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Local Versus Systemic Control of Bone and Skeletal Muscle Mass by Components of the Transforming Growth Factor-Β Signaling Pathway

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Local Versus Systemic Control of Bone and Skeletal Muscle Mass by Components of the Transforming Growth Factor-Β Signaling Pathway Local versus systemic control of bone and skeletal muscle mass by components of the transforming growth factor-β signaling pathway Yewei Liua, Adam Lehara, Renata Rydzikb, Harshpreet Chandoka, Yun-Sil Leec,d, Daniel W. Youngstromb, Joshy Georgea, Martin M. Matzuke,f, Emily L. Germain-Leeg,h,i, and Se-Jin Leea,j,1 aThe Jackson Laboratory for Genomic Medicine, Farmington, CT 06032; bDepartment of Orthopaedic Surgery, University of Connecticut School of Medicine, Farmington, CT 06030; cDepartment of Molecular Genetics, Seoul National University, Seoul 08826, Republic of Korea; dDental Research Institute, School of Dentistry, Seoul National University, Seoul 08826, Republic of Korea; eDepartment of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030; fCenter for Drug Discovery, Baylor College of Medicine, Houston, TX 77030; gDepartment of Pediatrics, University of Connecticut School of Medicine, Farmington, CT 06030; hCenter for Regenerative Medicine and Skeletal Development, University of Connecticut School of Dental Medicine, Farmington, CT 06030; iDivision of Endocrinology, Center for Rare Bone Disorders, Connecticut Children’s, Farmington, CT 06032; and jDepartment of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT 06030 Contributed by Se-Jin Lee, July 8, 2021 (sent for review June 20, 2021; reviewed by Mark W. Hamrick and Kunihiro Tsuchida) Skeletal muscle and bone homeostasis are regulated by members and signaling components with MSTN. Indeed, one of these of the myostatin/GDF-11/activin branch of the transforming components, FST, was originally identified for its ability to in- growth factor-β superfamily, which share many regulatory com- hibit secretion of follicle-stimulating hormone (FSH) by cultured ponents, including inhibitory extracellular binding proteins and pituitary cells (28), and subsequent work showed that FST is receptors that mediate signaling. Here, we present the results of capable of binding and inhibiting activins (29), which are capable genetic studies demonstrating a critical role for the binding pro- of signaling to pituitary gonadotrophs to induce FSH secretion tein follistatin (FST) in regulating both skeletal muscle and bone. (30). FST undergoes alternative splicing to generate two iso- Using an allelic series corresponding to varying expression levels forms, the full-length FST315 and a carboxyl-terminal truncated Fst of endogenous , we show that FST acts in an exquisitely dose- FST288 (31). A third form, FST303, is derived from proteolytic dependent manner to regulate both muscle mass and bone den- cleavage of the C-terminal domain. All of the FST isoforms PHYSIOLOGY sity. Moreover, by employing a genetic strategy to target Fst ex- contain a heparin-binding domain that mediates binding to cell- pression only in the posterior (caudal) region of the animal, we surface proteoglycans. The presence of the C-terminal acidic tail show that the effects of Fst loss are mostly restricted to the pos- terior region, implying that locally produced FST plays a much in FST315, however, appears to neutralize the basic residues more important role than circulating FST with respect to regula- present in the heparin-binding domain and, as a result, FST315 tion of muscle and bone. Finally, we show that targeting receptors binds poorly to proteoglycans and is the predominant form of – for these ligands specifically in osteoblasts leads to dramatic in- FST in the circulation. FST288, which lacks the C-terminal 26 creases in bone mass, with trabecular bone volume fraction being amino acid extension, tends to remain locally sequestered increased by 12- to 13-fold and bone mineral density being in- following secretion. creased by 8- to 9-fold in humeri, femurs, and lumbar vertebrae. Based on the existence of these multiple isoforms and their These findings demonstrate that bone, like muscle, has an enor- differential biodistribution following secretion, a major question mous inherent capacity for growth that is normally kept in check by this signaling system and suggest that the extent to which this Significance regulatory mechanism may be used throughout the body to reg- ulate tissue mass may be more significant than previously Maintenance of skeletal muscle and bone is regulated by key appreciated. signaling proteins belonging to the transforming growth factor family. Here, we present genetic studies in mice showing that follistatin | myostatin | activin | bone mineral density | chalone follistatin, which normally blocks the activities of these pro- teins, acts locally in an exquisitely dose-dependent manner to yostatin (MSTN) is a transforming growth factor-β (TGF- control muscle and bone growth. Moreover, our studies tar- Mβ) superfamily member that normally acts to limit skeletal geting the receptors for these proteins reveal the enormous muscle mass (1). Mice lacking MSTN exhibit dramatic increases capacity for bone growth that is normally kept in check by this in skeletal muscle mass throughout the body, with individual signaling system. These findings have implications for strate- muscles growing to about twice the normal size. The amino acid gies to target this signaling pathway for clinical applications to sequence of MSTN has been strongly conserved through evolu- treat patients with muscle and bone loss. tion (2), and engineered or naturally occurring mutations in the MSTN gene have been shown to lead to increased muscling in Author contributions: E.L.G.