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Adrenomedullin and truncated peptide adrenomedullin(22-52) affect chondrocyte response to apoptotis in vitro: downregulation of FAS protects chondrocyte from cell death

Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.


Expression of functional AM receptor complex CLR/RAMP2 is upregulated under hypoxia in BACs in vitro.
To mimic physiological conditions in vitro, we first validated 3% O 2 culture model as a hypoxic culture model for BACs. We followed HIF-1α translocation through the nuclei of BACs as a marker of cellular response to hypoxia. In normoxia-cultured cells, HIF-1α expression was detected in cytoplasm, which was reinforced under hypoxia (Fig. 1A). Moreover, hypoxia promoted nuclear localization, reaching a significant threefold mean increase (p = 0.004) in HIF-1α/nucleus colocalization (Fig. 1B).
We then investigated the expression and activity of CLR/RAMP2 AM receptor on BACs. Immunofluorescent staining revealed a more sustained signal for both CLR and RAMP2 under hypoxia versus normoxia (mean increase of 1.73-and 2.43-fold, respectively, p = 0.001 for both) ( Fig. 2A-C). In addition, hypoxic culture increased the colocalization cluster size (mean 51% increase, p = 0.027) and widespread distribution at the cell surface (mean 2.32-fold increase, p < 0.001) (Fig. 2D,E).
In both normoxic and hypoxic conditions, AM could induce cyclic adenosine monophosphate (cAMP) production in BACs via the CLR/RAMP2 receptor (Fig. 3A). Moreover, we found a dose-dependent effect of AM on cAMP production between 10 −8 and 10 −6 M under both normoxia (p = 0.016) and hypoxia (p = 0.031), with maximal effect at 10 −7 M (mean 2.31-and 3.54-fold increase, respectively). At this concentration, cAMP production was higher under hypoxia than normoxia (mean 55% increase, p = 0.050). Conversely, AM(22-52) did not induce cAMP production at any concentration tested (p > 0.05) (Fig. 3B). Moreover, when added before AM 10 −7 M stimulation, AM(22-52) blocked cAMP production (Fig. 3C). Such an effect was visible from 10 −10 M AM  concentration, with a mean 48% reduction in cAMP content (p = 0.031), and culminated at 10 −7 M, with a mean 83% reduction. Finally, we assessed the production of AM by BACs and found no significant difference between normoxia and hypoxia, with median concentrations of 10 and 20 pg mL −1 , respectively (Fig. 3D), corresponding to approximately 10 −11 M. Therefore, the intrinsic production of AM will not be a bias in a model in which we use 10 −7 M of exogenous AM in BACs.

AM regulates BAC apoptosis after Fas-L stimulation in vitro.
Under hypoxia, BACs were responsive to Fas-L-induced cell death as demonstrated by increased activity of caspase-3 and -8 (mean 95% and 48% increase; p = 0.002 and p = 0.003, respectively), the major markers of apoptosis induced by death receptors (Fig. 4A,B). Of importance, caspase-9 activity (involved in the apoptosis intrinsic pathway) remained ineffective in non-stimulated or Fas-L-stimulated BACs (p > 0.05) (Fig. 4C). AM had no effect on caspase-3, -8 or -9 activity in the basal culture condition (p > 0.05), whereas AM(22-52) induced a slight but significant increase in caspase-3 activity [mean 2.48 and 2.79 for control and AM , respectively, p = 0.05]. Nevertheless, AM acted on Fas-L-induced apoptosis, as assessed by a significant decrease in caspase-3 activity (mean 35% decrease; p = 0.031), to the control level (p > 0.05). Such an effect of AM could be observed with caspase-8 activity, which also showed a mean 31% decrease compared to Fas-L-stimulated BACs (p < 0.05) but failed to return to the control level. AM  had no significant ability to decrease Fas-L induced caspase-3 activity (p > 0.05) and allowed for the increased caspase-3 activity versus the control condition (mean 27% increase; p = 0.016). AM  had triggered nor a decrease in Fas-L-induced caspase-8 activity, neither an increase as compared to control (p > 0.05).
To evaluate the effects of both AM and AM(22-52) on late stages of apoptosis, TUNEL experiments were performed to detect fragmented DNA in BACs. BACs exhibited a basal apoptosis rate (red cells, control condition), which was increased in the presence of Fas-L (Fig. 5). On quantifying the ratio of TUNEL-positive cells to total cells, we found a significant mean 2.7-fold increase in dead cell proportion versus the control condition (p = 0.001, Fig. 5B). Under basal culture conditions, AM induced a 36% decrease in apoptotic cells (p = 0.021), whereas AM(22-52) did not induce such an effect (p > 0.05). Of note, AM decreased the apoptotic cell number as compared with AM(22-52) (mean 40% decrease; p = 0.046). Of note, both AM and AM  reduced the Fas-L effect on BAC apoptosis (mean 65% and 50%, respectively; p < 0.001 for both). In addition, AM reduced the dead cell number to the basal level, that (22-52)AM failed to do. Moreover, the AM effect was higher than the AM(22-52) effect (mean 29% fewer apoptotic cells; p = 0.005). These results for AM and AM(22-52) effects on BAC apoptosis are summarized in Fig. 6.
To understand how AM and AM(22-52) could enhance cellular resistance to Fas-L, we tested the Fas dysregulation hypothesis. First, the hypoxic cell culture model was tested against the classical normoxic cell culture model. Immunofluorescent staining for Fas revealed more intense cytoplasmic signal in hypoxic as compared to www.nature.com/scientificreports/ normoxic BACs (Fig. 7A). In line, this observation was sustained by reduced Fas distribution in the cytoplasm (mean 42% decrease; p = 0.031, Fig. 7B). FAS mRNA expression in BACs was decreased by a mean of 40% under hypoxia versus normoxia (p = 0.031, Fig. 7C), which could corroborate the decrease observed at the protein level. Then, we used immunofluorescent staining for experiments of the AM and AM(22-52) effects on Fas expression under hypoxia (control condition) (Fig. 8A). A more sustained production of Fas was observed in AM(22-52)-versus AM-treated cells (mean 2.7-fold increase; p = 0.047). In addition, AM treatment decreased Fas production as compared with the control (mean 67% decrease; p = 0.016) (Fig. 8B). Those observations were strengthened by a mean 24% decrease in FAS mRNA expression with AM treatment (p = 0.031), but AM(22-52) had no effect (p > 0.05, Fig. 8C). www.nature.com/scientificreports/

