Rab5a activates IRS1 to coordinate IGF-AKT-mTOR signaling and myoblast differentiation during muscle regeneration.

Rab5 is a master regulator for endosome biogenesis and transport while its in vivo physiological function remains elusive. Here, we find that Rab5a is upregulated in several in vivo and in vitro myogenesis models. By generating myogenic Rab5a-deficient mice, we uncover the essential roles of Rab5a in regulating skeletal muscle regeneration. We further reveal that Rab5a promotes myoblast differentiation and directly interacts with insulin receptor substrate 1 (IRS1), an essential scaffold protein for propagating IGF signaling. Rab5a interacts with IRS1 in a GTP-dependent manner and this interaction is enhanced upon IGF-1 activation and myogenic differentiation. We subsequently identify that the arginine 207 and 222 of IRS1 and tyrosine 82, 89, and 90 of Rab5a are the critical amino acid residues for mediating the association. Mechanistically, Rab5a modulates IRS1 activation by coordinating the association between IRS1 and the IGF receptor (IGFR) and regulating the intracellular membrane targeting of IRS1. Both myogenesis-induced and IGF-evoked AKT-mTOR signaling are dependent on Rab5a. Myogenic deletion of Rab5a also reduces the activation of AKT-mTOR signaling during skeletal muscle regeneration. Taken together, our study uncovers the physiological function of Rab5a in regulating muscle regeneration and delineates the novel role of Rab5a as a critical switch controlling AKT-mTOR signaling by activating IRS1.

Cepharanthine Prevents Estrogen Deficiency-Induced Bone Loss by Inhibiting Bone Resorption.

Osteoporosis is a common health problem worldwide caused by an imbalance of bone formation vs. bone resorption. However, current therapeutic approaches aimed at enhancing bone formation or suppressing bone resorption still have some limitations. In this study, we demonstrated for the first time that cepharanthine (CEP, derived from Stephania cepharantha Hayata) exerted a protective effect on estrogen deficiency-induced bone loss. This protective effect was confirmed to be achieved through inhibition of bone resorption in vivo, rather than through enhancement of bone formation in vivo. Furthermore, the in vitro study revealed that CEP attenuated receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast formation, and suppressed bone resorption by impairing the c-Jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways. The inhibitory effect of CEP could be partly reversed by treatment with anisomycin (a JNK and p38 agonist) and/or SC79 (an AKT agonist) in vitro. Our results thus indicated that CEP could prevent estrogen deficiency-induced bone loss by inhibiting osteoclastogenesis. Hence, CEP might be a novel therapeutic agent for anti-osteoporosis therapy.

Activation of AKT-mTOR Signaling Directs Tenogenesis of Mesenchymal Stem Cells.

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries. Stem Cells 2018;36:527-539.

Sophocarpine attenuates wear particle-induced implant loosening by inhibiting osteoclastogenesis and bone resorption via suppression of the NF-κB signalling pathway in a rat model.

BACKGROUND AND PURPOSE: Aseptic prosthesis loosening, caused by wear particles, is one of the most common causes of arthroplasty failure. Extensive and over-activated osteoclast formation and physiological functioning are regarded as the mechanism of prosthesis loosening. Therapeutic modalities based on inhibiting osteoclast formation and bone resorption have been confirmed to be an effective way of preventing aseptic prosthesis loosening. In this study, we have investigated the effects of sophocarpine (SPC, derived from Sophora flavescens) on preventing implant loosening and further explored the underlying mechanisms. EXPERIMENTAL APPROACH: The effects of SPC in inhibiting osteoclastogenesis and bone resorption were evaluated in osteoclast formation, induced in vitro by the receptor activator of NF-κB ligand (RANKL). A rat femoral particle-induced peri-implant osteolysis model was established. Subsequently, micro-CT, histology, mechanical testing and bone turnover were used to assess the effects of SPC in preventing implant loosening. KEY RESULTS: In vitro, we found that SPC suppressed osteoclast formation, bone resorption, F-actin ring formation and osteoclast-associated gene expression by inhibiting NF-κB signalling, specifically by targeting IκB kinases. Our in vivo study showed that SPC prevented particle-induced prosthesis loosening by inhibiting osteoclast formation, resulting in reduced periprosthetic bone loss, diminished pseudomembrane formation, improved bone-implant contact, reduced bone resorption-related turnover and enhanced stability of implants. Inhibition of NF-κB signalling by SPC was confirmed in vivo. CONCLUSION AND IMPLICATIONS: SPC can prevent implant loosening through inhibiting osteoclast formation and bone resorption. Thus, SPC might be a novel therapeutic agent to prevent prosthesis loosening and for osteolytic diseases.

