Loss of Mob1a/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD-dependent repression of SOX9.

Hippo signaling is modulated in response to cell density, external mechanical forces, and rigidity of the extracellular matrix (ECM). The Mps one binder kinase activator (MOB) adaptor proteins are core components of Hippo signaling and influence Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ), which are potent transcriptional regulators. YAP1/TAZ are key contributors to cartilage and bone development but the molecular mechanisms by which the Hippo pathway controls chondrogenesis are largely unknown. Cartilage is rich in ECM and also subject to strong external forces - two upstream factors regulating Hippo signaling. Chondrogenesis and endochondral ossification are tightly controlled by growth factors, morphogens, hormones, and transcriptional factors that engage in crosstalk with Hippo-YAP1/TAZ signaling. Here, we generated tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficient mice and show that hyperactivation of endogenous YAP1/TAZ impairs chondrocyte proliferation and differentiation/maturation, leading to chondrodysplasia. These defects were linked to suppression of SOX9, a master regulator of chondrogenesis, the expression of which is mediated by TEAD transcription factors. Our data indicate that a MOB1-dependent YAP1/TAZ-TEAD complex functions as a transcriptional repressor of SOX9 and thereby negatively regulates chondrogenesis.

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells.

Focal articular cartilage damage can eventually lead to the onset of osteoarthritis with degradation around healthy articular cartilage. Currently, there are no drugs available that effectively repair articular cartilage damage. Several surgical techniques exist and are expected to prevent progression to osteoarthritis, but they do not offer a long-term clinical solution. Recently, regenerative medicine approaches using human pluripotent stem cells (PSCs) have gained attention as new cell sources for therapeutic products. To translate PSCs to clinical application, appropriate cultures that produce large amounts of chondrocytes and hyaline cartilage are needed. So too are assays for the safety and efficacy of the cellular materials in preclinical studies including animal transplantation models. To confirm safety and efficacy, transplantation into the subcutaneous space and articular cartilage defects have been performed in animal models. All but one study we reviewed that transplanted PSC-derived cellular products into articular cartilage defects found safe and effective recovery. However, for most of those studies, the quality of the PSCs was not verified, and the evaluations were done with small animals over short observation periods. Large animals and longer observation times are preferred. We will discuss the recent progress and future direction of the animal transplantation studies for the treatment of focal articular cartilage damages using PSCs.

Statin treatment rescues FGFR3 skeletal dysplasia phenotypes.

Gain-of-function mutations in the fibroblast growth factor receptor 3 gene (FGFR3) result in skeletal dysplasias, such as thanatophoric dysplasia and achondroplasia (ACH). The lack of disease models using human cells has hampered the identification of a clinically effective treatment for these diseases. Here we show that statin treatment can rescue patient-specific induced pluripotent stem cell (iPSC) models and a mouse model of FGFR3 skeletal dysplasia. We converted fibroblasts from thanatophoric dysplasia type I (TD1) and ACH patients into iPSCs. The chondrogenic differentiation of TD1 iPSCs and ACH iPSCs resulted in the formation of degraded cartilage. We found that statins could correct the degraded cartilage in both chondrogenically differentiated TD1 and ACH iPSCs. Treatment of ACH model mice with statin led to a significant recovery of bone growth. These results suggest that statins could represent a medical treatment for infants and children with TD1 and ACH.

Changes in acetyl-CoA mediate Sik3-induced maturation of chondrocytes in endochondral bone formation.

The maturation of chondrocytes is strictly regulated for proper endochondral bone formation. Although recent studies have revealed that intracellular metabolic processes regulate the proliferation and differentiation of cells, little is known about how changes in metabolite levels regulate chondrocyte maturation. To identify the metabolites which regulate chondrocyte maturation, we performed a metabolome analysis on chondrocytes of Sik3 knockout mice, in which chondrocyte maturation is delayed. Among the metabolites, acetyl-CoA was decreased in this model. Immunohistochemical analysis of the Sik3 knockout chondrocytes indicated that the expression levels of phospho-pyruvate dehydrogenase (phospho-Pdh), an inactivated form of Pdh, which is an enzyme that converts pyruvate to acetyl-CoA, and of Pdh kinase 4 (Pdk4), which phosphorylates Pdh, were increased. Inhibition of Pdh by treatment with CPI613 delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture. These results collectively suggest that decreasing the acetyl-CoA level is a cause and not result of the delayed chondrocyte maturation. Sik3 appears to increase the acetyl-CoA level by decreasing the expression level of Pdk4. Blocking ATP synthesis in the TCA cycle by treatment with rotenone also delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture, suggesting the possibility that depriving acetyl-CoA as a substrate for the TCA cycle is responsible for the delayed maturation. Our finding of acetyl-CoA as a regulator of chondrocyte maturation could contribute to understanding the regulatory mechanisms controlling endochondral bone formation by metabolites.