-L. and S.-J.L. conceptualized the project; Y.L. and S.-J.L. de- many other species as well, including cattle (2–4), sheep (5), dogs signed research; Y.L., A.L., R.R., H.C., Y.-S.L., and S.-J.L. performed research; M.M.M. con- tributed new reagents/analytic tools; Y.L., A.L., R.R., H.C., D.W.Y., J.G., E.L.G.-L., and S.-J.L. (6), rabbits (7), rats (8), swine (9), goats (10), and humans (11). analyzed data; Y.L., E.L.G.-L., and S.-J.L. wrote the paper; and D.W.Y., J.G., E.L.G.-L., and MSTN is regulated by various extracellular binding proteins, S.-J.L. provided project administration. including follistatin (FST) (12), FSTL-3 (13), GASP-1 (14), and Reviewers: M.W.H., Augusta University; and K.T., Fujita Ika Daigaku. GASP-2 (15, 16), as well as the MSTN propeptide, which Competing interest statement: E.L.G.-L. and S.-J.L. are in the process of filing a patent maintains MSTN in an inactive, latent state (12, 17–19). MSTN application based on findings reported in this paper. signals initially by binding to the activin type 2 receptors ACVR2 This open access article is distributed under Creative Commons Attribution-NonCommercial- and ACVR2B (12, 20–22), followed by engagement of the type 1 NoDerivatives License 4.0 (CC BY-NC-ND). receptors ALK4 and ALK5 (22, 23). 1To whom correspondence may be addressed. Email: [email protected]. The function of MSTN as a negative regulator of muscle This article contains supporting information online at https://www.pnas.org/lookup/suppl/ growth is partially redundant with that of another TGF-β family doi:10.1073/pnas.2111401118/-/DCSupplemental. member, activin A (20, 24–27), which shares many regulatory Published August 12, 2021. PNAS 2021 Vol. 118 No. 33 e2111401118 https://doi.org/10.1073/pnas.2111401118 | 1of12 Downloaded by guest on September 26, 2021 has been whether the mode of action of FST is primarily local, with mice lacking cre, demonstrating that myofiber-derived FST regulating signaling by target ligands at or near the site of FST does contribute to the circulating pool (Fig. 1B); similar trends synthesis, or whether circulating FST can influence signaling to were seen in female mice, although the individual comparisons tissues distant from its site of synthesis. This question is relevant were not statistically significant. Targeting Fst in myofibers resul- not only in terms of understanding the mechanism of action of ted in decreases in muscle weights in both males and females; FST but also in terms of interpreting human studies seeking although the effects were relatively small, many of the differences associations between circulating FST levels and various physio- were statistically significant in both sexes (Fig. 1C and Table 1). logical and pathological states. Here, we use genetic approaches Hence, myofiber-derived FST does play some role in regulating in mice to address this fundamental question with respect to the muscle mass. roles of FST and this regulatory system in regulating two tissues, Because the effects of targeting Fst in myofibers were small, skeletal muscle and bone. however, we also examined the effects of more global targeting of Fst using the Cdx2-cre transgene, which is expressed specifi- Results cally in the posterior (caudal) but not anterior (cranial) region of A number of studies have shown that FST, by binding and the animal (36); in particular, Cdx2-cre is expressed in all cells inhibiting both MSTN and activin A, plays an important role in posterior to the umbilicus but not in any cells anterior to the regulating muscle growth. Specifically, we showed that transgenic umbilicus. In order to eliminate as much Fst expression as pos- +/− flox/flox overexpression of FST in skeletal muscle leads to muscle hy- sible, we set up crosses of Fst ; Cdx2-cre males with Fst flox/− pertrophy, consistent with inhibition of the MSTN/activin A females and analyzed Fst offspring that were either negative flox/− signaling pathway (12) and, conversely, that heterozygous loss of or positive for Cdx2-cre. Fst ; Cdx2-cre mice were viable, Fst in mice leads not only to reductions in muscle weights (by which allowed us to analyze mice at adulthood. As shown in about 15 to 20%) but also to a shift toward oxidative fiber types, Table 1 and Fig. 1D, muscles located in the posterior region of flox/− an impairment of muscle regeneration following cardiotoxin- Fst ; Cdx2-cre mice (quadriceps and gastrocnemius), where induced injury, and reduced tetanic force production (25), all Cdx2-cre is expressed, exhibited significant decreases in muscle −/− +/− consistent with overactivity of this signaling pathway.
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