Molecular basis of individual locomotor function: Integrated understanding of gene expression regulation in the development and homeostasis of the musculoskeletal system

Abstract

This review examines the molecular mechanisms controlling the development and homeostasis of the musculoskeletal system through gene expression regulation. It introduces key discoveries from basic transcriptional control to advanced mechanotransduction pathways, focusing on our contributions including the EMBRYS database for transcription factor expression analysis and the identification of RP58 in muscle development and Mohawk (Mkx) in tendon formation. We also elucidated the role of miR-140 as a critical regulator in cartilage development and homeostasis. This microRNA is specifically expressed in cartilage, promotes chondrogenesis, and is involved in protective mechanisms against cartilage degenerative diseases such as osteoarthritis. Our discovery of the PIEZO1-Mkx pathway provides a molecular mechanism linking mechanical stimuli to gene expression in tendons, explaining tissue adaptation and differences in motor abilities. Understanding these pathways offers new therapeutic strategies for tendon and ligament injuries, age-related decline, and cartilage diseases. Currently, we are proposing the concept of “tenopenia” to complement sarcopenia, addressing the mechanisms of age-related tendon deterioration. This integrated approach to the musculoskeletal system as an environment-responsive entity advances both fundamental science and clinical applications aimed at maintaining mobility throughout life.

Piezo1–Mkx Axis in Tendon Mechanotransduction and Locomotor Function

Mechanical stress activates the Piezo1 channel in tendon cells, leading to calcium influx and subsequent activation of the transcription factor Mkx. This Piezo1–Mkx axis promotes tendon development and supports locomotor function. Genetic polymorphisms in Piezo1 may modulate this pathway in both mice and humans.

1. Introduction: The musculoskeletal system and the significance of functional research into this system

Locomotion in animals has fundamental significance for life processes, enabling the acquisition of food for survival and the continuation of life to the next generation. In humans particularly, the musculoskeletal system and its functions play crucial roles in personal and social activities (Fig. 1).

Figure 1.

The significance of the musculoskeletal system and its functional research. (A) Conceptual framework illustrating the life cycle of motor function research from development to aging, highlighting areas such as pediatric diseases, athletic performance, and age-related decline. (B) Medically accurate illustrations depicting key musculoskeletal regions vulnerable to injury. The right panel highlights the knee joint—featuring cartilage, ligaments including the anterior cruciate ligament—and the ankle joint, with a detailed view of Achilles tendon rupture. Major associated soft tissues such as muscles and tendons are also shown.

The population of the world is rapidly aging, with the World Health Organization (WHO) predicting that the global population aged 60 and over will double from 1 billion to 2.2 billion by 2050. This demographic shift highlights the universal social challenge of extending healthy lifespans and maintaining quality of life (QOL) throughout life. The WHO’s “Decade of Healthy Aging (2020–2030)” aims to create a world where everyone can live longer and healthier lives, promoting efforts to maintain and improve the functional capabilities of older adults. Maintaining and reconstructing the musculoskeletal system and its functions remains a central challenge. The United Nations’ Sustainable Development Goals (SDGs) also emphasize ensuring healthy lives and promoting well-being for people of all ages.

Human motor function is also connected to the foundations of worldwide culture and civilization through sports. The outstanding performances demonstrated by athletes at the Olympic Games showcase the possibilities of human motor function to the world, while the Paralympic Games inspire us by showing that even with partial loss of physical function, remarkable abilities can be demonstrated through ingenuity and effort. In general society, maintaining, competing, and improving motor function is an important theme for individual health and the development of human society.

The “musculoskeletal system”, which forms the foundation of motor function, is a complex tissue consisting of muscles, cartilage, bones, tendons, ligaments, and nerves, forming an intricate network with organs throughout the body.¹⁾ Cartilage is a specialized connective tissue that covers the ends of bones in joints, providing lubrication and enabling smooth motion. Tendons are strong, fibrous tissues that connect muscles to bones, transmitting the force of muscle contraction to enable movement. The synovium is a thin layer of tissue that lines the inner surface of the joint capsule and produces synovial fluid, which reduces friction in the joint. While dysfunction or breakdown of the musculoskeletal system contributes to the pathogenesis of multi-organ diseases such as dementia and metabolic syndrome, maintaining its physiological activity directly leads to extending healthy lifespans and activating social activities.^2,3) Notably, unlike internal organs, both functional (use) and morphological (aesthetic) aspects of the limbs and joints that appear on the body surface are important elements of the musculoskeletal system. Additionally, bone system diseases, bone and soft tissue tumors, and limb injuries often occur in childhood, and in such cases, they continue to affect individuals throughout their lives as carryovers.⁴⁾

Despite significant advances in regenerative medicine and drug development in modern medicine, major challenges remain in developing medical technologies to protect the musculoskeletal system from disease, maintain its homeostasis, and restore lost function.⁵⁾ Particularly in tendon tissue, recovery from age-related degeneration and injuries is difficult, necessitating new therapeutic approaches. Addressing these challenges requires integrated musculoskeletal research from the molecular to individual levels.

This paper places the “musculoskeletal system” as its central axis and comprehensively discusses it from the perspectives of maintaining motor function health and overcoming disease throughout the life cycle, contributing to life science through elucidating development and genetic programs, and analyzing human diversity and motor abilities, all based on the molecular mechanisms of gene expression.

2. From extracellular signals to gene expression regulation: Molecular foundations of musculoskeletal research

2.1. Interface between external signal reception and transcriptional control.

To understand the development, differentiation, and homeostasis maintenance of the musculoskeletal system, it is necessary to grasp the basic principles of how extracellular signals are linked to nuclear gene expression regulation. The discovery of cyclic adenosine monophosphate (cAMP; Sutherland, 1971 Nobel Prize in Physiology or Medicine) and the identification of protein kinase A (PKA; Krebs & Fischer, 1992 Nobel Prize in Physiology or Medicine) functioning downstream, which laid the foundation for signal transduction research, revolutionized the understanding of cellular information transmission mechanisms.⁶⁾ As the search for targets of the cAMP-PKA signaling pathway in nucleated cells progressed, it was revealed that cAMP response element-binding protein (CREB) controls the expression of a series of genes containing the cAMP response element (CRE).⁷⁾

Based on this discovery, gene expression regulation research entered a new transition period from the late 1980s to the early 1990s. The decisive turning point was the discovery by Montminy and colleagues of the activation mechanism of CREB through phosphorylation of Ser133 by PKA.⁸⁾ This discovery was the first example showing that extracellular signals directly connect to nuclear transcriptional control. In addition to control by basal transcription factors,⁹⁾ a consistent molecular mechanism from external stimulus signals to nuclear transcriptional control was elucidated in detail for the first time, significantly advancing understanding of the molecular basis of cellular responses.

2.2. Development into chromatin control.

The next turning point in transcription factor research was the discovery of the histone modification enzyme p300/CREB-binding protein (CBP).¹⁰⁾ This discovery revealed for the first time the specific molecular mechanism by which transcription factors change chromatin state. In particular, Goodman and colleagues showed that phosphorylated CREB specifically binds to the nuclear protein CBP,¹¹⁾ revealing the molecular basis that directly links environmental responses and chromatin control. Furthermore, Karin’s group demonstrated that CBP is a nuclear factor commonly required for the activation of cAMP-responsive and mitogen-responsive genes,¹²⁾ revealing that diverse extracellular signals are integrated through CBP. Kouzarides and Nakatani demonstrated that p300/CBP possesses histone acetyltransferase (HAT) activity,^10,13) bringing new understanding to the molecular mechanism of transcriptional activation.

