Self-demixing of mRNA copies buffers mRNA:mRNA and mRNA:regulator stoichiometries.

Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.

NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients.

The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra-Bosch-Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss-of-function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria-like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long-term self-renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area-specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.

Arrest of WNT/ß-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries.

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic ß-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in ß-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of ß-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/ß-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that ß-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

Modulation of the inflammatory response to LPS by the recruitment and activation of brown and brite adipocytes in mice.

Numerous studies have shown that the recruitment and activation of thermogenic adipocytes, which are brown and beige/brite, reduce the mass of adipose tissue and normalize abnormal glycemia and lipidemia. However, the impact of these adipocytes on the inflammatory state of adipose tissue is still not well understood, especially in response to endotoxemia, which is a major aspect of obesity and metabolic diseases. First, we analyzed the phenotype and metabolic function of white and brite primary adipocytes in response to lipopolysaccharide (LPS) treatment in vitro. Then, 8-wk-old male BALB/c mice were treated for 1 wk with a ß3-adrenergic receptor agonist (CL316,243, 1 mg/kg/day) to induce recruitment and activation of brown and brite adipocytes and were subsequently injected with LPS (Escherichia coli lipopolysaccharide, 100 µg/mouse ip) to generate acute endotoxemia. The metabolic and inflammatory parameters of the mice were analyzed 6 h later. Our results showed that in response to LPS, thermogenic activity promoted a local anti-inflammatory environment with high secretion of IL-1 receptor antagonist (IL-1RA) without affecting other anti- or proinflammatory cytokines. Interestingly, activation of brite adipocytes reduced the LPS-induced secretion of leptin. However, thermogenic activity and adipocyte function were not altered by LPS treatment in vitro or by acute endotoxemia in vivo. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes specifically in response to endotoxemia. This encourages potential therapy involving brown and brite adipocytes for the treatment of obesity and associated metabolic diseases.NEW & NOTEWORTHY Recruitment and activation of brown and brite adipocytes in the adipose tissue of mice lead to a local low-grade anti-inflammatory phenotype in response to acute endotoxemia without alteration of adipocyte phenotype and function.

Preventive cancer stem cell-based vaccination reduces liver metastasis development in a rat colon carcinoma syngeneic model.

Cancer stem cells (CSCs) represent a minor population of self-renewing cancer cells that fuel tumor growth. As CSCs are generally spared by conventional treatments, this population is likely to be responsible for relapses that are observed in most cancers. In this work, we analyzed the preventive efficiency of a CSC-based vaccine on the development of liver metastasis from colon cancer in a syngeneic rat model. We isolated a CSC-enriched population from the rat PROb colon carcinoma cell line on the basis of the expression of the aldehyde dehydrogenase-1 (ALDH1) marker. Comparative analysis of vaccines containing lysates of PROb or ALDH(high) cells by mass spectrometry identifies four proteins specifically expressed in the CSC subpopulation. The expression of two of them (heat shock protein 27-kDa and aldose reductase) is already known to be associated with treatment resistance and poor prognosis in colon cancer. Preventive intraperitoneal administration of vaccines was then performed before the intrahepatic injection of PROb cancer cells. While no significant difference in tumor occurrence was observed between control and PROb-vaccinated groups, 50% of the CSC-based vaccinated animals became resistant to tumor development. In addition, CSC-based vaccination induced a 99.5% reduction in tumor volume compared to the control group. To our knowledge, this study constitutes the first work analyzing the potential of a CSC-based vaccination to prevent liver metastasis development. Our data demonstrate that a CSC-based vaccine reduces efficiently both tumor volume and occurrence in a rat colon carcinoma syngeneic model.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

miR-483-3p controls proliferation in wounded epithelial cells.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

Identification of Human Breast Adipose Tissue Progenitors Displaying Distinct Differentiation Potentials and Interactions with Cancer Cells.