Discussion
As compared with treating BACs with AM(22-52), AM treatment reduced caspase activities and cell death, accompanied by downregulated Fas expression and production. We bring the first piece of evidence that AM reduces articular chondrocyte apoptosis under hypoxic environment, as observed in other cellular models 1,13,[18][19][20] . AM was already demonstrated to exhibit immunomodulatory and anti-apoptotic properties. In the murine CIA model, AM contributes to decreased articular pro-inflammatory cytokine production and increased interleukin 10 level, a potent anti-inflammatory cytokine 17 . In addition, AM-stimulated bone-marrow-derived dendritic cells favoured regulatory T-lymphocyte induction 21 . AM was found an anti-apoptotic agent in human fibroblastlike synoviocytes and a murine osteoblastic cell line and in articular cartilage from CIA mice 8,13,17 . In many cell types, AM has also been found a critical regulator of cell survival in physiologic and pathologic environments [22][23][24][25] . To our knowledge, this paper is the first to show that AM inhibits Fas-induced apoptosis in BACs under hypoxia.
Cartilage is a non-innervated and non-vascularized tissue that is under constant hypoxia. Among classical pathways activated by low oxygen tension, hypoxia-inducible factor (HIF) is one of the most prevalent. HIF-1α is one of the two subunits of the HIF-1 complex. It is sequestered and destroyed in cell cytoplasm when O 2 is > 5% because of its interaction with the von Hippel Lindau tumour suppressor protein (pVHL), a ubiquitin www.nature.com/scientificreports/ E3 ligase complex. This interaction is due to proline hydroxylation of HIF-1α by proline hydroxylase 1-3, which is reversed under 5% O 2 26 . Additionally, pVHL is S-nitrosylated at low oxygen tension, which prevents it from interacting with HIF-1α 27 . We have validated a new culture model using 3% O 2 . The choice of this concentration was motivated by contradictory results for cell viability at 1% O 2 and possible lack of HIF pathway expression at 5% O 2 28,29 . Our model revealed clear nuclear translocation of HIF-1α, which suggests a functional hypoxic culture model for BACs.
In such an environment, AM production and activity could be enhanced in several cell types [23][24][25] , especially in endothelial cells, where CLR and AM levels were increased in response to hypoxic stress, thereby suggesting a regulatory loop to favour the proangiogenic effect of AM 7,30 . Of interest, patients with OA showed elevated levels of circulating AM, with a direct correlation with the severity of joint damage 31 . AM and AM receptor expression was previously demonstrated in chondrocytes, but to date, no information has been available on lack of oxygen modulating AM and AM receptor complex expression 5 . In the present work, we bring some evidence for AM receptor CLR/RAMP2 upregulation under hypoxia, but CLR/RAMP3 expression remained undetectable (data not shown). In addition, we demonstrated that AM production was not modified by lack of oxygen, which suggests that only CLR/RAMP2 upregulation could be considered an oxygen stress response. We also reveal that Of importance in our hypoxia model, the AM(22-52) truncated peptide did not induce any effect. This peptide lacks the intracellular loop (because of a disulfide bond between cysteine residues 16-21) known to mediate the major part of the AM biological effects 3 . Moreover, AM(22-52) inhibited AM-induced cAMP production, which reveals its role as an AM antagonist. This role has been previously proposed, but results were controversial because AM(22-52) could also be an AM agonist 8,13 .  www.nature.com/scientificreports/ With the anti-apoptotic features of AM, this natural peptide could be used as a potential therapeutic agent against the chondrocyte apoptosis observed in OA 32,33 . In such a condition, cell death program is mainly engaged through cell death receptors such as TRAIL and Fas, which account for 20% of the cell death in OA 34,35 . Seol et al. 34 also demonstrated that TRAIL-mediated apoptosis may be reduced under hypoxia. For the first time, we revealed a similar mechanism for Fas, which is downregulated under hypoxia, but we identified that AM but not AM(22-52) may accentuate the downregulation of the Fas receptor. This result may explain part how AM exerts its anti-apoptotic effect on Fas-L-induced BAC apoptosis.