A-674563 increases chondrocyte marker expression in cultured chondrocytes by inhibiting Sox9 degradation.

The implantation of autologous chondrocytes is a therapeutic treatment for articular cartilage damage. However, the benefits are limited due to the expansion of chondrocytes in monolayer culture, which causes loss of chondrocytic characters. Therefore, culture conditions that enhance chondrocytic characters are needed. We screened 5822 compounds and found that A-674563 enhanced the transcription of several chondrocyte marker genes, including Col2a1, Acan and Col11a2, in mouse primary chondrocytes. Experiments using cycloheximide, MG132 and bafilomycin A1 have revealed that Sox9 is degraded through the ubiquitin-proteasome pathway and that A-674563 inhibits this degradation, resulting in larger amount of Sox9 protein. RNA sequencing transcriptome analysis showed that A-674563 increases the expression of the gene that encodes ubiquitin-specific peptidase 29, which is known to induce the deubiquitination of proteins. Although the precise mechanism remains to be determined, our findings indicated that A-674563 could contribute to culture conditions that expand chondrocytes without losing chondrocytic characters.

[Cartilage/chondrocyte research and osteoarthritis. Regeneration of articular cartilage damage using iPS cell-derived cartilage.]

Induced pluripotent stem (iPS)cells have capacities of self-renewal and pluripotency. We have developed a method to differentiate human iPS cells toward chondrocytes, followed by the creation of cartilage tissue composed of chondrocytes and cartilage extracellular matrix. The mechanism through which tissue transplantation repairs cartilage defects involves the transplant itself constituting the repair tissue. Human iPS cell-derived cartilage has low immunogenicity and can be transplanted in an allogeneic manner. We are conducting pre-clinical tests on iPS cell-derived cartilage to verify efficacy and safety that will act as a basis for clinical tests.

Modeling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cells.

Type II collagen is a major component of cartilage. Heterozygous mutations in the type II collagen gene (COL2A1) result in a group of skeletal dysplasias known as Type II collagenopathy (COL2pathy). The understanding of COL2pathy is limited by difficulties in obtaining live chondrocytes. In the present study, we converted COL2pathy patients' fibroblasts directly into induced chondrogenic (iChon) cells. The COL2pathy-iChon cells showed suppressed expression of COL2A1 and significant apoptosis. A distended endoplasmic reticulum (ER) was detected, thus suggesting the adaptation of gene expression and cell death caused by excess ER stress. Chondrogenic supplementation adversely affected the chondrogenesis due to forced elevation of COL2A1 expression, suggesting that the application of chondrogenic drugs would worsen the disease condition. The application of a chemical chaperone increased the secretion of type II collagen, and partially rescued COL2pathy-iChon cells from apoptosis, suggesting that molecular chaperons serve as therapeutic drug candidates. We next generated induced pluripotent stem cells from COL2pathy fibroblasts. Chondrogenically differentiated COL2pathy-iPS cells showed apoptosis and increased expression of ER stress-markers. Finally, we generated teratomas by transplanting COL2pathy iPS cells into immunodeficient mice. The cartilage in the teratomas showed accumulation of type II collagen within cells, a distended ER, and sparse matrix, recapitulating the patient's cartilage. These COL2pathy models will be useful for pathophysiological studies and drug screening.

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

[iPS cells for the generation of cartilage and for regenerative medicine and disease modeling of cartilage diseases].