Effects of multi-environmental factors on physiological and biochemical responses of large yellow croaker, Larimichthys crocea.

Land-based recirculating aquaculture systems (RAS) and cage culture are important methods of Larimichthys crocea production. The effects of environmental factors on physiological and biochemical aspects of L. crocea require clarification. Temperature and salinity are controlled in RAS and directly affect L. crocea growth and survival. To explore optimal parameters, the oxygen consumption rate (RO), ammonium excretion rate (RN), and O/N ratio at different temperatures (8, 14, 20, 26, and 32 °C) and salinities (5, 15, 25, and 35‰) were determined. RO, RN, and O/N first increased and then decreased with elevated temperature and salinity, peaking at 26 °C and 25‰, respectively. This suggests that the metabolism of L. crocea was maximal at 26 °C and 25‰ salinity, which promote its growth and survival. Additionally, hypoxia affects cage culture, and has significant effects on enzymatic activities and stress-inducible gene expression. To accelerate the selective breeding of hypoxia-tolerant L. crocea in cage culture, we measured adenosine triphosphatase (ATPase), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) activities, and hypoxia-inducing factor 1 (HIF-1) mRNA expression in the myocardium under hypoxia (2.5, 3.5, and 4.5 mg L-1). ATPase and SDH activities first decreased and then increased under hypoxia, whereas LDH activity and HIF-1α expression first increased and then decreased. Thus, under hypoxia, the myocardial mitochondria switched from being susceptible to being resistant to injury induced by energy metabolism, and respiration in L. crocea likely converted from aerobic to anaerobic during adaptation. Furthermore, the upregulation of HIF-1α mRNA suggests it has an active role in protection against anoxic damage.

Pharmacological Regulation of In Situ Tissue Stem Cells Differentiation for Soft Tissue Calcification Treatment.

Calcification of soft tissues, such as heart valves and tendons, is a common clinical problem with limited therapeutics. Tissue specific stem/progenitor cells proliferate to repopulate injured tissues. But some of them become divergent to the direction of ossification in the local pathological microenvironment, thereby representing a cellular target for pharmacological approach. We observed that HIF-2alpha (encoded by EPAS1 inclined form) signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Proinflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation and promoted osteochondrogenic differentiation of tendon stem/progenitor cells (TSPCs). Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. Notably, Scleraxis (Scx), an essential tendon specific transcription factor, was suppressed on constitutive activation of HIF-2alpha and mediated the effect of HIF-2alpha on TSPCs fate decision. Moreover, pharmacological inhibition of HIF-2alpha with digoxin, which is a widely utilized drug, can efficiently inhibit calcification and enhance tenogenesis in vitro and in the Achilles's tendinopathy model. Taken together, these findings reveal the significant role of the tissue stem/progenitor cells fate decision and suggest that pharmacological regulation of HIF-2alpha function is a promising approach for soft tissue calcification treatment.

Nanoparticle delivery of stable miR-199a-5p agomir improves the osteogenesis of human mesenchymal stem cells via the HIF1a pathway.

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.

Electrospun scaffolds for multiple tissues regeneration in vivo through topography dependent induction of lineage specific differentiation.

Physical topographic cues from various substrata have been shown to exert profound effects on the growth and differentiation of stem cells due to their niche-mimicking features. However, the biological function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells (MSCs) and neo-tissue formation trigged by aligned and randomly-oriented fibrous scaffolds, both in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent bone tissue formation through ossification. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on randomly-oriented fiber scaffolds displayed enhanced osteogenic differentiation compared with cells cultured on aligned fiber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these findings illustrate the relationship between topographic cues of the scaffold and their inductive role in tissue regeneration; thus providing an insight into future development of smart functionalized bio-scaffold design and its application in tissue engineering.

