MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

A proteomic approach to investigate the role of the MECP2 gene mutation in Rett syndrome redox regulatory pathways.

Mutations in the X-linked methyl-CpG-binding 2 (MECP2) gene lead to Rett Syndrome (RTT; OMIM 312750), a devasting neurodevelopmental disorder. RTT clinical manifestations are complex and with different degrees of severity, going from autistic-like behavior to loss of acquired speech, motor skills and cardiac problems. Furthermore, the correlation between the type of MECP2 mutation and the clinical phenotype is still not fully understood. Contextually, different genotypes can differently affect the patient's phenotype and omics methodologies such as proteomics could be an important tool for a molecular characterization of genotype/phenotype correlation. The aim of our study was focused on evaluating RTT oxidative stress (OS) responses related to specific MECP2 gene mutations by using proteomics and bioinformatics approaches. Primary fibroblasts isolated from patients affected by R133C and R255× mutations were compared to healthy controls (HC). After clustering primary dermal fibroblasts based on their specific MECP2 mutations, fibroblast-derived protein samples were qualitative and quantitative analyzed, using a label free quantification (LFQ) analysis by mass spectrometry (MS), achieving a preliminary correlation for RTT genotype/phenotype. Among the identified proteins involved in redox regulation pathways, NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) was found to be absent in R255× cells, while it was present in R133C and in HC fibroblasts. Moreover, NQO1 aberrant gene regulation was also confirmed when cells were challenged with 100 µM hydrogen peroxide (H2O2). In conclusion, by employing a multidisciplinary approach encompassing proteomics and bioinformatics analyses, as well as molecular biology assays, the study uncovered phenotypic responses linked to specific MECP2 gene mutations. These findings contribute to a better understanding of the complexity of RTT molecular pathways, confirming the high heterogeneity among the patients.

Hydroxyanthracene derivates citotoxicity: A differential evaluation between single molecule and whole plant extract.

Hydroxyanthracene derivates (HADs) are a group of natural or synthetic compounds with a wide range of biological activities (for instance, anti-inflammatory, antibacterial, and antiarthritic). In addition, because of their properties for helping the normal bowel function, HADs are widely used in constipation as pharmacological drugs and nutritional supplements. Nevertheless, during the past years, a safety usage of HAD products has been under consideration because some studies reported that HADs are not lacking toxicity (i.e., genotoxic and carcinogenic activity). Thus, the first objective of this study is to shed light on the large variability in composition of botanical food supplements containing HAD by a systematic analysis of the qualitative and quantitative composition of a cohort of extracts and raw materials of plants with high levels of anthraquinones commercially available (Cassia angustifolia, Rhamnus purshiana, Rhamnus frangula, Rheum palmatum, and Rheum raponticum). To date, the investigation of HAD toxicity was based on in vitro and in vivo studies conducted mainly on the use of the single molecules (emodin, aloe-emodin, and rhein) rather than on the whole plant extract. The qualitative-quantitative characterization was the starting point to select the most appropriate products to be used as treatment for our in vitro cell studies. Thus, the second objective of this study is the investigation, for the first time, of the toxic events of HAD used as single molecule in comparison with the whole plant extracts containing HAD in an intestinal in vitro model using human colorectal adenocarcinoma cells (Caco-2). In addition, a shotgun proteomics approach was applied to profile the differential protein expression in the Caco-2 cells after a single-HAD or whole-plant extract treatment to fully understand the potential targets and signaling pathways. In conclusion, the combination of a detailed phytochemical characterization of HAD products and a largely accurate analysis of the proteomic profile of intestinal cells treated with HAD products provided the opportunity to investigate their effects in the intestinal system.

Dimerization of the C-type lectin-like receptor CD93 promotes its binding to Multimerin-2 in endothelial cells.

