Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans

Skeletal muscle from strength- and endurance-trained individuals represents diverse adaptive states. In this regard, AMPK-PGC-1α signaling mediates several adaptations to endurance training, while up-regulation of the Akt-TSC2-mTOR pathway may underlie increased protein synthesis after resistance exercise. We determined the effect of prior training history on signaling responses in seven strength-trained and six endurance-trained males who undertook 1 h cycling at 70% VO2peak or eight sets of five maximal repetitions of isokinetic leg extensions. Muscle biopsies were taken at rest, immediately and 3 h postexercise. AMPK phosphorylation increased after cycling in strength-trained (54%; P<0.05) but not endurance-trained subjects. Conversely, AMPK was elevated after resistance exercise in endurance- (114%; P<0.05), but not strengthtrained subjects. Akt phosphorylation increased in endurance- (50%; P<0.05), but not strengthtrained subjects after cycling but was unchanged in either group after resistance exercise. TSC2 phosphorylation was decreased (47%; P<0.05) in endurance-trained subjects following resistance exercise, but cycling had little effect on the phosphorylation state of this protein in either group. p70S6K phosphorylation increased in endurance- (118%; P<0.05), but not strength-trained subjects after resistance exercise, but was similar to rest in both groups after cycling. Similarly, phosphorylation of S6 protein, a substrate for p70 S6K, was increased immediately following resistance exercise in endurance- (129%; P<0.05), but not strength-trained subjects. In conclusion, a degree of “response plasticity” is conserved at opposite ends of the endurancehypertrophic adaptation continuum. Moreover, prior training attenuates the exercise specific signaling responses involved in single mode adaptations to training.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds

Lithium (Li) has been widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders. Li+ ions are thought to enhance the remyelination of peripheral nerves and also stimulate the proliferation of neural progenitor cells and retinoblastoma cells via activation of the Wnt/β-catenin signalling pathway. Until now there have been no studies reporting the biological effects of released Li+ in bioactive scaffolds on cemetogenesis in periodontal tissue engineering applications. In this study, we incorporated parts of Li+ ions into the mesoporous bioactive glass (MBG) scaffolds and showed that this approach yielded scaffolds with a favourable composition, microstructure and mesopore properties for cell attachment, proliferation, and cementogenic differentiation of human periodontal ligament-derived cells (hPDLCs). We went on to investigate the biological effects of Li+ ions themselves on cell proliferation and cementogenic differentiation. The results showed that 5% Li+ ions incorporated into MBG scaffolds enhanced the proliferation and cementogenic differentiation of hPDLCs on scaffolds, most likely via activation of Wnt/β-catenin signalling pathway. Further study demonstrated that Li+ ions by themselves significantly enhanced the proliferation, differentiation and cementogenic gene expression of PDLCs. Our results indicate that incorporation of Li+ ions into bioactive scaffolds is a viable means of enhancing the Wnt canonical signalling pathway to stimulate cementogenic differentiation of PDLCs.

The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation

Topographically and chemically modified titanium implants are recognized to have improved osteogenic properties; however, the molecular regulation of this process remains unknown. This study aimed to determine the microRNA profile and the potential regulation of osteogenic differentiation following early exposure of osteoprogenitor cells to sand-blasted, large-grit acid-etched (SLA) and hydrophilic SLA (modSLA) surfaces. Firstly, the osteogenic characteristics of the primary osteoprogenitor cells were confirmed using ALP activity and Alizarin Red S staining. The effect of smooth (SMO), SLA and modSLA surfaces on the TGF-β/BMP (BMP2, BMP6, ACVR1) and non-canonical WNT/Ca2+ (WNT5A, FZD6) pathways, as well as the integrins ITGB1 and ITGA2, was determined. It was revealed that the modified titanium surfaces could induce the activation of TGF-β/BMP and non-canonical WNT/Ca2+ signaling genes. The expression pattern of microRNAs (miRNAs) related to cell differentiation was evaluated. Statistical analysis of the differentially regulated miRNAs indicated that 35 and 32 miRNAs were down-regulated on the modSLA and SLA surfaces respectively, when compared with the smooth surface (SMO). Thirty-one miRNAs that were down-regulated were common to both modSLA and SLA. There were 10 miRNAs up-regulated on modSLA and nine on SLA surfaces, amongst which eight were the same as observed on modSLA. TargetScan predictions for the down-regulated miRNAs revealed genes of the TGF-β/BMP and non-canonical Ca2+ pathways as targets. This study demonstrated that modified titanium implant surfaces induce differential regulation of miRNAs, which potentially regulate the TGF-β/BMP and WNT/Ca2+ pathways during osteogenic differentiation on modified titanium implant surfaces.

The effect of silicate ions on proliferation, osteogenic differentiation and cell signalling pathways (WNT and SHH) of bone marrow stromal cells

Silicon (Si) is a trace element, which plays an important role in human bone growth. Si has been incorporated into biomaterials for bone regeneration in order to improve their osteogenic potential, both in vitro and in vivo. Little is known, however, as to how Si ions elicit their biological response on bone-forming cells. The aim of this study was to investigate the effect of Si ions on the proliferation, differentiation, bone-related gene expression and cell signalling pathways of bone marrow stromal cells (BMSCs) by comparing the BMSC responses to different concentrations of NaCl and Na2SiO3, while taking into account and excluding the effect of Na ions. Our study showed that Si ions at a concentration of 0.625 mM significantly enhanced the proliferation, mineralization nodule formation, bone-related gene expression (OCN, OPN and ALP) and bone matrix proteins (ALP and OPN) of BMSCs. Furthermore, Si ions at 0.625 mM could counteract the effect of the WNT inhibitor (W.I.) cardamonin on the osteogenic genes expression, (OPN, OCN and ALP), WNT and SHH signalling pathway-related genes in BMSCs. These results suggest that Si ions by themselves play an important role in regulating the proliferation and osteogenic differentiation of BMSCs, with the involvement of WNT and SHH signalling pathways. Our study provides evidence to explain possible molecular mechanisms whereby Si ions released from Si-containing biomaterials can acquire enhanced bioactivity at desired concentration.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

Signaling of receptor tyrosine kinases in the nucleus

Since the discovery of the first receptor tyrosine kinase (RTK) proteins in the late 1970s and early 1980s, many scientists have explored the functions of these important cell signaling molecules. The finding that these proteins are often deregulated or mutated in diseases such as cancers and diabetes, together with their potential as clinical therapeutic targets, has further highlighted the necessity for understanding the signaling functions of these important proteins. The mechanisms of RTK regulation and function have been recently reviewed by Lemmon & Schlessinger (2010) but in this review we instead focus on the results of several recent studies that show receptor tyrosine kinases can function from subcellular localisations, including in particular the nucleus, in addition to their classical plasma membrane location. Nuclear localisation of receptor tyrosine kinases has been demonstrated to be important for normal cell function but is also believed to contribute to the pathogenesis of several human diseases.