The development of induced pluripotent stem cells(iPSCs)has enabled the acquisition of patient-specific chondrocytes by converting somatic cells, such as dermal fibroblasts or blood cells, from patients to iPSCs and then differentiating them toward chondrocytes. We can further generate cartilage tissue from iPSC-derived chondrocytes. Studies on iPSC-derived chondrocytes/cartilage for the regeneration of articular cartilage injury are ongoing. These studies will in the future use autologous iPSCs and allogenic iPSCs from an iPSC stock prepared from donor cells. Drug discovery research for related diseases such as skeletal dysplasia is also being conducted.

SIK3 is essential for chondrocyte hypertrophy during skeletal development in mice.

Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.

[Analysis of Musculoskeletal Systems and Their Diseases. Use of iPS cells in regenerative therapy and drug discovery for cartilage diseases].

Induced pluripotent stem (iPS) cells can be generated by transiently expressing defined factors in somatic cells such as dermal fibroblasts and blood cells and culturing them in specific medium. iPS cells are expected to provide new tools for research and development of therapies for various diseases because of two important properties : 1) they can be differentiated into any type of somatic cell (pluripotency) and 2) they can be expanded infinitely (self renew). Research for the transplantation of iPS cell-derived cartilage/chondrocytes and drug discovery by iPS cell-based disease modeling are ongoing.

Cyp26b1 within the growth plate regulates bone growth in juvenile mice.

Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo. However, it has not been addressed whether a RA gradient also exists and functions in organs after birth. We found localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1(Δchon) cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. These results collectively suggest that Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.

Cartilage intermediate layer protein promotes lumbar disc degeneration.

Lumbar disc disease (LDD) is one of the most common musculoskeletal disorders, and accompanies intervertebral disc degeneration. CILP encodes cartilage intermediate layer protein, which is highly associated with LDD. Moreover, CILP inhibits transcriptional activation of cartilage matrix genes in nucleus pulposus (NP) cells in vitro by binding to TGF-ß1 and inhibiting the phosphorylation of Smads. However, the aetiology and mechanism of pathogenesis of LDD in vivo are unknown. To demonstrate the role of CILP in LDD in vivo, we generated transgenic mice that express CILP specifically in the intervertebral disc tissues and assessed whether CILP exacerbates disc degeneration. Degeneration of the intervertebral discs was assessed using magnetic resonance imaging (MRI) and histology. The level of phosphorylation of Smad2/3 in intervertebral discs was measured to determine whether overexpressed CILP suppressed TGF-beta signalling. Although the macroscopic skeletal phenotype of transgenic mice appeared normal, histological findings revealed significant degeneration of lumbar discs. MRI analysis of the lumbar intervertebral discs indicated a significantly lower signal intensity of the nucleus pulposus where CILP was overexpressed. Intervertebral disc degeneration was also observed. The number of phosphorylation of Smad2/3 immuno-positive cells in the NP significantly was decreased in CILP transgenic mice compared with normal mice. In summary, overexpression of CILP in the NP promotes disc degeneration, indicating that CILP plays a direct role in the pathogenesis of LDD.

Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways.

During endochondral bone formation, Sox9 expression starts in mesenchymal progenitors, continues in the round and flat chondrocyte stages at high levels, and ceases just prior to the hypertrophic chondrocyte stage. Sox9 is important in mesenchymal progenitors for their differentiation into chondrocytes, but its functions post-differentiation have not been determined. To investigate Sox9 function in chondrocytes, we deleted mouse Sox9 at two different steps after chondrocyte differentiation. Sox9 inactivation in round chondrocytes resulted in a loss of Col2a1 expression and in apoptosis. Sox9 inactivation in flat chondrocytes caused immediate terminal maturation without hypertrophy and with excessive apoptosis. Inactivation of Sox9 in the last few cell layers resulted in the absence of Col10a1 expression, suggesting that continued expression of Sox9 just prior to hypertrophy is necessary for chondrocyte hypertrophy. SOX9 knockdown also caused apoptosis of human chondrosarcoma SW1353 cells. These phenotypes were associated with reduced Akt phosphorylation. Forced phosphorylation of Akt by Pten inactivation partially restored Col10a1 expression and cell survival in Sox9(floxdel/floxdel) mouse chondrocytes, suggesting that phosphorylated Akt mediates chondrocyte survival and hypertrophy induced by Sox9. When the molecular mechanism of Sox9-induced Akt phosphorylation was examined, we found that expression of the PI3K subunit Pik3ca (p110α) was decreased in Sox9(floxdel/floxdel) mouse chondrocytes. Sox9 binds to the promoter and enhances the transcriptional activities of Pik3ca. Thus, continued expression of Sox9 in differentiated chondrocytes is essential for subsequent hypertrophy and sustains chondrocyte-specific survival mechanisms by binding to the Pik3ca promoter, inducing Akt phosphorylation.