Further work is needed to fully investigate pathways involved in AM control of chondrocyte apoptosis. However, AM may represent a potential therapeutic agent against cartilage degradation in OA.
BAC culture. BACs were isolated from the carpal-metacarpal joint of daily slaughtered cows at the SOVIAM slaughterhouse (Meaux, France). Briefly, small pieces of cartilage were harvested from joints, rinsed 3 times before being minced. Small pieces (< 2 mm) were rinsed again before enzymatic digestion overnight in bacterial collagenase II (2% in DMEM w/v) at 37 °C 36 . Cells were passed through a nylon cell strainer (100 µm porosity) to avoid collection of non-digested cartilage before being rinsed, counted and seeded at high density (10 7 cells in 14-cm diameter TPP Petri dishes) in 15 mL DMEM supplemented with 10% (v/v) FCS, 2 mM l-glutamine, 1% antibiotics and fungizone (v/v). Cells were cultured in a dedicated incubator (Binder CB150) at 37 °C in humidified atmosphere containing 5% CO 2 and 3% O 2 (hypoxic condition) or atmospheric O 2 content (normoxic basal condition). Medium was changed twice a week for 2 weeks to obtain confluent cell culture. Cells were then starved overnight, detached by using trypsin/EDTA, rinsed twice, then counted and plated (3.10 5 cells/well in 24-well culture plates) in starvation medium before treatment. Cells were cultured directly on plastic except for TUNEL and immunostaining experiments, for which they were cultured on glass coverslips. For TUNEL and caspase activity measurements, cells were treated for 24 h with 20 ng mL −1 Fas-L. In all cases, cells were pretreated for 30 min with AM or AM (22- In vitro apoptosis. BAC early apoptosis was first determined by caspase-3, -8, and -9 activity measurements by using specific protease substrates as described 19 . Briefly, cells were lysed in 200 µL lysis buffer [tris(hydroxymethyl)aminomethane 10 mM, NaCl 200 mM, EDTA 5 mM, glycerol 10% (v/v) and NP40 1% (v/v) at pH 7.4] for at least 30 min at 4 °C after 24 h of Fas-L treatment with or without AM or AM . Lysates were then frozen and stored at − 20 °C. After thawing, they were centrifuged at 10,000 rpm for 10 min at 4 °C, and supernatants were collected. Caspase activity was determined by specific fluorogenic substrate cleavage (DEVD-AFC, LEHD-AFC, IETD-AFC, for caspase-3, -8 and -9, respectively). Substrates were conjugated with a fluorophore (7-amino-4-trifluoromethyl coumarin) and fluorescence was measured after its release at 505-nm emission wavelength (excitation at 400 nm) on a GloMax-Multi fluorimeter (PROMEGA). The assay was performed with 50 µL samples in duplicate incubated for 2 h at 37 °C with 50 µL reaction buffer mixed in reaction buffer [DTT 10 mM, PMSF 0.1 mM, Hepes 10 mM at pH 7.4] containing 5 µL specific substrate (1 mM). Results are expressed as arbitrary units and normalized to total protein content (BCA Protein Assay kit, PIERCE). TUNEL assay was performed to evaluate late apoptosis. BACs cultured on glass coverslips were stained for TUNEL assay by using a commercial kit (ApopTag Red In Situ Apoptosis Detection Kit, MILLIPORE), according to the manufacturer's protocol. Briefly, chondrocytes were fixed first with 1% paraformaldehyde (w/v), then with an acetic acid/ethanol solution. Cells were incubated with terminal deoxynucleotidyl transferase enzyme for 1 h at 37 °C in a humidified dark chamber and with rhodamine-coupled digoxigenin at room temperature. Finally, cells were rinsed and mounted with the appropriate medium. Image acquisition was performed on an inverted Zeiss AxioImager Z1 microscope with a × 20 objective. Cell counts were performed on four randomly chosen fields containing at least 100 cells. Results are expressed as percentage of TUNEL-positive cells versus total cell number.

Statistical analysis.
All experiments were performed in duplicate with BACs derived at least from 6 independent animals for each experiment. The significance of results was assessed first with a non-parametric Kruskal-Wallis test followed by a post-hoc exact non-parametric and stratified Wilcoxon-Mann-Whitney test as appropriate (StatXact 7.0, Cytel Inc.). We used non-parametric statistics because of lack of normal distribution of the assessed variables (due to small number of samples), and the stratification allowed for taking into account the impact of individual variability. Difference between conditions was considered significant at p < 0.05.