Montminy and colleagues elucidated the essential role of secondary structure in transcriptional activation through analysis of activator and co-activator complexes.¹⁴⁾ Through structural analysis of the phosphorylated region (KID) of CREB and the interaction domain (KIX) of CBP, they showed that this interaction depends on the formation of specific secondary structures. Furthermore, Montminy and colleagues demonstrated the importance of chromatin-dependent cooperation in transcriptional activation in their research using CREB.¹⁵⁾ They revealed that the constitutive activation domain (Q2 domain) and inducible activation domain (KID domain) of CREB function cooperatively in a chromatin environment. This research elucidated the molecular mechanism by which multiple activation domains of environment-responsive transcription factors function cooperatively in a chromatin environment, significantly advancing understanding of the relationship between transcriptional control and chromatin structure.

2.3. Molecular basis of cell fate determination.

Identification of the transcription factor MyoD, which controls the differentiation of muscle cells, by Weintraub and colleagues provided a breakthrough in the gene-level mechanism of cell differentiation, which forms the basis for tissue and organ formation.^16–18) This discovery showed that transcription factors can determine cell fate, establishing the innovative concept of master transcription factors and bringing a revolutionary perspective to understanding tissue-specific differentiation programs. Subsequently, a muscle differentiation regulatory factor family consisting of MyoD, Myf5, myogenin, and MRF4 was identified,^19–21) showing that these cooperatively act to precisely regulate the muscle differentiation program.²²⁾ Similarly, various tissue-specific master transcription factors were successively identified, such as NeuroD by Weintraub and colleagues²³⁾ and PU.1 for blood cell differentiation by Singh and colleagues.²⁴⁾ This series of studies established the important concept that tissue-specific gene expression programs are hierarchically controlled by a small number of master transcription factors.

This concept later became an important guideline in research on the development and differentiation of musculoskeletal tissues. In particular, the discovery of musculoskeletal tissue-specific master transcription factors, such as Sox9 for cartilage differentiation control²⁵⁾ and Mkx for tendon differentiation control,^26,27) were important achievements that applied the concept of cell fate determination to musculoskeletal research. These discoveries are significant not only in elucidating molecular mechanisms but also in terms of application to tissue regenerative medicine and disease treatment.^28,29)

3. Transcriptional regulation in cartilage development, metabolism, and homeostasis maintenance

Endochondral ossification is a process through which most of the bones in the body are formed. It begins with a cartilage template that gradually becomes mineralized and is replaced by bone tissue. This process is essential for the formation of long bones and the development of the growth plate, and which involves a well-orchestrated series of chondrocyte proliferation, hypertrophy, and eventual replacement by osteoblasts.

3.1. Importance of cartilage in the musculoskeletal system.

Cartilage tissue plays a central role in the formation of the skeletal system and is essential for maintaining joint function. The development, differentiation, and homeostasis maintenance of cartilage are precisely regulated by a network of strictly controlled transcription factors. The breakdown of this transcriptional control network causes pathological conditions such as osteochondrodysplasia and osteoarthritis (OA).^5,30,31)

Cartilage tissue not only forms the basis for the entire body’s skeletal formation but also plays an essential role in load distribution and smooth movement in joints. In particular, articular cartilage, with its low friction coefficient and viscoelastic properties, forms the foundation for all motor functions from daily activities to sports. Deterioration of cartilage function due to aging, trauma, or metabolic abnormalities causes musculoskeletal diseases such as OA, resulting in significant QOL decline.³²⁾ Against this background, molecular biological understanding of cartilage is an extremely important issue not only for basic life science but also for clinical medicine.

3.2. Transcriptional control of cartilage development centered on Sox9.

Sox9 is known as the master transcription factor for cartilage development. Sox9 is essential for the differentiation induction of mesenchymal stem cells (MSCs) into chondrocytes (cartilage precursor cells) in the early stages and directly controls the expression of cartilage-specific extracellular matrix genes, including type II collagen (COL2A1).^33,34) It is known that Sox9 dysfunction causes severe skeletal diseases such as campomelic dysplasia (CD).^35,36)

Akiyama and colleagues showed that Sox9 directly activates the transcription of cartilage-specific matrix proteins such as Col2a1, Col11a2, and aggrecan.²⁵⁾ Additionally, research by Benko and colleagues showed complex genomic reorganization in the 5′ upstream region of the SOX9 gene in patients with Pierre Robin syndrome and left scapular dysplasia, indicating that chromosomal regions more than 1.16 Mb away from SOX9 are involved in controlling SOX9 expression.³⁷⁾

Furthermore, using CRISPR/Cas9 technology, we successfully elucidated the function of a complex distal enhancer that control the tissue-specific expression of SOX9.³⁸⁾ Specifically, we identified genomic regions that function as distal enhancers controlling SOX9 expression, and we also clarified the transcription factors that regulate this enhancer, revealing that they precisely control SOX9 expression patterns (Fig. 2). This research revealed the functional significance of long-distance genomic interactions that were difficult to analyze with conventional gene modification technologies, providing a new paradigm for tissue-specific gene expression control in the developmental process.

Figure 2.

Identification of an ultra-distal switch system in cartilage Sox9. (A) The development of limbs and the skeleton is determined by the expression of the master transcription factor Sox9, which governs cartilage cell differentiation. Abnormalities in this process can lead to congenital skeletal disorders like campomelic dysplasia. Insights from studying this disease indicated that enhancers regulating the cartilage-specific expression of Sox9 are exceptionally distal to the gene. However, accurately identifying these elements remained challenging. (B) By applying deadCas9, we managed to concentrate cis-elements and transcription factors that cluster around the Sox9 promoter, identifying Stat3 as a key regulator of the distal cartilage enhancer. (Modified from Mochizuki et al., Dev. Cell 2018.³⁸⁾)

3.3. Epigenetic control and cartilage transcription complexes.

Gene expression in cartilage differentiation is also precisely regulated by epigenetic control through changes in chromatin structure. Sox9 cooperates with CBP/p300, which has HAT activity, in the transcriptional activation of cartilage-specific genes such as COL2A1 on chromatin. We found that Sox9 directly interacts with CBP/p300, and this Sox9-CBP/p300 complex induces the expression of cartilage-specific genes.^39,40)

Transforming growth factor β (TGF-β) signaling plays an important role in cartilage development and homeostasis maintenance.⁴¹⁾ We showed that Smad3, which is activated in a TGF-β-dependent manner, strongly stimulates Sox9-dependent transcription.⁴²⁾ Namely, the TGF-β/Smad3 signaling pathway is thought to change the chromatin structure of cartilage-specific genes and activate transcription by recruiting Sox9 and CBP/p300.