Blocking the signaling activated by the plasma membrane receptor CD93 has recently been demonstrated a useful tool in antiangiogenic treatment and oncotherapy. In the proliferating endothelium, CD93 regulates cell adhesion, migration, and vascular maturation, yet it is unclear how CD93 interacts with the extracellular matrix activating signaling pathways involved in the vascular remodeling. Here for the first time we show that in endothelial cells CD93 is structured as a dimer and that this oligomeric form is physiologically instrumental for the binding of CD93 to its ligand Multimerin-2. Crystallographic X-ray analysis of recombinant CD93 reveals the crucial role played by the C-type lectin-like and sushi-like domains in arranging as an antiparallel dimer to achieve a functional binding state, providing key information for the future design of new drugs able to hamper CD93 function in neovascular pathologies.

In Vitro Anti-Proliferative and Apoptotic Effects of Hydroxytyrosyl Oleate on SH-SY5Y Human Neuroblastoma Cells.

The antitumor activity of polyphenols derived from extra virgin olive oil and, in particular the biological activity of HTyr, has been studied extensively. However, the use of HTyr as a therapeutic agent for clinical applications is limited by its low bioavailability and rapid excretion in humans. To overcome these limitations, several synthetic strategies have been optimized to prepare lipophenols and new compounds derived from HTyr to increase lipophilicity and bioavailability. One very promising ester is hydroxytyrosyl oleate (HTyr-OL) because the chemical structure of HTyr, which is responsible for several biological activities, is linked to the monounsaturated chain of oleic acid (OA), giving the compound high lipophilicity and thus bioavailability in the cellular environment. In this study, the in vitro cytotoxic, anti-proliferative, and apoptotic induction activities of HTyr-OL were evaluated against SH-SY5Y human neuroblastoma cells, and the effects were compared with those of HTyr and OA. The results showed that the biological activity of HTyr was maintained in HTyr-OL treatments at lower dosages. In addition, the shotgun proteomic approach was used to study HTyr-OL-treated and untreated neuroblastoma cells, revealing that the antioxidant, anti-proliferative and anti-inflammatory activities of HTyr-OL were observed in the unique proteins of the two groups of samples.

Reduced miR-184-3p expression protects pancreatic ß-cells from lipotoxic and proinflammatory apoptosis in type 2 diabetes via CRTC1 upregulation.

The loss of functional ß-cell mass in type 2 diabetes (T2D) is associated with molecular events that include ß-cell apoptosis, dysfunction and/or dedifferentiation. MicroRNA miR-184-3p has been shown to be involved in several ß-cell functions, including insulin secretion, proliferation and survival. However, the downstream targets and upstream regulators of miR-184-3p have not been fully elucidated. Here, we show reduced miR-184-3p levels in human T2D pancreatic islets, whereas its direct target CREB regulated transcription coactivator 1 (CRTC1) was increased and protects ß-cells from lipotoxicity- and inflammation-induced apoptosis. Downregulation of miR-184-3p in ß-cells leads to upregulation of CRTC1 at both the mRNA and protein levels. Remarkably, the protective effect of miR-184-3p is dependent on CRTC1, as its silencing in human ß-cells abrogates the protective mechanism mediated by inhibition of miR-184-3p. Furthermore, in accordance with miR-184-3p downregulation, we also found that the ß-cell-specific transcription factor NKX6.1, DNA-binding sites of which are predicted in the promoter sequence of human and mouse MIR184 gene, is reduced in human pancreatic T2D islets. Using chromatin immunoprecipitation analysis and mRNA silencing experiments, we demonstrated that NKX6.1 directly controls both human and murine miR-184 expression. In summary, we provide evidence that the decrease in NKX6.1 expression is accompanied by a significant reduction in miR-184-3p expression and that reduction of miR-184-3p protects ß-cells from apoptosis through a CRTC1-dependent mechanism.

Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.

Homogentisic acid induces cytoskeleton and extracellular matrix alteration in alkaptonuric cartilage.

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease.

