Pro-inflammatory protein S100A9 alters membrane organization by dispersing ordered domains

Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.     

The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.     

MRP14 (S100A9) protein interacts with Alzheimer beta-amyloid peptide and induces its fibrillization

Increasing evidence supports the contribution of local inflammation to the development of Alzheimer's disease (AD) pathology, although the precise mechanisms are not clear. In this study, we demonstrate that the pro-inflammatory protein S100A9 interacts with the Aβ1-40 peptide and promotes the formation of fibrillar β-amyloid structures. This interaction also results in reduced S100A9 cytotoxicity by the binding of S100A9 toxic species to Aβ1-40 amyloid structures. These results suggest that secretion of S100A9 during inflammation promotes the formation of amyloid plaques. By acting as a sink for toxic species, plaque formation may be the result of a protective response within the brain of AD patients, in part mediated by S100A9.     

S100A8 and S100A9 in Cancer

S100A8 and S100A9 are Ca²⁺ binding proteins that belong to the S100 family. Primarily expressed in neutrophils and monocytes, S100A8 and S100A9 play critical roles in modulating various inflammatory responses and inflammation-associated diseases. Forming a common heterodimer structure S100A8/A9, S100A8 and S100A9 are widely reported to participate in multiple signaling pathways in tumor cells. Meanwhile, S100A8/A9, S100A8, and S100A9, mainly as promoters, contribute to tumor development, growth and metastasis by interfering with tumor metabolism and the microenvironment. In recent years, the potential of S100A8/A9, S100A9, and S100A8 as tumor diagnostic or prognostic biomarkers has also been demonstrated. In addition, an increasing number of potential therapies targeting S100A8/A9 and related signaling pathways have emerged. In this review, we will first expound on the characteristics of S100A8/A9, S100A9, and S100A8 in-depth, focus on their interactions with tumor cells and microenvironments, and then discuss their clinical applications as biomarkers and therapeutic targets. We also highlight current limitations and look into the future of S100A8/A9 targeted anti-cancer therapy.     

Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

The calcium binding protein S100A9 is essential for pancreatic leukocyte infiltration and induces disruption of cell-cell contacts

Leukocyte infiltration is an early and critical event in the development of acute pancreatitis. However, the mechanism of leukocyte transmigration into the pancreas and the function of leukocytes in initiating acute pancreatitis are still poorly understood. Here, we studied the role of S100A9 (MRP14), a calcium binding protein specifically released by polymorph nuclear leukocytes (PMN), in the course of acute experimental pancreatitis. Acute pancreatitis was induced by repeated supramaximal caerulein injections in S100A9 deficient or S100A9 wild-type mice. We then determined S100A9 expression, trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities, and tissue myeloperoxidase (MPO) activity. Cell-cell contact dissociation was analyzed in vitro with biovolume measurements of isolated acini after incubation with purified S100A8/A9 heterodimers, and in vivo as measurement of Evans Blue extravasation after intravenous application of S100A8/A9. Pancreatitis induced increased levels of S100A9 in the pancreas. However, infiltration of leukocytes and MPO activity in the lungs and pancreas during acute pancreatitis was decreased in S100A9-deficient mice and associated with significantly lower serum amylase and lipase activities as well as reduced intrapancreatic TAP-levels. Incubation of isolated pancreatic acini with purified S100A8/A9-heterodimers resulted in a rapid dissociation of acinar cell-cell contacts which was highly calcium-dependent. Consistent with these findings, in vivo application of S100A8/A9 in mice was in itself sufficient to induce pancreatic cell-cell contract dissociation as indicated by Evans Blue extravasation. These data show that the degree of intrapancreatic trypsinogen activation is influenced by the extent of leukocyte infiltration into the pancreas which, in turn, depends on the presence of S100A9 that is secreted from PMN. S100A9 directly affects leukocyte tissue invasion and mediates cell contact dissociation via its calcium binding properties.     

Effect of the C-terminus of murine S100A9 protein on experimental nociception

Calcium-binding protein S100A9 induces antinociception in mice evaluated by the writhing test. Similarly, a peptide identical to the C-terminus of murine S100A9 (mS100A9p) inhibits the hyperalgesia induced by jararhagin, a metalloprotease. Thus, we investigated the effect of mS100A9p on different models used to evaluate nociception. mS100A9p induced a dose-dependent inhibitory effect on the writhing test, and on mechanical hyperalgesia induced by carrageenan. mS100A9p inhibited thermal hyperalgesia induced by carrageenan. mS100A9p did not modify the nociceptive response in hot plate or tail-flick tests. These data demonstrate that the C-terminus of S100A9 protein interferes with control mechanisms of inflammatory pain.     

Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

BACKGROUND: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice. AIM: In an attempt to broaden the concept that neutrophils and MRP-14 controls inflammatory pain induced by different type of irritants, in the present study, after demonstrating that carrageenan (Cg) also induces atinociception in mice, we investigated the participation of both neutrophils and MRP-14 in the phenomenon. METHODS: Male Swiss mice were injected intraperitoneally with Cg and after different time intervals, the pattern of cell migration of the peritoneal exudate and the nociceptive response of animals submitted to the writhing test were evaluated. The participation of neutrophils and of the MRP-14 on the Cg effect was evaluated by systemic inoculation of monoclonal antibodies anti-granulocyte and anti-MRP-14. RESULTS: Our results demonstrate that the acute neutrophilic peritonitis evoked by Cg induced antinociception 2, 4 and 8 h after inoculation of the irritant. Monoclonal antibodies anti-granulocyte or anti-MRP-14 reverts the antinociceptive response only 2 and 8 h after Cg injection. The antibody anti-MRP-14 partially reverts the antinociception observed after 4 h of Cg injection while the anti-granulocyte antibody enhances this effect. This effect is reverted by simultaneous treatment of the animals with both antibodies. After 4 h of Cg injection in neutrophil-depleted mice a significant expression of the calcium-binding protein MRP-14 was detected in the cytoplasm of peritoneal macrophages. This suggests that the enhancement of the effect observed after treatment with the anti-neutrophil antibody may be due to secretion of MRP-14 by macrophages. It has also been demonstrated that endogenous opioids and glucocorticoids are not involved in the antinociception observed at the 4th hour after Cg injection. CONCLUSION: These data support the hypothesis that neutrophils and the calcium-binding protein MRP-14 are participants of the endogenous control of inflammatory pain in mice despite the model of acute inflammation used.     

S100A8 and S100A9—oxidant scavengers in inflammation

S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys₄₂ residue. Met₇₈(O) and Trp₅₄(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp₅₄(+16). Evidence for oxidation of the single Cys₃ residue in recS100A9 HOCl was weak; Met₆₃, Met₈₁, Met₈₃, and Met₉₄ were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met₆₃, Met₈₁, and Met₉₄ were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.     

Oxidation regulates the inflammatory properties of the murine S100 protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.     

A comparison of human S100A12 with MRP-14 (S100A9)

MRP-8 and -14 are two S100 proteins highly expressed as a complex by neutrophils, and to a lesser extent by monocytes and certain squamous epithelia. However, less is known about the close homologue S100A12. This S100 protein is expressed by neutrophils and here we show that it is also expressed by monocytes, but not lymphocytes. An absence of coimmunoprecipitation of MRP-14 and S100A12 indicates that S100A12 is not associated with the MRP proteins in vivo. When directly compared to MRP-14, S100A12 expression by squamous epithelia is more restricted. In esophagus and psoriatic skin, S100A12 is differentially regulated, like MRP-14, but the expression pattern of the two S100 proteins is quite different.     

S100A8/S100A9 and their association with cartilage and bone

S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.     

Antinociceptive effect of the C-terminus of murine S100A9 protein on experimental neuropathic pain

The synthetic peptide identical to the C-terminus of murine S100A9 protein (mS100A9p) has antinociceptive effect on different acute inflammatory pain models. In this study, the effect of mS100A9p was investigated on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. Hyperalgesia, allodynia, and spontaneous pain were assessed to evaluate nociception. These three signs were detected as early as 2 days after sciatic nerve constriction and lasted for over 14 days after CCI. Rats were treated with different doses of mS100A9p by intraplantar, oral, or intrathecal routes on day 14 after CCI, and nociception was evaluated 1h later. These three routes of administration blocked hyperalgesia, allodynia and spontaneous pain. The duration of the effect of mS100A9p depends on the route used and phenomenon analyzed. Moreover, intraplantar injection of mS100A9p in the contralateral paw inhibited the hyperalgesia on day 14 days after CCI. The results obtained herein demonstrate the antinociceptive effect of the C-terminus of murine S100A9 protein on experimental neuropathic pain, suggesting a potential therapeutic use for it in persistent pain syndromes, assuming that tolerance does not develop to mS100A9p.     

Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis

Previous studies suggest that up-regulation of Ras signaling in neurons promotes gliosis and astrocytoma formation in a cell nonautonomous manner. However, the underlying mechanisms remain unknown. To address this question, we generated compound mice (LSL Kras G12D/+;CamKII-Cre) that express oncogenic Kras from its endogenous locus in postmitotic neurons after birth. These mice developed progressive gliosis, which is associated with hyperactivation of Ras signaling pathways. Microarray analysis identified S100A8 and S100A9 as two secreted molecules that are significantly overexpressed in mutant cortices. In contrast to their usual predominant expression in myeloid cells, we found that overexpression of S100A8 and S100A9 in the mutant cortex is primarily in neurons. This neuronal expression pattern is associated with increased infiltration of microglia in mutant cortex. Moreover, purified S100A8-S100A9 but not S100A8 or S100A9 alone promotes growth of primary astrocytes in vitro through both TLR4 and receptor of advanced glycation end product receptors. In summary, our results identify overexpression of S100A8-S100A9 in neurons as an early step in oncogenic Kras-induced gliosis. These molecules expressed in nonhematopoietic cells may be involved in tumorigenesis at a stage much earlier than what has been reported previously.