4. Control of musculoskeletal development and homeostasis by microRNAs and RNA binding proteins and their therapeutic applications

4.1. Post-transcriptional regulation mechanisms by RNA binding protein.

Post-transcriptional regulatory mechanisms play important roles in the precise control of gene expression. Among them, RNA binding proteins (RBPs) and microRNAs (miRNAs; miRs) are major factors that control the stability, translation efficiency, and localization of target RNAs. RBPs recognize and bind to specific RNA sequences or motifs, exhibiting various regulatory functions. Additionally, the RBP ZCCHC24 promotes cancer progression by targeting specific RNAs,⁴³⁾ and LIN28A regulates the expression of the hypoxia-inducible factor (HIF) 1α through binding to the UGAU motif.⁴⁴⁾ These findings indicate that RBPs play central roles in post-transcriptional regulation in various tissues including the musculoskeletal system, forming complex regulatory networks involved in development, homeostasis maintenance, and disease onset.

4.2. Skeletal and cartilage formation by microRNAs.

The history of miRNA research began with the discovery of the first miRNA (lin-4) from the nematode Caenorhabditis elegans by Dr. Victor Ambros and Dr. Gary Ruvkun and colleagues in 1993.^45,46) For this discovery of miRNAs, both were awarded the Nobel Prize in Physiology or Medicine in 2024.

miRNAs are highly conserved short non-coding RNAs of about 22 nucleotides that suppress gene expression by binding mainly to the 3′UTR of mRNAs.⁴⁷⁾ It is said that more than half of human genes are controlled by miRNAs, which are closely involved in development and pathogenesis. Bartel systematically showed that these small non-coding RNAs play important roles in gene expression regulation.⁴⁷⁾

We focused on the reciprocal expression mechanism of let-7 and Lin28a, finding that in Lin28a knockout mice, let-7 family members are ectopically overexpressed, greatly disrupting the edge pattern of Hox gene expression, resulting in phenotypes affecting Hox-mediated pattern formation, such as changes in the number of ribs⁴⁸⁾ (Fig. 3). We also discovered a new expression control mechanism for let-7, in which the tRNA modification enzyme TruB1, in a role different from its original tRNA pseudouridination function, controls the maturation process of let-7 miRNAs.⁴⁹⁾

Figure 3.

Lin28/let-7 regulates edge of Hox gene expression. To understand the downstream network of mammalian Hox genes that remains largely unknown, we created mouse lines with tagged full Hox genes and analyzed them using techniques such as Cut and Tag to reveal the gene expression program that determines patterning. Gene expression regulation at the RNA level also holds a significant impact. MicroRNAs, as short non-coding RNAs, play a crucial role in suppressing gene expression. We reported a reciprocal regulatory mechanism between the microRNA Let7 and the RNA-binding protein Lin28 that controls the edge of Hox gene expression. (Modified from Sato et al., eLife 2020.⁴⁸⁾)

4.3. miRNAs in cartilage.

Representative musculoskeletal diseases include arthritis such as rheumatoid arthritis and OA. In joints, cartilage maintains its form throughout life, and its smooth surface function is responsible for joint movement. However, in arthritis, this cartilage is destroyed, and patients are restricted in their daily lives any may experience severe pain.

In previous research, however, sufficient treatment methods have not been developed, and the development of modalities based on new ideas remains an unmet need. Rheumatoid arthritis has a major pathology of eroding articular cartilage due to chronic inflammation of the synovial tissue, but we found that miR-146a, which had been identified as an anti-inflammatory miRNA, was highly expressed in rheumatoid arthritis synovial tissue.^50,51) When we created respective KO mice for miR-146a and miR-146b, we found that their deficiency caused B cell malignancy.⁵²⁾ Furthermore, research on the relationship between miR-146a and the pathogenesis of OA continues globally.^50,53)

We also found that miR-140 has a key function in the development and homeostasis maintenance of cartilage.⁵⁴⁾ miR-140 is strongly expressed specifically in chondrocytes, and its expression is markedly decreased in knee OA.⁵⁵⁾ miR-140 knockout mice showed a mild reduction in bone formation. Furthermore, miR-140 knockout mice showed strong age-related knee cartilage damage, and at 12 months of age, significant cartilage degeneration similar to the pathology of OA was observed.⁵⁶⁾ This was one of the first reports to demonstrate that miRNAs are involved not only in development and differentiation but also in tissue homeostasis maintenance.

Notably, miR-140 exists in the intron of the E3 ubiquitin ligase WWP2. In a report on the creation of a knockout mouse for WWP2 using the exon trap method, the same bone and cartilage development phenotype as ours was reported,⁵⁷⁾ but since this method also reduced the expression of miR-140 in the intron at the same time, for a long time, no conclusion was reached on whether miR-140 or WWP2 has a significant function in this phenotype, or whether both are important.

We created mice with a frameshift in WWP2 using Crispr/Cas9, mice with only a small deficiency in miR-140, and mice deficient in both, and found that the bone phenotype was seen only in mice with miR-140 deficiency. This was the same result as with mutants that inactivated WWP2.⁵⁸⁾

These results were the first paper to sound an alarm for the analysis of knockout mice by exon trapping, as only the intron was involved in the phenotype (Fig. 4).

Figure 4.

Cartilage development phenotype is only shown in miR-140 knockout. (A) miR-140 is located within the intron of the E3 ubiquitin ligase WWP2. It remained inconclusive for a long time whether miR-140 or WWP2 played a significant role in this phenotype. To address this issue, we used Crispr/Cas9 to create mice with a frame-shift mutation in WWP2, mice with a small deletion in miR-140 alone, and mice with both genes deleted. (B) Surprisingly, we found that only miR-140 exhibited the bone-related phenotype. This result was consistent even in mutants with inactivated WWP2. These findings highlight that the intron alone is involved in the phenotype, providing the first apparent evidence challenging the previous analyses of exon-trap knockout mice. (Modified from Inui et al., Nat. Cell. Biol. 2018.⁵⁸⁾)

We also found that miR-140 and WWP2 work cooperatively in cartilage tissue homeostasis maintenance, unlike during the developmental period. We had found that the target of miR-140 is Adamts5, a cartilage matrix degrading enzyme that induces joint destruction,⁵⁶⁾ but we found that WWP2, as an E3 ubiquitin ligase, suppresses Adamts5 through ubiquitination of Runx2.⁵⁹⁾ This means that we found a program that performs robust regulation by suppressing Adamts5 both pre-transcriptionally (WWP2) and post-transcriptionally (miR-140) (Fig. 5).

Figure 5.

Medical applications of miRNA and pseudouridine mRNA. Sox9 transcription creates WWP2 protein and miR-140 miRNA, suppressing pre- and post-transcription ADAMTS5 production. Sox9 also generates miR-455 5P/3P, countering osteoarthritis degenerative signals.

4.4. Discovery of miR-455 and dual control in cartilage differentiation.

In parallel with miR-140 research, another important miRNA was identified: miR-455. We found that miR-455, in the intron of the COL27A1 gene, is specifically expressed in cartilage tissue, and has abnormal expression in OA cartilage.^60,61) Notably, while miRNAs generally select either the -5p or -3p strand to mature and function, we found that in cartilage, miR-455 exceptionally matures both strands (-5p and -3p) at almost the same level, and both play important roles in cartilage homeostasis maintenance. Both strands are directly controlled by Sox9 and are highly expressed in chondrocytes of normal articular cartilage.

