The role of TGF-ß signaling in muscle atrophy, sarcopenia and cancer cachexia.

Skeletal muscle, comprising a significant proportion (40 to 50 percent) of total body weight in humans, plays a critical role in maintaining normal physiological conditions. Muscle atrophy occurs when the rate of protein degradation exceeds protein synthesis. Sarcopenia refers to age-related muscle atrophy, while cachexia represents a more complex form of muscle wasting associated with various diseases such as cancer, heart failure, and AIDS. Recent research has highlighted the involvement of signaling pathways, including IGF1-Akt-mTOR, MuRF1-MAFbx, and FOXO, in regulating the delicate balance between muscle protein synthesis and breakdown. Myostatin, a member of the TGF-ß superfamily, negatively regulates muscle growth and promotes muscle atrophy by activating Smad2 and Smad3. It also interacts with other signaling pathways in cachexia and sarcopenia. Inhibition of myostatin has emerged as a promising therapeutic approach for sarcopenia and cachexia. Additionally, other TGF-ß family members, such as TGF-ß1, activin A, and GDF11, have been implicated in the regulation of skeletal muscle mass. Furthermore, myostatin cooperates with these family members to impair muscle differentiation and contribute to muscle loss. This review provides an overview of the significance of myostatin and other TGF-ß signaling pathway members in muscular dystrophy, sarcopenia, and cachexia. It also discusses potential novel therapeutic strategies targeting myostatin and TGF-ß signaling for the treatment of muscle atrophy.

Handelin alleviates cachexia- and aging-induced skeletal muscle atrophy by improving protein homeostasis and inhibiting inflammation.

BACKGROUND: Handelin is a bioactive compound from Chrysanthemum indicum L. that improves motor function and muscle integrity during aging in Caenorhabditis elegans. This study aimed to further evaluate the protective effects and molecular mechanisms of handelin in a mouse muscle atrophy model induced by cachexia and aging. METHODS: A tumour necrosis factor (TNF)-α-induced atrophy model was used to examine handelin activity in cultured C2C12 myotubes in vitro. Lipopolysaccharide (LPS)-treated 8-week-old model mice and 23-month-old (aged) mice were used to examine the therapeutic effects of handelin on cachexia- and aging-induced muscle atrophy, respectively, in vivo. Protein and mRNA expressions were analysed by Western blotting, ELISA and quantitative PCR, respectively. Skeletal muscle mass was measured by histological analysis. RESULTS: Handelin treatment resulted in an upregulation of protein levels of early (MyoD and myogenin) and late (myosin heavy chain, MyHC) differentiation markers in C2C12 myotubes (P < 0.05), and enhanced mitochondrial respiratory (P < 0.05). In TNF-α-induced myotube atrophy model, handelin maintained MyHC protein levels, increased insulin-like growth factor (Igf1) mRNA expression and phosphorylated protein kinase B protein levels (P < 0.05). Handelin also reduced atrogin-1 expression, inhibited nuclear factor-κB activation and reduced mRNA levels of interleukin (Il)6, Il1b and chemokine ligand 1 (Cxcl1) (P < 0.05). In LPS-treated mice, handelin increased body weight (P < 0.05), the weight (P < 0.01) and cross-sectional area (CSA) of the soleus muscle (P < 0.0001) and improved motor function (P < 0.05). In aged mice, handelin slightly increased the weight of the tibialis anterior muscle (P = 0.06) and CSA of the tibialis anterior and gastrocnemius muscles (P < 0.0001). In the tibialis anterior muscle of aged mice, handelin upregulated mRNA levels of Igf1 (P < 0.01), anti-inflammatory cytokine Il10 (P < 0.01), mitochondrial biogenesis genes (P < 0.05) and antioxidant-related enzymes (P < 0.05) and strengthened Sod and Cat enzyme activity (P < 0.05). Handelin also reduced lipid peroxidation and protein carbonylation, downregulated mRNA levels of Fbxo32, Mstn, Cxcl1, Il1b and Tnf (P < 0.05), and decreased IL-1ß levels in serum (P < 0.05). Knockdown of Hsp70 or using an Hsp70 inhibitor abolished the ameliorating effects of handelin on myotube atrophy. CONCLUSIONS: Handelin ameliorated cachexia- and aging-induced skeletal muscle atrophy in vitro and in vivo, by maintaining homeostasis of protein synthesis and degradation, possibly by inhibiting inflammation. Handelin is a potentially promising drug candidate for the treatment of muscle wasting.

Amphibian pore-forming protein ßγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency.

Nutrient acquisition is essential for animal cells. ßγ-CAT is a pore-forming protein (PFP) and trefoil factor complex assembled under tight regulation identified in toad Bombina maxima. Here, we reported that B. maxima cells secreted ßγ-CAT under glucose, glutamine, and pyruvate deficiency to scavenge extracellular proteins for their nutrient supply and survival. AMPK signaling positively regulated the expression and secretion of ßγ-CAT. The PFP complex selectively bound extracellular proteins and promoted proteins uptake through endolysosomal pathways. Elevated intracellular amino acids, enhanced ATP production, and eventually prolonged cell survival were observed in the presence of ßγ-CAT and extracellular proteins. Liposome assays indicated that high concentration of ATP negatively regulated the opening of ßγ-CAT channels. Collectively, these results uncovered that ßγ-CAT is an essential element in cell nutrient scavenging under cell nutrient deficiency by driving vesicular uptake of extracellular proteins, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

Handelin extends lifespan and healthspan of Caenorhabditis elegans by reducing ROS generation and improving motor function.

Aging and aging-related disorders contribute to formidable socioeconomic and healthcare challenges. Several promising small molecules have been identified to target conserved genetic pathways delaying aging to extend lifespan and healthspan in many organisms. We previously found that extract from an edible and medicinal plant Chrysanthemum indicum L. (C. indicum L.) protect skin from UVB-induced photoaging, partially by reducing reactive oxygen species (ROS) generation. Thus, we hypothesized that C. indicum L. and its biological active compound may extend lifespan and health span in vivo. We find that both water and ethanol extracts from C. indicum L. extended lifespan of Caenorhabditis elegans, with better biological effect on life extending for ethanol extracts. As one of the major biological active compounds, handelin extended lifespan of C. elegans too. RNA-seq analysis revealed overall gene expression change of C. elegans post stimulation of handelin focus on several antioxidative proteins. Handelin significantly reduced ROS level and maintained the number and morphology of mitochondria. Moreover, handelin improveed many C. elegans behaviors related to healthspan, including increased pharyngeal pumping and body movement. Muscle fiber imaging analyses revealed that handelin maintains muscle architecture by stabilizing myofilaments. In conclusion, our present study finds a novel compound handelin, from C. indicum L., which bring about biologically beneficial effects by mild stress response, termed as hormetin, that can extend both lifespan and healthspan in vivo on C. elegans. Further study on mammal animal model of natural aging or sarcopenia will verify the potential clinical value of handelin.

Animal secretory endolysosome channel discovery.

Secretory pore-forming proteins (PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs (af-PFPs) and trefoil factors (TFFs). As a toad af-PFP, BmALP1 can be reversibly regulated between active and inactive forms, with its paralog BmALP3 acting as a negative regulator. BmALP1 interacts with BmTFF3 to form a cellular active complex called ßγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of ßγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence, we hereby propose a secretory endolysosome channel (SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with ßγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.

Isolation and characterization of the major centipede allergen Sco m 5 from Scolopendra subspinipes mutilans.

BACKGROUND: Allergic reactions have been observed following both direct centipede bites and the clinical use of centipede-containing medicines, such as traditional Chinese medicines utilizing Scolopendra subspinipes mutilans; however, no natural centipede allergen has yet been characterized. METHODS: An allergen was purified from S. s. mutilans venom using Superdex 75 gel filtration and RESOURCE S ion chromatography, and its primary structure was determined via a combination of LC-MS-MS, MALDI-TOF/TOF and protein sequencing techniques. Its potential allergenicity was evaluated by immunoblotting, ELISAs, skin prick tests (SPTs) and mast cell activation assays. RESULTS: A novel allergen Sco m 5 (210 amino acids long) was successfully purified from crude S. s. mutilans venom. Sco m 5 could promote the degranulation of a human mast cell line, HMC-1. Among centipede-allergic patients, Sco m 5 showed an 83.3% IgE-binding frequency and a 66.7% positive reaction frequency, as detected by immunoblotting and SPTs, respectively. Sco m 5 IgE-binding frequencies of common Chinese population was found to be 9%-16%. Sera positive for Sco m 5 IgE-binding was cross-reactive against venom from the wasp Vespa mandaeinia. CONCLUSIONS: The present study isolated and characterized a novel allergen termed as Sco m 5 from the centipede S. s. mutilans. The use of Sco m 5 to identify centipede-allergic individuals could be important, given the high potential allergenicity of Sco m 5 among the general Chinese population, along with the likely possibility of cross-reactivity against wasp venom among centipede-allergic patients.

A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that ßγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of ßγ-CAT. The cellular action of ßγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of ßγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.

Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.

Tissue repair is a highly dynamic process, and the immediate onset of acute inflammation has been considered necessary for repair. Pore-forming proteins are important, both in pathogen invasion and host immunity. However, their roles in wound healing and tissue repair are unclear. ßγ-crystallin fused aerolysin-like protein (α-subunit) and trefoil factor (ß-subunit) complex (ßγ-CAT) is a complex of a bacterial pore-forming toxin aerolysin-like protein and trefoil factor identified in the frog Bombina maxima. In this study, we established mouse cutaneous wound models to explore the effects of ßγ-CAT on skin wound healing. ßγ-CAT accelerated the healing of full-thickness wounds by improving re-epithelialization. This complex relieved dermal edema and promoted scarless healing. ßγ-CAT treatment resulted in a rapid release of IL-1ß, which initiated an acute inflammation response in the early stage of healing. Meanwhile, the expression levels of TGF-ß1, VEGF, and bFGF and the recruitment of M2 macrophages around the wound significantly increased after ßγ-CAT treatment. ßγ-CAT protected skin wounds against methicillin-resistant Staphylococcus aureus by improving neutrophil recruitment at the site of the wound. Overall, our results suggest that ßγ-CAT can promote tissue repair and protect skin wounds against antibiotic-resistant bacterial infection by triggering the acute inflammatory response. This is the first example that aerolysin-like pore-forming proteins widely existing in plants and animals may act in wound healing and tissue repair.-Gao, Z.-H., Deng, C.-J., Xie, Y.-Y., Guo, X.-L., Wang, Q.-Q., Liu, L.-Z., Lee, W.-H., Li, S.-A., Zhang, Y. Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.

Host Pore-Forming Protein Complex Neutralizes the Acidification of Endocytic Organelles to Counteract Intracellular Pathogens.

Many intracellular pathogens invade cells via endocytic organelles and have adapted to the drop in pH along the endocytic pathway. However, the strategy by which the host cell counteracts this pathogen adaptation remains unclear. ßγ-CAT is an aerolysin-like pore-forming protein and trefoil factor complex in the frog Bombina maxima. We report here that ßγ-CAT, as a host-secreted factor with an intrinsic channel-forming property, is the first example of a molecule that actively neutralizes the acidification of endocytic organelles to counteract Listeria monocytogenes infection. Immunodepletion of endogenous ßγ-CAT largely impaired the control of L. monocytogenes by frog cells. ßγ-CAT elevates the pH of L. monocytogenes-containing vacuoles to limit the vacuole escape of L. monocytogenes to cytosol. Furthermore, ßγ-CAT promotes intracellular L. monocytogenes clearance via autophagy and by that the nonlytic expulsion of the bacteria from host cells. Finally, ßγ-CAT attenuated the dissemination of L. monocytogenes in vivo. These findings reveal a novel host strategy and effectors that combat pathogen adaptation to acidic conditions along the endocytic pathway.