Breast adipose tissue (AT) participates in the physiological evolution and remodeling of the mammary gland due to its high plasticity. It is also a favorable microenvironment for breast cancer progression. However, information on the properties of human breast adipose progenitor cells (APCs) involved in breast physiology or pathology is scant. We performed differential enzymatic dissociation of human breast AT lobules. We isolated and characterized two populations of APCs. Here we report that these distinct breast APC populations selectively expressed markers suitable for characterization. The population preferentially expressing ALPL (MSCA1) showed higher adipogenic potential. The population expressing higher levels of INHBA and CD142 acquired myofibroblast characteristics upon TGF-ß treatment and a myo-cancer-associated fibroblast profile in the presence of breast cancer cells. This population expressed the immune checkpoint CD274 (PD-L1) and facilitated the expansion of breast cancer mammospheres compared with the adipogenic population. Indeed, the breast, as with other fat depots, contains distinct types of APCs with differences in their ability to specialize. This indicates that they were differentially involved in breast remodeling. Their interactions with breast cancer cells revealed differences in the potential for tumor dissemination and estrogen receptor expression, and these differences might be relevant to improve therapies targeting the tumor microenvironment.

Regulation of Adipose Progenitor Cell Expansion in a Novel Micro-Physiological Model of Human Adipose Tissue Mimicking Fibrotic and Pro-Inflammatory Microenvironments.

The expansion of adipose progenitor cells (APCs) plays an important role in the regeneration of the adipose tissue in physiological and pathological situations. The major role of CD26-expressing APCs in the generation of adipocytes has recently been highlighted, revealing that the CD26 APC subtype displays features of multipotent stem cells, giving rise to CD54- and CD142-expressing preadipocytes. However, a relevant human in vitro model to explore the regulation of the APC subpopulation expansion in lean and obese adipose tissue microenvironments is still lacking. In this work, we describe a novel adipose tissue model, named ExAdEx, that can be obtained from cosmetic surgery wastes. ExAdEx products are adipose tissue units maintaining the characteristics and organization of adipose tissue as it presents in vivo. The model was viable and metabolically active for up to two months and could adopt a pathological-like phenotype. The results revealed that inflammatory and fibrotic microenvironments differentially regulated the expansion of the CD26 APC subpopulation and its CD54 and CD142 APC progenies. The approach used significantly improves the method of generating adipose tissue models, and ExAdEx constitutes a relevant model that could be used to identify pathways promoting the expansion of APCs in physiological and pathological microenvironments.

Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle.

Skeletal muscle cells constitute a heterogeneous population that maintains muscle integrity through a high myogenic regenerative capacity. More unexpectedly, this population is also endowed with an adipogenic potential, even in humans, and intramuscular adipocytes have been found to be present in several disorders. We tested the distribution of myogenic and adipogenic commitments in human muscle-derived cells to decipher the cellular basis of the myoadipogenic balance. Clonal analysis showed that adipogenic progenitors can be separated from myogenic progenitors and, interestingly, from myoadipogenic bipotent progenitors. These progenitors were isolated in the CD34(+) population on the basis of the expression of CD56 and CD15 cell surface markers. In vivo, these different cell types have been found in the interstitial compartment of human muscle. In vitro, we show that the proliferation of bipotent myoadipogenic CD56(+)CD15(+) progenitors gives rise to myogenic CD56(+)CD15(-) progenitors and adipogenic CD56(-)CD15(+) progenitors. A cellular hierarchy of muscle and fat progenitors thus occurs within human muscle. These results provide cellular bases for adipogenic differentiation in human skeletal muscle, which may explain the fat development encountered in different muscle pathological situations.

Impact of thermogenesis induced by chronic ß3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice.

White adipocytes store energy differently than brown and brite adipocytes which dissipate energy under the form of heat. Studies have shown that adipocytes are able to respond to bacteria thanks to the presence of Toll-like receptors at their surface. Despite this, little is known about the involvement of each class of adipocytes in the infectious response. We treated mice for one week with a ß3-adrenergic receptor agonist to induce activation of brown adipose tissue and brite adipocytes within white adipose tissue. Mice were then injected intraperitoneally with E. coli to generate acute infection. The metabolic, infectious and inflammatory parameters of the mice were analysed during 48 hours after infection. Our results shown that in response to bacteria, thermogenic activity promoted a discrete and local anti-inflammatory environment in white adipose tissue characterized by the increase of the IL-1RA secretion. More generally, activation of brown and brite adipocytes did not modify the host response to infection including no additive effect with fever and an equivalent bacteria clearance and inflammatory response. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes in response to acute bacterial infection and open a way to characterize their effect along more chronic infection as septicaemia.

Diet Supplementation in ω3 Polyunsaturated Fatty Acid Favors an Anti-Inflammatory Basal Environment in Mouse Adipose Tissue.