Functional analysis of miR-455 revealed that this miRNA directly targets HIF-2α, a factor with catabolic action in cartilage homeostasis maintenance. miR-455 knockout mice showed OA-like cartilage degeneration and increased expression of cartilage degeneration-related genes at 6 months of age. Conversely, overexpression of both miR-455-5p and -3p in a mouse OA model was shown to suppress cartilage degeneration (Fig. 5).

Such control of the same target by both strands arising from one miRNA precursor is a mechanism that ensures the reliable execution of important development and homeostasis maintenance programs, forming the molecular basis for the robustness of biological systems.⁶²⁾

4.5. Nucleic acid therapy strategies for the musculoskeletal system.

RNA biology has shown further innovative developments entering the 2020s. The 2023 Nobel Prize in Physiology or Medicine was awarded to Dr. Katalin Karikó and Dr. Drew Weissman, who demonstrated the importance of mRNA modification. They discovered that chemical modification of mRNA, particularly the substitution of uridine with N1-methylpseudouridine (m1Ψ), reduces the immunogenicity of RNA while simultaneously improving translation efficiency.⁶³⁾ This research on RNA modification brought new perspectives for therapeutic applications. We had found that WWP2 suppresses arthritis, but the induction of inflammation by mRNA introduction was a major barrier to therapeutic application. However, the modified RNA technology developed by Karikó and colleagues showed the possibility of fundamentally solving this problem.⁶⁴⁾ In fact, expression control of WWP2 using chemically modified mRNA showed significant therapeutic effects in an arthritis model mouse.⁵⁹⁾ We have also confirmed the therapeutic effect by intra-articular injection of miR-455.⁶¹⁾ Notably, miRNAs, which were the subject of the Nobel Prize in Physiology or Medicine, have not yet been realized in clinical applications despite their widely recognized fundamental importance. The cartilage-specific miRNA system miR-140/miR-455 is expected to have clinical applications for OA treatment (Fig. 5).

For the practical application of RNA therapy, the development of delivery technology is an important challenge. Recent research has advanced the development of liposomes and nanoparticles for selectively delivering RNA to specific cells and tissues, and these technological innovations are expected to realize more effective and safer RNA therapy.

5. From comprehensive expression analysis to functional analysis of the musculoskeletal system

5.1. Establishment and significance of the EMBRYS database, a catalog of transcription factor expression in embryonic development.

In the early 2000s, during the post-genome era, comprehensive analysis of gene expression patterns in developmental processes was a major challenge. To address this challenge, our research group began constructing a whole mount in situ hybridization (WISH) system that comprehensively analyzes the expression of about 1,600 transcription factors and cofactors during embryonic development. This project gave birth to the Embryonic Molecular Biology Research Yield System (EMBRYS) database⁶⁵⁾ (Fig. 6). EMBRYS includes the meaning of comprehensive research on EMBRYO and also includes the anecdote from “Kojiki” and “Nihon Shoki” that Ebisu-sama, despite having physical disabilities, was active as a god of fortune.

Figure 6.

EMBRYS construction revealed the musculoskeletal development transcriptional network including Rp58 and Mkx. (A) We constructed the EMBRYS database to elucidate gene expression during the developmental process, and (B) identified Mkx as the master transcription factor for tendons and ligaments, crucial not only in rodents but also in human tendons. (Modified from Yokoyama et al., Dev. Cell 2009,⁶⁵⁾ Ito et al. PNAS 2010,²⁷⁾ Nakahara et al., Arthritis Rheum. 2013.⁸⁷⁾)

EMBRYS is one of the world’s largest comprehensive resources that systematically collects and publishes high-resolution images of the spatial expression patterns of transcription factors and epigenome regulatory factors in mouse embryos (E9.5, E10.5, E11.5). The greatest feature of this database is that it covers more than 1,600 genes specializing in transcription and epigenome regulatory factors, whereas conventional gene expression databases were limited to hundreds of genes.

Furthermore, by introducing the AERO system, a three-dimensional image reconstruction technology, we succeeded in precisely recording the spatiotemporal patterns of gene expression in complex developmental processes.⁶⁶⁾ This AERO system generates pseudo-3D representation from conventional 2D images, enabling three-dimensional understanding of the developmental process. Particularly groundbreaking was the ability to integratively visualize both the time and space axes, which are important in tissue and organ formation processes.

The EMBRYS database differs from existing databases such as the Edinburgh Mouse Atlas Project (EMAP)⁶⁷⁾ and Allen Brain Atlas⁶⁸⁾ in that it focuses particularly on transcription and epigenome regulatory factors and achieves comprehensive expression analysis in the early embryonic development period. The Edinburgh Mouse Atlas constructed by Baldock and colleagues has become an important digital resource in developmental biology research,⁶⁷⁾ and EMBRYS developed this in a form specialized for musculoskeletal research. Additionally, by establishing complementary relationships with genome functional annotation projects such as the VISTA Enhancer Browser⁶⁹⁾ and ENCODE Project,⁷⁰⁾ it was internationally highly evaluated as a foundation for promoting comprehensive understanding of transcriptional regulatory networks.

The construction of this comprehensive database made it possible to realize spatial expression analysis of developmental regulatory factors, which had previously relied on individual in situ hybridization experiments, on a single integrated platform. This made it easier to compare expression patterns of multiple genes and track expression changes along the time axis, dramatically improving the efficiency of developmental biology research.

One of the most important contributions of the EMBRYS database is that it enabled the identification of tissue-specific developmental program regulatory factors that had previously been identified. In conventional approaches, genes were often individually analyzed based on known phenotypes and existing knowledge, but the comprehensive approach of EMBRYS led to the discovery of tissue-specific regulatory factors that were not readily predictable. This “hypothesis-independent” approach later played a decisive role in our identification of important transcription factors such as RP58⁶⁵⁾ and Mkx.²⁷⁾ The Edinburgh Mouse Atlas Project developed by Baldock and colleagues was developed for a similar purpose,⁶⁷⁾ but the detailed analysis from EMBRYS was unique in focusing particularly on the developmental control of the musculoskeletal system.

5.2. Discovery of RP58 and spatial expression analysis of transcription factors in limb development.

Analysis using the EMBRYS database revealed dynamic expression patterns of transcription factors governing the three-dimensional axes of limb development.⁷¹⁾ This research identified hierarchical expression patterns of gene groups controlling the three-dimensional structure formation of the mouse forelimb bud, including the anteroposterior axis, proximodistal axis, and dorsoventral axis. Specifically, it revealed spatial expression gradients of proximal region-specific transcription factors such as Meis1, Meis2, and Pknox1, intermediate region-specific transcription factors such as Irx3 and Irx5, and distal region-specific transcription factors such as Dlx5 and Dlx6.

A notable discovery was the identification of the novel transcriptional repressor RP58 (Zfp238). Yokoyama and colleagues identified RP58 from the EMBRYS database as a transcription factor specifically expressed in developing skeletal muscle.⁶⁵⁾ Detailed functional analysis clearly showed from knockout mouse analyses that RP58 is expressed downstream of MyoD and is essential for muscle differentiation. Particularly important was the discovery of an elaborate control mechanism in which RP58 promotes muscle differentiation by suppressing the expression of muscle differentiation inhibitors such as Id2 and Id3.

