Christophe Chauveau

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AIMS Joint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs). MAIN METHODS In MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct. KEY FINDINGS TNF-alpha (from 0.1 to 10 ng/ml) and IL-1beta (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-alpha-treated cultures did not reverse TNF-alpha effects, indicating that IL-1 was not involved in TNF-alpha-stimulated TNAP activity. Both TNF-alpha and IL-1beta decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures. SIGNIFICANCE The different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.

Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes.

In osteoporosis, bone loss is accompanied by greater adiposity in the marrow. Given the cellular proximity within the bone marrow, we wondered whether adipocytes might have a paracrine impact on osteoblast differentiation. To test this hypothesis, we cocultured adipocytes with osteoblasts derived from mesenchymal stem cells (MSCs) in the absence of direct cell contact and then analyzed gene expression changes in the osteoblastic population by using real-time reverse transcription polymerase chain reaction. We found that, upon coculture, MSC-derived osteoblasts showed appearance of adipogenic (lipoprotein lipase, leptin) and decrease of osteogenic (osteocalcin) mRNA markers. Our results indicate that in vitro, MSC-derived adipocytes are capable of inducing MSC-derived osteoblasts to differentiate to an adipocyte phenotype. These new data suggest that (i) transdifferentiation of committed osteoblasts into adipocytes may contribute to the increase in marrow fat content at the expense of bone-forming cells and (ii) this switch might be initiated by the adipocytes themselves.

Ghrelin: Central and Peripheral Implications in Anorexia Nervosa

Increasing clinical and therapeutic interest in the neurobiology of eating disorders reflects their dramatic impact on health. Chronic food restriction resulting in severe weight loss is a major symptom described in restrictive anorexia nervosa (AN) patients, and they also suffer from metabolic disturbances, infertility, osteopenia, and osteoporosis. Restrictive AN, mostly observed in young women, is the third largest cause of chronic illness in teenagers of industrialized countries. From a neurobiological perspective, AN-linked behaviors can be considered an adaptation that permits the endurance of reduced energy supply, involving central and/or peripheral reprograming. The severe weight loss observed in AN patients is accompanied by significant changes in hormones involved in energy balance, feeding behavior, and bone formation, all of which can be replicated in animals models. Increasing evidence suggests that AN could be an addictive behavior disorder, potentially linking defects in the reward mechanism with suppressed food intake, heightened physical activity, and mood disorder. Surprisingly, the plasma levels of ghrelin, an orexigenic hormone that drives food-motivated behavior, are increased. This increase in plasma ghrelin levels seems paradoxical in light of the restrained eating adopted by AN patients, and may rather result from an adaptation to the disease. The aim of this review is to describe the role played by ghrelin in AN focusing on its central vs. peripheral actions. In AN patients and in rodent AN models, chronic food restriction induces profound alterations in the « ghrelin » signaling that leads to the development of inappropriate behaviors like hyperactivity or addiction to food starvation and therefore a greater depletion in energy reserves. The question of a transient insensitivity to ghrelin and/or a potential metabolic reprograming is discussed in regard of new clinical treatments currently investigated.

Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (EVs)

BackgroundIn osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model.ResultsWe observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts.ConclusionsHere, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

BackgroundOsteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages.In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation.ResultsGene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage.ConclusionThis model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.

Identification of CBFA1-regulated genes on SaOs-2 cells.

Current knowledge about mechanisms controlling osteoblast-specific gene expression has led to the identification of Cbfa1 as a key regulator of osteoblast differentiation. Several essential questions about this transcription factor remain to be addressed, e.g., the nature of stimuli that may modulate its own expression, as well as the genetic repercussions following alterations in Cbfa1 levels. To identify such Cbfa1-responsive genes, the SaOs-2 cell line was stably transfected with a dominant negative mutant of Cbfa1 (ΔCbfa1). Comparison of gene expression patterns by differential display on selected SaOs-2 clones allowed the identification of four new genes that may be under the control of Cbfa1. Three of them, SelM, elF-4AI, and RPS24, seemed to be linked to a global change in cellular metabolism and cell growth. The fourth, the CD99/MIC2 gene, was strongly overexpressed (around tenfold) in cells presenting high levels of Δcbfa1. This observation adds evidence to show that this marker of Ewing family tumors is linked to the osteoblast lineage. The exact function of CD99 remains largely undefined, and this is the first time that its regulation by an essential transcription factor involved in osteoblast differentiation has been observed.

Physical activity: benefit or weakness in metabolic adaptations in a mouse model of chronic food restriction?