Scleraxis-overexpressed human embryonic stem cell-derived mesenchymal stem cells for tendon tissue engineering with knitted silk-collagen scaffold.

AIM: Despite our previous study that demonstrates that human embryonic stem cells (hESCs) can be used as seed cells for tendon tissue engineering after stepwise induction, suboptimal tendon regeneration implies that a new strategy needs to be developed for tendon repair. We investigated whether overexpression of the tendon-specific transcription factor scleraxis (SCX) in hESC-derived mesenchymal stem cells (hESC-MSCs) together with knitted silk-collagen sponge scaffold could promote tendon regeneration. METHODS AND RESULTS: hESCs were initially differentiated into MSCs and then engineered with scleraxis (SCX+hESC-MSCs). Engineered tendons were constructed with SCX+hESC-MSCs and a knitted silk-collagen sponge scaffold and then mechanical stress was applied. SCX elevated tendon gene expression in hESC-MSCs and concomitantly attenuated their adipogenic and chondrogenic potential. Mechanical stress further augmented the expression of tendon-specific genes in SCX+hESC-MSC-engineered tendon. Moreover, in vivo mechanical stimulation promoted the alignment of cells and increased the diameter of collagen fibers after ectopic transplantation. In the in vivo tendon repair model, the SCX+hESC-MSC-engineered tendon enhanced the regeneration process as shown by histological scores and superior mechanical performance compared with control cells, especially at early stages. CONCLUSION: Our study offers new evidence concerning the roles of SCX in tendon differentiation and regeneration. We demonstrated a novel strategy of combining hESCs, genetic engineering, and tissue-engineering principles for tendon regeneration, which are important for the future application of hESCs and silk scaffolds for tendon repair.

The effect of decellularized matrices on human tendon stem/progenitor cell differentiation and tendon repair.

It is reported that decellularized collagen matrices derived from dermal skin and bone have been clinically used for tendon repair. However, the varying biological and physical properties of matrices originating from different tissues may influence the differentiation of tendon stem cells, which has not been systematically evaluated. In this study, the effects of collagenous matrices derived from different tissues (tendon, bone and dermis) on the cell differentiation of human tendon stem/progenitor cells (hTSPCs) were investigated, in the context of tendon repair. It was found that all three matrices supported the adhesion and proliferation of hTSPCs despite differences in topography. Interestingly, tendon-derived decellularized matrix promoted the tendinous phenotype in hTSPCs and inhibited their osteogenesis, even under osteogenic induction conditions, through modulation of the teno- and osteolineage-specific transcription factors Scleraxis and Runx2. Bone-derived decellularized matrix robustly induced osteogenic differentiation of hTSPCs, whereas dermal skin-derived collagen matrix had no apparent effect on hTSPC differentiation. Based on the specific biological function of the tendon-derived decellularized matrix, a tissue-engineered tendon comprising TSPCs and tendon-derived matrix was successfully fabricated for Achilles tendon reconstruction. Implantation of this cell-scaffold construct led to a more mature structure (histology score: 4.08 ± 0.61 vs. 8.51 ± 1.66), larger collagen fibrils (52.2 ± 1.6 nm vs. 47.5 ± 2.8 nm) and stronger mechanical properties (stiffness: 21.68 ± 7.1 Nm m(-1) vs.13.2 ± 5.9 Nm m(-1)) of repaired tendons compared to the control group. The results suggest that stem cells promote the rate of repair of Achilles tendon in the presence of a tendinous matrix. This study thus highlights the potential of decellularized matrix for future tissue engineering applications, as well as developing a practical strategy for functional tendon regeneration by utilizing TSPCs combined with tendon-derived decellularized matrix.

Force and scleraxis synergistically promote the commitment of human ES cells derived MSCs to tenocytes.