Oxylipins are metabolized from dietary ω3 and ω6 polyunsaturated fatty acids and are involved in an inflammatory response. Adipose tissue inflammatory background is a key factor of metabolic disorders and it is accepted that dietary fatty acids, in terms of quality and quantity, modulate oxylipin synthesis in this tissue. Moreover, it has been reported that diet supplementation in ω3 polyunsaturated fatty acids resolves some inflammatory situations. Thus, it is crucial to assess the influence of dietary polyunsaturated fatty acids on oxylipin synthesis and their impact on adipose tissue inflammation. To this end, mice fed an ω6- or ω3-enriched standard diet (ω6/ω3 ratio of 30 and 3.75, respectively) were analyzed for inflammatory phenotype and adipose tissue oxylipin content. Diet enrichment with an ω3 polyunsaturated fatty acid induced an increase in the oxylipins derived from ω6 linoleic acid, ω3 eicosapentaenoic, and ω3 docosahexaenoic acids in brown and white adipose tissues. Among these, the level of pro-resolving mediator intermediates, as well as anti-inflammatory metabolites, were augmented. Concomitantly, expressions of M2 macrophage markers were increased without affecting inflammatory cytokine contents. In vitro, these metabolites did not activate macrophages but participated in macrophage polarization by inflammatory stimuli. In conclusion, we demonstrated that an ω3-enriched diet, in non-obesogenic non-inflammatory conditions, induced synthesis of oxylipins which were involved in an anti-inflammatory response as well as enhancement of the M2 macrophage molecular signature, without affecting inflammatory cytokine secretion.

Plasmidic CpG sequences induce tumor microenvironment modifications in a rat liver metastasis model.

Bacterial DNA contains unmethylated cytosine-phosphate-guanine (CpG) motifs which are recognized by mammalian immune cells as a danger signal indicating an infection. These immunostimulatory properties led to the use of oligodeoxynucleotides bearing CpG motifs (CpG-ODN) for cancer treatment in preclinical and clinical studies. Although naked DNA administration presently represents 18% of the gene therapy clinical trials worldwide, most of the work regarding the effects of unmethylated CpG sequences was performed using CpG-ODN. In the present study, we analyzed early induced tumor microenvironment modifications in a rat liver metastasis model after intratumoral injection of a plasmid used in suicide gene therapy. We first showed that plasmidic CpG motifs were active, i.e. able to induce IFN-gamma secretion by rat splenocytes. Then, we compared tumor-infiltrating immune cells 24 h after injection of native or SssI-treated plasmid, in which immunostimulatory CpG motifs have been inactivated by methylation. The presence of active plasmidic CpG sequences within the tumor was associated with a decrease in the number of tumor-infiltrating conventional dendritic cells and an upregulation of the CCR7 chemokine receptor responsible for lymph node homing. We also observed an increase in plasmacytoid dendritic cells and natural killer cell infiltration within the tumors as well as an increased mRNA expression of three cytokines/chemokines (IL-1beta, IL-10 and IL-18). These data suggest that, although suicide plasmid injection without prodrug treatment is not sufficient to observe a therapeutic effect, the presence of plasmidic CpG motifs within the tumor induces the recruitment and activation of the immune cells involved in antitumor response. These early cellular and molecular events should facilitate the induction of the immune response against tumor antigens released after in situ drug production.

Clues to CD2-associated protein involvement in cytokinesis.

Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis.

Saccharomyces boulardii prevents TNF-alpha-induced apoptosis in EHEC-infected T84 cells.

