes?
In the case of S100s such as S100B, also a specific ligand of RAGE, S100B may exist in a soluble form in the intracellular space, and in association with intracellular membranes, centrosomes, microtubules, and type III intermediate filaments (28
). In the intracellular space, S100B’s chief role is as a calcium sensor (28
). Yet, much evidence indicates that S100B may be actively released from cells during various forms of stress, such as metabolic stress, physical exercise, and ischemia. Also, S100B may be “leaked” from damaged cells (30
An analogous situation has been presented for the case of HMGB1. Although usually found in the nucleus in homeostasis, HMGB1 has been located at the leading edge of transformed cells and in neurite outgrowth (32
). Further, cells damaged by necrosis or autophagy, and those beset by PARP activation may release HMGB1 (33
). It is also possible that release of HMGB1 may be associated with cell death mechanisms initiated by apoptosis (36
Irrespective of the mode of release from the intracellular space, experiments from multiple laboratories suggested that S100/calgranulin or HMGB1 interaction with RAGE-expressing cells modulated properties of inflammatory cells (monocytes/macrophages, T lymphocytes and dendritic cells), vascular cells, epithelial cells, terminally-differentiated cells such as neurons and cardiomyocytes, and transformed cells (37
). These data supported the hypothesis that once “outside” the cell, S100/calgranulin and HMGB1 ligands may exert novel and profound effects on cellular phenotype. Via autocrine and/or paracrine mechanisms, such ligands may engage RAGE or other cellular receptors, thereby activating inflammatory and stress signaling pathways. Importantly, although suggested in vitro
but not proved in vivo
, the “dose” of these molecules may determine whether they mediate injury or contribute to repair. We predict that beyond the local concentrations available in the extracellular environment, the monomeric vs. oligomeric forms of the ligands and their mode of presentation may be more important. For example, we recently demonstrated that multimeric forms of S100, particularly octameric forms, were most apt to stimulate RAGE in retinal pigment epithelial (RPE) cells, activate NF-kB and increase expression of VEGF (42
Yet, given the balance of evidence, is it counter-intuitive that evolution would perpetuate the expression and release of such molecules linked to injury? We propose that the timing, doses and forms of released S100/calgranulins and HMGB1 may be part of an exquisitely regulated system in which in acute stress, such ligand release may facilitate repair. However, once in the extracellular space, the vulnerability of these molecules to modification by oxidative, hyperglycemic and hypoxic stresses may result in their oligomerization, thereby increasing the likelihood that RAGE becomes their chief cell surface target.
In this context, certain S100/calgranulins and HMGB1 have been reported to bind to toll receptors as well as RAGE. Thus, it is conceivable that their occupancy of specific receptors may be mediated, at least in part, via the extracellular microenvironment into which they are cast consequent to cellular stress. The balance of evidence, to date, suggests that RAGE engagement amplifies inflammatory stress, down-regulates repair mechanisms and unless interrupted, causes cellular and tissue damage. Thus, it is not surprising that RAGE is expressed by multiple types of inflammatory cells, as these cells play central roles in the immediate responses to a diverse array of environmental stresses. In the sections to follow, we present the evidence linking RAGE to the immune response.