The discovery of RP58 established several important concepts in developmental differentiation control. First, it was revealed that transcriptional repression plays an equally important role as transcriptional activation in MyoD-mediated muscle differentiation control, which was previously thought to be mainly mediated through transcriptional activation. Second, it was shown that muscle differentiation proceeds through a sophisticated molecular mechanism of a double-negative gate, where RP58 suppresses the action of Id proteins, which Weintraub had found to inhibit development and differentiation. Third, by analyzing the expression pattern of RP58 in detail, the importance of spatiotemporal gene expression control in the developmental period was demonstrated.

These discoveries greatly advanced the understanding of developmental biology by proposing a control mechanism through a double-negative gate for the major challenge of “how tissue-specific gene expression programs are established”, as a mechanism that guarantees the robustness of gene expression in the developmental process.

5.3. Identification and functional analysis of the mohawk transcription factor.

Tenogenesis refers to the process of tendon development, which begins with the condensation of mesenchymal progenitor cells under the regulation of key transcription factors such as Scleraxis (Scx). Scx is essential for the early specification of tendon progenitors, whereas Mohawk (Mkx) plays a critical role in the maturation and maintenance of tendon identity in later stages. This sequential transcriptional program ensures the proper formation of functional tendons capable of mechanical load-bearing.

Following success with the identification of RP58, we further advanced this comprehensive analysis using the EMBRYS database and conducted identification and functional analysis of the transcription factor Mkx, which is specifically expressed in tendon tissue.²⁷⁾ At that time, the developmental control mechanism of tendon tissue was not as advanced compared with other connective tissues. Scx, identified by Schweitzer and colleagues, was known to be involved in tendon and ligament development,⁷²⁾ but since its expression is strong in early development and decreases after birth, the existence of transcription factors involved in tendon maturation and homeostasis maintenance was predicted.

In 2010, we discovered that the homeodomain transcription factor Mkx is specifically expressed in tendon and ligament tissues from the developmental period to adulthood.²⁷⁾ Spatiotemporal expression analysis using the EMBRYS database revealed that Mkx begins to be expressed in tendon precursor tissue from E12.5 in embryonic development and its expression increases with the progression of development. Particularly notable was the finding that Scx is strongly expressed during development and decreases in adulthood, whereas Mkx maintains high expression levels even in adulthood. These differences in expression pattern suggested functional differences between the two transcription factors, indicating the possibility that they control different phases in tendon tissue development and maturation (Fig. 6).

The identification of Mkx brought a breakthrough to the understanding of tendon tissue-specific gene expression programs, which had been little known until then. Despite tendons being important tissues in the motor system connecting muscles and bones, understanding of their molecular biological processes had long been limited. The discovery of Mkx accelerated the elucidation of molecular mechanisms controlling tendon tissue differentiation and homeostasis maintenance.

5.4. Functional analysis of Mkx.

Analysis of Mkx knockout mice revealed that this homeodomain transcription factor is essential for the development and maturation of tendon and ligament tissues.^27,73) Mice lacking Mkx showed significant reduction in tendon tissue size and decreased mechanical strength. Particularly important findings included decreased width of the Achilles tendon, reduced wet weight of the anterior tibialis tendon, and reduced tail tendon cells and collagen fiber diameter. These findings molecularly supported the conventional knowledge that the arrangement and diameter of collagen fibers, which are the basic structure of tendon tissue, are extremely important in determining the biomechanical properties of tendons.^74,75)

Molecular level analysis determined that Mkx directly regulates the expression of important extracellular matrix molecules that determine tendon characteristics, such as type I collagen (Col1a1/Col1a2), tenomodulin (Tnmd), and fibromodulin (Fmod).^27,73) This discovery provided important evidence that the expression of extracellular matrix molecules and the construction of their hierarchical structure are subject to strict transcriptional control, deepening the understanding of the formation mechanism of connective tissue. In particular, in addition to the regulation of Tnmd expression by Scx reported by Shukunami and colleagues,⁷⁶⁾ it was clarified that Mkx also directly regulates the expression of tendon differentiation markers, contributing to the maturation and maintenance of tendon tissue. This elucidates the molecular basis of the structural hierarchy of tendon tissue described in detail by Kannus.⁷⁴⁾

To further elucidate the function of Mkx, we created Mkx knockout rats using genome editing technology.⁷⁷⁾ Surprisingly, in rats lacking Mkx, heterotopic ossification occurred in the Achilles tendon, showing a dramatic phenotype where tendon tissue was replaced by bone tissue. This phenomenon strongly indicated the importance of Mkx in the tendon development process, suggesting that Mkx not only promotes tendon formation but also plays a role in suppressing heterotopic ossification of tendon tissue. This dual function was an important discovery in terms of maintaining tissue identity, greatly contributing to the elucidation of molecular mechanisms that maintain the boundary between tendon tissue and bone tissue (Fig. 7). Recently, Chen and colleagues reported that disturbed glycolipid metabolism in tendon stem cells of elderly mice activates the CXCL13-CXCR5 pathway, promoting heterotopic ossification,⁷⁸⁾ and the relationship between age-related tendon degeneration and heterotopic ossification is attracting attention.

Figure 7.

Dysplasia and ossification of tendons in Mkx knockout animals. (A, B) We created Mkx knockout mice and rats, revealing that these animals exhibited tendon dysplasia. In particular, Mkx knockout rats showed heterotopic ossification in the Achilles tendon. (C) From the analysis of Mkx knockout animals, it was revealed that Mkx has the function of inducing the expression of tendon-related genes and suppressing cartilage gene expression. (Modified from Ito et al., PNAS 2010,²⁷⁾ Suzuki et al., PNAS 2016.⁷⁷⁾)

5.5. Molecular mechanisms of Mkx.

Mkx target genes were comprehensively identified through ChIP-seq and RNA-seq analyses. Mkx was confirmed to activate the expression of tendon-specific genes such as Col1a1, Col1a2, Tnmd, and Fmod. Notably, it was also revealed that Mkx suppresses the expression of bone formation-related genes (Ibsp, Bglap, Spp1, etc.).^77,79) These results indicated that Mkx functions not only as an activator but also as a repressor, having a dual control mechanism that promotes tendon differentiation while simultaneously suppressing ossification.

Particularly important in the control mechanism of Mkx is its interaction with the TGF-β signaling pathway. We revealed that Mkx is involved in regulation of the TGF-β signaling pathway.⁸⁰⁾ Normally, TGF-β signaling promotes the development and maintenance of tendon and ligament tissues, but in mice lacking Mkx, the balance of this signaling pathway is disrupted, with decreased Smad3 phosphorylation and increased Smad1/5/8 phosphorylation. This indicated that Mkx controls the switching mechanism between the TGF-β/Smad2/3 pathway (promoting tendon differentiation) and the BMP/Smad1/5/8 pathway (promoting bone differentiation). The breakdown of this regulatory mechanism is thought to lead to heterotopic ossification of tendon tissue due to Mkx deficiency.

5.6. Expression of Mkx in tendon-like tissues.

As research progressed, we discovered that Mkx is highly expressed not only in tendon tissue but also in tendon-like tissues such as the outer layer of the annulus fibrosus of the intervertebral disc and the periodontal ligament.^80,81) In particular, functional analysis in the intervertebral disc, using a mouse model of lumbar disc herniation, revealed that Mkx has a protective effect against intervertebral disc degeneration. The intervertebral disc is a disc-shaped structure between vertebral bodies, consisting of the nucleus pulposus in the central part and the annulus fibrosus surrounding it. The annulus fibrosus has a concentric layer structure mainly composed of type I collagen, with the outer layer in particular showing similar molecular composition to tendon tissue. Mkx is specifically expressed in this outer layer of the annulus fibrosus, and its deficiency was shown to increase the risk of intervertebral disc degeneration.

Functional analysis of Mkx in the periodontal ligament revealed that Mkx plays an important role in the mechanical stress response of the periodontal ligament and bone remodeling control. The periodontal ligament is a tendon-like tissue connecting the tooth and alveolar bone, constantly exposed to mechanical stresses such as mastication forces. It was also found that Mkx affects bone metabolism and controls bone resorption associated with orthodontic tooth movement.^82,83) Furthermore, single-cell RNA-seq analysis showed that Mkx-expressing cells form characteristic subpopulations within the periodontal ligament and proliferate and differentiate in response to mechanical stress.⁸⁴⁾ This finding indicates that Mkx is an important factor linking the mechanical environment of tissues and gene expression programs.

These Mkx-expressing tissues all have the common feature of being exposed to strong mechanical stress. This suggests that Mkx may play an important role in tissue adaptation in response to mechanical stimuli, leading to the pioneering discovery of functional linkage with PIEZO1, as described in the next chapter.

5.7. Clinical significance and applications in regenerative medicine.

From a clinical perspective, the importance of Mkx has also become clear. Recently, it has been reported that aging and injury affect the morphological heterogeneity of tendons, and the molecular mechanisms of tendon tissue homeostasis maintenance are being reported.^85,86) Analysis of human anterior cruciate ligament samples revealed that Mkx expression significantly decreases in patients with arthritis and as part of aging.⁸⁷⁾ This decrease in expression is thought to be associated with increased risk of tendon and ligament tissue degeneration and injury. Particularly in tendon and ligament tissues of patients with rheumatoid arthritis, Mkx expression is suppressed due to the influence of inflammatory cytokines, promoting tissue fragility. Mkx has also been suggested to be involved in collagen cross-linking formation,⁸⁸⁾ which determines the mechanical properties of tendons, and further analysis is expected.⁸⁹⁾

Tendon regenerative medicine is a rapidly developing field in recent years. Russo and colleagues have pointed out the importance of interactions between stem cells and immune cells in tendon regeneration, indicating the possibility of treatment strategies utilizing stem cells.⁹⁰⁾ Furthermore, for the decline in tendon regenerative capacity associated with aging, as shown by Wang and colleagues, rejuvenation of aged tendon stem cells by specific compounds may become an effective therapeutic approach.⁹¹⁾ Zhang and colleagues have also reported that metformin improves tendon degeneration in elderly mice,⁹²⁾ and pharmacological approaches to maintain tendon tissue homeostasis and promote regeneration are attracting attention.

Research on the application of Mkx to regenerative medicine is also progressing. We demonstrated that introducing Mkx into bone marrow-derived MSCs can induce differentiation into tendon-like tissue in vitro.⁹³⁾ Furthermore, it was also confirmed that transplanting these Mkx-introduced MSCs into a mouse model of outer annulus fibrosus injury of the intervertebral disc promotes tissue repair.⁸⁰⁾ We have also succeeded in creating tissue closer to living tendons and ligaments by combining a three-dimensional culture system with cyclic mechanical stretching stimulation.⁹⁴⁾ In this method, tendon-like tissue with aligned collagen fibers can be created by culturing Mkx-expressing cells in a three-dimensional collagen gel and applying cyclic mechanical stretching stimulation. Recently, technology to create tendon-like tissue from human induced pluripotent stem cells using Mkx has also been established.⁹⁵⁾ This technology is expected as a new treatment for tendon injuries caused by sports injuries and age-related tendon degeneration (Fig. 8).

Figure 8.

Technology for creating tendon-like tissue from human induced pluripotent stem cells using Mkx. The figure illustrates the innovative process of generating tendon-like tissue by introducing Mkx into human induced pluripotent stem cells for treating tendon injuries and age-related degeneration through tissue engineering approaches. (Modified from Tsutsumi et al., J. Tissue Eng. 2022.⁹⁵⁾)

This series of studies is expected to lead to the development of new treatments for tendon and ligament diseases by targeting Mkx signaling and applications in regenerative medicine for tendon and ligament tissues in the future.

6. Discovery of the PIEZO1-Mkx pathway: New understanding of mechanical response

6.1. Historical context of mechanical response research and applications to the musculoskeletal system.

In the history of exercise physiology, tendons were long viewed as merely passive elastic elements connecting muscles to bones. In this classical view, the mechanical properties of tendons were thought to be determined solely by the physical properties of collagen fibers.^74,75)

This understanding began to change in the 1980s. Research by Woo and colleagues⁹⁶⁾ demonstrated the ability of tendon tissue to adaptively remodel according to loading conditions. This led to the recognition of tendons as biological entities that dynamically respond to environmental changes.⁷⁵⁾

The origin of mechanical response research in the musculoskeletal system can be traced back to A.V. Hill’s thermodynamic study of muscle contraction in 1938.⁹⁷⁾ Hill proposed the concept that muscle contraction energy and tendon elastic energy are arranged in series, which became the foundation for understanding mechanical energy control in the musculoskeletal system.⁹⁸⁾

From the 1990s to 2000s, tendon research further progressed. Elucidation of the microstructure and cell biological characteristics of tendons and ligaments by Benjamin and Ralphs⁹⁹⁾ and the demonstration of metabolic activity and plasticity of tendon tissue by Kjaer and colleagues¹⁰⁰⁾ deepened the understanding of the biological importance of tendons. However, the fundamental mechanism of how tendon tissue senses mechanical stimuli and converts them into biochemical and molecular biological signals remained unexplored for a long time.^101,102)

This situation was significantly changed by the discovery of PIEZO1/2 by Patapoutian and colleagues, which was the subject of the 2021 Nobel Prize in Physiology or Medicine.¹⁰³⁾ Patapoutian and colleagues identified PIEZO1 as an ion channel that opens in response to mechanical stimuli and that this channel is the molecular basis of touch and pressure sensation.¹⁰⁴⁾ Li and colleagues, through functional analysis of PIEZO1 in the vascular system, showed that this mechanosensor channel plays an important role in integrating physiological forces and tissue architecture.¹⁰¹⁾ This discovery dramatically advanced understanding of the sensing mechanism of mechanical stimuli at the cellular level, but many questions remained about tissue-specific functions and physiological significance.

6.2. Discovery of a mechanical response system in tendons: The PIEZO1-Mkx pathway.

The discovery of PIEZO1 brought an innovative perspective to musculoskeletal research. In previous musculoskeletal research, although tissue adaptation to external mechanical stimuli (such as Wolff’s law) was widely recognized, the molecular mechanism was unknown. The identification of PIEZO1 revealed the molecular entity by which cells directly sense mechanical stimuli, greatly advancing understanding of the conversion from mechanical stimuli to biochemical signals (mechanotransduction).^101,105,106)

As we continued our analysis of Mkx identified from the EMBRYS database, we discovered an interesting property that this transcription factor responds to mechanical stimuli. Systematic analysis combining in vivo experiments in tendon tissue and in vitro mechanical stretching stimulation experiments revealed that Mkx expression shows a positive correlation with mechanical loading. Furthermore, evidence showed that PIEZO1 is involved in this mechanical response.