Induction of apoptosis and necrosis by enterohemorrhagic Escherichia coli (EHEC) has been reported in vivo and in vitro, but features of cell death were not noted in those reports. Since tumor necrosis factor-alpha (TNF-alpha) has been implicated in the apoptosis of invasive bacteria, we investigated the role of this cytokine in EHEC-induced apoptosis. We hypothesize that the probiotic yeast strain Saccharomyces boulardii that interferes with EHEC-induced pro-inflammatory pathways delays EHEC-induced apoptosis. By 6 h of infection, flow cytometry analysis of T84 cells demonstrated that 40% of cells were FITC-annexin-V-positive and 40% of cells incorporated both annexin and propidium iodide (PI). Simultaneously, western blot analysis demonstrated that procaspases-8 and -3 were cleaved. Fragmentation of internucleosomal DNA revealed evidence of apoptotic leader formation after 8 and 9 h of infection. Procaspase-9 activation and 3',3-dihexyloxacarbocyanine iodide (DiOC(6)) incorporation were observed at 3 h of infection. In cells preincubated with S. boulardii and infected with EHEC in the presence of yeast, the quantities of procaspases-8, -9 and -3 did not vary, and no DNA fragmentation was observed. The TNF-alpha transcript level and the level of secreted TNF-alpha increased considerably (P<0.001vs control cells) at 6 h of infection in EHEC-alone-infected cells, but were significantly reduced in cells infected in the presence of S. boulardii (P<0.001vs EHEC-alone-infected cells). The presence of anti-TNF-alpha antibody during infection reduced by 30% the level of FITC-annexin V-positive cells. Altogether, these findings demonstrated that: (i) EHEC infection stimulated TNF-alpha synthesis that is implicated in apoptosis of T84 cells; and (ii) S. boulardii induced a decrease in TNF-alpha and related apoptosis in EHEC-infected T84 cells.

SIGMAR1 Regulates Membrane Electrical Activity in Response to Extracellular Matrix Stimulation to Drive Cancer Cell Invasiveness.

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/ß1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

The protective effect of phorbol esters on Fas-mediated apoptosis in T cells. Transcriptional and postranscriptional regulation.

Phorbol esters are tumor promoters that bind and activate both conventional and new Protein kinase C (PKC) isoforms. In various circumstances, PKC-dependent signaling pathways can promote cell survival and protect against cell death. This was first analysed in Jurkat T cells where Phorbol Myristate Acetate (PMA) was found to inhibit Fas-mediated apoptosis as judged by DiOC6(3) staining, caspase activation and DNA fragmentation, indicating that PMA exerts its protective effect upstream or at the mitochondrial level in these cells. PMA activated most of the main kinase pathways in T cells such as PKCs, p42/44MAPK, p38MAPK and p90Rsk but not JNK and Akt. A pharmacological approach allowed us to identify that nPKCs are both necessary and likely sufficient to promote T cell survival. Besides this post-transcriptional regulation, nPKCs may also regulate apoptosis at the transcriptional level. cDNA arrays were used to identify a set of genes whose expression was modulated in death versus survival conditions. Following PMA treatment, expression of Mcl-1 and Bcl-x increased while that of c-Myc was significantly reduced. Moreover, survivin expression decreased upon CH11 or PMA treatment. c-Myc, survivin and Bcl-x modulation seems to be regulated at the transcriptional level while decrease in Mcl-1 protein in CH11-treated cells resulted especially from a caspase-dependent proteolysis. Taken together, our data demonstrate that PMA-mediated inhibition of apoptosis is a complex process that is integrated at both the transcriptional and post-transcriptional level and point out to the potential role of Mcl-1, Bcl-x, c-Myc and survivin in this process.

AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF-alpha in Jurkat leukemic cells.

NF-kappaB transcription factors promote survival in numerous cell types via induction of antiapoptotic genes. Pharmacological blockade of the IKK2 kinase with AS602868, a specific inhibitor that competes with ATP binding, prevented TNF-alpha-induced NF-kappaB activation in Jurkat leukemic T cells. While TNF-alpha by itself had no effect on Jurkat survival, the addition of AS602868 induced cell death, visualized by DNA fragmentation and sub-G1 analysis. A disruption of the mitochondrial potential followed by activation of caspases 9 and 3 was observed in cells treated by the combination TNF-alpha+AS602868. Quantitative real-time PCR demonstrated that AS602868 prevented TNF-alpha induction of the antiapoptotic genes coding for c-IAP-2, Bclx, Bfl-1/A1 and Traf-1. The use of a specific IKK2 inhibitor appears, therefore, as an interesting pharmaceutical strategy to increase the cell's sensitivity towards apoptotic effectors.

Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction.

UNLABELLED: Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. INTRODUCTION: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. METHODS: Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. RESULTS: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. CONCLUSIONS: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.

Dynamic characterization of the molecular events during in vitro epidermal wound healing.

The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal keratinocytes as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified keratinocyte cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (growth factors, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human keratinocytes: mechanical injuries (i) immediately stimulate the tyrosine phosphorylation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p21ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2, c-Jun NH2 terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription factor 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription factor complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human keratinocyte cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers.