Molecular characteristic of activin receptor IIB and its functions in growth and nutrient regulation in Eriocheir sinensis

Activin receptor IIB (ActRIIB) is a serine/threonine-kinase receptor binding with transforming growth factor-β (TGF-β) superfamily ligands to participate in the regulation of muscle mass in vertebrates. However, its structure and function in crustaceans remain unknown. In this study, the ActRIIB gene in Eriocheir sinensis (Es-ActRIIB) was cloned and obtained with a 1,683 bp open reading frame, which contains the characteristic domains of TGF-β type II receptor superfamily, encoding 560 amino acids. The mRNA expression of Es-ActRIIB was the highest in hepatopancreas and the lowest in muscle at each molting stage. After injection of Es-ActRIIB double-stranded RNA during one molting cycle, the RNA interference (RNAi) group showed higher weight gain rate, higher specific growth rate, and lower hepatopancreas index compared with the control group. Meanwhile, the RNAi group displayed a significantly increased content of hydrolytic amino acid in both hepatopancreas and muscle. The RNAi group also displayed slightly higher contents of saturated fatty acid and monounsaturated fatty acid but significantly decreased levels of polyunsaturated fatty acid compared with the control group. After RNAi on Es-ActRIIB, the mRNA expressions of five ActRIIB signaling pathway genes showed that ActRI and forkhead box O (FoxO) were downregulated in hepatopancreas and muscle, but no significant expression differences were found in small mother against decapentaplegic (SMAD) 3, SMAD4 and mammalian target of rapamycin. The mRNA expression s of three lipid metabolism-related genes (carnitine palmitoyltransferase 1β (CPT1β), fatty acid synthase, and fatty acid elongation) were significantly downregulated in both hepatopancreas and muscle with the exception of CPT1β in muscles. These results indicate that ActRIIB is a functionally conservative negative regulator in growth mass, and protein and lipid metabolism could be affected by inhibiting ActRIIB signaling in crustacean.

Altered Structure and Function of Mesenchymal Stromal Cell-Derived Extracellular Matrix in MDS Can be Restored By Luspatercept

Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p< 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Myostatin inhibitors as pharmacological treatment for muscle wasting and muscular dystrophy

Myostatin, a member of the transforming growth factor beta (TGF-β) superfamily that is highly expressed in skeletal muscle, was first described in 1997. It has been known that loss of myostatin function induces an increase in muscle mass in mice, cow, dogs and humans. Therefore, myostatin and its receptor have emerged as a therapeutic target for loss of skeletal muscle such as sarcopenia and cachexia, as well as muscular dystrophies. At the molecular level, myostatin binds to and activates the activin receptor IIB (ActRIIB)/Alk 4/5 complex. Therapeutic approaches therefore are being taken both pre-clinically and clinically to inhibit the myostatin signaling pathway. Several myostatin inhibitors , including myostatin antibodies, anti-myostatin peptibody, activin A antibody, soluble (decoy) forms of ActRIIB (ActRⅡB-Fc), anti-myostatin adnectin, ActRⅡB antibody have been tested in the last decade. The current review covers the present knowledge of several myostatin inhibitors as therapeutic approach for patients with loss of skeletal muscle however, the available information about compounds in development is limited.