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Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.

Objective— Aldosterone (Aldo) is involved in arterial stiffness and heart failure, but the mechanisms have remained unclear. Galectin-3 (Gal-3), a β-galactoside-binding lectin, plays an important role in inflammation, fibrosis, and heart failure. We investigated here whether Gal-3 is involved in Aldo-induced vascular fibrosis. Methods and Results— In rat vascular smooth muscle cells Gal-3 overexpression enhanced specifically collagen type I synthesis. Moreover Gal-3 inhibition by modified citrus pectin or small interfering RNA blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, inflammation, fibrosis, and increased aortic Gal-3 expression. Spironolactone or modified citrus pectin treatment reversed all the above effects. Wild-type and Gal-3 knock-out mice were treated with Aldo for 6 hours or 3 weeks. Aldo increased aortic Gal-3 expression, inflammation, and collagen type I in wild-type mice at both the short- and the long-term, whereas no changes occurred in Gal-3 knock-out mice. Conclusion— Our data indicate that Gal-3 is required for inflammatory and fibrotic responses to Aldo in vascular smooth muscle cells in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis.

Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl4) toxicity was similar in wild-type (WT), PAR-1−/−, and PAR-1+/−mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl4or its solvent for 6 wk (300 μl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl4treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1+/−and PAR-1−/−mice, respectively ( P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1+/−and PAR-1−/−mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-β receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.

International audience; PURPOSE: The purpose of this study is to describe the time course of gene expression in a skeletal muscle local injury induced by an intramuscular (IM) injection, and to compare the dynamics of gene expression with pathological events. MATERIALS AND METHODS: Ten piglets received 4 IM injections of propylene glycol in the longissimus dorsi muscles 6 h, 2, 7, and 21 days before euthanasia, where control and injected muscle sites were sampled for RNA isolation and microscopic examination. The hybridization of nylon cDNA microarrays was carried out with radioactive probes obtained from the muscle RNA. RESULTS: 153 genes were found under- or over-expressed at least once among the investigated time-conditions. The eight most discriminant genes were also identified: Two genes (GTP-binding protein RAD and Ankyrin repeat domain protein) were over-expressed at 6 h and six genes between 2 and 21 days (Osteonectin, Fibronectin, Matrix metalloproteinase-2, Collagen alpha 1(I) chain, Collagen alpha 2(I) chain, and Thymosin beta-4). Necrosis, inflammation and regeneration were observed through both the dynamics of gene expression profiles and through the microscopic examinations. CONCLUSION: Our data demonstrate that several pathways are involved in post-injection muscle injury, and that necrosis, inflammation and regeneration are not sequential but occur in parallel.

International audience; The molecular mechanisms underlying the progression of cirrhosis toward hepatocellular carcinoma were investigated by a combination of DNA microarray analysis and literature data mining. By using a microarray screening of suppression subtractive hybridization cDNA libraries, we first analyzed genes differentially expressed in tumor and nontumor livers with cirrhosis from 15 patients with hepatocellular carcinomas. Seventy-four genes were similarly recovered in tumor (57.8% of differentially expressed genes) and adjacent nontumor tissues (64% of differentially expressed genes) compared with histologically normal livers. Gene ontology analyses revealed that downregulated genes (n = 35) were mostly associated with hepatic functions. Upregulated genes (n = 39) included both known genes associated with extracellular matrix remodeling, cell communication, metabolism, and post-transcriptional regulation gene (e.g., ZFP36L1), as well as the tumor suppressor gene menin (multiple endocrine neoplasia type 1; MEN1). MEN1 was further identified as an important node of a regulatory network graph that integrated array data with array-independent literature mining. Upregulation of MEN1 in tumor was confirmed in an independent set of samples and associated with tumor size (P = .016). In the underlying liver with cirrhosis, increased steady-state MEN1 mRNA levels were correlated with those of collagen alpha2(I) mRNA (P < .01). In addition, MEN1 expression was associated with hepatic stellate cell activation during fibrogenesis and involved in transforming growth factor beta (TGF-beta)-dependent collagen alpha2(I) regulation. In conclusion, menin is a key regulator of gene networks that are activated in fibrogenesis associated with hepatocellular carcinoma through the modulation of TGF-beta response.

Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

International audience; Type I collagen mediates melanoma cells invasion through upregulation of matrix metalloproteinases-1 and -2 (MMP-1 and -2) expression and activation. We investigated here the contribution of elastin-derived peptides (ED), degradation products of elastin, the main component of elastic fibers in melanoma cells invasion and MMP-1 and -2 expression. Our results evidenced fragmentation of elastin at the invasive front of melanoma, particularly in the most invasive tumors where those fibers nearly totally vanished. By electron microscopy, elastolysis was found to occur mainly at the periphery of melanoma cells, where close contact between elastic fibers and cells could be noticed. Therefore, we showed in vitro that plating melanoma cells high tumorigenic potential on ED-coated dishes, selectively enhanced MMP-2, as membrane-type MMP-1 (MT1-MMP) production and activation. Nevertheless, pro-MMP-2 activation was not observed owing to the parallel increase in tissue inhibitor of metalloproteinase (TIMP)-2 expression. The effects of ED on melanoma cells were found to be mediated by splicing form of beta-galactosidase (S-Gal) occupancy, as being suppressed by lactose. Supplementing collagen lattices with ED led to consistent activation of MMP-2 that can be attributed to TIMP-2 downregulation. Upregulation of MMP-2 activation by ED led to enhanced melanoma cells invasion through S-Gal occupancy. Immunohistochemistry studies, confirmed that S-Gal expression was more prominent at the melanoma invasion site associated with a strong expression of MMP-2 and MT1-MMP. We hypothesize that ED following interactions with S-Gal elastin receptor can favor melanoma cells invasion through a three-dimensional type I collagen matrix by upregulating MMP-2 activation.

International audience; The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, whereas for COL1A2-C-propeptide variants, the phenotypic outcome is milder than predicted.

International audience; Chronic sun exposure and especially UVA wavelengths are responsible for long-term clinical skin changes such as photoageing and photocancers. The objectives of the present study were to analyse the contractile activity of fibroblasts irradiated with several doses of UVA and to evaluate the preventive, protective and restoring effects of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. The forces generated by fibroblasts in tense collagen lattices were quantified using Glasbox device before and after UVA irradiation and the addition of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. The production of collagen was also evaluated before and after irradiation and with and without the presence of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate. A dose of 3 J cm(-2) of UVA showed more than 50% of mortality in fibroblast population after 48 h and significant decreases in contractile forces developed by irradiated fibroblasts and collagen I production. One percentage of a mixture of monomethylsilanetriol mannuronate and dimethylsilanediol salicylate protected fibroblasts from UVA irradiation and made it possible to restore their capacity to the same level as fibroblasts that were not irradiated. It also tended to restore the capacity to synthesize collagen I. These results show that the use of the new device Glasbox makes it possible to evaluate a possible preventive and repairing effect of a cosmetic functional active on photoageing.

International audience; Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-gamma induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.

International audience; BACKGROUND AND OBJECTIVE: Low-Level Laser Therapy (LLLT) has been suggested to improve bone tissue healing. The cellular and molecular mechanisms involved in this effect are still unclear but bone cell proliferation and differentiation alteration have been proposed. The aim of the present study was to investigate, in vitro, the effect of LLLT on bone cell proliferation, osteoblastic and osteoclastic differentiation, both involved in bone remodeling and regeneration. STUDY DESIGN/MATERIALS AND METHODS: Murine bone marrow cells, which contain both osteoblast and osteoclast progenitors, were cultured and induced to differentiate in the absence or in the presence of LLLT. Laser exposition parameters were determined using a powermeter and consisted in an 808 nm infrared wavelength laser light in continuous mode, with an energy density of 4 J/cm(2) administered three times a week. Cell proliferation and differentiation were assessed after specific staining and microscopic analysis of the cultures after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteoclast markers after nucleic acid extraction. RESULTS: The use of a powermeter revealed that the power emitted by the optical fiber of the laser device was markedly reduced compared to the displayed power. This allowed to adjust the LLLT parameters to a final energy density exposure of 4 J/cm(2). In these conditions, proliferation of bone marrow mesenchymal stem cells as well as osteoclast or osteoblast differentiation of the corresponding progenitors were found similar in control and LLLT conditions. CONCLUSION: Using the present experimental protocol, we concluded that an 808 nm wavelength infrared LLLT does not alter murine bone progenitor cell proliferation and differentiation. Moreover our results confirm the necessary use of a powermeter to fix LLLT protocol parameters.

International audience; This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.

