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Up until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this "scar-free" healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19 days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair. Refereed/Peer-reviewed

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-β1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of β-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair. Refereed/Peer-reviewed

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities,which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of over expression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis,and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how down regulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis.

Craniosynostosis is a developmental disorder of the skull arising from premature bony fusion of cranial sutures, the sites of skull bone growth. In a recent gene microarray study, we demonstrated that retinol-binding protein ₄ (RBP₄) was the most highly downregulated gene in suture tissue during the pathological process of premature bony fusion. To gain insight into the function of RBP₄ in cranial sutures, we analysed primary cells cultured from human cranial suture mesenchyme. These cells express RBP₄ but not CRBP₁, cellular retinol-binding protein ₁, the typical cytoplasmic retinol storage protein. Using flow cytometry, we showed that suture mesenchymal cells express the RBP₄ receptor, STRA6, on the cell surface. In a cell culture model of cranial osteogenesis, we found that RBP₄ was significantly downregulated during mineralization, analogous to its decrease in pathological suture fusion. We found that cranial suture cells do not secrete detectable levels of RBP₄, suggesting that it acts in a cell-autonomous manner. High-resolution confocal microscopy with a panel of antibody markers of cytoplasmic organelles demonstrated that RBP₄ was present in several hundred cytoplasmic vesicles of about 300 nm in diameter which, in large part, were conspicuously distinct from the ER, the Golgi and endosomes of the endocytic pathway. We speculate that in suture mesenchymal cells, endogenous RBP₄ receives retinol from STRA6 and the RBP₄-retinol complex is stored in vesicles until needed for conversion to retinoic acid in the process of osteogenesis. This study extends the role of RBP₄ beyond that of a serum transporter of retinol and implicates a broader role in osteogenesis. Refereed/Peer-reviewed

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Background Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. Results A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. Conclusions A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.