Project description:To investigate the fibrotic effect on BMSCs by CTGF and TGFβ1 treatment, and the anti-fibrotic effect of docetaxel on fibrotic chondrocytes. We established BMSCs primary culture and treated CTGF and TGFβ1, then treated by docetaxel.
Project description:Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3 and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 co-immunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that while insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates re-distribution of CLASP2 and G2L1 from exclusive plus-end tracking to “trailing” behind the growing tip of the microtubule. Insulin treatment increases -tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR, and also led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.
Project description:Full thickness articular cartilage lesions with penetration into the subchondral bone fill with fibrocartilage-like repair tissue. However, the repair tissue has compromised structural and biomechanical properties relative to normal articular cartilage. The objective of this study was to evaluate transcriptome differences between normal articular cartilage and repair tissue. Bilateral one-cm2 full-thickness lesions were made in the articular surface of the distal femurs of four adult horses followed by subchondral microfracture. Four months postoperatively, repair tissue from the lesion site and grossly normal articular cartilage from each stifle were collected. Total RNA was isolated from tissue samples, linearly amplified, and applied to a 9367-probeset equine-specific cDNA microarray. Eight paired comparisons matched by limb and horse were made with a dye-swap experimental design. Comparisons were validated by histological analysis and quantitative real-time polymerase chain reaction (qPCR). Statistical analysis revealed 3,327 (35.2%) differentially expressed probesets. Biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue corroborate earlier studies. Other changes in gene expression previously unassociated with cartilage repair were also revealed and validated by qPCR. The magnitude of divergence in transcriptional profiles between normal chondrocytes and the cells that populate repair tissue reveal substantial functional differences between these two cell populations. At the four-month postoperative time point, the relative deficiency within repair tissue of transcripts from genes which typically define articular cartilage indicate that while cells occupying the lesion might be of mesenchymal origin, they have not recapitulated differentiation to the chondrogenic phenotype of normal articular chondrocytes.
Project description:Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injury to the articular surface is a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.
Project description:We report a high-efficiency differentiation protocol to tendon and fibrocartilage perform molecular profiling of tendon and fibrocartilage fatse in relation to developmetnal in vivo analogues. We also the molecular induction of tendon and fibrocartilage by examining fate dependent and independent gene modules
Project description:Accumulating evidence indicates the close association between the circadian clock and the aging process. However, it is unclear whether and how the circadian clock proteins regulate stem cell aging. Here, we identified a noncanonical transcriptional activator-independent role of CLOCK, a core component of the molecular circadian clock machinery, in counteracting human mesenchymal stem cell (hMSC) senescence. CLOCK expression was decreased during hMSC aging. In addition, CLOCK deficiency accelerated hMSC senescence, whereas CLOCK overexpression attenuated physiological and pathological hMSC senescence. Mechanistic studies revealed that CLOCK formed complexes with nuclear lamina proteins and KAP1, thus maintaining heterochromatin architecture and stabilizing genomic repetitive sequences. Finally, gene therapy with lentiviral vectors encoding CLOCK promoted cartilage regeneration and attenuated age-related osteoarthritis in mice. These findings reveal a novel role of CLOCK independent of its transcriptional activation activity in the stabilization of heterochromatin and alleviation of hMSC senescence, providing a potential therapeutic target for treating aging-associated articular degeneration.
Project description:We report the transcriptomic profiling of tendon and fibrocartilage induction by day, examining both the fate dependent and independent modules
Project description:Tau is a microtubule-associated protein that ensures neuronal shape and function. Besides, Tau is a central player in Alzheimer’s disease (AD) and related Tauopathies where it is found aggregated in degenerating neurons. Mechanisms leading to Tau pathology and its progression are far from being elucidated. Among Tau species found in AD brains, several yet unidentified truncated Tau fragments are showed. A major step forward in the understanding of the role of Tau truncation would be to identify the precise cleavage sites of Tau species. This key step is mandatory to generate appropriate experimental tools in order to investigate the impact of each identified truncated-species on Tau function/dysfunction. Here, we achieved an optimized proteomics approach and succeed in identifying a number of new N-terminally truncated-Tau species from human brain. As N-terminal residues of these fragments are located broadly across Tau sequence, one could expect to have different effects on Tau. We initiated cell-based functional studies by analyzing biochemical characteristics of two N-terminally truncated Tau species starting at residues Met11 and Gln124 (accordingly to the longest Tau isoform) regarding Tau microtubule function. Our results surprisingly showed that the ability of Tau to bind and stabilize microtubules was greater when the first 123 residues are truncated, suggesting that Tau N-terminus would have a role in regulation of microtubule stabilization. Overall, future studies based on our new N-terminally truncated-Tau species will provide new knowledge on the role of truncation in Tau biology as well as in AD pathological process.
Project description:To understand the effect of semaphorin 4D on articular chondrocytes, articular chondrocytes were stimulated with semaphorin 4D. Total RNA was analyzed by RNA-seq.