International audience; We previously showed that grape extracts enriched in different polyphenolic families were similarly able to prevent reactive oxygen species (ROS) production, although having differential effects on various features of metabolic syndrome when administered at a dose of 21 mg/kg to the fructose (60%)-fed rat (a model of metabolic syndrome). In the present work, we analyzed on the same model the effect of pure polyphenolic molecules (catechin, resveratrol, delphinidin, and gallic acid) administered at a dose of 2.1 mg/kg. Delphinidin and gallic acid prevented insulin resistance, while gallic acid prevented the elevation of blood pressure. All molecules prevented cardiac ROS overproduction and NADPH overexpression. We also showed that fructose feeding was associated with cardiac fibrosis (accumulation of collagen I) and expression of osteopontin, a factor induced by ROS and a collagen I expression inducer. Collagen I and osteopontin expressions were prevented by the administration of all polyphenolic molecules. The potential use of polyphenols in the prevention of cardiac fibrosis should be further explored.

International audience; In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.

International audience; The immunosuppressive drug mycophenolate mofetil (MMF) is used to prevent organ rejection after transplantation and has shown some efficacy to prevent the fibrotic complications that occur during autoimmune diseases such as systemic sclerosis or during graft-versus-host disease (GVHD). We tested the hypothesis that MMF may exert direct effects on fibroblast extracellular matrix remodeling. Incubation of human lung fibroblast cultures with MMF led to dose- and time-dependent reduction in the synthesis and expression of type I collagen. Inhibition of COL1A1 and COL1A2 mRNA steady-state levels occurred at the level of transcription via repression of their promoters. In contrast, MMF significantly enhanced the expression and the synthesis of interstitial collagenase (matrix metalloproteinase-1). MMF was also found to diminish the capacity of fibroblast to contract mechanically unloaded collagen lattices and to reduce the synthesis of alpha-smooth muscle actin, a marker of the contractile myofibroblast phenotype. In addition, MMF diminished the fibroblasts motility. In conclusion, we provide novel mechanism by which MMF alters fibroblast functions important for wound healing and implicated in the development of tissue fibrosis, e.g., collagen production, extracellular matrix contraction, and cell migration. Such properties may contribute to the beneficial therapeutic effects of MMF against fibrotic lesions developing in systemic sclerosis or during GVHD.

International audience; We have examined whether rapamycin, an immunosuppressive drug, may exert part of its antifibrotic activity by directly targeting fibroblast extracellular matrix deposition. Incubation of human lung fibroblast (WI-26) cultures with rapamycin led to dose- and time-dependent reduction in the expression of types I and III collagens, both at the protein and mRNA levels. Rapamycin had no effect on collagen promoter activity but accelerated mRNA decay, indicating post-transcriptional control of collagen gene expression. In contrast, rapamycin significantly enhanced the expression of interstitial collagenase (MMP-1) at the protein and mRNA levels and transcriptionally. We determined that rapamycin efficiently activates AP-1-driven transcription by rapidly inducing c-jun/AP-1 phosphorylation with activation of the c-Jun N-terminal kinase (JNK) cascade, resulting in enhanced binding of AP-1.DNA complex formation and AP-1-dependent gene transactivation. Conversely, the JNK inhibitor SP600125 inhibited rapamycin-induced MMP-1 gene transactivation and AP-1/DNA interactions. A c-jun antisense expression vector efficiently prevented rapamycin-induced MMP-1 gene transcription. Pharmacological inhibition of either ERK or p38 MAPK pathways was without effect on rapamycin-induced MMP-1 gene expression. It thus appears that rapamycin may exert direct antifibrotic activities independent from its immunosuppressive action.

International audience; Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties. Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film. However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks. In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends. The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating. Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films. The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes. Only keratinocyte proliferation was significantly reduced by the presence of cavities. Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts. In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time. In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests.

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Introduction: Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. Materials and Methods: C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10−7 M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Results and Conclusions: Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

International audience; Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.

HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

International audience; Second Harmonic Generation (SHG) microscopy offers the opportunity to image collagen of type I without staining. We recently showed that a simple scoring method, based on SHG images of histological human liver biopsies, correlates well with the Metavir assessment of fibrosis level (Gailhouste et al., J. Hepatol., 2010). In this article, we present a detailed study of this new scoring method with two different objective lenses. By using measurements of the objectives point spread functions and of the photomultiplier gain, and a simple model of the SHG intensity, we show that our scoring method, applied to human liver biopsies, is robust to the objective's numerical aperture (NA) for low NA, the choice of the reference sample and laser power, and the spatial sampling rate. The simplicity and robustness of our collagen scoring method may open new opportunities in the quantification of collagen content in different organs, which is of main importance in providing diagnostic information and evaluation of therapeutic efficiency.

International audience; This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.