Passini and colleagues and we independently revealed through detailed biochemical and molecular biological analyses that PIEZO1 functions upstream of Mkx, forming a new pathway that converts mechanical stimuli into changes in gene expression.^107,108)

The detailed mechanism of the PIEZO1-Mkx pathway was elucidated as follows: When mechanical stimuli are applied to tendon tissue, PIEZO1 channels on the cell membrane are activated, inducing Ca²⁺ influx into the cell. This Ca²⁺ signal activates calcineurin, promoting dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T cells, cytoplasmic (NFATc). NFATc translocates into the nucleus and binds to the promoter region of the Mkx gene, activating its transcription. Furthermore, Mkx induces the expression of target genes such as Col1a1, Col1a2, and Tnmd, promoting the synthesis of tendon-specific extracellular matrix.

The importance of this discovery is also evaluated from the perspective of biomechanics. In conventional biomechanics research, tissue properties were mainly understood as physical characteristics, and their regulatory mechanisms were mainly interpreted as results of mechanical adaptation.^75,102) Our research presents a new paradigm that tissue mechanical properties are actively controlled at the molecular level. This suggests the possibility of biologically controlling tissue mechanical properties, opening the way for new treatment strategies for tendon and ligament disorders. As also pointed out by Martinac and colleagues, mechanosensitive ion channels in the cell membrane play an important role in controlling the mechanical properties of living tissues,¹⁰⁹⁾ and PIEZO1 is a representative example of this.

6.3. Unexpected discoveries from PIEZO1 gene polymorphism research.

Research on PIEZO1 gene polymorphism was initially noted in relation to malaria resistance. Ma, Patapoutian, and colleagues reported that a PIEZO1 gene polymorphism (E756del), present in about 30% of African Americans and Jamaicans of West African descent, confers malaria resistance by affecting red blood cell morphology.^110,111) This discovery became an important example supporting the relationship between human genetic diversity and adaptive evolution proposed by Tishkoff and colleagues.¹¹²⁾ This is also important in the context of sports gene polymorphism research, having significance similar to reports that ACE gene polymorphism is associated with elite swimmer performance¹¹³⁾ and findings that actin-3 gene polymorphism affects muscle performance.¹¹⁴⁾

We noted that the distribution of this PIEZO1 gene polymorphism (E756del) corresponds very closely with the country distribution of athletes holding the top 100 records in the 100-meter sprint, and we conducted an examination of human motor abilities. As a collaborative study with the Athlome Consortium, which analyzes the genomes of international athletes, with the cooperation of Drs. Pitsiladis, Morrison, and Fuku, we investigated the frequency of active-type PIEZO1 E756del in Olympic-level sprinters and the general population in Jamaica. The results showed that 54% of Jamaican sprinters possess the active-type polymorphism, a significantly increased rate compared with about 30% in the general population (Fig. 9). This ratio was the same when the data were divided by gender.

Figure 9.

PIEZO1 E756del polymorphism prevalence and athletic performance correlation. (A) Geographical distribution of PIEZO1 E756del polymorphism matches the distribution of countries producing top 100-meter sprint record holders. The polymorphism appears in approximately 30% of West Africans, Jamaicans, and African Americans. (Modified from Ma et al., Cell 2018.¹¹⁰⁾) (B) Sample collection through collaboration with the Athlome Consortium, analyzing the genomes of international athletes with cooperation from Drs. Pitsiladis, Morrison, and Fuku in Jamaica. (C) Analysis showing significantly higher prevalence of active-type PIEZO1 E756del polymorphism in Olympic-level sprinters (54%) compared with the general Jamaican population (approximately 30%). (Modified from Nakamichi et al., Sci. Trans. Med. 2022.¹⁰⁸⁾)

To examine what influence this PIEZO1 gene polymorphism has on tendon tissue, we created mice expressing Piezo1(R2482H), which mimics this mutation, specifically in tendons. First, we created mice with active-type Piezo1 introduced throughout the body and analyzed their motor function. Surprisingly, without any training, the whole-body type jumped about 1.6 times higher. To identify the tissue that is the factor for this jumping power, we created muscle-specific and tendon-specific active-type Piezo1 mice. The results were even more surprising, as tendon-specific mutant mice had acquired great jumping power similar to whole-body mutant mice (Fig. 10, Supplementary Movie 1), but there was no change in muscle-specific mutant mice. This high jump phenotype was observed in both males and females. Furthermore, maximum speed was also improved in both males and females in whole-body mutant and tendon-specific mutant mice. More notably, we found that even when the active-type Piezo1 gene is replaced after maturation of the musculoskeletal system with tamoxifen administration, jumping ability was enhanced along with tendon tissue enhancement, suggesting that tendons can be drug targets. However, no changes in the muscle itself have been observed in tendon-specific active-type Piezo1 mice so far.

Figure 10.

Impact of PIEZO1 gene polymorphism on tendon tissue and motor function in mouse models. This figure demonstrates the functional effects of active-type PIEZO1 on motor performance through engineered mouse models. The data shows that mice expressing Piezo1(R2482H) throughout their body exhibited approximately 1.6 times higher jumping ability without training compared with control mice. Further tissue-specific analysis revealed that tendon-specific Piezo1(R2482H) expression produced jumping capabilities similar to the whole-body mutant model, whereas muscle-specific expression did not. These findings suggested that the PIEZO1 polymorphism enhances motor function primarily through its effects on tendon tissue rather than muscle. (Modified from Nakamichi et al., Sci. Trans. Med. 2022.¹⁰⁸⁾)

Detailed histological and molecular biological analyses revealed the essence of this phenotype. First, in the tendon tissue of tendon-specific Piezo1(R2482H) mice, the tendons were significantly thicker macroscopically, and the collagen fiber diameter constituting the tendons was significantly thicker at the electron microscope level (Fig. 11).

Figure 11.

Morphological changes in tendon tissue of tendon-specific Piezo1(R2482H) mice. This figure demonstrates the structural adaptations observed in tendon tissue following tendon-specific expression of active-type Piezo1(R2482H). (A) Macroscopic images of tendons from wild-type and mutant mice revealing significantly increased tendon thickness in the Piezo1(R2482H) mice. (B) Electron microscopy images illustrating the enlarged diameter of collagen fibers in mutant tendons compared with controls. (Modified from Nakamichi et al., Sci. Trans. Med. 2022.¹⁰⁸⁾)

Furthermore, in the tendon tissue of tendon-specific Piezo1(R2482H) mice, the expression levels of tendon-specific markers Scx, Mkx, and Tnmd were significantly increased, confirming the molecular basis supporting qualitative improvement of the extracellular matrix (Fig. 12).

Figure 12.

Mechanistic pathway of Piezo1-mediated tendon enhancement: Wild-type vs. gain-of-function. Wild-type (WT) Piezo1 under mechanical stimulation permits moderate Ca²⁺ influx into tendon cells, resulting in baseline expression of tendon-specific transcription factors (Scx, Mkx, Tnmd) and normal tendon development and maintenance. In gain-of-function (GOF) Piezo1, the Piezo1(R2482H) mutation creates hypersensitive channels that allow enhanced Ca²⁺ influx in response to the same mechanical stimuli. This amplified Ca²⁺ signaling substantially increases Mkx expression and other tendon markers, leading to more robust tendon development with thicker collagen fibers and improved extracellular matrix composition, ultimately resulting in enhanced functional performance.

