CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis

Volunteering and well-being : do self-esteem, optimism, and perceived control mediate the relationship?

Volunteers play a vital role in modern societies by boosting the labor force within both the public and private sectors. While the factors that may lead people to volunteer have been investigated in a number of studies, the means by which volunteering contributes to the well-being of such volunteers is poorly understood. It has been suggested through studies that focus on the absence of depression in volunteers that self-esteem and sense of control may be major determinants of the increased well-being reported by volunteers. This is consistent with the homeostatic model of subjective well-being, which proposes that self-esteem, optimism, and perceived control act as buffers that mediate the relationship between environmental experience and subjective well-being (SWB). Using personal well-being as a more positive measure of well-being than absence of depression, this study further explored the possible mediating role of self-esteem, optimism, and perceived control in the relationship between volunteer status and well-being. Participants (N = 1,219) completed a 97-item survey as part of the Australian Unity Wellbeing project. Variables measured included personal well-being, self-esteem, optimism, and a number of personality and psychological adjustment factors. Analyses revealed that perceived control and optimism, but not self-esteem, mediated the relationship between volunteer status and personal well-being.<br /

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Signaling through the Smad Pathway by insulin-like growth factor-binding protein-3 in breast cancer cells : relationship to transforming growth factor-β1 signaling

We previously demonstrated in T47D cells transfected to express the transforming growth factor-β receptor type II (TGF-βRII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate TGF-β1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-β1 in cell growth inhibition (Fanayan, S., Firth, S. M., Butt, A. J., and Baxter, R. C. (2000) J. Biol. Chem. 275, 39146–39151). This study further explores IGFBP-3 signaling through the Smad pathway. Like TGF-β1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-βRI as well as Smad2 and Smad3. This effect required the presence of TGF-βRII. IGFBP-3 mutated in carboxyl-terminal nuclear localization signal residues retained activity in TGF-βR1 and Smad phosphorylation, whereas IGFBP-5 was inactive. Immunoneutralization of endogenous TGF-β1 suggested that TGF-β1 was not essential for IGFBP-3 stimulation of this pathway, but it increased the effect of IGFBP-3. IGFBP-3, like TGF-β1, elicited a rapid decline in immunodetectable Smad4 and Smad4·Smad2 complexes. IGFBP-3 and nuclear localization signal mutant IGFBP-3 stimulated the activation of the plasminogen activator inhibitor-1 promoter but was not additive with TGF-β, suggesting that this end point is not a direct marker of the IGFBP-3 effect on cell proliferation. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-βRII but not dependent on the nuclear translocation of IGFBP-3. The precise mechanism by which IGFBP-3 interacts with the TGF-β receptor system remains to be established.7 page(s

Structural and Functional Evidence for the Interaction of Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins with Vitronectin

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and 125iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells

Development of resistance to insulin-like growth factor binding protein-3 in transfected T47D breast cancer cells

Insulin-like growth factor binding protein-3 (IGFBP-3) has antiproliferative effects in many cell types but paradoxical growth stimulation has also been reported. In early passages following transfection of T47D breast cancer cells with IGFBP-3 cDNA, the proliferation rate and serum-stimulated DNA synthesis were significantly reduced compared to control cells. Cell cycle analysis indicated that growth-inhibited IGFBP-3-producing cells accumulated in G1phase. After several passages, the transfected cells became resistant to the inhibitory effects of IGFBP-3 and showed transiently enhanced proliferation rates despite an increased IGFBP-3 concentration in the medium. IGFBP-3 proteolysis did not account for its decreased antiproliferative activity in resistant cells. We hypothesize that development of resistance to the antiproliferative action of IGFBP-3 might be an important step in the malignant progression of breast cancer cells.5 page(s

Growth inhibition by insulin-like growth factor-binding protein-3 in T47D breast cancer cells requires transforming growth factor-β (TGF-β) and the Type II TGF-β receptor

This study explores the relationship between anti-proliferative signaling by transforming growth factor-β (TGF-β) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-β1. To investigate the mechanism, we used T47D cells that lack type II TGF-β receptor (TGF-βRII) and are insensitive to TGF-β1. After introducing the TGF-βRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-β1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-β1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-βRII-expressing cells when exogenous TGF-β1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-β1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-βRII and exogenous TGF-β1. To investigate this synergism, the phosphorylation of TGF-β signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-β1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-β signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.6 page(s