As tendon stem/progenitor cells were reported to be rare in tendon tissues, tendons as vulnerable targets of sports injury possess poor self-repair capability. Human ESCs (hESCs) represent a promising approach to tendon regeneration. But their teno-lineage differentiation strategy has yet to be defined. Here, we report that force combined with the tendon-specific transcription factor scleraxis synergistically promoted commitment of hESCs to tenocyte for functional tissue regeneration. Force and scleraxis can independently induce tendon differentiation. However, force alone concomitantly activated osteogenesis, while scleraxis alone was not sufficient to commit, but augment tendon differentiation. Scleraxis synergistically augmented the efficacy of force on teno-lineage differentiation and inhibited the osteo-lineage differentiation by antagonized BMP signaling cascade. The findings not only demonstrated a novel strategy of directing hESC differentiation to tenocyte for functional tendon regeneration, but also offered insights into understanding the network of force, scleraxis and bmp2 controlling tendon-lineage differentiation.

Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles.

Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon tissue engineering has not yet been adequately explored. This study incorporated hESC-derived mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon regeneration in situ, as evidenced by better histological scores and superior mechanical performance characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen sponge scaffold followed by mechanical stimulation.

[Growth inhibition of MG-63 cells by cyclin A2 gene-specific small interfering RNA].

OBJECTIVE: To study the impact of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of MG-63 and HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma. METHODS: One pair of siRNA targeting the cyclin A2 mRNA and a pair of nonsense siRNA were designed according the current criteria. SiRNA was chemically synthesized and purified. The siRNA was transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast (HSF) cells via oligofectamine. Cells transfected with nonsense siRNA served as the negative control and those only treated with PBS as the blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and colony-forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA treated MG-63 cells were examined. RESULTS: 1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L cyclin A2-siRNA can reduced cyclin A2 mRNA and protein expression respectively by 9.43%, 56.35%, 79.17% and 84.30% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. After 48 h treatment with 10 nmol/L siRNA, MG-63 cells were arrested in G0/G1 phase and the proliferation of this tumor cell was suppressed by 39.06% 48 h after transfection. Furthermore, the treated MG-63 cells showed less colony-forming ability. Increasing the siRNA concentration to 50 nmol/L can further inhibit the proliferation of MG-63 cells by 54.94%. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 mRNA and protein expression in HSF reduced 58.13% 48 h after treatment by 50 nmol/L siRNA, these cells exhibited only a slight change in cell cycle, and no clear inhibition of proliferation and impaired plate colony-forming ability was observed. CONCLUSION: Cyclin A2 gene maybe served as a potential target for tumor therapy. RNA interference induces obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently downregulate the proliferation of MG-63 cells. There is few inhibitory effect on the proliferation by siRNAs for HSF cells. These results indicate that siRNAs against cyclin A2 could become a potential antiproliferative tool in future antitumor therapy.

[Knockdown of cyclin A2 expression by small interfering RNA in MG-63 cells].

OBJECTIVE: To study the inhibitory effect of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of osteosarcoma MG-63 and human normal skin fibroblast HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma. METHODS: Three pairs of siRNAs targeting cyclin A2 mRNA and a pair of nonsense siRNA were designed according to the current criteria. SiRNAs were chemically synthesized and purified. The siRNAs were transfected into MG-63 cells and HSF cells via oligofectamine. The cells transfected with nonsense siRNA served as negative control group and those only treated with PBS as blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clone forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA-treated MG-63 cells were examined. RESULTS: Although all three siRNAs could reduce the cyclin A2 expression, siRNA, appeared to be the most effective. After 48 h treatment with siRNA1, cyclin A2 mRNA and protein expression in MG-63 cells was significantly reduced by nearly 80% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. MG-63 cells treated with siRNA1 were arrested at G0/G1 phase by 80.1% and the proliferation of these tumor cells was suppressed 48 h after transfection. Furthermore, MG-63 cells showed a decreased colony forming ability after siRNA1 treatment. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 expression in HSF reduced by nearly 60% after treatment by siRNA1 for 48h, these cells exhibited only a slight change in cell cycling, and neither clear inhibition of proliferation nor impaired colony forming ability was observed. CONCLUSION: Cyclin A2 is critical for proliferation of MG-63 cells. Cyclin A2-siRNAs can induce obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently down-regulate the proliferation of MG-63 cells. There is little effect on the proliferation of siRNA-treated HSF cells. Those results indicate that siRNAs against cyclin A2 may become a potential antiproliferative tool in future antitumor therapy.