Yap/Taz activity is associated with increased expression of phosphoglycerate dehydrogenase that supports myoblast proliferation.

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (Phgdh). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Effect of fibrin glue on the healing efficacy of deproteinized bovine bone and autologous bone in critical-sized calvarial defects in rats.

OBJECTIVES: In order to verify the hypothesis that fibrin glue (FG) is able to seal the area of bone grafting and facilitate bone regeneration. MATERIALS AND METHODS: Twenty-one Sprague-Dawley rats with critical-sized calvarial bone defects were randomly assigned to three groups: (A) co-administrated deproteinized bovine bone (DBB) and autologous bone grafts with FG [fibrin ( +)], (B) co-administrated DBB and autologous bone grafts without FG [fibrin ( -)], and (C) no graft as control. Four weeks and 8 weeks later, micro-CT analysis and histomorphometric analysis were carried out to evaluate following parameters: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp), percentage of new bone area (Pe.NB), average thickness of bone defect (Th.BD), average thickness of basal bone (Th.BB), and percentage of new bone in center of the skull defect (Pe.NBc). RESULTS: BV/TV, Tb.Th, and Tb.N in fibrin ( -) group were significantly higher than that of fibrin ( +) group (p = 0.008, 0.000, 0.007, respectively) and control group (p = 0.004, 0.001, and 0.007, respectively) at 8 weeks. Pe.NB in fibrin ( -) group (33.67 ± 11.72%) was significantly higher than that of fibrin ( +) group (12.33 ± 3.21%) (p = 0.038) and control group (9.66 ± 8.50%) (p = 0.045) at 8 weeks. Pe.NBc in fibrin ( -) group (12.05 ± 3.91%) was significantly higher than that of fibrin ( +) group (4.79 ± 1.21%) (p = 0.005) and control group (0.00 ± 0.00%) (p = 0.000) at 4 weeks. CONCLUSIONS: Administration of both DBB and autograft stimulates calvarial bone defect regeneration, while combination of FG does not additionally accelerate new bone formation. CLINICAL RELEVANCE: The use of fibrin to cement traditional bone graft materials in oral clinical practice requires caution.

Synergistic short-term and long-term effects of TGF-ß1 and 3 on collagen production in differentiating myoblasts.

Skeletal muscle fibrosis and regeneration are modulated by transforming growth factor ß (TGF-ß) superfamily. Amongst them, TGF-ß1 is a highly potent pro-fibrotic factor, while TGF-ß3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. However, little is known about the individual and combined short- and long-term effects of TGF-ß1 and TGF-ß3 on collagen expression in myoblasts and myotubes. Here we show that in C2C12 myoblasts TGF-ß1 and/or TGF-ß3 increased mRNA expression of Ctgf and Fgf-2 persistently after 3 h and of Col1A1 after 24 h, while TGF-ß1+TGF-ß3 mitigated these effects after 48 h incubation. Gene expression of Tgf-ß1 was enhanced by TGF-ß1 and/or TGF-ß3 after 24 h and 48 h. However, Tgfbr1 mRNA expression was reduced at 48 h. After 48 h incubation with TGF-ß1 and/or TGF-ß3, Col3A1 and Col4A1 mRNA expression levels were decreased. Myoblasts produced collagen after three days incubation with TGF-ß1 and/or TGF-ß3 in a dose independent manner. Collagen deposition was doubled when myoblasts differentiated into myotubes and TGF-ß1 and/or TGF-ß3 did not stimulate collagen production any further. TGF-ß type I receptor (TGFBR1) inhibitor, LY364947, suppressed TGF-ßs-induced collagen production. Collagen I expression was higher in myotubes than in myoblasts. TGF-ß1 and/or TGF-ß3 inhibited myotube differentiation which was antagonized by LY364947. These results indicate that both C2C12 myoblasts and myotubes produce collagen. Whereas TGF-ß1 and TGF-ß3 individually and simultaneously stimulate collagen production in C2C12 differentiating myoblasts, in myotubes these effects are less prominent. In muscle cells, TGF-ß3 is ineffective to antagonize TGF-ß1-induced collagen production.

Lack of Tgfbr1 and Acvr1b synergistically stimulates myofibre hypertrophy and accelerates muscle regeneration.

In skeletal muscle, transforming growth factor-ß (TGF-ß) family growth factors, TGF-ß1 and myostatin, are involved in atrophy and muscle wasting disorders. Simultaneous interference with their signalling pathways may improve muscle function; however, little is known about their individual and combined receptor signalling. Here, we show that inhibition of TGF-ß signalling by simultaneous muscle-specific knockout of TGF-ß type I receptors Tgfbr1 and Acvr1b in mice, induces substantial hypertrophy, while such effect does not occur by single receptor knockout. Hypertrophy is induced by increased phosphorylation of Akt and p70S6K and reduced E3 ligases expression, while myonuclear number remains unaltered. Combined knockout of both TGF-ß type I receptors increases the number of satellite cells, macrophages and improves regeneration post cardiotoxin-induced injury by stimulating myogenic differentiation. Extra cellular matrix gene expression is exclusively elevated in muscle with combined receptor knockout. Tgfbr1 and Acvr1b are synergistically involved in regulation of myofibre size, regeneration, and collagen deposition.

The NLRP3 inflammasome contributes to inflammation-induced morphological and metabolic alterations in skeletal muscle.

BACKGROUND: Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement. METHODS: Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1ß production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function. RESULTS: NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1ß concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL-1ß caused similar alterations in cell morphology, and MCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation. CONCLUSIONS: LPS-induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association.

Human salivary histatin-1 (Hst1) promotes bone morphogenetic protein 2 (BMP2)-induced osteogenesis and angiogenesis.

Large-volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin-1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2-induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 µg Hst1 per sample and 0 or 2 µg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2-induced new bone formation in a dose-dependent manner. Co-administration of 500 µg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2-induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large-volume bone defects.

All-trans retinoic acid and human salivary histatin-1 promote the spreading and osteogenic activities of pre-osteoblasts in vitro.

Cell-based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large-volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co-administration of all-trans retinoic acid (ATRA) and human salivary peptide histatin-1 (Hst1) on the spreading and osteogenic activities of pre-osteoblasts on bio-inert glass surfaces. Pre-osteoblasts (MC3T3-E1 cell line) were seeded onto bio-inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point-counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, ß, and γ, such as ER-50891, LE-135, and MM-11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short-term (2 h) co-administration of 10 µm ATRA and Hst1 to pre-osteoblasts resulted in significantly higher spreading of pre-osteoblasts compared to ATRA or Hst1 alone. ER-50891 and LE-135 both nullified these effects of ATRA. Co-administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co-administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre-osteoblasts on bio-inert glass surfaces in vitro.