Amyloid-ß Precursor Protein APP Down-Regulation Alters Actin Cytoskeleton-Interacting Proteins in Endothelial Cells.

The amyloid-ß precursor protein (APP) is a ubiquitous membrane protein often associated with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Despite its role in the development of the pathogenesis, APP exerts several physiological roles that have been mainly investigated in neuronal tissue. To date, the role of APP in vasculature and endothelial cells has not been fully elucidated. In this study, we used molecular and proteomic approaches to identify and investigate major cellular targets of APP down-regulation in endothelial cells. We found that APP is necessary for endothelial cells proliferation, migration and adhesion. The loss of APP alters focal adhesion stability and cell-cell junctions' expression. Moreover, APP is necessary to mediate endothelial response to the VEGF-A growth factor. Finally, we document that APP propagates exogenous stimuli and mediates cellular response in endothelial cells by modulating the Scr/FAK signaling pathway. Thus, the intact expression and processing of APP is required for normal endothelial function. The identification of molecular mechanisms responsible for vasoprotective properties of endothelial APP may have an impact on clinical efforts to preserve and protect healthy vasculature in patients at risk of the development of cerebrovascular disease and dementia including AD and CAA.

A proteomics approach to further highlight the altered inflammatory condition in Rett syndrome.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene. RTT patients show multisystem disturbances associated with perturbed redox homeostasis and inflammation, which appear as possible key factors in RTT pathogenesis. In this study, using primary dermal fibroblasts from control and RTT subjects, we performed a proteomic analysis that, together with data mining approaches, allowed us to carry out a comprehensive characterization of RTT cellular proteome. Functional and pathway enrichment analyses showed that differentially expressed proteins in RTT were mainly enriched in biological processes related to immune/inflammatory responses. Overall, by using proteomic data mining as supportive approach, our results provide a detailed insight into the molecular pathways involved in RTT immune dysfunction that, causing tissue and organ damage, can increase the vulnerability of affected patients to unknown endogenous factors or infections.

Proteomic profiling reveals mitochondrial alterations in Rett syndrome.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder associated with mutation in MECP2 gene. Despite a well-defined genetic cause, there is a growing consensus that a metabolic component could play a pivotal role in RTT pathophysiology. Indeed, perturbed redox homeostasis and inflammation, i.e. oxinflammation, with mitochondria dysfunction as the central hub between the two phenomena, appear as possible key contributing factors to RTT pathogenesis and its clinical features. While these RTT-related changes have been widely documented by transcriptomic profiling, proteomics studies supporting these evidences are still limited. Here, using primary dermal fibroblasts from control and patients, we perform a large-scale proteomic analysis that, together with data mining approaches, allow us to carry out the first comprehensive characterization of RTT cellular proteome, showing mainly changes in expression of proteins involved in the mitochondrial network. These findings parallel with an altered expression of key mediators of mitochondrial dynamics and mitophagy associated with abnormal mitochondrial morphology. In conclusion, our proteomic analysis confirms the pathological relevance of mitochondrial dysfunction in RTT pathogenesis and progression.

Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (-30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preß-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.

In vitro effects of VA441, a new selective cyclooxygenase-2 inhibitor, on human osteoarthritic chondrocytes exposed to IL-1ß.