In restrictive-type anorexia nervosa (AN) patients, physical activity is usually associated with food restriction, but its physiological consequences remain poorly characterized. In female mice, we evaluated the impact of voluntary physical activity with/without chronic food restriction on metabolic and endocrine parameters that might contribute to AN. In this protocol, FRW mice (i.e., food restriction with running wheel) reached a crucial point of body weight loss (especially fat mass) faster than FR mice (i.e., food restriction only). However, in contrast to FR mice, their body weight stabilized, demonstrating a protective effect of a moderate, regular physical activity. Exercise delayed meal initiation and duration. FRW mice displayed food anticipatory activity compared with FR mice, which was strongly diminished with the prolongation of the protocol. The long-term nature of the protocol enabled assessment of bone parameters similar to those observed in AN patients. Both restricted groups adapted their energy metabolism differentially in the short and long term, with less fat oxidation in FRW mice and a preferential use of glucose to compensate for the chronic energy imbalance. Finally, like restrictive AN patients, FRW mice exhibited low leptin levels, high plasma concentrations of corticosterone and ghrelin, and a disruption of the estrous cycle. In conclusion, our model suggests that physical activity has beneficial effects on the adaptation to the severe condition of food restriction despite the absence of any protective effect on lean and bone mass.

Long-Term Physiological Alterations and Recovery in a Mouse Model of Separation Associated with Time- Restricted Feeding: A Tool to Study Anorexia Nervosa Related Consequences

Background Anorexia nervosa is a primary psychiatric disorder, with non-negligible rates of mortality and morbidity. Some of the related alterations could participate in a vicious cycle limiting the recovery. Animal models mimicking various physiological alterations related to anorexia nervosa are necessary to provide better strategies of treatment. Aim To explore physiological alterations and recovery in a long-term mouse model mimicking numerous consequences of severe anorexia nervosa. Methods C57Bl/6 female mice were submitted to a separation-based anorexia protocol combining separation and time-restricted feeding for 10 weeks. Thereafter, mice were housed in standard conditions for 10 weeks. Body weight, food intake, body composition, plasma levels of leptin, adiponectin, IGF-1, blood levels of GH, reproductive function and glucose tolerance were followed. Gene expression of several markers of lipid and energy metabolism was assayed in adipose tissues. Results Mimicking what is observed in anorexia nervosa patients, and despite a food intake close to that of control mice, separation-based anorexia mice displayed marked alterations in body weight, fat mass, lean mass, bone mass acquisition, reproductive function, GH/IGF-1 axis, and leptinemia. mRNA levels of markers of lipogenesis, lipolysis, and the brown-like adipocyte lineage in subcutaneous adipose tissue were also changed. All these alterations were corrected during the recovery phase, except for the hypoleptinemia that persisted despite the full recovery of fat mass. Conclusion This study strongly supports the separation-based anorexia protocol as a valuable model of long-term negative energy balance state that closely mimics various symptoms observed in anorexia nervosa, including metabolic adaptations. Interestingly, during a recovery phase, mice showed a high capacity to normalize these parameters with the exception of plasma leptin levels. It will be interesting therefore to explore further the central and peripheral effects of the uncorrected hypoleptinemia during recovery from separation-based anorexia.

Leptin Receptors and β2-Adrenergic Receptor mRNA Expression in Brain Injury-Related Heterotopic Ossification

Heterotopic ossification (HO) frequently occurs after brain injury. Recently, we found that leptin levels were decreased in the serum of patients with HO. Data suggest two mechanisms mediating leptin effects: a central suppressive mechanism acting via the β2-adrenergic system and a direct stimulatory action starting when leptin binds to its receptors in osteoblastic cells. In this study, we analyzed leptin and β2-adrenergic receptors mRNA expression in osteocytes originated from normal or heterotopic bone biopsies to investigate whether direct or indirect pathway signaling might be implicated in this pathological bone formation. We report for the first time the mRNA expression of the leptin receptor isoforms in osteocytes isolated from all biopsies. Moreover, quantitative reverse transcription-polymerase chain reaction allowed us to measure a significant decrease in the level of β2-adrenergic receptor mRNA in cells isolated from heterotopic bone biopsies. These results could suggest an association between hypothalamic leptin signaling and brain injury-related HO.

Marrow adiposity and bone: Review of clinical implications

There is growing interest in the relationship between bone marrow fat (BMF) and skeletal health. Progress in clinical studies of BMF and skeletal health has been greatly enhanced by recent technical advances in our ability to measure BMF non-invasively. Magnetic resonance imagery (MRI) with or without spectroscopy is currently the standard technique for evaluating BMF content and composition in humans. This review focuses on clinical studies of marrow fat and its relationship with bone. The amount of marrow fat is associated with bone mineral density (BMD). Several studies have reported a significant negative association between marrow fat content and BMD in both healthy and osteoporotic populations. There may also be a relationship between marrow fat and fracture (mostly vertebral fracture), but data are scarce and further studies are needed. Furthermore, a few studies suggest that a lower proportion of unsaturated lipids in vertebral BMF may be associated with reduced BMD and greater prevalence of fracture. Marrow fat might be influenced by metabolic diseases associated with bone loss and fractures, such as diabetes mellitus, obesity and anorexia nervosa. An intriguing aspect of bariatric (weight loss) surgery is that it induces bone loss and fractures, but with different impacts on marrow fat depending on diabetic status. In daily practice, the usefulness for clinicians of assessing marrow fat using MRI is still limited. However, the perspectives are exciting, particularly in terms of improving the diagnosis and management of osteoporosis. Further studies are needed to better understand the regulators involved in the marrow fat-bone relationship and the links between marrow fat, other fat depots and energy metabolism.