Notably, these changes can be similarly induced not only during the developmental period but also by tendon-specific gene editing in mature mice, suggesting that this pathway could be a therapeutic target. However, no notable changes were observed in muscle tissues such as muscle fiber type distribution or muscle size in tendon-specific Piezo1(R2482H) mice.

This PIEZO1 mutant mimics a polymorphism existing in nature and is positioned as an adaptive mutation rather than a pathological mutation. This point is important, indicating the possibility that genetic diversity in motor ability is neutral or adaptive. This is a finding that matches the research by Suminski and colleagues who investigated the relationship between skeletal muscle characteristics and diseases in risk differences by race and ethnicity.¹¹⁵⁾

Research by Mendias and colleagues comparing the structure of tendons between athletes and the general population showed that sustained exercise loading increases Scx expression in tendon tissue,¹¹⁶⁾ suggesting a relationship between exercise and tendon transcriptional control. Recent research by Götschi and colleagues reported that elite alpine skiers show higher shear wave velocity in the patellar tendon than the general population, with region-specific patterns observed,¹¹⁷⁾ suggesting that adaptive changes in tendons occur according to the type of sports competition.

These studies provide a new perspective on individual differences in motor ability. Conventionally, individual differences in motor ability were often explained by differences in muscle fiber type distribution or cardiopulmonary function, but our research suggests that qualitative differences in tendon tissue are also important factors (Fig. 13).

Figure 13.

Mechanistic pathway of Piezo1-mediated tendon enhancement. This schematic illustration depicts the molecular cascade triggered by mechanical stimulation in tendons. In normal tendons (left panel), mechanical stimuli activate wild-type Piezo1 channels, resulting in moderate Ca²⁺ influx, which induces baseline expression of tendon-specific transcription factors Mkx. In contrast, tendons expressing the gain-of-function (GOF) Piezo1(R2482H) mutation exhibit enhanced mechanosensitivity, leading to increased Ca²⁺ influx upon the same mechanical stimulation, promoting anabolic effects in tendon tissue including increased collagen fiber diameter and improved extracellular matrix quality. This pathway functions both during development and in mature tissue, highlighting its potential as a therapeutic target for tendon enhancement.

In particular, the discovery that changes in tendon biomechanical properties due to PIEZO1 gene polymorphism may directly affect exercise performance is an important finding in sports science.^98,117,118)

We propose the concept of ‘tenopenia’ to describe the age-related decline in tendon quantity and quality, analogous to ‘sarcopenia’ for muscle loss. Tenopenia may underlie tendon-related functional impairments in aging, such as reduced elasticity, injury susceptibility, and joint instability. While sarcopenia has established diagnostic criteria, tenopenia is a new concept that awaits clinical characterization. Similarly, ‘tenokine’ is a proposed term referring to bioactive factors secreted from tendons that may exert paracrine or endocrine effects. Although no specific tenokines have been definitively identified yet, ongoing proteomic and transcriptomic studies aim to uncover such factors with potential roles in systemic physiology.

The discovery of enhanced motor ability through tendon-specific PIEZO1 activation also suggests the possibility of new therapeutic strategies for age-related decline in motor function.

7. Prospects for musculoskeletal research as integrated life science

This paper has outlined the expression and maintenance of motor function, starting from the elucidation of gene expression mechanisms by transcription factors in the late 1980s, through the construction of the EMBRYS database, the identification of RP58 and Mkx, the progress of miRNA research, to the elucidation of the PIEZO1-Mkx pathway. Musculoskeletal research is developing rapidly through the integration of diverse fields such as molecular biology, biomechanics, and regenerative medicine.^88,109)

In particular, tendon tissue has traditionally been viewed as a passive connective tissue simply connecting muscle and bone, but research on the PIEZO1-Mkx pathway seems to be beginning a shift from the static view of “musculoskeletal system as structure” to the dynamic view of “musculoskeletal system as environment-responsive dynamic system”.^109,118)

In addition, while it is known that myokines, physiologically active factors, are released from muscle and affect the whole body, it is not yet known whether there are physiologically active factors from tendon tissue that show paracrine action on surrounding tissues. We are attempting to identify this unknown factor, which we have tentatively named “tenokine”.

Muscle atrophy (sarcopenia), frailty, and locomotive syndrome due to aging are widely recognized as health problems for the elderly.¹¹⁹⁾ However, these disease concepts do not yet sufficiently include the important element of tendon and ligament degeneration. Our research indicates that tendon and ligament tissues play important roles in age-related decline in motor function, both in terms of disease mechanisms and health maintenance. Therefore, we would like to propose “tenopenia” as a new concept to express the decrease and functional decline of tendon tissue, corresponding to sarcopenia, which means muscle reduction. Currently, we are conducting detailed investigations on the relationship between tenopenia and sarcopenia, working on elucidating the molecular mechanisms focusing on the PIEZO1-Mkx pathway. This research may lead to the development of new prevention and treatment strategies for maintaining motor function in the elderly.^85,86,92)

The development of musculoskeletal research requires interdisciplinary collaboration from basic biology to clinical medicine, and further to exercise physiology and sports medicine. Through such an integrated approach, the creation of scientific findings that respond to the social demand for extending healthy lifespans in a super-aging society is expected. It provides answers at the molecular level to the fundamental question of “why exercise is good for health”.⁹⁰⁾

This perspective is thought to contribute to the understanding and respect of biological diversity related to the core of sustainable development goals, the realization of a society with healthy longevity, and the development of science and technology that contributes to improving QOL for the next generation.

Profile

Hiroshi Asahara graduated from Okayama University School of Medicine and completed clinical training in orthopedics before earning his PhD in orthopedic surgery and molecular biology. He conducted postdoctoral research in the laboratory of Dr. Marc Montminy at Harvard University, Cambridge, MA, where he investigated chromatin-mediated gene regulation, and later worked as a staff scientist at the Salk Institute, La Jolla, CA. He established his independent research laboratory as a Principal Investigator at Scripps Research, San Diego, CA, where he continues to lead research into musculoskeletal biology.

From 2004 to 2011, Dr. Asahara served as Director of the Research Institute at the National Center for Child Health and Development (NCCHD) in Tokyo. He currently holds a professorship at Tokyo Medical and Dental University, which was reorganized in 2024 as the Institute of Science Tokyo (IST). There, he leads a laboratory focused on molecular biology and educates medical students and researchers in developmental and regenerative biology.

His research explores the molecular and cellular mechanisms underlying musculoskeletal development, regeneration, and homeostasis, with an emphasis on transcriptional and epigenetic regulation in stem/progenitor cell populations.

Dr. Asahara has received numerous honors, including the Young Scientist Award from the Minister of Education, Culture, Sports, Science and Technology (MEXT) in 2006, the Novartis Prize in Rheumatology (2012), the Japan Osteoporosis Society Scientific Award (2017), the NAM Catalyst Award (2020), the Japan College of Rheumatology Award (2022), and the MEXT Science and Technology Award in 2023. He is also active in international collaborations and plays leadership roles in several research initiatives related to aging and tissue regeneration.