The aim of this in vitro study was to examine the possible effect of [2-methyl-5-(4-methylsulphonyl)phenyl-1-phenyl-3-(2-n-propyloxyethyl)]-1H-pyrrole (VA441), a new selective cyclooxygenase (COX)-2 inhibitor, on human osteoarthritic (OA) chondrocyte cultivated in the presence or absence of interleukin-1ß (IL-1ß). In particular, we assessed the effects of 1 and 10 µM of VA441, celecoxib, and indomethacin on cell viability, COX-2 and inducible nitric oxide synthase (iNOS) gene expression, prostaglandin E(2) (PGE(2)) production, and nitric oxide (NO) and metalloproteinase-3 (MMP-3) release. Furthermore, we carried out morphological assessment by transmission electron microscopy (TEM). The presence of IL-1ß led to a significant increase in PGE(2), MMP-3, and NO production, as well as a significant increase in gene expression of COX-2 and iNOS. All the drugs tested had a statistically significant inhibitory effect on PGE(2) production and gene expression of COX-2 stimulated by IL-1ß. VA441 and celecoxib significantly suppressed IL-1ß-stimulated MMP-3 and NO and iNOS gene expression in a dose-dependent manner, while indomethacin did not show any significant effect on MMP-3 and NO production or on iNOS gene expression. TEM demonstrated that IL-1ß severely alters the structure of chondrocytes; after co-incubation with VA441 or celecoxib, the cells recovered their ultrastructure. Our data suggest that VA441 and celecoxib may have a beneficial effect on chondrocyte metabolism.

Alkaptonuria is a novel human secondary amyloidogenic disease.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Biochemical and proteomic characterization of alkaptonuric chondrocytes.

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as "black" AKU chondrocytes, while those coming from the white portion were referred to as "white" AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both "white" and "black" AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in "black" AKU chondrocytes.

Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria.

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy.

Raloxifene protects cultured human chondrocytes from IL-1ß induced damage: a biochemical and morphological study.

It is well known that estrogens are implicated in the pathogenesis of osteoarthritis. Raloxifene is a selective estrogen receptor modulator used in the treatment of osteoporosis, though little is known about the possible effects of raloxifene on cartilage metabolism. The aim of our study was to evaluate the possible in vitro effects of raloxifene in human osteoarthritis chondrocytes cultivated in the presence or absence of Interleukin-1 beta (IL-1ß) (5 ng/ml). The effects of 0.1 µM and 1 µM of raloxifene in the culture medium were assessed using an immuno-enzymatic method for proteoglycans and metalloproteinase-3 (MMP-3), and the Griess method for nitrite. Gene expression of inducible Nitric Oxide Synthase (iNOS) was detected by real-time PCR. A morphological analysis was performed by transmission electron microscopy (TEM). Cell viability was significantly (P<0.01) reduced by the IL-1ß, and restored to basal levels by raloxifene at both of the concentrations used. The presence of IL-1 ß led to a significant decrease (P<0.01) in proteoglycan levels as well as a significant increase of MMP-3 and NO (P<0.01). When the cells were co-incubated with IL-1ß and raloxifene, a significant and dose-dependent increase in proteoglycans and a reduction of MMP-3 and nitric oxide (NO) were detected. iNOS was noticeably expressed in IL-1ß stimulated chondrocytes, while raloxifene decreased in a very significant manner the gene expression of iNOS at both of the concentrations used. The results of the biochemical evaluation were confirmed by TEM. Our data suggest that raloxifene may have a potential chondroprotective role in osteoarthritis.

Development of an in vitro model to investigate joint ochronosis in alkaptonuria.

OBJECTIVES: Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. METHODS: Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 µM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorl's stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. RESULTS: There was a dose-related deposition of pigment in cells and associated matrix from 33 µM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. CONCLUSION: We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.

Proteomic and redox-proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes.

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues up to the deposition of melanin-like pigments (ochronosis). Since little is known on the effects of HGA and its metabolites on articular cells, we carried out a proteomic and redox-proteomic analysis to investigate how HGA and ascorbic acid (ASC) affect the human chondrocytic protein repertoire. We settled up an in vitro model using a human chondrocytic cell line to evaluate the effects of 0.33 mM HGA, alone or combined with ASC. We found that HGA and ASC significantly affect the levels of proteins with specific functions in protein folding, cell organization and, notably, stress response and cell defense. Increased protein carbonyls levels were found either in HGA or ASC treated cells, and evidences produced in this paper support the hypothesis that HGA-induced stress might be mediated by protein oxidation. Our finding can lay the basis towards the settling up of more sophisticated models to study AKU and ochronosis.