Subpopulations of fibroblasts derived from human iPS cells.

Organ fibrosis causes collagen fiber overgrowth and impairs organ function. Cardiac fibrosis after myocardial infarction impairs cardiac function significantly, pulmonary fibrosis reduces gas exchange efficiency, and liver fibrosis disturbs the natural function of the liver. Its development is associated with the differentiation of fibroblasts into myofibroblasts and increased collagen synthesis. Fibrosis has organ specificity, defined by the heterogeneity of fibroblasts. Although this heterogeneity is established during embryonic development, it has not been defined yet. Fibroblastic differentiation of induced pluripotent stem cells (iPSCs) recapitulates the process by which fibroblasts acquire diversity. Here, we differentiated iPSCs into cardiac, hepatic, and dermal fibroblasts and analyzed their properties using single-cell RNA sequencing. We observed characteristic subpopulations with different ratios in each organ-type fibroblast group, which contained both resting and distinct ACTA2+ myofibroblasts. These findings provide crucial information on the ontogeny-based heterogeneity of fibroblasts, leading to the development of therapeutic strategies to control fibrosis.

Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification.

Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

[Cartilage regeneration using cell reprogramming technologies].

Injuries to articular cartilage do not heal spontaneously, and when left untreated, result in the diffuse degeneration of cartilage. Autologous chondrocyte transplantation has been performed to treat focal articular cartilage defects, but the repaired tissue includes fibrous tissue. Healing with hyaline cartilage has been difficult to achieve. The generation of induced pluripotent stem (iPS) cells has enabled us to rejuvenate somatic cells and provide them with pluripotency. This technology may contribute to producing hyaline cartilage. In addition, fibroblasts can be converted toward chondrogenic cells by directed reprogramming technology. Research aimed at the realization of cartilage regeneration using cell reprogramming technologies is underway.

[Stem cell aging and the implications for stem cell-based therapies for aging-related diseases and aged tissues].

Adult stem cells exist in most mammalian tissues to maintain their homeostasis and help repair them. Reductions in adult stem cell function and/or number are clearly associated with aging, however, the causal correlations between such findings and the effects of aging are largely unknown. Some stem cell functional changes, such as the loss of lineage specificity and self-renewal capacity, senescence and transformation, arise in stem cells autonomously during the aging process. These autonomous changes of stem cell functions reflect the damaging effects of age on the genome, epigenome, and proteome. Other stem cell functional changes are influenced by the age-related changes in the local microenvironments (niches) or systemic environments. If stem cell-based therapy can be used not only for age-related degenerative diseases, but also normal functional declines associated with aging, consideration of the behavior of stem cells based on effects from the local microenvironments (niches) and systemic environments in older individuals will therefore be needed.

Regeneration of joint surface defects by transplantation of allogeneic cartilage: application of iPS cell-derived cartilage and immunogenicity.

BACKGROUND: Because of its poor intrinsic repair capacity, articular cartilage seldom heals when damaged. MAIN BODY: Regenerative treatment is expected for the treatment of articular cartilage damage, and allogeneic chondrocytes or cartilage have an advantage over autologous chondrocytes, which are limited in number. However, the presence or absence of an immune response has not been analyzed and remains controversial. Allogeneic-induced pluripotent stem cell (iPSC)-derived cartilage, a new resource for cartilage regeneration, reportedly survived and integrated with native cartilage after transplantation into chondral defects in knee joints without immune rejection in a recent primate model. Here, we review and discuss the immunogenicity of chondrocytes and the efficacy of allogeneic cartilage transplantation, including iPSC-derived cartilage. SHORT CONCLUSION: Allogeneic iPSC-derived cartilage transplantation, a new therapeutic option, could be a good indication for chondral defects, and the development of translational medical technology for articular cartilage damage is expected.

Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone.

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes. Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture. Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell-cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels. Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.